Significance of PLCE1 gene polymorphism detection in children with primary nephrotic syndrome in Guangxi Zhuang Autonomous Region / 中华实用儿科临床杂志

Objective:To investigate the correlation between the single nucleotide polymorphism (SNP) of the PLCE1 gene and children with primary nephrotic syndrome (PNS) in Guangxi Zhuang Autonomous Region. Methods:This study was a retrospective study, a case-control study was used to select 155 cases of PNS in Guangxi Zhuang children attending the Affiliated Hospital of Youjiang Medical University for Nationalities from January 2017 to January 2021 (PNS group), and 100 healthy Guangxi Zhuang children who were physically examined during the same period (healthy control group). Genotyping of PLCE1 SNP rs3765524, and rs2274223 were performed using the second-generation gene sequencing technology, and their correlation with the development of PNS was analyzed. Logistic regression analysis was used for correlation analysis, and Chi- square test or Fisher′ s exact probability method was used for comparison between groups. Results:(1)Compared with the healthy control group, PLCE1 rs3765524 was correlated with the risk of PNS in children of PNS group, and the TT genotype may reduce the risk of PNS in the co-dominant model ( OR=0.435, 95% CI: 0.238-0.794, P=0.007). There were no significant differences in the genotype of PLCE1 rs2274223 and the frequency of allele distribution between PNS group and healthy control group (all P>0.05). (2) A strong linkage disequilibrium existed at PLCE1 SNP rs3765524 and rs2274223.(3) There were no significant differences in the frequency of the distribution of haplotypes AC, AT and GT between PNS group and healthy control group (all P>0.05). Conclusions:PLCE1 SNP rs3765524 is correlated with the risk of PNS in children in Guangxi Zhuang Autonomous Region, and the TT genotype may be a protective factor for PNS in children in Guangxi Zhuang Autonomous Region.

Intervention Effect of Qufeng Gutong Babu Ointment on Rat Model of Osteoarthritis with Cold-dampness Obstruction Based on PI3K/Akt Signal Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the intervention effect of Qufeng Gutong Babu ointment （QFGT） on rats with osteoarthritis （OA） with cold-dampness obstruction， and preliminarily clarify its mechanism. MethodSD male rats were divided into 6 groups， namely， the blank group， model group， positive control drug Huoxue Zhitong ointment （HXZTG） group （1.26 cm2·d-1）， and low， medium， and high-dose QFGT group （75， 150， 300 mg·d-1）. OA model was prepared by joint cavity injection of papain and L-cysteine. On the second day of modeling， climate factors were applied to establish an animal model of combination of disease and syndrome of OA rats with cold-dampness obstruction. Standard VonFrey fiber was used to evaluate the threshold of mechanical pain. Weight bearing difference score and joint function score of both hind limbs were recorded. Hematoxylin-eosin （HE） staining and safranine fixation green staining were used to observe the pathological changes and cartilage degeneration of rat knee joint. Immunohistochemistry （IHC） was used to detect the expression of interleukin-1β （IL-1β）， interleukin-8 （IL-8）， tumor necrosis factor-α （TNF-α）， matrix metalloproteinase-9 （MMP-9）， and cathepsin K （CTSK）. Western blot was used to detect the protein expression of kinase B （Akt）， phosphorylated protein kinase B （p-Akt）， phosphatidylinositol 3-kinase （PI3K）， nuclear factor 1 （NFATc1）， MMP-9， and CTSK in T cells. ResultCompared with the normal group， the model group showed significant mechanical pain sensitivity reaction after modeling （P<0.01）， and the weight bearing difference of both hind limbs and joint function score were significantly increased （P<0.05， P<0.01）. Compared with the model group， both the high-dose QFGT group and the HXZTG group significantly reduced the mechanical pain sensitivity， weight difference， and joint function score of rats （P<0.05， P<0.01）， and the medium-dose QFGT group also improved the joint function to a certain extent， and the degeneration of the knee joint cartilage of rats was significantly reduced （P<0.05， P<0.01）. QFGT and HXZTG both inhibited the protein expression of IL-1β， IL-8， TNF-α， MMP-9， CTAK， PI3K， p-Akt， Akt， and other related proteins in articular cartilage of rats with OA to a certain extent （P<0.05， P<0.01）. ConclusionQFGT can inhibit the release of inflammatory factors and matrix metalloproteinases by inhibiting the PI3K/Akt signal pathway in articular articular cartilage of rats with OA with cold-dampness obstruction， thus ultimately weakening local cartilage degeneration and improving joint function.

Repair Mechanism of Jianpi Huogu Prescription on Vascular Injury in Alcohol-induced Osteonecrosis of Femoral Head Based on VEGF/VEGFR2/PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

MethodIn the experiment， 46% vol Red Star Erguotou （10 mL·kg·d-1） was used to establish the AONFH rat model， and the intervention effect of JPHGP at different doses （2.5， 5.0， 10.0 g·kg-1） was observed. Jiangusheng pill （JGS， 1.53 g·kg-1） was selected as the positive control. After 8 weeks of administration， the bone histomorphometry of the femoral head was analyzed by Micro-CT imaging， and the area of medullary microvessels in the femoral head was detected by ink perfusion. The pathological change was observed by hematoxylin and eosin （HE） staining. The protein expressions of Platelet endothelial cell adhesion molecule-1 （CD31）， VEGF， VEGFR2， PI3K， phosphor-Akt （p-Akt） and phosphatase and Tensin homologue deleted on chromosome 10 （PTEN） in the femoral head were determined by immunohistochemistry and Western blot. ResultCompared with normal group， the model group presented the fracture and thinning of trabeculae in the femoral head， increased empty bone lacunae， and elevated number and diameter of adipocytes （P<0.01）. Micro-CT imaging revealed a decrease in bone mineral density （BMD）， bone volume fraction （BV/TV）， trabecular thickness （Tb.Th） and trabecular number （Tb.N） （P<0.05， P<0.01） while an increase in bone surface-to-volume ratio （BS/BV） and trabecular separation （Tb.Sp） （P<0.01）. The results of ink perfusion showed that the area of medullary microvessels in the femoral head was reduced （P<0.01）. Compared with model group， JPHGP lowered the empty bone lacunae rate as well as the number and diameter of adipocytes in the femoral head of AONFH rats. Micro-CT imaging indicated that JPHGP low-dose group had elevated BV/TV， Tb.Th and Tb.N （P<0.05， P<0.01） while decreased BS/BV （P<0.01）， and there was an upward trend in BMD while a downward trend in Tb.Sp， but without statistical difference. In addition， JPHGP medium- and high-dose groups had a rise in BMD， BV/TV， Tb.Th and Tb.N （P<0.05， P<0.01）， a decrease in BS/BV and Tb.Sp （P<0.05， P<0.01） and enlarged area of medullary microvessels in the femoral head （P<0.05， P<0.01）. The expressions of CD31， VEGF， VEGFR2， PI3K， p-Akt in the model group were lower than those in the normal group （P<0.01）， and after medium and high doses of JPHGP treatment， the expressions of CD31， PI3K and p-Akt in the femoral head of rats were up-regulated （P<0.01） while the protein expression of PTEN was down-regulated （P<0.01）. Moreover， JPHGP up-regulated the expressions of VEGF and VEGFR2 （P<0.05， P<0.01）. ConclusionJPHGP can repair the vascular injury in AONFH， and its mechanism may be related to the activation of VEGF/VEGFR2/PI3K/Akt signaling pathway. This study provides certain scientific basis and reference for the clinical application of JPHGP. ObjecctiveTo observe the repair effect of Jianpi Huogu prescription （JPHGP） on vascular injury in experimental alcohol-induced osteonecrosis of femoral head （AONFH）， and to explore its mechanism based on vascular endothelial growth factor （VEGF）/VEGFR2/phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway.

Exploration on "Efficacy-toxicity" Association Mechanisms of Tripterygium wilfordii Polyglycoside Tablets Against Rheumatoid Arthritis Based on Multi-omics Integrated Regulatory Network / 中国实验方剂学杂志

ObjectiveTo explore the "efficacy-toxicity" association mechanisms of Tripterygium wilfordii polyglycoside tablets （TWPT） by establishing and analyzing an interaction network associated with the clinical efficacy of TWPT in the treatment of rheumatoid arthritis （RA） and TWPT-induced liver injury. MethodOn the basis of the TWPT efficacy-related gene expression profile and TWPT-induced liver injury-related protein expression profile which were both obtained from our clinical cohorts， the "efficacy-toxicity" association network of TWPT was constructed， and the key network targets were identified by calculating the topological values of the nodes， including the degree， closeness and betweenness. After that， the biological functions and pathways of the key network targets were investigated by enrichment analysis. ResultA total of 119 differentially expressed genes （58 up-regulated and 61 down-regulated） between RA patients with TWPT well and weak response were identified as TWPT efficacy-related genes by clinical transcriptomics， and 49 differentially expressed proteins （36 up-regulated and 13 down-regulated） were demonstrated to be TWPT-induced liver injury-related proteins by clinical proteomics. In addition， the clinical symptom enrichment analysis indicated that the TWPT efficacy-related genes were significantly associated with various clinical symptoms of arthralgia in traditional Chinese medicine and clinical phenotypes of modern medicine， and most of the TWPT-induced liver injury-related proteins were involved in digestive system abnormalities. Therefore， the aforementioned multi-omics data represented the main clinical symptoms of TWPT treating RA and inducing liver injury. Mechanically， the "efficacy-toxicity" association network revealed that both TWPT efficacy-related genes and TWPT-induced liver injury-related core proteins were involved in the "immune-inflammatory" imbalance， especially playing an important role in neutrophil degranulation， complement cascade reaction， and immune-inflammatory response mediated by protein post-translational modification. Notably， the above genes and proteins were also enriched in various signaling pathways related to cell proliferation and cell cycle regulation， such as RAS and mitogen-activated protein kinase （MAPK） signaling pathway， and in several liver functional processes， such as glycogen metabolism and redox reaction. ConclusionThis study systematically explained the "efficacy-toxicity" association characteristics and molecular mechanisms of TWPT by applying a research strategy integrating clinical phenomics， transcriptomics and proteomics， laying a good data foundation for exploring the "efficacy enhancing and toxicity-reducing" mechanisms of TWPT.

Mechanism of Glycyrrhizae Radix et Rhizoma Alleviating Tripterygium wilfordii Polyglycoside Tablets-induced Liver Injury / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of cytochrome P4502D6 （CYP2D6） and cytochrome P4503A4 （CYP3A4）， key enzymes of drug metabolism in liver， on acute liver injury in water extract of Glycyrrhizae Radix et Rhizoma （WEOGRR）. MethodHealthy male Kunming mice were divided into normal group， model group， WEOGRR low-， medium- and high-dose groups （5， 10， 15 g·kg-1·d-1） and positive drug group （diammonium glycyrrhizinate， 75 mg·kg-1·d-1）， with 10 in each group. One week after preventive administration， acute liver injury model was induced by single intragastric administration of 270 mg·kg-1 Tripterygium Glycosides tablets， and samples were collected after 18 h. The pathological changes of liver were observed by hematoxylin-eosin （HE） staining. Serum liver function indexes including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptadase （γ-GT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） as well as the levels of oxidative stress indexes including malondialdehyde （MDA） and superoxide dismutase （SOD） in hepatocytes were determined by biochemical method. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to detect the mRNA and protein expression levels of CYP2D6 and CYP3A4， respectively. ResultCompared with normal group， model group had significant hepatocyte swelling and inflammatory cell infiltration （P<0.01）， increased AST， ALT， γ-GT， ALP and TBIL （P<0.05）， elevated MDA and decreased SOD （P<0.01） as well as down-regulated mRNA and protein expression levels of CYP2D6 and CYP3A4 （P<0.05）. Compared with the model group， the normal group had intact liver structure without obvious abnormality， and the WEOGRR groups and positive drug group presented alleviated hepatocyte swelling and inflammatory cell infiltration （P<0.01）， reduced AST， ALT， γ-GT， ALP and TBIL （P<0.01）， lowered MDA and increased SOD （P<0.01） as well as up-regulated expression levels of CYP2D6 and CYP3A4 （P<0.01）. ConclusionThe protective effect of WEOGRR on acute liver injury induced by Tripterygium glycosides tablets may be related to reducing the contents of AST， ALT， γ-GT， ALP and TBIL in serum， inhibiting MDA and increasing the activity of SOD in liver cells， and enhancing the activities of CYP2D6 and CYP3A4， thus accelerating the metabolism of toxic substances.

Effect and Mechanism of Cuscutae Semen Total Flavonoids Combined with Tripterygium wilfordii Polyglycoside Tablets on Ovarian Germline Stem Cells of Physiological Mice by Notch Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Cuscutae Semen total flavonoids combined with Tripterygium wilfordii polyglycoside tablets （TWPT） on ovarian germline stem cells of female physiological mice through neurogenic locus notch homolog （Notch） signaling pathway. MethodSixty female Kunming mice （5 weeks old） were randomly divided into normal group， Tripterygium wilfordii polyglycoside tablets low-， high-dose groups （13.65 mg·kg-1·d-1 and 27.3 mg·kg-1·d-1， 1 and 2 times clinical equivalent dose）， Cuscutae Semen total flavonoids low- and high-dose groups （150 mg·kg-1·d-1 and 300 mg·kg-1·d-1）， and combination group （13.65 mg·kg-1·d-1 TWPT and 150 mg·kg-1·d-1 Cuscutae Semen total flavonoids）， with 10 in each group. After 3 weeks of continuous administration， the uterus/brain and ovarian/brain indexes were calculated， and the pathological changes of ovarian tissue were observed under light microscope. The content of estradiol in serum was determined by enzyme linked immunosorbent assay （ELISA）. Immunofluorescence assay was performed to observe the expressions of germline stem cell markers in ovarian epithelium， including mouse vasa homologue （Mvh）， octamer-binding transcription factor 4 （Oct4）， tyrosine-protein kinase receptor （c-kit）， Nanog， Notch signaling pathway molecules， neurogenic locus notch homolog protein 1 （Notch1）， hes family BHLH transcription factor 1（Hes1）， and jagged canonical Notch ligand 1 （JAG1）. ResultCompared with the normal group， low and high doses of TWPT had no significant effect on the uterus/brain and ovary/brain indexes and the uterus and ovary morphologies of mice， while only the number of atretic follicles was increased （P<0.01）. The expressions of ovarian germline stem cell markers and Notch signaling pathway molecules had a decreasing trend in TWPT low-dose group， while the expressions of Mvh， c-kit， and Nanog were down-regulated （P<0.05， P<0.01） and the expressions of Notch1 and Hes1 were also reduced （P<0.01） in TWPT high-dose group. However， the above indexes were increased in Cuscutae Semen total flavonoids low-dose group （P<0.05， P<0.01）. Compared with the low does of TWPT group， the combination group had a decrease in the increased number of atretic follicles （P<0.01）， an improvement in the down-regulated expressions of Mvh and Nanog （P<0.01）， and an increase in the expressions of Notch1 and Hes1 （P<0.05， P<0.01）. ConclusionOvarian germline stem cells are the source target of the reproductive toxicity of TWPT. Cuscutae Semen total flavonoids participate in the regulation of the germline stem cell pathways to alleviate the reproductive toxicity caused by TWPT， and its mechanism of action may be related to the Notch signaling pathway.

Systematic Review on Safety of Chinese Medicines Combined with Tripterygium wilfordii Preparations in Treatment of Rheumatoid Arthritis and Exploration on Underlying Network Regulatory Mechanisms of Enhancing Efficacy and Reducing Toxicity / 中国实验方剂学杂志

ObjectiveTo systematically evaluate the safety of Chinese medicines combined with Tripterygium wilfordii polyglycoside tablets/Tripterygium wilfordii tablets （TWPT/TWT） in the treatment of rheumatoid arthritis （RA）， and to explore the network regulatory mechanisms of enhancing efficacy and reducing toxicity of commonly used combination regimes. MethodThe literature involving the adverse reactions of TWPT/TWT in treating RA was searched and collected from three Chinese databases （CNKI， Wanfang Data， VIP） and three English databases （PubMed， Cochrane Library， Embase） from the inception of the databases to July 2021. All studies were assessed by the Cochrane risk of bias tool， and the data were extracted and analyzed by Stata 15.0. Furthermore， Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine 2.0 （TCMIP v2.0，http：//www.tcmip.cn/） was used to construct a "drug target-symptom gene of efficacy and toxicity" interaction network， to explore the underlying network regulatory mechanisms of enhancing efficacy and reducing toxicity of common T. wilfordii preparation combinations. ResultA total of 2 132 articles on Chinese medicines combined with TWPT/TWT in the treatment of RA were retrieved， and 18 of them were finally included. The systematic review showed that the adverse reactions of TWPT/TWT against RA mainly occurred in the digestive system， blood system， and reproductive system， of which digestive system had the highest incidence of damages. However， the combination with Chinese medicines effectively alleviated the adverse reactions caused by TWPT/TWT ［RR （95% CI）=0.45 （0.30， 0.66）， P<0.01］. In addition， the subgroup analysis indicated that the age of RA patients， course of disease， combination regimen， medication dosage and duration of treatment all affected the occurrence of adverse reactions of TWPT/TWT. It was found in clinical studies that total glucosides of paeony （TGP） and TWPT/TWT was most widely combined， and the effect of TGP in reducing TWPT/TWT-induced hepatotoxicity was also more significant than that of other Chinese medicines. Moreover， taking this combination regime as an example， this paper explored the "efficacy-toxicity" association mechanisms of TGP-TWPT/TWT against RA. The "drug target-symptom gene of efficacy and toxicity" interaction network revealed that the core network targets of TGP-TWPT/TWT enhanced efficacy and reduced toxicity mainly through regulating immunity-inflammation-related pathways， metabolic pathways and cell signal transduction. Especially， interleukin-4 （IL-4） and interleukin-13 （IL-13）， which were involved in the "immunity-inflammation" module， were the common targets of TGP-TWPT/TWT to enhance efficacy and reduce toxicity. The endogenous sterols， bile acids and bile salts， insulin secretion and other metabolic pathways in the "body metabolism" module were closely associated with the mechanisms of TWPT/TWT inducing hepatotoxicity and TGP reducing hepatotoxicity. While cell function regulation pathways， such as stem cell factor （SCF）/tyrosine kinase receptor （KIT） signaling pathway and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）signaling pathway were involved in both anti-RA effects and hepatotoxicity of TWPT/TWT. ConclusionClinical application of suitable Chinese medicines combined with TWPT/TWT in the treatment of RA can effectively improve the rheumatism and reduce the adverse reactions of TWPT/TWT， and TGP-TWPT/TWT has the most significant toxicity-reducing effect. Further biological network-based investigation indicates that the toxicity-reducing mechanism of TGP-TWPT/TWT may be related to the regulation of interleukin signaling pathway and bile acid metabolism pathway， and the synergistic efficacy-enhancing effect of the combination may be achieved by acting on interleukin signaling pathway and cell function regulation pathway.

Intervention effect of Qufeng Gutong Cataplasm on myofascial pain syndrome in rats and its mechanism / 中国中药杂志

This paper aims to investigate the intervention effect of Qufeng Gutong Cataplasm(QFGT) on myofascial pain syndrome(MPS) in rats and to preliminarily explain its mechanism from the perspective of improving muscle inflammation and pain. Male SD rats were divided into 6 groups, namely normal group, model group, positive control drug(Huoxue Zhitong Ointment, HXZT) group, and low, medium, and high-dose QFGT groups(75, 150, and 300 mg·d~(-1)). The rat model of MPS was established by striking combined with centrifugation for 8 weeks, during which QFGT and HXZT were used for corresponding intervention. Standard VonFrey fiber was used to evaluate the mechanical pain threshold, and acetone was used to detect the cold pain threshold. The electrophysiological activity of muscle at trigger point was detected, and the electromuscular analysis of trigger point was performed. CatWalk gait analyzer was used to detect pain-induced gait adaptation changes. The hematoxylin-eosin(HE) staining was used to observe the pathological changes in muscle and skin tissues at the trigger point of rats. Immunohistochemistry was used to detect the expression of capsaicin receptor transient receptor potential vanilloid 1(TRPV1) in muscle tissues and interleukin(IL)-33 in skin tissues at the trigger point. The protein expression levels of TRPV1, protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), IL-1β, and tumor necrosis factor-α(TNF-α) in muscle tissues at the trigger point were detected by Western blot. The results showed that as compared with the model group, the mechanical pain threshold and cold pain threshold of rats in other groups were increased after treatment with QFGT. The spontaneous electromyography(EMG) activity was observed in the model group, but QFGT alleviated the EMG activity in a dose-dependent manner. Gait analysis showed that standing duration, average intensity, swing speed, maximum contact point, maximum contact area, paw print length, paw print width, and paw print area were significantly improved in all QFGT groups. Pathological results showed that the disorder of muscle arrangement at the trigger point was decreased, muscle fiber adhesion and atrophy were reduced, and inflammatory cell infiltration was alleviated after treatment with QFGT. In addition, QFGT and HXZT both inhibited the protein expression of TRPV1, PI3K, Akt, p-Akt, IL-1β, and TNF-α in the muscle tissues of rats with MPS. However, there was no significant difference in the pathological structure and expression of IL-33 in the treated skin as compared with the normal group. The related results have proved that QFGT can inhibit the release of inflammatory factors by inhibiting the TRPV1/PI3K/Akt signaling pathway in the muscle trigger point of rats with MPS and finally attenuate the atrophy and adhesion of local muscles and inflammatory infiltration, thereby relieving the muscle pain of rats with MPS, and local administration has no skin irritation.

Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology / 中国中药杂志

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.

Comparative study on pathological characteristics of four different antigen-induced rheumatoid arthritis mouse models / 药学学报

Rheumatoid arthritis (RA) is an autoimmune disease driven by antigens and mediated by T cells. Collagen II (CII) and fibrinogen (Fib) are the two main antigens in the pathogenesis of RA. The antigen produced after citrulline modification (Cit) is also one of the inducements to induce the body to produce a pathogenic anti-citrulline protein antibody (ACPA). To provide a reference for RA-related research, this study intends to establish an RA animal model by using CII, Cit-CII, Fib, and Cit-Fib antigens, emulsification with complete Freund's adjuvant and immunization with DBA/1 mice, respectively, to compare the pathological characteristics of RA models induced by different antigens from the aspects of pathology, imaging and serum biochemistry. Animal welfare and experimental process are in accordance with the regulations of the Experimental Animal Ethics Committee of the China Academy of Chinese Medical Sciences. The results showed that the CII, Cit-CII, and Cit-Fib induced mice all had symptoms such as joint redness and swelling, and toe deformation and the clinical score and incidence rate were higher than those of the normal group. The CII group had the most serious lesions, with a incidence rate of 100%, and the Cit-CII and Cit-Fib groups had mild symptoms, with a incidence rate of 25% and 37.5%, respectively; pathological and imaging examination results showed that the joints of mice in CII-induced group showed severe synovial inflammation, cartilage and bone destruction, while those in Cit-CII and Cit-Fib group showed only slight inflammatory infiltration, joint cavity stenosis and bone destruction; the results of serum antibody detection showed that CII, Cit-CII and Cit-Fib groups all produced high levels of anti-cyclic citrullinated peptide (CCP) antibodies, among which, Cit-Fib group > Cit-CII group > CII group > Fib group, and both Cit-CII and Cit-Fib groups produced high levels of citrullinated epitope-specific antibodies, while the total IgG level was the highest in CII group; serum ELISA and RT-PCR analysis of joint tissue showed that the expression of pro-inflammatory factors and bone destruction-related molecules increased most significantly in the CII-induced group, followed by Cit-Fib and Cit-CII. The above results showed that among the four different antigens, the symptoms and conditions of arthritis in RA mice induced by CII were the most serious, and IgG instead of anti-CCP antibody was its typical immunological feature, and CII could be the first choice for the model of RA mice; Cit-Fib has certain immunogenicity, can partially induce the symptoms and conditions of RA arthritis in mice, and produce high-level anti-CCP antibody and anti-Cit-Fib antibody, which is more suitable for the study of citrulline-related RA; although Cit-CII has certain immunogenicity, the incidence, and severity of RA arthritis induced by Cit-CII in mice are low.

Investigation of pharmacological characteristics and mechanisms of Qufeng Gutong Cataplasm based on a "disease-syndrome-symptom-formula" association network analysis / 药学学报

Growing clinical evidence shows that Qufeng Gutong Cataplasm may exert a significant analgesic effect. However, the pharmacological characteristics and mechanisms underlying this prescription are still unclear. In the current study, a "disease-syndrome-symptom-formula" association network analysis was performed to explore the pharmacological characteristics and mechanisms of Qufeng Gutong Cataplasm against osteoarthritis (OA), neuropathic pain (NP), chronic inflammatory pain (CIP) and myofascial pain syndrome (MPS) by integrating clinical phenomics data, transcriptomics data and biological interaction network mining. As a result, the three functional modules (Qufeng Sanhan-QFSHG, Shujin Huoxue-SJHXG and Xiaozhong Zhitong-XZZTG) enriched by the drug network targets were all related to the pharmacological effects of Qufeng Gutong Cataplasm, including dispersing cold and relieving pain, activating blood and relieving pain, reducing swelling and relieving pain. In addition, the main pharmacological effects of QFSHG and XZZTG were dispelling wind and dispersing cold and dehumidifying, promoting Qi and reducing swelling and relieving pain, respectively. In terms of reversing the imbalance of "immune-inflammation-vascular axis", the main pharmacological effects of SJHXG were regulating the liver and promoting Qi, activating blood circulation and removing stasis. Mechanically, the key network targets of Qufeng Gutong Cataplasm against OA, NP, CIP and MPS may play a therapeutic role in relieving hyperalgesia and paresthesia by reversing the "neuro-endocrine-immune" imbalance system during the occurrence and progression of diseases. In conclusion, our data indicate that Qufeng Gutong Cataplasm may relieve the pain and wind-cold-dampness arthralgia syndrome related symptoms by regulating the "neuro-endocrine-immune" system, neurological and endocrine disorders and reversing the imbalance of "immunity-inflammation". The relevant results may provide a network-based evidence for clinical positioning of Qufeng Gutong Cataplasm, and offer a direction for further clinical and experimental validation.

Exploring the potential mechanism of artesunate in intervening with U87 cells and U251 cells with distinct therapeutic effects on the basis of transcriptome sequencing and network pharmacology / 药学学报

Artesunate possesses the potential of intervening with glioma, however, its pharmacological mechanisms remain unclarified. Firstly, the effects of artesunate on cell activity, proliferation and apoptosis of U87 and U251 human glioma cells were explored. It was found that artesunate exerted stronger inhibitory effects on the activity and proliferation of U87 cells than U251 cells. It could significantly promote apoptosis in U87 cells (P < 0.05), while only high dose of artesunate can promote that of U251 cells (P < 0.01), detected by Hoechst and TUNEL cell apoptosis staining. Further, the differential expression gene sets between artesunate-sensitive and non-sensitive cell line, as well the therapeutic effects-related genes of artesunate were obtained through transcriptome sequencing and differential data analysis by using the lysates of U87 and U251 cells before and after artesunate treatment, aiming to explore the molecular mechanism of distinct artesunate sensitivity to two types of cells. Then, key putative targets that related to therapeutic effects were screened by constructing the interaction network of differential genes of three above comparison groups, and calculating their topological characteristics. Pathway enrichment analysis showed that those key putative targets were significantly enriched in several signaling pathways that were closely associated with the main pathological changes of glioma, among which apoptosis-related activating transcription factor 4 (ATF4)-DNA damage induced transcript 3 (DDIT3)- polyadenosine diphosphate ribose polymerase 1 (PARP1) signaling axis was the most enriched in. Molecular docking indicated that artesunate had fine binding affinities with ATF4 and DDIT3. Above all, this study preliminarily revealed that ATF4-DDIT3-PARP1 signaling axis is the target pathway of artesunate intervening with U87 glioma cells, and PARP1 may be an important gene for U251 cells to develop resistance to artesunate. Our results not only provide fundamental experimental evidence for artesunate as a potential therapeutic drug in glioma treatment, but shed light into overcoming drug resistance in its clinical therapy.

Tenghuang Jiangu Tablets Combined with Non-steroidal Anti-inflammatory Drugs for Osteoarthritis of Knee： A Real-world Study / 中国实验方剂学杂志

ObjectiveTo investigate the clinical efficacy of Tenghuang Jiangu tablets （THJGT） combined with oral non-steroidal anti-inflammatory drugs （NSAIDs） in the treatment of osteoarthritis of the knee and its applicable stage based on real-world data， and provide a basis for the rational clinical use of THJGT. MethodA total of 218 cases treated with THJGT combined with oral NSAIDs included in the "THJGT for knee osteoarthritis case registry" from September 2019 to January 2021 were selected as the observation group， and 126 cases treated with oral NSAIDs alone as the control group （CG）. The data of gender， age， body mass index， Kellgren-Lawrence grading scale （K-L scale） score， visual analogue score （VAS score）， Western Ontario and McMaster Universities Osteoarthritis Index （WOMAC） score， swelling grade， joint fear of cold score， back pain and weakness score， and occurrence of adverse events/reactions of the patients in both groups were used for the evaluation of efficacy with full analysis set. The propensity score matching method was used to exclude the influence of confounding factors between groups， and the sub-data sets were established， with which the repeated measures analysis of variance （ANOVA） was carried out to evaluate the efficacy. Visit points were at registration， 4 weeks and 8 weeks after registration. The data were statistically analyzed in Excel 2019 and SPSS 23.0. ResultThe proportion of females in the observation group was 66.06% （144/218）， which was higher than that （58.73%， 74/126） in the control group （χ2=1.846）. The average age in the observation group was （61.12±7.01） years， which was higher than that ［（59.38±5.99） years］ in the control group （W=19 918.50， P<0.05）. The remission rate in the observation group was 98.17% （214/218）. In the observation group， the proportions of the patients at K-L grades Ⅱ and Ⅲ were 64.22% （144/218） and 25.23% （55/218）， respectively. The effect analysis of the whole data set for enrollment and treatment for 8 weeks showed that the VAS score of the experimental group decreased by （3.27±1.24） points on average， which was better than that of the control group [（2.75±1.20）， W=34 179.00， P<0.05]. The average WOMAC score decreased （23.43±11.46） points， which was better than that of the control group [（16.71±8.86）， W=32 387.00， P<0.05]. The average swelling grade decreased （0.63±0.64）， which was better than the control group [（0.33±0.59）， W=33 847.50， P<0.05]. The average score of joint chills decreased （1.90±1.84）， points， which was better than that of control group [（1.40±1.28）， W=35 165.00， P<0.05]. The average lumbar acid fatigue score decreased by （2.02±1.64） points, which was better than that of the control group [（1.10±1.28）， W=32 986.50， P<0.05]. Efficacy analysis of subdata sets for enrollment， 4 weeks of medication and 8 weeks of medication showed that VAS scores of both groups showed a downward trend after treatment， and the improvement of experimental group was more significant than that of control group at 4 weeks， with statistical significance （P<0.05）. After treatment， the total WOMAC score of both groups showed a downward trend， and the improvement of experimental groups was more significant at 4 weeks and 8 weeks （P<0.05）. After treatment， swelling， cold fear grade and lumbar acid fatigue score of both groups showed a decreasing trend,， and the improvement of experimental group was more significant at 8 weeks （P<0.05）. The therapeutic effect analysis of patients in the attack stage and remission stage of the experimental group showed that the total WOMAC score of the two groups showed a downward trend after treatment， and the trend was basically the same， and there was no statistical difference between the two groups at enrollment， 4 weeks after treatment， and 8 weeks after treatment （t=1.675， t=2.068， t=2.364）. The total WOMAC score of the patients in remission stage in the experimental group with K-L grading between grade 0 and grade Ⅲ had statistical significance at 4 weeks after treatment compared with the time of entry （P<0.05， P<0.01）. Group of adverse event rate was 4.13% （9/218）， lower than the control group 10.32% （13/126） （χ2= 5.109， P<0.05）. ConclusionThe population receiving THJGT combined with oral NSAIDs is mostly female， old， in remission， and with K-L grades Ⅱ and Ⅲ. THJGT can enhance the anti-inflammatory and analgesic effects of oral NSAIDs and keep the drug effect in improving joint function and alleviating fear of cold， swelling， and back pain and weakness. The drug combination can be applied to patients in both attack and remission， and the clinical application should take patient's disease stage and degree of osteoarthritis into account. Furthermore， the combination has the potential to reduce the incidence of adverse events caused by NSAIDs.

Anti-swelling and Analgesic Mechanism of Jianpi Tongluo Prescription from MAPKs Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the anti-swelling and analgesic effects of Jianpi Tongluo prescription （JPTL） and to explore its mechanism initially. MethodA total of 120 ICR mice were divided into normal group， model group， JPTL low-， medium- and high-dose groups （5， 10， 20 g·kg-1） and positive drug （celecoxib， 0.03 g·kg-1） group， with 10 in each group （po，once a day）. Complete freund's adjuvant （CFA） was used to induce the model of chronic inflammatory pain， and xylene-induced ear swelling test， hot plate test and acetic acid writhing test were performed to observe the anti-swelling and analgesic effects of different doses of JPTL in these four acute and chronic models. Further， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of prostaglandin E2 （PGE2）， interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in serum and inflammatory paw of mice with chronic inflammatory pain， and the expressions of aquaporin 1 （AQP1）， aquaporin 3 （AQP3）， cyclooxygenase 1 （COX1）， cyclooxygenase 2 （COX2） and mitogen-activated protein kinases （MAPKs） in inflammatory paw were detected by Western blot， to explore the preliminary mechanism of JPTL. ResultCompared with the conditions in the normal group， there was a significant increase in the ear swelling of xylene-induced model mice， a shortened paw withdrawal latency in the hot plate test （P<0.01）. Compared with the model group， JPTL remarkably increased the inhibition rate of xylene-induced ear swelling （P<0.05， P<0.01）， prolonged the latency period of writhing caused by acetic acid and reduced the number of writhing （P<0.05， P<0.01）. Compared with normal group, the degree of feet swelling in chronic inflammatory pain mice was significantly increased， the threshold of mechanical pain was decreased and the threshold of cold pain was increased （P<0.05， P<0.01）， the protein contents of AQP1 and AQP3 in inflammatory feet were increased， and the contents of IL-1β， IL-6， TNF-α， PGE2 and COX2 in inflammatory feet were increased in serum and/or inflammatory feet. The protein expression levels of p-p38 MAPK， p-JNK and p-ERK in inflammatory feet were increased （P<0.01）. Compared with the model group， JPTL relieved paw swelling of mice with chronic inflammatory pain， elevated mechanical withdrawal threshold while decreased cold withdrawal threshold， with analgesia lasting for 4 h and the optimal time point for analgesia being 2 h after administration （P<0.05， P<0.01）. Moreover， JPTL down-regulated AQP1， AQP3， COX2， p-p38 MAPK， p-JNK and p-ERK in inflammatory paw of mice with chronic inflammatory pain and reduced IL-1β， IL-6， TNF-α， and PGE2 in serum and/or inflammatory paw， but it had no significant effect on COX1 （P<0.05， P<0.01）. ConclusionJPTL has anti-swelling and analgesic effects， and its mechanism is related to inhibiting the production of cytokines and inflammatory mediators via the down-regulation of MAPKs signaling pathway， which provides an experimental basis for the clinical application of JPTL.

Exploring biological connotation of blood stasis syndrome of rheumatoid arthritis and establishment of improved animal models based on syndrome-symptom mapping / 药学学报

Blood stasis syndrome is one of the core clinical syndrome of rheumatoid arthritis (RA), but the biological connotation of this syndrome is not clear, and there is a lack of disease improved animal models that match the characteristics of this disease and syndrome. The aim of this study was to screen the candidate biomarker gene set of blood stasis syndrome of RA, reveal the biological connotation of this syndrome, and explore and evaluate the preparation method of the improved animal model based on the characteristics of "disease-syndrome-symptom". The study was approved by the ethics committee of Guang'anmen Hospital, Chinese Academy of Traditional Chinese Medicine (No. 2019-073-KY-01) and the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (No. TYLL2021[K]018), and the study subjects gave their informed consent. Animal welfare and experimental procedures followed the regulations of the Experimental Animal Ethics Committee of the Chinese Academy of Traditional Chinese Medicine (No. IBTCMCACMS21-2207-01). The whole blood samples were collected clinically from RA patients with blood stasis syndrome (3 cases) or other syndromes (7 types, 3 cases/type), and healthy volunteers (4 cases), and then transcriptome sequencing, KEGG, gene set enrichment analysis (GSEA) and weighted correlation network analysis (WGCNA) analysis were performed. 126 pivotal genes were screened, and their functional annotation results were significantly enriched in "immune-inflammation" related pathways and lipid metabolism regulation (sphingolipids, ether lipid metabolism and steroid biosynthesis). Syndrome-symptom mapping of hub gene set to the TCM primary and secondary symptoms, Western phenotypic symptoms and pathological links showed that joint tingling, abnormal joint morphology, petechiae and abnormal blood circulation are representative of blood stasis syndrome of RA. The results of the improved animal model showed that the rats in the collagen-induced arthritis + adrenaline hydrochloride (CIA+Adr) 3 model group had increased blood rheology, coagulation, platelet function and endothelial function abnormalities compared with the CIA-alone model group, suggesting that the rats with blood stasis syndrome of RA may be in a state of "blood stasis". The results of the study can help to advance the objective study of the evidence of blood stasis syndrome in RA, and provide new ideas for the establishment of an animal model that reflects the clinical characteristics of the disease and syndrome.

Clinical Study of Osteoking Combined with Non-Steroidal Anti-inflammatory Drugs in Treatment of Knee Osteoarthritis / 中国实验方剂学杂志

ObjectiveTo explore the clinical efficacy of Osteoking combined with non-steroidal anti-inflammatory drugs in the treatment of knee osteoarthritis based on real-world data and provide a basis for clinical medication. MethodFrom May 2020 to December 2021， the data of a total of 1 002 patients with knee osteoarthritis who did not undergo knee joint replacement surgery was collected through the registration method. 952 patients were ultimately included， including 133 cases orally taking Osteoking combined with non-steroidal anti-inflammatory drugs as the observation group and 73 cases orally taking non-steroidal anti-inflammatory drugs alone as the control group. Statistical analysis was conducted on the baseline data， VAS scores， WOMAC scores， and other items. The visit point is the 4th and 8th weeks after registration. In order to further elucidate the clinical efficacy of Osteoking combined with non-steroidal anti-inflammatory drugs in the treatment of knee osteoarthritis， the effective components of Osteoking and the relevant gene sets of non-steroidal anti-inflammatory drugs and knee osteoarthritis were obtained through network pharmacology methods and retrieval in bone injury cross database， TCMSP， and other databases. Venn analysis was performed on the relevant gene sets， and a PPI network diagram was constructed. Then key core targets were screened out， and enrichment GO and KEGG enrichment analyses were conducted. ResultThe VAS score of the observation group decreases by an average of （-2.79±1.206） scores in the 4th week， which is better than the control group ［（-2.73±1.575） scores， P<0.05］. The VAS score of the observation group decreases by an average of （-3.97±1.308） scores in the 8th week， which is better than the control group ［（-3.89±1.822） scores， P<0.05］. The total WOMAC score of the observation group decreases by an average of （-52.07±21.677） scores points in the 8th week， which is significantly better than the control group ［（-46.75±25.368） scores， P<0.05］. The observation group has an average decrease of （-10.99±4.229） scores in WOMAC （pain） score in the 8th week， which is better than the control group ［（-10.03±5.535） scores， P<0.05］. The observation group has an average decrease of （-1.49±2.901） in WOMAC （stiffness） score in the 4th week， which is better than the control group ［（-0.92±1.998） scores， P<0.05］， and the observation group has an average decrease of （-1.90±3.200） scores in WOMAC （stiffness） score in the 8th week， which is better than the control group ［（-1.26±2.230） scores， P<0.05］. The observation group shows an average decrease of （-39.17±16.562） scores in WOMAC （joint function） score in the 8th week， which is significantly better than the control group ［（-35.47±20.098） scores， P<0.05］. According to network pharmacology analysis， the core network target of Osteoking in treating knee osteoarthritis is manifested as regulating signal pathways such as signal transduction transcription activator 3（STAT3）， vascular endothelial growth factor A（VEGFA）， tumor necrosis factor （TNF） to regulate cell signaling， angiogenesis， chondrocyte proliferation and migration， and inflammatory cells， thereby inhibiting inflammatory reactions， reducing damage， and delaying the development of the disease. ConclusionAfter a 4-week and 8-week course of treatment for knee osteoarthritis with Osteoking combined with non-steroidal anti-inflammatory drugs， there is a significant therapeutic effect on relieving pain and joint stiffness and improving joint function. In network pharmacology， Osteoking is involved in regulating inflammatory factors， metabolic response-related biological processes， the proliferation and apoptosis of chondrocytes， etc. in the treatment of knee osteoarthritis， resulting in anti-inflammatory and analgesic effects and improving joint mobility and joint stiffness. Therefore， it is worthy of clinical promotion and application.

A Real-World Clinical Study of Osteoking Combined with Intra-Articular Injection of Sodium Hyaluronate in Treatment of Knee Osteoarthritis / 中国实验方剂学杂志

ObjectiveTo investigate the improvement of the efficacy of Osteoking in patients with knee osteoarthritis in the onset and remission stage and to systematically explore its potential intervention mechanism， so as to provide a certain reference for improving the clinical application value of Osteoking and guiding its clinical rational drug use. MethodThrough the real-world study of the treatment of knee osteoarthritis with Osteoking， the data was obtained and entered into the "Osteoking for the treatment of knee osteoarthritis case registration system"， and 105 patients with episodic and remission knee osteoarthritis from the outpatient or inpatient orthopedic department of 20 medical institutions， including the Third Affiliated Hospital of Beijing University of Chinese Medicine， Peking Union Medical College Hospital， Wangjing Hospital of the Chinese Academy of Chinese Medical Sciences and Hunan Aerospace Hospital， from May 1， 2020 to December 31， 2021， were selected in the system. It included 60 patients treated with Osteoking and joint injection， and 45 patients treated with joint injection alone. The WOMAC osteoarthritis index score， visual analogue （VAS） pain score， individual types of pain symptoms （cold pain， hot pain， tingling， dull pain， soreness） and other TCM symptoms were observed and compared between the two groups， and statistically analyzed. In order to further elucidate the potential molecular mechanism of Osteoking combined with joint injection in the treatment of knee osteoarthritis in the treatment of onset and remission， this study used the "Bone Injury Cross Database （http：//bone-xtrans.com/database，BX-Data）" to collect the gene set of knee osteoarthritis disease， the traditional Chinese medicinal materials， chemical composition， material base， candidate target， candidate target， sodium hyaluronate candidate target data for screening， and constructed an interaction network of "disease target". ResultsAmong the 105 patients with knee osteoarthritis enrolled， 15.24% （16/105） were in the episodic period， 84.76% （89/105） were in remission， and there were no convalescent patients. There were 72 cases （68.57%） in women， 33 cases （31.43%） more than men， 60 cases in the observation group and 45 cases in the control group in 105 patients. There were 20 patients with a VAS score of 5 and 19 patients with a score of 6 in the observation group， accounting for 65.00% of the observation group. The comparative results of VAS scores between groups before and after treatment showed that the scores of the two groups were （4.42±1.01） scores， （5.00±1.02） scores.4 weeks after treatment， and （3.12±1.04） scores and （3.56±1.08） scores，8 weeks after treatment， respectively， which were lower than those before treatment （6.23±1.28） scores，（ 6.02±1.22） scores （P<0.05）， and the comparative results of the pain properties of the two groups showed that the improvement rates before and after thermal pain and tingling in the observation group were 3.3%（2/60） and 16.7%（10/60）， respectively. The control group was 2.2% （1/45）and 15.6%（7/45）［（χ2=4.034、13.583，P<0.05）］， respectively， and the improvement rate of cold pain and soreness in the observation group was 5.0%（3/60） and 3.3%（2/60）， which was higher than that of the control group . The results of comparing the WOMAC scores before and after treatment of the two groups showed that the difference between the stiffness score before and after treatment in the observation group was （1.68±1.42） scores， the difference between the score before and after treatment in the control group was （1.20±1.60） scores （P<0.05）， and the pain score before and after treatment was （3.43±2.88） scores， the difference before and after daily activity score was （12.37±10.21） scores， and the total score before and after treatment was （17.48±12.76） scores， which were also higher than those in the control group （2.82±3.29）， （10.80±9.63），（14.82±12.62） scores. The results of comparing the improvement of other symptoms before and after treatment showed that the improvement rate of less sleep and more dreams in the observation group was 28.3%（17/60）， which was significantly higher than that of the control group of 2.2%（1/45）（χ2=5.914，P<0.05）， and the improvement rates of the five symptoms of thirst and drinking， irritability， dry mouth and pharynx， dull complexion and hand， foot and mouth fever in the observation group were 3.3%（2/60）， 10.0%（6/60）， 8.3%（5/60）， 10.0%（6/60） and 5.0%（3/60）， respectively， which were higher than those in the control group -2.2%（1/45）， 2.2%（1/45）， 2.2%（1/45）， 4.5%（2/45）， -6.7%（3/45）. Through network analysis， it was found that the enrichment pathway of Henggu bone wound healing agent mainly acted on the three mechanisms of bone improvement， energy metabolism and anti-inflammatory and analgesic， and the sodium hyaluronate enrichment pathway mainly acted on the anti-inflammatory and analgesic mechanism. ConclusionThe efficacy of Osteoking combined with intra-articular injection of sodium hyaluronate in the treatment of patients with knee osteoarthritis in attack and remission is better than that of sodium hyaluronate alone， especially in anti-inflammatory and analgesic， and the two drugs have synergistic effect. Osteoking may play its role in relieving the symptoms of joint stiffness， tingling， heat pain， and less sleep and more dreams by improving bone quality and regulating the body's energy metabolism pathways， which is worthy of clinical promotion.

Clinical Efficacy and Mechanism of Osteoking in Treatment of Knee Osteoarthritis Based on Real-world Data / 中国实验方剂学杂志

ObjectiveTo investigate the clinical efficacy and mechanisms of Osteoking in the treatment of knee osteoarthritis （KOA） in real-world practice， so as to provide a basis for the rational clinical use of Osteoking. MethodFrom the Osteoking for knee osteoarthritis case registration system， 638 KOA cases treated with Osteoking were selected and analyzed in SPSS 26.0. The clinical data were collected from 20 hospitals in China from May 2020 to December 2021. Descriptive analyses of patient age， gender， body mass index， course of treatment and other parameters were performed. The Mann-Whitney U test was performed to compare the visual analogue scale （VAS） and Western Ontario and McMaster universities arthritis index （WOMAC） scores before and after treatment. The integrative pharmacology-based research platform of traditional Chinese medicine （TCMIP） v2.0 was used for network analysis of the core targets of Osteoking in treating knee osteoarthritis. Furthermore， 20 KOA patients treated with Osteoking in the Third Affiliated Hospital of Beijing University of Chinese Medicine from October to December in 2022 were enrolled in the treatment group， and 20 healthy volunteers in the control group. The enzyme-linked immunosorbent assay was employed to measure the serum levels of related indicators to verify the prediction results. ResultA total of 638 KOA patients were treated with Osteoking， including 429 （67.24%） receiving Osteoking alone and 209 （32.76%） receiving Osteoking combined with other therapies. The female patients （415， 65.05%） were more than the male patients （223， 34.95%）. The patients showed the mean age of （63.48±13.51） years， mean body mass index of （24.09±2.98） kg·m-2， and mean course of treatment of （15.78±9.66） days. Most of the patients were rated as grades Ⅱ （46.24%） and Ⅲ （34.64%） in Kellgren-Lawrence （K-L） grading and in the relief stage （82.45%） in clinical staging. There was no significant correlation between clinical staging and K-L grading results. The cluster analysis identified three TCM syndromes： Qi stagnation and blood stasis， cold-dampness obstruction， and liver-kidney deficiency. The overall clinical efficacy evaluation showed that VAS score decreased from （6.01±0.85） scores before treatment to （2.54±1.73） scores after treatment （P<0.05）， and the WOMAC score decreased from （93.25±25.91） scores before treatment to （50.73±25.14） scores after treatment （P<0.05）. The network analysis predicted that Osteoking might regulate the transforming growth factor-beta （TGF-β）， tumor necrosis factor-alpha （TNF-α）， and nuclear factor-kappa B （NF-κB） signaling pathways to exert the therapeutic effect. The clinical trial showed elevated TGF-β1 level （P<0.01） and lowered NF-κB subunit RELA and tumor necrosis factor receptor superfamily， member 1A （TNFRSF1A） levels （P<0.05） after treatment. The synergistic effects of these changes provide a multidimensional and comprehensive therapeutic efficacy for KOA， alleviating the joint pain and limited mobility in patients. ConclusionOsteoking showed significant therapeutic efficacy in treating KOA. Osteoking may act on multiple pathways involved in cartilage metabolism and inflammation. The findings provide experimental evidence and theoretical support for elucidating the multi-target mechanism of Osteoking in treating KOA.

Mechanism of Osteoking in Improving Energy Metabolism in Rats with Myofascial Pain Syndrome Based on cAMP/PKA/PGC1α Signaling Pathway / 中国实验方剂学杂志

ObjectiveFrom the perspective of energy metabolism， the mechanism of Osteoking （OK） in the treatment of myofascial pain syndrome （MPS） was revealed through systems biology prediction combined with holistic animal experimental validation methods. MethodFirstly， the key targets of MPS and their related molecular mechanisms were predicted by the systems biology method， and the core network targets were screened. Then， the network-predicted targets were verified by animal experiments. Specifically， 60 SD rats were randomly divided into normal group， model group， low， medium， and high dose OK groups （0.66， 1.31， 2.63 mL·kg-1）， and positive celecoxib group （21 mg·kg-1）. The MPS model was established by beating combined with a centrifugal exercise method for eight weeks. Except for two days after modeling， the intervention of OK or celecoxib was performed. After the completion of the model， the drug was administered for two weeks. The histopathological changes of trigger point muscle tissue were observed by hematoxylin-eosin staining. The content/activity of Na-K-ATP enzyme （Na+-K+-ATPase）， Ca2+ pump （Ca2+ATPase）， Ca2+， lactate dehydrogenase （LDH）， glutathione （GSH）， malondialal （MDA）， superoxide dismutase （SOD）， cyclic adenosine phosphate （cAMP）， and protein kinase A （PKA） in serum and/or trigger point muscle tissue in MPS rats was detected by enzyme-linked immunosorbent assay. Protein expression levels of PKA and the peroxisome proliferator-activated receptor γ coactivator 1α （PGC1α） in MPS rats were detected by immunohistochemistry. The protein expression levels of PKA， PGC1α， and mitochondrial transcription factor A （TFAM） in MPS rats were detected by Western blot. ResultThe network prediction results suggest that OK acts on the key target of energy metabolism related to the occurrence and development of MPS and may participate in the activation of the cAMP/PKA/PGC1α signaling pathway. The experimental validation results show that compared with the normal group， contracture nodules and disordered arrangement of muscle fibers appear in the trigger point muscle tissue of MPS rats. Na+-K+-ATPase， Ca2+ATPase， SOD activity， Ca2+， and GSH contents in serum and/or trigger point muscle tissue are significantly decreased （P<0.01）. Both LDH activity and MDA contents are significantly increased （P<0.01）， and the protein expression levels of cAMP， PKA， PGC1α， and TFAM are significantly decreased （P<0.01）. Compared with the model group， OK improves the histopathological morphology of trigger point muscle fibers in MPS rats， and after the intervention of OK， Na+-K+-ATPase， Ca2+ATPase， SOD activity， Ca2+， and GSH contents in serum and/or trigger point muscle tissue in MPS rats are significantly increased （P<0.05， P<0.01）. LDH activity and MDA contents are significantly reduced （P<0.05， P<0.01）. The protein expression levels of cAMP， PKA， PGC1α， and TFAM are significantly increased （P<0.05， P<0.01）. ConclusionThe mechanism of OK's intervention in MPS rats may be related to its effective activation of the cAMP/PKA/PGC1α signaling pathway， thus promoting mitochondrial energy metabolism and trigger point muscle fiber damage repair in muscle cells.

Investigation on Mechanisms of Osteoking Against Four Types of Bone Injury Diseases Based on Heterogeneous Information Network with Concept of "Treating Different Diseases with an Identical Treatment" / 中国实验方剂学杂志

ObjectiveTo elucidate the mechanism of Osteoking against fracture， femoral head necrosis， osteoarthritis， and lumbar disc herniation by integrating heterogeneous information network mining and experimental validation. MethodOn the basis of the disease-related database and transcriptome expression profiling dataset， as well as the ETCM database， the gene sets related to four target diseases and the candidate target spectrum of Osteoking were obtained through the integration and analysis of bioinformatics data， and a "disease-syndrome-formula-target-pathway-effect" heterogeneous information network was constructed. In addition， by functional enrichment analysis， the core targets of Osteoking in interfering with the imbalance network of four kinds of bone injury diseases， the biological pathways involved， and the corresponding clinical symptoms were screened， and they were verified in animal experiments. ResultHeterogeneous information network mining indicates that Osteoking may commonly reverse the imbalance networks of fracture， femoral head necrosis， osteoarthritis， and lumbar disc herniation via regulating cell function and activity， inhibiting inflammatory response， reducing bone destruction， and improving the immune function of the body by modulating relevant core candidate targets such as RAC-alpha serine/threonine-protein kinase （Akt1）， catenin beta-1 （CTNNB1）， epidermal growth factor receptor （EGFR）， heat shock protein 90-alpha （HSP90AA1）， and phosphatidylinositol 3-kinase catalytic subunit alpha isoform （PI3KCA）， as well as related biological pathways such as phosphatidylinositide 3-kinases/protein kinase B （PI3K/Akt）， janus kinase/signal transducer and activator of transcription （JAK/STAT）， tumor necrosis factor （TNF）， nuclear factor kappa-B （NF-κB）， and Toll-like receptors. In particular， Osteoking may improve the blood supply of the fracture end by regulating blood circulation at the target site of the disease， and it may maintain the balance of bone metabolism by regulating hormone-related pathways to promote fracture healing. In addition， Osteoking may relieve lipid metabolism disorders by targeting and regulating lipid-related pathways， accelerate bone formation and bone repair， and delay the progression of femoral head necrosis. Osteoking may relieve the symptoms of pain by acting on neurological pathways to reduce local nociceptive stimulation in patients with osteoarthritis and lumbar disc herniation. Further experimental validation demonstrates that the PI3K/Akt signaling pathway is the most significantly enriched pathway for the key network targets of Osteoking for the four diseases. The candidate target of Osteoking may have the strongest association with the network of fracture-related genes. Therefore， this study chooses fracture as the target disease to verify the efficacy of Osteoking. The results show that Osteoking can accelerate bone formation and promote fracture healing by inhibiting the activation of the PI3K/Akt signaling axis. ConclusionThe study shows that the main mechanism of "treating different diseases with an identical treatment" of four bone injury diseases with Osteoking involves cell function regulation and immune inflammation-related signaling pathways. Further experimental validation identifies that the PI3K/Akt signaling axis may be one of the key pathways of Osteoking to promote bone regeneration， bone reconstruction， and bone metabolism homeostasis.