Danggui Shaoyaosan Regulates Autophagy via AMPK/mTOR/ULK1 Signaling Pathway in Rat Model of Metabolism-associated Fatty Liver Disease / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase-1 （ULK1） signaling pathway in the rat model of metabolism-associated fatty liver disease （MAFLD）. MethodSixty SD rats were randomized into control， model， western medicine （polyene phosphatidylcholine capsules，0.144 g·kg-1）， and low-， medium-， and high-dose （2.44， 4.88， 9.76 g·kg-1， respectively） Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks， the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment， serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group， the model group showed increased contents of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C） and activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum， increased contents of TC， TG， and free fatty acids （FFAs） in the liver （P<0.01）， and decreased content of high-density lipoprotein cholesterol （HDL-C） in the serum （P<0.01）. Furthermore， the model group showed down-regulated protein levels of p-AMPK， microtubule-associated protein 1 light chain 3B （LC3B） Ⅱ， Beclin1， and ULK1 （P<0.01） and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver （P<0.01）. The hepatic steatosis was obvious and the NAFLD activity score （NAS） and oil red O staining area increased in the model group， （P<0.05， P<0.01）. Compared with the model group， Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum， lowered the levels of TC， TG， and FFA in the liver， down-regulated the protein levels of p-mTOR and p62 （P<0.01）， elevated the serum HDL-C level， and up-regulated the protein levels of p-AMPK， LCBⅡ， Beclin1， and ULK1 in the liver （P<0.05， P<0.01）. Moreover， it alleviated hepatic steatosis and decreased the NAS and oil red O staining area （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.

History and Clinical Application of Classic Famous Formula Danggui Sinitang / 中国实验方剂学杂志

Danggui Sinitang is first recorded in the Treatise on Cold Damage written by ZHANG Zhongjing in the Han dynasty. It is composed of Angelicae Sinensis Radix， Cinnamomi Ramulus， Paeoniae Radix Alba， Asari Radix et Rhizoma， Glycyrrhizae Radix et Rhizoma， Tetrapanacis Medulla， and Jujubae Fructus and serves as a classic formula for treating the syndrome of blood deficiency and cold reversal. This study systematically reviews the records of Danggui Sinitang in ancient Chinese medicine books of various dynasties and the modern clinical applications to probe into the composition， plant species， processing， dosage， decocting method， and indications of Danggui Sinitang， aiming to provide a reference for the development and clinical application of this classic formula. The review of the records showed that there were a variety of records of Danggui Sinitang with different composition， and the composition of this formula listed in the Treatise on Cold Damage has a significant impact on later generations and has been used by medical practitioners throughout history. Although the dosage of some drugs decreased during the Ming and Qing dynasties， the medical practitioners continued to use the original formula. In terms of processing， although there were slight changes in the processing of Angelicae Sinensis Radix， Paeoniae Radix Alba， Glycyrrhizae Radix et Rhizoma， and Tetrapanacis Medulla， the original processing method was inherited. In terms of indications， Danggui Sinitang was designed to treat cold reversal due to blood deficiency and dysentery. Furthermore， it was used to treat headache， convulsive disease， infantile convulsion， and private part adduction in the Ming and Qing dynasties. Nowadays， this formula is mostly used to treat diabetes peripheral neuropathy， rheumatoid arthritis， dysmenorrhea， Raynaud's disease and other diseases. In terms of precautions， ancient physicians believed that Danggui Sinitang should not be taken by pregnant women and should only be used for limb chills caused by blood deficiency and cold coagulation. For limb chills caused by other reasons， this formula should not be used indiscriminately. Modern research has not reported any serious adverse reactions related to this formula. Danggui Sinitang has a definite therapeutic effect. In subsequent research and development， quality control standards of Danggui Sinitang should be established while its safety is ensured， and the related preparations should be developed and applied.

Study of the improvement effect of "DuZhong-DangGui" on osteoarthritis rats via regulating NLPR3 inflammasome / 中国临床药理学与治疗学

AIM: To investigate the protective effect of "DuZhong-DangGui" (DZ-DG) on the knee tissue of rats with osteoarthritis (OA) and its regulation role on NLPR3 inflammasome. METHODS: Twenty-four SPF male SD rats, eighteen rats were randomly selected to establish OA model by anteri- or cruciate ligament amputation (ACLT) method, and rats were divided into control group, OA model group, DZ-DG group and positive drug group, 6 in each group, treatment for 8 weeks. The peripheral blood were collected, ELISA was used to detect the levels of IL-1β, IL-6, IL-18, and TNF-α; cartilage tissue of knee joint were collected, HE staining was used to observe pathology changes, OARSI staining was used to observe cartilage calcification and preform quantitative OARSI scoring; immunohistochemistry and TUNEL staining were used to detect the contents of Caspase1 and Collagen II and the number of apoptosis in the tissue, respectively, and western blot was used to detect the protein expressions of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), p53, p21, NLPR3, apoptosis-associated speck-like protein containing a CARD (ASC) and Pro-Caspase-1. RESULTS: Compared to control group, OA model group rats serum levels of IL-1β, IL-6, IL-18, and TNF - α significantly increased (P<0.01), OARSI scores significantly increased (P<0.01), chondrocyte apoptosis was increased (P<0.01), Caspase-1 content and MMP-13, p53, p21, NLPR3, ASC, Pro-Caspase-1 protein expressions significantly increased (P <0.01), while Collagen II content and TIMP-1 protein expression significantly decreased (P<0.01). Compared with the OA model group, DZ-DG group and positive drug group rats serum levels of IL-1β, IL-6, IL -18 and TNF - α significantly decreased (P< 0.05), chondrocyte apoptosis were significantly decreased (P<0.01), Caspase1 content, MMP-13, p53, NLPR3, Pro-Caspase-1 significantly decreased (P< 0.05), Collagen Ⅱ content and TIMP- 1 protein expression (P<0.01); DZ-DG group rats protein expression of p21 and ASC were decreased (P<0.01). CONCLUSION: The DZ-DG have protection role on cartilage of OA rats, its effect may related to mediation of NLPR3/ASC/Pro-Caspase-1 pathway, to decrease of IL-1β, and inhibition of cell apoptosis.

Mechanism of Modified Danggui Shaoyao San in treatment of chronic atrophic gastritis based on network pharmacology and experimental verification / 中国药理学通报

Aim To predict the targets of Modified Danggui Shaoyao San ( MDSS) in the treatment of chronic atrophic gastritis ( CAG) based on network pharmacology and vertify the results based on experim-ention. Methods TCMSP, SWISS TARGETS, GENE CARDS and OMIM databases were used to screen the therapeutic targets of MDSS for CAG. STRING database and Cytoscape software were used to construct the protein interaction network and screen the core targets. Metascape database was used for GO analysis and KEGG enrichment pathways. And molecular docking was used for target validation. CAG rat model was pre¬pared by N-methyl-N'-nitroso-N-nitroguanidine free drink combined with sodium salicylate gastric lavage. The pathology of rat gastric mucosa was observed by hematoxylin-eosin staining,and the ultrastructure of ep¬ithelial cells was observed by transmission electron mi-croscopy. The serum IL-6 and IL-10 content was detected by enzyme-linked immunosorbent assay, and the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in rats was detected by qPCR and Western blot. Results MDSS acted on 189 targets, mainly involved in response to oxidative stress and apoptotic signaling pathway. KEGG analysis related to pathways in cancer and JAK-STAT signaling pathway. The experimental results showed that the MDSS could improve the degree of atrophy of gastric mucosa in CAG rats and improve the status of epithelial cells, down-regulate the serum IL-6 content of CAG rats, up-regulate the IL-10 content, and reduce the expression of JAK2, STAT3 , p-STAT3 , c-MYC mRNA and protein in gastric mucosa with statistical significance. Conclusions MDSS treats CAG through multiple active ingredients, targets, and pathways, the mechanism of which may be related to the inhibition of the JAK2/STAT3 signaling pathway.

Effect of Danggui Niantongtang on Intestinal Flora in Adjuvant-induced Arthritis Rats of Wind-dampness-heat Arthralgia：Based on 16S rDNA Sequencing / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Danggui Niantongtang （DGNTT） against adjuvant-induced arthritis （AA） in rats with wind-dampness-heat arthralgia （FSR） based on the variation of intestinal flora. MethodA total of 60 SD rats were randomized into normal （control） group， FSR group， low-， medium-， and high-dose DGNTT （5.67， 11.34， 22.68 g·kg-1） groups， and methotrexate （MTX） group （1.35 mg·kg-1）， with 10 rats in each group. The rats， except the control group， were injected with Mtb adjuvant and then exposed to artificial climatic chamber （hot and humid with wind） for 64 h for modeling. The rats were treated with water， DGNTT or MTX for 28 days from the day of injection. Arthritis index （AI） of rats was measured and paw volume was determined with a volume meter. The morphology of synovial tissues of the knees was observed based on hematoxylin-eosin （HE） staining and the changes of intestinal flora were analyzed based on 16S rRNA sequencing. ResultDGNTT can alleviate the hyperplasia of synovial tissue and inflammation of AA rats with FSR and inhibit the formation of pannus. The results of 16S rRNA sequencing showed that the relative abundance of Firmicutes， Lactobacillus， Prevotella 9， and Alloprevotella decreased （P<0.05， P<0.01） and the relative abundance of Bacteroidetes and Bacteroides increased （P<0.01） in FSR group compared those in the control group. Compared with the FSR group， all DGNTT groups and MTX group had high relative abundance of Lactobacillus （P<0.05， P<0.01） and low relative abundance of Bacteroidetes （P<0.01） and medium-dose and high-dose DGNTT groups and MTX group showed high abundance of Firmicutes， Prevotella 9， and Alloprevotella and low abundance of Bacteroides （P<0.05， P<0.01）. Spearman's correlation analysis suggested that the abundance of Bacteroides and Helicobacter was in positive correlation with AI （P<0.05）， while the abundance of Prevotella 9 and Candidatus Saccharimonas was in negative correlation with AI （P<0.01， P<0.05）. There was a negative correlation between the abundance of Prevotella 9 and paw volume （P<0.01）， and the abundance of Ruminococcaceae NK4A214 group， Christensenellaceae R-7 group， and Bacteroides was in negative correlation with spleen index （P<0.05）. The abundance of Prevotella 9 was in negative correlation with spleen index （P<0.01）. ConclusionDGNTT is effective for arthritis with FSR， as it can regulate the composition of intestinal flora in AA rats by increasing the abundance of probiotics and inhibiting the growth of pathogenic bacteria. The mechanism is the likelihood that it improves intestinal immune metabolism to ensure intestinal homeostasis.

Historical Evolution and Modern Clinical Application of Danggui Liuhuangtang / 中国实验方剂学杂志

Danggui Liuhuangtang is the 47th of the 100 famous classical formulas published by the National Administration of Traditional Chinese Medicine， and is known as the holy medicine for night sweat. By bibliometrics， the authors collected the ancient books on Danggui Liuhuangtang and screened out 269 valid data， involving 156 ancient books of traditional Chinese medicine. The analysis on the historical origin， disease syndromes， pathogenesis， composition， dosage， preparation， usage， and processing of Danggui Liuhuangtang found that this famous classical formula originated from Secret Book of the Orchid Chamber （《兰室秘藏》） written by LI Dongyuan， and is composed of Angelicae Sinensis Radix， Rehmanniae Radix， Rehmanniae Radix Praeparata， Phellodendri Chinensis Cortex， Scutellariae Radix， Coptidis Rhizoma and Astragali Radix. It has the functions of nourishing Yin， reducing fire， consolidating exterior and stopping sweating， and mainly treats night sweat due to Yin deficiency and fire exuberance. In the later generations， disease syndromes are mostly treated based on LI Dongyuan's theory， and have expanded to more than 30 kinds （339 in total）， among which night sweat （208） was the most， accounting for 61.36% of the total disease syndromes， followed by spontaneous sweating （38）， accounting for 11.21%. Additionally， it was found that Danggui Liuhuangtang was widely used in modern clinical practice for various disease syndromes. Among them， endocrine disease （77， 28.21%） was predominant， followed by gynecological disease （48， 17.58%）， and pediatric disease （24， 8.79%）. Although Danggui Liuhuangtang treats many disease syndromes， their pathogenesis was always yin deficiency and fire exuberance. Through the systematic excavation of the ancient books on Danggui Liuhuangtang and the analysis of its modern clinical application， this paper probed into the historical evolution and confirmed the key information of the formula， providing detailed literature basis for the research and development application of famous classical formulas.

Mechanism of Danggui Sini Decoction in improving kidney injury caused by blood stasis syndrome based on metabolomics and network pharmacology / 中国中药杂志

This article analyzed the mechanism of Danggui Sini Decoction(DSD) in improving kidney injury caused by blood stasis syndrome(BSS) in rats. Firstly, 32 female SD rats were randomly divided into the following four groups: a normal group and a BSS group, both receiving an equal amount of distilled water by gavage; a normal+DSD group and a BSS+DSD group, both receiving 5.103 g·kg~(-1) DSD orally for a total of 14 days. Daily cold water bath was given to establish the BSS model, and on the 14th day, BSS rats were subcutaneously injected with 0.8 mg·kg~(-1) adrenaline. Normal rats were subjected to the water bath at 37 ℃ and injected with an equal volume of distilled water. After the experiment, 24-hour urine, serum, and kidney samples were collected for metabolomic analysis, biochemical measurements, and hematoxylin-eosin(HE) staining. The study then employed ~1H-NMR metabolomic technology to reveal the metabolic network regulated by DSD in improving BSS-induced kidney injury and used network pharmacology to preliminarily elucidate the key targets of the effectiveness of DSD. Pathological and biochemical analysis showed that DSD intervention significantly reduced inflammation and abnormal levels of blood creatinine, blood urea nitrogen, and urine protein in the kidneys. Metabolomic analysis indicated that DSD attenuated BSS-induced kidney injury primarily by regulating 10 differential metabolites and three major metabolic pathways(taurine and hypotaurine metabolism, citrate cycle, and acetaldehyde and dicarboxylic acid metabolism). Network pharmacology analysis suggested that the protective effect of DSD against BSS-induced kidney injury might be related to two key genes, ATP citrate lyase(ACLY) and nitric oxide synthase 2(NOS2), and two main metabolic pathways, i.e., arginine biosynthesis, and arginine and proline metabolism. This study, from the perspective of network regulation, provides initial insights and evidence into the mechanism of DSD in improving kidney injury induced by BSS, offering a basis for further investigation into the molecular mechanisms underlying its efficacy.

Effect and mechanism of Danggui Buxue Decoction-containing serum in mitigating H9c2 cell injury caused by exposure to intermittent low oxygen / 中国中药杂志

This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.

Danggui Sinitang Mitigates Gouty Arthritis in Rats by Regulating Autophagy via PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study the mechanism of Danggui Sinitang in mitigating gouty arthritis （GA） in rats by regulating autophagy via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodSixty male SD rats were randomly assigned into normal， model， colchicine （0.3 mg·kg-1）， and low-， medium-， and high-dose Danggui Sinitang （6.54， 13.08， and 26.16 g·kg-1） groups （n=10） and administrated with corresponding drugs by gavage. The rats in the normal group and model group were administrated with equal volume of normal saline by gavage for 7 days. One hour after administration on day 5， the GA model was established by injecting sodium urate suspension （50 g·L-1） into the right ankle joint of rats in other groups except the normal group， and the rats in the normal group were injected with sterile normal saline of the same volume. The swelling and pathological changes of the ankle joint were observed. The serum levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and IL-1β were determined. Western blot was employed to determine the protein levels of PI3K， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， mTOR， phosphorylated mTOR （p-mTOR）， microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ （LC3Ⅱ/Ⅰ）， autophagy effector Beclin-1， and ubiquitin-binding protein p62 in the synovial tissue. Real-time fluorescent quantitative PCR （Real-time PCR） was employed to determine the mRNA levels of PI3K， Akt， mTOR， LC3， Beclin-1 and p62. ResultCompared with the normal control， the model group showed increased joint swelling index （P<0.01）， elevated serum levels of TNF-α， IL-6， and IL-1β， inflammatory cell infiltration， and fibrous tissue hyperplasia. In addition， the model group showed up-regulated protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue， while it showed down-regulated protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and mRNA levels of LC3 and Beclin-1 （P<0.01）. Compared with the model group， medium- and high-dose Danggui Sinitang alleviated the joint swelling （P<0.01）， lowered the serum levels of TNF-α， IL-6， and IL-1β （P<0.05）， and relieved the inflammatory cell infiltration in the synovial tissue of the ankle joint and the fibrous tissue hyperplasia. Moreover， they down-regulated the protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and the mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue （P<0.05）， while they up-regulated the protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and the mRNA levels of LC3 and Beclin-1 （P<0.05）. ConclusionDanggui Sinitang， especially at a high dose， can inhibit PI3K/Akt/mTOR signaling pathway to improve autophagy in the synovial tissue， thereby mitigating GA.

Historical Evolution and Textual Research on Classic Prescription Danggui Buxuetang / 中国实验方剂学杂志

Danggui Buxuetang， derived from Clarifying Doubts about Damage from Internal and External Causes （Volume 2）： Treatise on Heat Injury to Stomach Qi（《内外伤辨惑论卷中·暑伤胃气论》） by LI Dongyuan in the Jin and Yuan dynasties， is a classic and famous formula for tonifying qi and generating blood that has been inherited and promoted by successive generations of medical practitioners and has been included in the "Catalogue of Ancient Classical Prescriptions （First Batch）" published by the National Administration of Traditional Chinese Medicine in 2018. The paper analyzed the historical origin， composition， dosage， processing， preparation， decocting， and taking methods， efficacy， and application of the classic formula Danggui Buxuetang by consulting ancient and modern literature and combining the key information examination principles of ancient classic prescriptions. A total of 604 pieces of information on relevant ancient literature were collected， including 186 ancient Chinese medical books， of which 40 （five in the Jin and Yuan dynasties， 19 in the Ming Dynasty， and 16 in the Qing Dynasty） had detailed records of composition， processing， and dosage. Danggui Buxuetang is mainly comprised of Astragali Radix and Angelicae Sinensis Radix. According to the ancient and modern dose conversion， there are 37.3-38.1 g of Astragali Radix and 7.5-7.6 g of Angelicae Sinensis Radix in the formula. Astragali Radix is preferably fried with honey and Angelicae Sinensis Radix with wine. Astragali Radix and Angelicae Sinensis Radix are decocted with 600 mL of water to 300 mL， and taken warm before meals. The main effect of this formula are described in ancient books as blood deficiency and fever， with symptoms of muscle fever， dryness and heat， irritability and thirst， red eyes and face， sleeplessness in daytime and night， and surging and feeble pulse which is weak under hard pressing， and it is a famous formula for replenishing qi and generating blood. Modern research shows that Danggui Buxuetang is commonly used in the treatment of various kinds of anemia， diabetic nephropathy， tumors， and cardiovascular and cerebrovascular diseases. The above research results can provide a reference for the subsequent development and research on the classic formula Danggui Buxuetang.

Effect on Danggui Shaoyao Powder on mitophagy in rat model of Alzheimer's disease based on PINK1-Parkin pathway / 中国中药杂志

This study investigated the mechanism of Danggui Shaoyao Powder(DSP) against mitophagy in rat model of Alzheimer's disease(AD) induced by streptozotocin(STZ) based on PTEN induced putative kinase 1(PINK1)-Parkin signaling pathway. The AD rat model was established by injecting STZ into the lateral ventricle, and the rats were divided into normal group, model group, DSP low-dose group(12 g·kg~(-1)·d~(-1)), DSP medium-dose group(24 g·kg~(-1)·d~(-1)), and DSP high-dose group(36 g·kg~(-1)·d~(-1)). Morris water maze test was used to detect the learning and memory function of the rats, and transmission electron microscopy and immunofluorescence were employed to detect mitophagy. The protein expression levels of PINK1, Parkin, LC3BⅠ/LC3BⅡ, and p62 were assayed by Western blot. Compared with the normal group, the model group showed a significant decrease in the learning and memory function(P<0.01), reduced protein expression of PINK1 and Parkin(P<0.05), increased protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05), and decreased occurrence of mitophagy(P<0.01). Compared with the model group, the DSP medium-and high-dose groups notably improved the learning and memory ability of AD rats, which mainly manifested as shortened escape latency, leng-thened time in target quadrants and elevated number of crossing the platform(P<0.05 or P<0.01), remarkably activated mitophagy(P<0.05), up-regulated the protein expression of PINK1 and Parkin, and down-regulated the protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05 or P<0.01). These results demonstrated that DSP might promote mitophagy mediated by PINK1-Parkin pathway to remove damaged mitochondria and improve mitochondrial function, thereby exerting a neuroprotective effect.

Textual research classic decoction Danggui Yinzi by ancient and modern literatures / 国际中医中药杂志

The Danggui Yinzi, as one of the classic prescriptions, was first recorded in Yan's Jisheng Prescription and is mainly used to treat various skin diseases with blood deficiency and wind dryness. By referring to ancient books and modern literature researches, this study analyzed and summarized the literature of Danggui Yinzi from the aspects of prescription origin, composition, addition and subtractive changes of flavor, dosage and decocting and taking method, discrimination of prescription and efficacy, raw material and processing of medicinal materials, and modern clinical application. Textual researches explored more than 80 ancient literature and 170 modern literature and showed its content included Angelicae Sinensis Radix, Paeoniae Radix Alba, Chuanxiong Rhizoma, Rehmannia Radix, Tribuli Fructus, Saposhnikoviae Radix, Schizonepetae Spica, Polygoni Multiflora Radix, Astragali Radix, Glycyrrhizae Radix et Rhizoma. It was cooked by water. It was used for the patients with skin diseases and Chinese pattern of blood deficiency wind drying. It has showed a wide range of applications, and similar application in ancient and modern time. This paper provides a more comprehensive reference for the research and development of compound preparation of Danggui Yinzi.

Molecular mechanism of Danggui Niantong Decoction in the treatment of gouty arthritis based on network pharmacology and molecular docking / 国际中医中药杂志

Objective:To explore the molecular mechanism of Danggui Niantong Decoction in the treatment of gouty arthritis (GA) based on network pharmacology and molecular docking.Methods:By selecting for the active components and targets of Danggui Niantong Decoction with TCMSP, and retrieving the GeneCards, OMIM, PharmGKB and DrugBank databases to obtain GA related targets. The potential targets of Danggui Niantong Decoction in the treatment of GA were obtained by the intersection of mappings. The regulation network of Chinese medicine compound and protein-protein interaction network of Danggui Niantong Decoction were constructed by Cytoscape software, and the targets of Danggui Niantong Decoction in the treatment of GA were analyzed by GO and KEGG enrichment by David Database. Finally, molecular docking was performed by using Autodock software.Results:There are 198 active components that could treat GA in Danggui Niantong Decoction. The key active components are Quercetin and Kaempferol. There are 46 key targets, the core targets are NFE2L2, HMOX1, PPARA, PTGS2, IL1β, CXCL8. GO enrichment suggests that the key genes are primarily involved in many biological processes such as Inflammatory response regulation, response to oxidative stress, Fatty acid metabolism process, steroid metabolism, lipopolysaccharide response and reactive oxygen species metabolism. KEGG pathway indicates that Danggui Niantong Decoction mainly acted on IL-17 signal pathway, HIF-1 signal pathway, TNF signal pathway and AGE-RAGE signal pathway. Molecular docking shows that the active components of Danggui Niantong Decoction and action target of GA can combine toghether with high efficiency, and the structure is stable.Conclusion:Danggui Niantong Decoction has multi-component, multi pathway and multi-protein characteristics. Danggui Niantong Decoction can treat GA by regulating immune inflammatory reaction and oxidative stress reaction.

Analysis of chemical constituents in classical prescription Danggui Buxue Decoction based on RP-Q-TOF-MS and HILIC-Q-TOF-MS / 中国中药杂志

The chemical constituents of classical prescription Danggui Buxue Decoction were analyzed by reversed-phase(RP) chromatography and hydrophilic interaction chromatography(HILIC) coupled with quadrupole time-of-flight mass spectrometry. RP separation of Danggui Buxue Decoction was performed on ACQUITY UPLC HSS T3(2.1 mm×100 mm, 1.8 μm), while HILIC separation was on Waters BEH Amide(2.1 mm×100 mm, 1.7 μm). Mass spectrometry(MS) data were acquired in both negative and positive ion modes. Chemical constituents of Astragali Radix and Angelicae Sinensis Radix were searched from Reaxys and thus the in-house library was established. MS data were further analyzed by MassLynx 4.1 combined with in-house library, HMDB, Reaxys, and comparison with reference substances. In conclusion, a total of 154 compounds were identified and characterized: 16 saponins, 44 flavonoids, 10 phthalides, 7 phenylpropanoids, 15 bases and the corresponding nucleosides, 30 oligosaccharides, and 32 other compounds. Among them, 65 compounds were detected by HILIC-MS/MS. This study provides experimental evidences for the material basis research, quality control, and preparation development of Danggui Buxue Decoction and a reference method for comprehensive characterization of Chinese medicine decoctions typified by classical prescriptions.

Establishment of the fingerprints for Danggui buxue pills and the method for the content determination of three indicative constituents / 中国药房

OBJECTIVE To establish the fingerprints of Danggui buxue pills a nd the method for the content determination of three indicative constituents （ferulic acid ，calycosin 7-O-β-D-glucoside and astragaloside Ⅳ）. METHODS Fifteen batches of Danggui buxue pills from two manufacturers were analyzed by high performance liquid chromatography （HPLC）. The determination was performed on a Hypersil ODS 2 C18 column with mobile phase consisted of acetonitrile- 0.2％ formic acid （gradient elution ）at the flow rate of 1.0 mL/min. The column temperature was set at 25 ℃，and the sample size was 20 μL. UV detector [detection wavelengths were 250 nm （calycosin 7-O-β-D-glucoside），323 nm （ferulic acid ）] and evaporative light scattering detector （astragaloside Ⅳ）were selected as detectors. HPLC fingerprints of 15 batches of Danggui buxue pills were established with Similarity Evaluation System of TCM Chromatographic Fingerprint （2012 edition）. The chromatographic peaks were identified and assigned by comparing with the chromatogram of the reference substance and reference medicinal material ；the contents of ferulic acid ，calycosin 7-O-β-D-glucoside and astragaloside Ⅳ were also determined. RESULTS There were 33 common peaks in the fingerprints of 15 batches of samples with the similarities not lower than 0.893. Ferulic acid and calycosin 7-O-β-D-glucoside were identified as peak 13 and 15，respectively. Compared with the chromatogram of reference medicinal material，it could be found that peaks 1-3，7，8，10，12，13（ferulic acid ），17-19，27-29，32 and 33 belonged to Angelica sinensis，and peak 14，15（calycosin 7-O-β-D-glucoside），20-23，25 belonged to Astragalus membranaceus . The methodology of content determination met the requirements. The mean contents of ferulic acid ，calycosin 7-O-β-D-glucoside and astragaloside Ⅳ in 15 batches of samples were 0.050 1，0.402 6，0.913 4 mg/g. CONCLUSIONS In this study ，HPLC fingerprints of Danggui buxue pills and the method of HPLC quantitative analysis for three indicative constituents are established. Established methods are accurate，reliable and repeatable.

Exploration of medication rules and mechanisms of traditional Chinese medicine in the treatment of periodontal disease based on data mining, network pharmacology, and molecular docking / 口腔疾病防治

Objective @#To explore the medication law and mechanism of traditional Chinese medicine compounds in the treatment of periodontal disease through data mining, network pharmacology, and molecular docking. @* Methods@#First, data mining was used to search single medicinal materials for the treatment of periodontal disease, and the active components and their action targets were screened. Second, the disease target database was employed to download the targets related to the pathogenesis of periodontal disease, map them with the action targets of traditional Chinese medicine, and obtain the targets that are considered potential targets of traditional Chinese medicine in the treatment of periodontal disease. Potential targets were analyzed for gene ontology function and signaling pathway. They were then screened to obtain the key targets for the treatment of periodontal disease. Finally, the active components were docked with key targets.@* Results@# Among the traditional Chinese medicine prescriptions for the treatment of periodontal disease, Shudihuang, Mudanpi, Danggui, Fuling, Jinyinhua, Shanyao and Zhimu had the highest frequencies. Forty-three active components and 118 action targets were screened, and 52 potential targets were obtained by intersection with 856 disease targets. The molecular functions and biological processes in which potential targets may participate mainly focus on vitamin D biosynthesis and RNA polymerase Ⅱ regulation and involve 96 signaling pathways. Through the analysis of network topology parameters, 11 key targets were obtained. The results of molecular docking showed that the active components and RAC-alpha serine/threonine-protein kinase (AKT1), cellular tumor antigen p53 (TP53), and mitogen-activated protein kinase-1 (MAPK-1) have good binding activity. @* Conclusion @#Traditional Chinese medicine compounds may play a role in the treatment of periodontal disease by inhibiting alveolar bone absorption, have antibacterial and anti-inflammatory properties, and promote tissue repair. The effective treatment of periodontal disease by traditional Chinese medicine compounds provides a more scientific reference to the sustainable development of traditional Chinese medicine.

Investigation of Intestinal Absorption Characteristics of Multi-index Components of Danggui Buxuetang Based on Drug Absorption Simulating System / 中国实验方剂学杂志

ObjectiveTo investigate the intestinal absorption characteristics of multi-index components in Danggui Buxuetang with drug absorption simulating system （DASS） established by everted intestinal sac model. MethodThe intestinal absorption solution at different time points after administration of Danggui Buxuetang was collected and detected by high performance liquid chromatography （HPLC）， acetonitrile （A）-0.2% glacial acetic acid solution （B） was used as the mobile phase for gradient elution （0-16 min， 15%-23%A； 16-20 min， 23%-28%A； 20-25 min， 28%-30%A； 25-30 min， 30%A； 30-35 min， 30%-65%A； 35-45 min， 65%-95%A）， the detection wavelength was 302 nm. HPLC fingerprint of intestinal absorption solution was established and the common peak was calibrated， and the relative cumulative absorption rate of each index component was calculated. The relative cumulative absorption curves of components were fitted with various mathematical models by DDSolver 1.0 to explore the absorption law of different components. ResultThe absorption process of C2 （calycosin-7-glucoside） and C6 in Danggui Buxuetang was in line with zero-order equation， C9 was best fitted by Weibull equation， and the remaining 7 components were in line with Makoid-Banakar equation. C1 with C2， C3， C5， C7 and C10， C2 with C5 and C7， C3 with C4， C5， C7 and C10， C4 with C6 and C10， C5 with C7， C6 with C10， C7 with C10， C8 with C9 were absorbed simultaneously during the absorption process. With the prolongation of time， the overall cumulative absorption rate of Danggui Buxuetang increased. At 120 min， the overall cumulative absorption rate of Danggui Buxuetang exceeded 38%， and reached 49.14% at 180 min. ConclusionTen ingredients in Danggui Buxuetang are absorbed in the jejunum， but absorption law of various components is different， which shows that the intestinal absorption of compound preparations of traditional Chinese medicine （TCM） has multiple characteristics. Intestinal absorption study of TCM compound preparations with chemical composition as the index can reveal some of its absorption law， but it is not complete.

Mechanism of Danggui Niantongtang on Adjuvant Arthritis Rats with Wind-dampness-heat Arthralgia Based on Quantitative Proteomics / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Danggui Niantongtang （DGNT） against adjuvant arthritis （AA） rats with wind-dampness-heat arthralgia by quantitative proteomics. MethodSixty SD rats were randomly divided into normal group， model group， angelica came pain soup low， medium and high dose group and methotrexate （MTX） group， each group of 10， only the rat tail root subcutaneously inactivated mycobacterium tuberculosis （Mtb） of adjuvant to build model of AA， artificial climate box intervention 16 d rheumatic fever bi syndrome model is set up， building the day began to drug intervention， The intervention lasted for 28 days. The proteins of synovial tissues in experimental rats were extracted. The differential proteins in the medium-dose DGNT group and the model group were detected and analyzed by 4D label-free quantification （4D-LFQ） proteomics. The differentially expressed proteins associated with mitochondrial pathway apoptosis were verified by immunohistochemistry and Western blot. ResultA total of 4 756 proteins were identified from rat synovial tissues， of which 4 234 proteins contained quantitative information. There were 814 differential proteins between the model group and the DGNT group. As revealed by Gene Ontology （GO） and Kyoto Encyclopedia of Gene and Genome （KEGG） enrichment analyses， DGNT had an effect on the synovial proteome of AA rats with wind-dampness-heat arthralgia， and the differential proteins were enriched in the regulation of the immune system， response to acute inflammation， and apoptosis regulation. As demonstrated by the results of immunohistochemistry and Western blot， compared with the model group， the DGNT groups and the MTX group showed increased protein expression of B-cell lymphoma 2 （Bcl-2）-associated X protein （Bax） and cytochrome C （Cyt C）（P<0.05， P<0.01）， reduced Bcl-2 level （P<0.05， P<0.01）， elevated level of cleaved cysteinyl aspartate-specific protease 9 （Caspase-9）/Caspase-9 （P<0.01）， and decreased level of phosphorylated protein kinase B （p-Akt）/Akt（P<0.05， P<0.01）. ConclusionDGNT involved multiple targets in the treatment of AA with wind-dampness-heat arthralgia and it may exert its effect in the prevention and treatment by regulating the Akt/Bax/Bcl-2 pathway and promoting the cell apoptosis in the mitochondrial pathway.

Effect of Danggui Niantongtang on Autophagy and Apoptosis of Rheumatoid Arthritis Fibroblast-like Synoviocyte / 中国实验方剂学杂志

ObjectiveTo explore the effect of Danggui Niantongtang （DGNTT） on cell apoptosis and autophagy in rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLS）. MethodRA-FLS were isolated and cultured from the synovial tissue of RA patients. The cells were treated with 10% blank serum （blank control group）， 10% sera containing low， medium and high doses of DGNTT. Wound healing assa and cell invasion test were applied to observe the effect of RA-FLS invasion technique. The apoptosis and autophagy level of RA-FLS cells was detected by Hoechst33342 method and monodansylcadaverine （MDC） staining. The mRNA and protein expression levels of B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， microtubule-associated protein 1 light chain 3 （LC3）， autophagy key molecular yeast Atg6 homolog 1 （Beclin1） were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）and Western blot. ResultCompared with the blank control group，each dose of serum could slow down the wound healing and significantly Reduce the number of RA-FLS cells invading the lower chamber（P<0.01），the mRNA and protein expression levels of Bcl-2，LC3，Beclin1 were significantly decreased（P<0.01）， and Bax were significantly increased（P<0.01）. Hoechst33342 results showed that low， medium and high doses DGNTT could promote RA-FLS cell apoptosis. After MDC staining，autophagosome in low， medium and high doses DGNTT decreased significantly（P<0.01）. ConclusionDanggui Niantongtang can effectively inhibit the proliferation of fibroblast-like synoviocytes. Its mechanism may be related to promote apoptosis and inhibit autophagy of fibroblast-like synoviocytes.

Effect of Danggui Buxuetang on Podocyte Pyroptosis in Diabetic Kidney Disease Rats： An Exploration Based on TXNIP/NLRP3/GSDMD Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease （DKD） rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group， and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg-1 streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling， they were randomized into the model group， low- （0.72 g·kg-1） and high-dose （1.44 g·kg-1） Danggui Buxuetang group， and irbesartan （0.017 g·kg-1） group and gavaged with the corresponding drugs， while those in the normal group and model group with an equal volume of normal saline， once per day， for 20 weeks. During the medication， the fasting blood glucose （FBG） and 24 h urine protein （24 h-UTP） were measured regularly. After administration， the pathological changes in renal tissues were observed by periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. Serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling （TUNEL） assay. The protein expression levels of thioredoxin interacting protein （TXNIP）， cysteine-dependent aspartate-directed protease-1 （Caspase-1）， and gasdermin D （GSDMD） in renal tissues of rats were detected by immunohistochemistry （IHC）， the expression levels of nucleotide binding domain like receptor protein 3 （NLRP3） and Wilms tumor protein-1 （WT-1） in podocytes by immunofluorescent （IF） staining， and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group， the model group exhibited increased FBG and 24 h UTP， glomerular hypertrophy， mesangial hyperplasia， increased extracellular matrix， thickened basement membrane， K-W nodules， vacuolar degeneration in renal tubular epithelial cells， foot process fusion or loss， elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue， enhanced NLRP3 but diminished WT-1 expression in podocytes， down-regulated Synaptopodin protein expression， and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group remarkably lowered FBG， 24-h UTP， and TUNEL-positive cells in renal tissue， improved renal histopathology and podocyte injury and loss， down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels， and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression （P<0.05， P<0.01）. ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.