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ObjectiveTo explore the effect of virtual reality on upper limb function in stroke patients through diffusion tensor imaging (DTI). MethodsFrom September, 2021 to March, 2023, 80 stroke patients in the Fuzhou Second General Hospital were randomly divided into control group (n = 40) and experimental group (n = 40). Both groups received routine rehabilitation, while the experimental group received virtual reality training additionally, for four weeks. They were assessed with Fugl-Meyer Assessment-Upper Extremities (FMA-UE) and Action Research Arm Test (ARAT) before treatment, after treatment and after four-week follow-up; and they were scaned with DTI to measure the fractional anisotropy (FA) and relative anisotropy (RA) of cerebral peduncle and posterior limb of inner capsule of the affected side before and after treatment. ResultsTwo cases dropped in each group. The FMA-UE and ARAT scores increased in both groups after treatment and follow-up (F > 2.790, P < 0.001), and increased more in the experimental group than in the control group (t > 2.297, P < 0.05). FA and RA in the posterior limb of inner capsule increased in both groups after treatment (t > 21.013, P < 0.001), and increased more in the experimental group (t > 2.006, P < 0.05). The d-value of FA of the posterior limb of internal capsule before and after treatment (ΔFA) was positively correlated with the d-value of FMA-UE score (r > 0.362, P < 0.05) in both groups, the ΔFA of the posterior limb of internal capsule was positively correlated with the d-value of ARAT score (r = 0.459, P < 0.01). ConclusionVirtual reality training can promote the recovery of upper limb function in stroke patients, which may associate with the conductivity of posterior limb of inner capsule.
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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.
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ObjectiveTo investigate the protective effect of Jianpi Huogu prescription （JPHGP） on the functional injury of vascular endothelial cells caused by alcohol and explore its mechanism based on protein kinase B/c-Jun amino-terminal kinase/p38 MAPK （Akt/JNK/p38 MAPK） signaling pathway. MethodThrough chick embryo allantoic membrane， thoracic aortic ring， and migration， invasion， adhesion， and lumen formation of human umbilical vein endothelial cells （HUVEC）， the effect of JPHGP with different concentrations （8， 16 and 32 μg·L-1） on angiogenesis was observed in the presence or absence of alcohol. The expression levels of phosphorylation of Akt， JNK， and p38 MAPK were determined by Western blot. ResultAs compared with the normal group， the number and length of capillaries around the arterial ring in the model group were decreased， and the migration， invasion， and lumen formation capacity of HUVEC were decreased （P<0.05， P<0.01）. After treatment with 16 and 32 μg·L-1 JPHGP， the length of neovascularization in chick embryo allantoic membrane was significantly increased （P<0.05， P<0.01）. Compared with the model group， the 8， 16， and 32 μg·L-1 JPHGP groups increased the number of capillaries around the thoracic aortic ring in a concentration-dependent manner （P<0.05， P<0.01）， and the 32 μg·L-1 JPHGP group increased the length of capillaries around the thoracic aortic ring （P<0.05）. The 16 and 32 μg·L-1 JPHGP groups enhanced the migration， invasion， and lumen formation capacity of HUVEC. The results of Western blot showed that， as compared with the normal group， the protein expression levels of p-JNK/JNK， p-p38 MAPK/p38 MAPK， and p-Akt/Akt were significantly decreased in the model group （P<0.01）， and as compared with the model group， the protein expression levels of p-p38 MAPK/p38 MAPK and p-Akt/Akt were significantly increased in the 8， 16， and 32 μg·L-1 JPHGP groups （P<0.01） and the protein expression level of p-JNK/JNK was increased significantly in the 16 and 32 μg·L-1 JPHGP groups （P<0.01）. ConclusionJPHGP has a protective effect on the functional injury of vascular endothelial cells caused by alcohol， and its mechanism may be related to the activation of Akt/JNK/p38 MAPK signaling pathway. Relevant research results will provide certain scientific basis for clarifying the effect of JPHGP on 'invigorating spleen and promoting blood circulation'.
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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.
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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.
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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.
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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.
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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.
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ObjectiveTo investigate the analgesic effect and mechanism of Osteoking （OK） on nerve compression in lumbar disc herniation. MethodThe rat model of chronic compression of dorsal root ganglion （CCD） was established to simulate clinical lumbar disc herniation. The CCD rats were randomly divided into model group， low， medium， and high dose OK groups （1.31， 2.63， 5.25 mL·kg-1·d-1）， and pregabalin group （5 mg·kg-1）， with eight rats in each group. Another eight SD rats were taken as the blank group， and the same volume of normal saline was given by gavage. Behavioral tests， side effect evaluation， network analysis， Western blot， immunofluorescence， and antagonist application were used to explore the effect. ResultCompared with the blank group， the mechanical hyperalgesia threshold， thermal hyperalgesia threshold， and the expression of inflammatory factors in the spinal dorsal horn of the model group are significantly increased （P<0.01）， and the related indicators of the affected foot footprints are significantly down-regulated （P<0.01）. The expression of signal transducer and activator of transcription 3 （STAT3）， vascular endothelial growth factor A （VEGFA）， and phosphorylated extracellular regulated protein kinase （p-ERK） in microglia in the spinal dorsal horn is significantly increased in the model group （P<0.01）. Compared with the model group， low， medium， and high dose OK groups can increase the mechanical hyperalgesia and thermal hyperalgesia thresholds of CCD rats （P<0.05， P<0.01） in a dose-dependent manner， improve the gait of CCD rats （P<0.05， P<0.01）， and reduce the expression of inflammatory factors in the spinal dorsal horn （P<0.05， P<0.01）. The expression of STAT3， VEGFA， and p-ERK in the spinal dorsal horn microglia of CCD rats is significantly decreased （P<0.05， P<0.01）， and the acetic acid-induced nociceptive response in rats is effectively reduced （P<0.05， P<0.01）. In addition， there is no tolerance. The results of the body mass test， organ index， forced swimming， and rotation show that OK has no obvious toxic or side effects. Further antagonist experiments show that MRS1523 and RS127445 can reverse the transient analgesic effect of OK compared with the high dose OK group （P<0.01）. ConclusionOK has a good analgesic effect on the CCD model without obvious toxic side effects， and its mechanism may be related to the activation of ADORA3 and HTR2B and the inhibition of STAT3， VEGFA， p-ERK， and other elements in microglia.
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ObjectiveTo clarify the intervention effect of Osteoking （OK） in rats with myofascial pain syndrome （MPS） and preliminarily explore the pharmacological mechanism of OK in relieving chronic pain from the perspective of anti-inflammatory disease. MethodThe 60 SD rats were 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 rat model was established by beating combined with the centrifugal exercise method， and the OK and celecoxib were given at the same time. SMALGO paw pressure pain manometer detected the shock pain point tenderness threshold of rats， and the Von-Frey needle and acetone stimulation method detected the mechanical hyperalgesia threshold and cold hyperalgesia stimulation response respectively. Eight weeks and 10 weeks after modeling， the spontaneous discharge state and convulsion response of MPS rats were determined by electromyograph （EMG） instrument. The gait changes of MPS rats were detected using a CatWalk gait analyzer. The expression levels of interleukin-1 β （IL-1β）， tumor necrosis factor-α （TNF-α）， substance P （SP）， and bradykinin （BK） were measured by enzyme-linked immunosorbent assay （ELISA）. The protein expression levels of nuclear transcription factor-κB （NF-κB） inhibiting protein α （IκBα）， phosphorylates （p）- IκBα， NF-κB p65， and p-NF-κB p65 were detected in MPS rats by Western blot. The positive expression of p-NF-κB p65 was detected by immunofluorescence. ResultCompared with the normal group， the model group shows 100% positive rates for EMG signal and local convulsions response at both the 8th and 10th weeks. The tenderness threshold and mechanical hyperalgesia threshold are significantly reduced. Cold hyperalgesia score is significantly increased， and gait is abnormal. The expression levels of serum and trigger points IL-1β， TNF-α， SP， BK， p-IκBα， and p-NF-κB p65， as well as the positive expression intensity of p-NF-κB p65 are significantly increased （P<0.01）. Compared with the model group， the positive rate of EMG detection and local convulsion response is significantly reduced in the medium and high dose OK groups （P<0.05）. The tenderness threshold and mechanical hyperalgesia threshold increase significantly in the medium and high dose OK groups， and the cold hyperalgesia score is significantly reduced in the high dose OK group （P<0.01）. The standing time， swing time， and walking period are significantly increased. The swing speed， maximum contact area， and maximum contact intensity are significantly decreased in the high dose OK group （P<0.05）. Moreover， the protein expression levels of p-IκBα/IκBα and p-NF-κB p65/NF-κB p65 are significantly reduced in the medium and high dose OK groups （P<0.05，P<0.01）. The positive expression intensity of p-NF-κB p65 is significantly decreased in the high dose OK group （P<0.01）. ConclusionThe mechanism of OK in relieving the pain in trigger points of MPS and improving gait abnormalities is related to the downregulation of the NF-κB p65 inflammatory signaling pathway to reduce the expression of inflammatory factors and pain mediators in blood and trigger point tissue.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.