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.

ObjectiveTo evaluate the animal model of rheumatoid arthritis （RA） induced by inactivated Mycobacterium tuberculosis （Mtb） from inflammation， apoptosis and autophagy， compare the rat RA model of wind-damp-heat arthralgia （FSR） induced by Mtb with the rat RA model of adjuvant arthritis （AA）， and provide experimental evidence for improving the disease-syndrome combined model and developing drugs for the prevention and treatment of RA. MethodThirty 6-week-old SD rats were randomly assigned into 3 groups： a normal group， an AA group and a FSR group， with 10 rats in each group. The RA rat model was established by injection of Mtb suspension （1 mg/rat）， and the rats in the normal group were injected with the same volume of normal saline. The invention in the FSR group lasted for 16 days. The general conditions， body weight， spleen index， swelling of ankle joints， arthritis index （AI）， and the symptoms of arthritis in the hind feet of the rats in each group were observed and measured. The expression levels of serum interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） were detected by enzyme-linked immunosorbent assay （ELISA）. The expressions of IL-1β， TNF-α， apoptosis-related protein B cell lymphoma-2 （Bcl-2） and Bcl-2 associated X protein （Bax）， autophagy-related protein 1 light chain 3 （LC3）， autophagy key molecule yeast Atg6 （Beclin1），and p62 mRNA expression levels were detected by Real-time polymerase chain reaction （Real-time PCR）. The protein expression levels of Bcl-2， Bax， LC3， Beclin1 and p62 were detected by Western blot. ResultCompared with those of the normal group， the rats of AA and FSR groups grew slowly and presented dull hair， soft or loose stool， slow movement， swelling of spleen， redness and swelling of ankle joints， increased AI， and histopathologic changes in the synovium and ankle joints. Moreover， the modeling elevated the levels of IL-1β and TNF-α in the serum， up-regulated the mRNA levels of IL-1β and TNF-α in the synovium， up-regulated the mRNA and protein levels of Bcl2， LC3， Beclin1， and down-regulated the mRNA and protein levels of Bax and p62. Compared with the AA group， the FSR group showed severe symptoms， slowly increased body weight （P<0.01）， early appearance of obvious redness and swelling of ankle joints， increased AI （P<0.05）， increased spleen index （P<0.05）， and obvious pathologic changes of synovial tissue and ankle joints， the inflammatory infiltration in the synovial tissue， and the damage of joint structure. Moreover， the FSR group had higher expression levels of IL-1β， TNF-α， Bcl-2， LC3， and Beclin1 （P<0.05） and lower expression levels of Bax and p62 than the AA group （P<0.05）. ConclusionThe animal model of RA （syndrome of wind-damp-heat arthralgia） can be induced successfully with the symptoms consistent with clinical manifestations of RA. The FSR group has lower apoptosis level and higher autophagy level than the AA group， which indicates that the FSR group is more appropriate for the research on the RA with the syndrome of wind-damp-heat arthralgia.

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.

ObjectiveThis study aims to explore the potential mechanism of Danggui Niantongtang in treating knee osteoarthritis （KOA） by regulating the intestinal flora through 16S rDNA analysis. MethodThirty-six C57BL/6J mice were subjected to anterior cruciate ligament transection （ACLT） to establish a KOA model and were randomly divided into the sham surgery group， model group， low-dose Danggui Niantongtang group （0.819 g·kg^-1）， medium-dose Danggui Niantongtang group （1.638 g·kg^-1）， high-dose Danggui Niantongtang group （3.276 g·kg^-1）， and Meloxicam group （0.975 mg·kg^-1）， with 6 mice in each group. Except for the treatment groups， the sham surgery group and model group were given normal saline by gavage. After 4 weeks of continuous intervention， feces and intact knee joints of the mice were collected. Hematoxylin-eosin （HE） staining and Safranin O-Fast Green staining were performed to observe the pathological changes in knee joint tissue morphology. The 16S rDNA sequencing was used to analyze changes in the abundance and diversity of intestinal microorganisms before and after treatment， along with corresponding functional predictions. ResultHigh-dose Danggui Niantongtang and Meloxicam significantly relieved pain symptoms in KOA mice， improved the disorder of joint structure， maintained the integrity of knee articular cartilage， increased the expression of type Ⅱ collagen alpha 1 （Col2a1） in articular cartilage， and decreased the expression of matrix metalloproteinase-13 （MMP-13）. The results of 16S rDNA sequencing showed that high-dose Danggui Niantongtang could adjust the abundance and structure of intestinal microbial species. Compared with the sham surgery group， the abundance of Proteobacteria， Actinobacteria， Ruminococcus， and Bacteroides was significantly increased in the model group （P<0.05）， while in the Danggui Niantongtang group， the abundance of these four flora was significantly reduced compared with the model group. Compared with the sham surgery group， the abundance of Verrucomicrobia， Oscillospira， and Akkermansia was significantly decreased in the model group （P<0.05）， while in the Danggui Niantongtang groups， the abundance of these three flora was significantly increased compared with the model group （P<0.05）. Functional pathway prediction of differential genera revealed that species differences among groups mainly involved metabolic pathways with high abundance associated with biosynthesis and precursors， as well as energy production， including amino acid biosynthesis， nucleotide and nucleoside biosynthesis， cofactors， prosthetic groups， electron carriers， and vitamin biosynthesis. ConclusionDanggui Niantongtang can effectively protect articular cartilage and delay the progression of KOA， possibly by regulating the structure of the intestinal flora， promoting probiotics， and inhibiting the growth of harmful pathogenic bacteria.