Study On Pharmacodynamic Substance Basis And Prescription Rule Of Bizhengning Decoction Against Wind-cold-dampness Type Rheumatoid Arthritis

Based on theoretical research,network pharmacology,molecular docking,metabolomics experiment and single cell sequencing data analysis,explore the material basis of Bizhengning decoction against wind-cold-dampness type rheumatoid arthritis.1.Theoretical researchTo understand Rheumatoid arthritis(RA)by reading modern literature and ancient books of Chinese medicine.Following the treatment principle of arthralgia theory in Huangdi Neijing and the theory of storing collagalgia disease,the cubic principle of Arthralgia formula is analyzed by absorbing Wang Mengying’s theory of "long illness and evil spirits".The compatibility characteristics of Bizhengning decoction were studied by combining the compatibility of Junchen and Zhushi with the compatibility of medicinal flavor.Based on previous studies and modern pharmacological experiments to understand the pharmacological basis of Bizhengning decoction against wind-cold-dampness type RA.A preliminary understanding of the theoretical origin of Bizhengning decoction’s resistance to wind-cold-dampness type RA;Again,the role of metabolites in the pathogenesis of RA was understood from the perspectives of pathopathology,early markers,pathogenesis and TCM syndroms of RA,providing theoretical support for the material basis research of Bizhengning decoction against wind-cold-dampness type RA.2.Based on network pharmacology to explore the molecular mechanism of Bizhengning decoction resisting rheumatoid arthritis.Objective: To explore the molecular mechanism of Bizhengning decoction against rheumatoid arthritis from the perspective of network pharmacology and molecular docking.Methods: Based on BATMAN-TCM and TCMSP databases,the main active components and targets of Bizhengning decoction were selected,and the relevant targets of Rheumatoid arthritis(RA)were obtained by Genecards and OMIM databases.The anti-RA targets of Bizhengning decoction were obtained by intersecting the target points of Bizhengning decoction with the target points of RA,and the mapping map of "prescription compatibility-effective components-action target-treatment of disease" was established in Cytoscape software.Next,the protein interaction network was constructed and GO and KEGG enrichment analysis was performed on the anti-RA related targets of Bizhengning decoction.Then,the key genes of Bizhengning decoction resisting RA were filtrated out and analyzed the biological process of it.Finally,through molecular docking technology verified the binding activities of the Bizhengning decoction active components and key genes.At the same time in the fourth part of the in vivo animal experiment verification.Results: The effective components of Bizhengning decoction against RA mainly included 34 kinds such as β-sterol,kaempferol,(+)catechin,etc.There were 56 potential targets of Bizhengning decoction resisting RA such as IL6,MMP2,FOS,CXCL8 and so on.The results of GO enrichment mainly involve biological processes such as reaction to bacterial molecule and lipopolysaccharide,inflammatory reaction,and metabolism of reactive oxygen species.The KEGG pathway enriched the main signaling pathways of TNF,AGE-RAGE,IL-17 and NF-κB.The results of molecular docking verified that the active components of Bizhengning decoction had good binding activity with key genes.In the fourth part,it was verified by in vivo animal experiments.Conclusion: Bizhengning decoction inhibits RA by regulating blood microenvironment,inflammatory response,local oxygen supply and metabolism,epigenetics,stress process and so on.It focuses on the delay of local joint structural damage and chronic pain,and has advantages in the treatment of comorbidities such as lipid metabolism disorder,atherosclerosis,diabetic vascular complications and inflammatory bowel disease.3.Based on metabonomics to explore the material basis of Bizhengning decoction’s resistance to wind-cold-dampness type RA.Objective: Based on the method of serum metabolomics to explore the material basis of Bizhengning decoction against wind-cold-dampness type RA.Methods: 24 Wistar rats were randomly divided into 3 groups:normal group,model group and Bizhengning decoction group,with 8rats in each group.The wind-cold-dampness type RA rat model was prepared by bovine type Ⅱ collagen emulsion and simulated wind-cold-dampness environment at the same time.The pathological changes of the right ankle joint of the rats in each group were observed by HE staining after 4 weeks of intervention treatment with Bizhengning decoction.Serum of rats was taken for liquid chromatograph-mass spectrometer(LC-MS)analysis,data dimension reduction analysis was performed by PCA combined with OPLS-DA pattern recognition method to screen the differential metabolites of the normal group,the model group and the Bizhengning decoction group.Finally,the pathway enrichment analysis of differential metabolites was carried out.Results: HE staining suggested that Bizhengning decoction could inhibit synovial cell proliferation,inflammatory cell infiltration,pannus formation of adjacent articular surface,improve articular cavity clarity and stenosis,joint bone destruction,and repair articular surface smoothness in wind-cold-dampness type RA rats.The main serum differential metabolites of wind-cold-dampness type RA were L-Glutaminic acid,Ascorbic acid,L-Phenylalanine,Anthranilate,L-Histidine,etc.The main pathways involved are steroid hormone biosynthesis,phenylalanine metabolism,arginine and proline metabolism,tryptophan metabolism,etc.Conclusion: Bizhengning decoction may up-regulate the relative expression levels of metabolites such as vitamin C,biliverdin,D-proline and corticosterone,and down-regulate the relative expression levels of metabolites such as thymine,creatine,5-hydroxytryptophan,stearamide and oleic acid amide.It affects the metabolism of arginine and proline,porphyrin and chlorophyll,glycine,serine and threonine,pyrimidine,steroid hormone biosynthesis,and thus plays the role of against wind-cold-dampness type RA rats.4.Based on the group effect relationship to explore the material basis of Bizhengning decoction’s resistance to wind-cold-dampness type RA.Objective: Based on the method of serum metabolomics,to explore the difference of metabolites of different Bizhengning decoction in against wind-cold-dampness type RA.Methods: 56 Wistar rats were randomly divided into 7 groups:normal group,model group,Bizhengning Quanfang group,Bizhengning Junyao group,Bizhengning Chenyao group,Bizhengning Zuoyao group,Bizhengning Shiyao group,8 rats in each group.The wind-cold-dampness type RA rat model was prepared by bovine type Ⅱcollagen emulsion and simulative wind-cold-dampness environment.After 4 weeks of intervention with Bizhengning decoction and split decoction of it,the pathological changes of the right ankle joint of rats in each group were observed by HE staining and saffron O-solid green staining,and serum cytokines IL-1B,IL-6 and VEGFA were detected by ELSIA method.Serum of rats was taken for liquid chromatograph-mass spectrometer(LC-MS)analysis,data dimension reduction analysis was performed by PCA,combined with OPLS-DA pattern recognition method to screen the differential metabolites between the groups,and finally the pathway enrichment analysis of differential metabolites was performed.Results: Compared with the model group,the degree of synovial cell proliferation,degree of inflammatory cell infiltration,joint bone destruction and articular surface smoothness,articular cavity clarity and narrowing,the number and volume of pannus in the adjacent articular surface were improved after intervention in the Bizhengning Quanfang group and its split decoction group.However,the improvement of the Bizhengning Quanfang group was particularly obvious,which was close to that of the normal group.The contents of IL-1B,IL-6 and VEGFA in serum of wind-cold-dampness type RA rats in Bizhengning Quanfang group and Bizhengning Chenyao group were effectively reduced.The contents of IL-1B and IL-6 in serum of RA rats with wind-cold-dampness type can be effectively reduced in the Bizhengning Junyao group.The content of IL-1B and VEGFA in serum of wind-cold-dampness type RA rats can be reduced effectively in Bizhengning Zuoyao group.The content of serum cytokine VEGFA in wind-cold-dampness type RA rats can be reduced effectively in Bizhengning Shiyao group.The metabolites regulated by resisting wind-cold-dampness type RA rats in Bizhengning Quanfang group were vitamin C,thymine and creatine,and the pathways of action were arginine and proline metabolism,porphyrin and chlorophyll metabolism,glycine,serine and threonine metabolism,etc.The metabolites regulated by resisting wind-cold-dampness type RA rats in Bizhengning Junyao group were cholesterol,stearamide,and prostaglandin I2,and the pathways of action were steroid hormone biosynthesis,arginine and proline metabolism,and aminoyl biosynthesis.The metabolites regulated by resisting wind-cold-dampness type RA rats in Bizhengning Chenyao group were 3-succinylpyridine,prostaglandin I2 and prostaglandin B2,and the pathways of action were aminoyl biosynthesis,histidine metabolism and beta-Alanine metabolism.The metabolites regulated by resisting wind-cold-dampness type RA rats in Bizhengning Zuoyao group were thymine,oleamide,oleanolic acid,etc,and the pathways of action were arachidonic acid metabolism,arginine and proline metabolism and pyrimidine metabolism.The metabolites regulated by resisting wind-cold-dampness type RA rats in Bizhengning Shiyao group were cholesterol,L-canine,thymine,etc.,and the pathways of action were steroid hormone biosynthesis,histidine metabolism,beta-Alanine metabolism,etc.Conclusion: Bizhengning Quanfang group and its split decoction group may affect the change of metabolic pathway by regulating the relative expression of metabolites in serum and play the role of resisting wind-cold-dampness type RA rats.There are differences in inflammatory factors,metabolites and metabolic pathway after different compatibility intervention,which also reveals the metabolic material basis under the compatibility rule of Bizhengning decoction.5.To explore the mechanism of Bizhengning decoction against wind-cold-dampness type RA based on cell heterogeneity.Objective: Based on single cell sequencing and molecular docking techniques to explore the mechanism of Bizhengning decoction against wind-cold-dampness type RA.Methods: Single cell peripheral blood sequencing data of rheumatoid arthritis was obtained from GEO database,and differential genes were screened.Through PCA and UMAP clustering analysis,cell annotation,Marker gene search and pseudo-time sequence analysis were carried out.The effective components of Bizhengning decoction were screened and the interaction between Marker and drug in vivo was simulated by molecular docking.Then,24 Wistar rats were randomly divided into normal group,model group and Bizhengning decoction group,with 8 rats in each group.The wind-cold-dampness type RA model was established in model group and Bizhengning decoction group.The joint histopathological changes of rats in each group were observed after drug administration,and the expression of Marker gene was detected by RT-PCR.Results: Ighm,Jchain,Elane and other differential genes were mainly distributed in B cells and red blood cells.The initial phase of rheumatoid arthritis involves B cells,CD8+T cells,and monocytes.The effective components Kaempferol,beta-sitosterol and Stigmasterol of Bizhengning decoction had good binding activity with Marker genes Ighm and Jchain of rheumatoid arthritis.The level of joint inflammation in Bizhengning decoction group was significantly lower than that in model group,and the relative expression level of Jchain m RNA in Bizhengning decoction group was significantly lower than that in model group(P<0.01).Conclusion: Bizhengning decoction has the effect of resisting wind-cold-dampness type RA rats,and may regulate immune function by affecting the expression level of Jchain in B cells,which is of great significance for the clinical prevention and treatment of RA.