Study On The Treatment Of Knee Osteoarthritis With Shaoyaogancao Decoction Under The Guidance Of The Theory Of "Gan Pi Tong Zhi"

Objective:The treatment of traditional Chinese medicine has the advantages of multiple targets and multiple approaches to treat the disease,and the theory of traditional Chinese medicine has been continuously developed by the treatment.After refering to related papers,it was found that the theory of treating knee osteoarthritis from the "liver" and "spleen" was the foundation for treating the knee osteoarthritis which the theory of " Gan Pi Tong Zhi".In this study,by summarizing the theory of treating liver and spleen together,the feasibility of treating knee osteoarthritis is explained.Based on this theory,traditional experimental methods are combined with bioinformatics technology to complete a series of researches such as theoretical construction,experimental verification,and network database mining,scientific organization and so on.Method:(1)Research through the classic prescription of "Gan Pi Tong Zhi"——SGT was selected as an example for research by screening classic prescriptions.(2)Searched the components and targets of SGT from the ETCM database,used Cytoscape software to build and analyze the network,used DAVID to analyze functional enrichment from targets。and perform molecular docking experiments to verify the compounds and targets;analyzd SGT based on the above results for the treatment of KOA rat models of multi-component,multi-target treatment mechanism,and a database of prescriptions for " Gan Pi Tong Zhi " was initially constructed(3)A model of IL-1β inducing inflammation of chondrocyte in rats was established and divided into normal group,model group,SGT with serum group and SGT with serum+PTDC group.MTT was used to detect the effects of SGT with serum on IL-1β-induced chondrocyte viability.ELISA was used to detect the effects of content about SGT with serum on DL-1βinducing chondrocyte supernatant IL-6 and IL-10.。WB was used to detect The effect of SGT with serum on the expression of MMP13,ADAMTS-5,and IL-6 in chondrocytes induced by IL-1β was detected by the method;The effect of SGT with serum on IL-1β-induced chondrocyte NF-κB related pathway was detected by WB.Based on the above results,the effect and mechanism of SGT with serum on IL-1β-induced chondrocytes were analyzed.(4)The rat KOA model was established by the ACLT and divided into normal group,model group,SGT group and SGT with+PTDC group.Pathological observation of the effect of SGT on KOA model in rats with light microscope.The effect of SGT on the contents of IL-6,IL-1β,TNF-α,SOD and MDA in KOA rats was measured by ELISA.WB detects the affects the expression of MMP13,ADAMTS-5,IL-6 protein in KOA rats for SGT.The effect of SGT on the expression of NF-κB related pathway proteins in KOA rats was detected by WB.Based on the above results,researched the effect and mechanism of SGT on KOA rats was analyzed.(5)The rat KOA model was established by the ACLT and divided into normal group,model group and SGT group.Metabolonics was used to analyze the rat KOA model sera,and the output was screened for differential screening and KEGG analysis.The effects and mechanisms of SGT With on KOA rats were analyzed through changes in metabolites.(6)Searched ETCM database for the composition and targeted of formula of "Gan Pi Tong Zhi",used Cytoscape software to build and analyze the network,and used DAVID to analyze functional enrichment of the target.Based on the above results,analyze GPTZF to treat the multi-component,multi-target treatment mechanism of the rats KOA model is composed of the treatment of KOA prescription under the guidance of the theory of " Gan Pi Tong Zhi ".Results:(1)32 potential targets of SGT are co-expressed with KOA disease genes,and important targets are mainly enriched in 25 signaling pathways,nine of them are highly related to KOA.The 11 compounds were molecularly docked with 14 targets,and 28 kinds of Chinese medicines containing these 11 compounds through the ECTM database,and the construction of the "Gan Pi Tong Zhi”formula database was initially completed.(2)MTT results showed that the inflammatory factor IL-1β(10 ng/ml)had no toxic effect on chondrocytes in 72 hours,and the medicine-containing serum of SGT had no cytotoxicity against chondrocytes.Chondrocyte activity was increased by serum with SGT 2.5 g/kg comparing with untreated.After adding 10%SGT with 2.5g/kg serum,the chondrocyte viability did not differ significantly with the extension of treatment time.The ELISA results shows that compared with the normal group,the IL-6 content in the model group was significantly increased and the IL-10 content was significantly reduced(P<0.05);compared with the model group,the Med serum of SGT IL-6 was significantly reduced and IL-10 content was significantly increased(P<0.05);compared with the model group,the IL-6 content of Med serum of SGT+PTDC group was significantly reduced,and the IL-10 content was significantly increased(P<0.05).Compared with the Med serum of SGT group,its IL-6 content was increased significantly,and the content of IL-10 decreased significantly(P<0.05).WB results shows that compared with the normal group,protein were significantly increased in the model group of MMP-13,ADAMTS-5 and IL-6(P<0.05),and compared with the model group,protein expressed of the groups of Med serum of SGT of MMP-13,ADAMTS-5 and IL-6 decreased significantly(P<0.05).The expression of total IκBα in chondrocytes did not increase with the addition of IL-1β,but the degree of phosphorylation of IKBa significantly increased(P<0.05)Compared with the model group,the concentration of p-IκBα in Med serum of SGT decreased significantly(P<0.05);Comparing with the model group,the content of p-IκBα decreased significantly in Med serum of SGT+PTDC group(P<0.05),and comparing with the Med serum of SGT group,the content of p-IκBα increased significantly(P<0.05)(3)Pathological results and assessment shows that SGT decreased cartilage degeneration in KOA rats by inhibiting cartilage matrix and cartilage surface degradation.The ELISA results shows that compared with the normal group,the IL-6、IL-1β、TNF-α content in the model group was significantly increased(P<0.01),the MDA content was significantly increased and the SOD content was significantly reduced(P<0.05).compared with the model group,the IL-6、IL-1β、TNF-α content in the SGT significantly reduced(P<0.01),the MDA content was significantly reduced and SOD content was significantly increased(P<0.05),compared with the model group,the IL-6、IL-1β、TNF-α content of SGT+PTDC group was significantly reduced(P<0.05),the MDA content was significantly reduced and SOD content was significantly increased(P<0.05).Compared with SGT group,its IL-6、IL-1β、TNF-α content was increased significantly(P<0.05),its SOD content was decreased significantly and its MDA content was increased significantly(P<0.05).WB results shows that compared with the normal group,protein were significantly increased in the model group of MMP-13,ADAMTS-5 and IL-6(P<0.05),and compared with the model group,protein expressed of the groups of SGT of MMP-13,ADAMTS-5 and IL-6 decreased significantly(P<0.05).The expression of total IκBα in synovium did not increase with the addition of IL-1β,but the degree of phosphorylation of IKBa significantly increased(P<0.05).Compared with the model group,the concentration of p-IκBαin SGT decreased significantly(P<0.05);Comparing with the model group,the content of p-IκBα decreased significantly in SGT+PTDC group(P<0.05),and comparing with SGT group,the content of p-IκBα increased decreased significantly(P<0.05)(4)Difference screening shows that 694 different metabolites were screened in SGT group vs.model group in positive mode,including 380 metabolites with up-regulated expression and 314 metabolites with down-regulated expression.754 different metabolites were screened in SGT normal vs.model group in positive mode,including 484 metabolites with up-regulated expression and 270 metabolites with down-regulated expression.678 different metabolites were screened in normal vs.model group in negative mode,including 351 metabolites with up-regulated expression and 327 metabolites with down-regulated expression.665 dif’ferent metabolites were screened in SGT group vs.model group in positive mode,including 507 metabolites with up-regulated expression and 158 metabolites with down-regulated expression Enrichment analysis of KEGG differential metabolites in the positive model:SGT group vs Model group differential metabolites were significantly enriched in aminoacyl-tRNA biosynthesis,arginine and proline metabolism,D-arginine and D-Ornithine metabolism and glycerophospholipid metabolism.Normal vs.Model group differential metabolites were significantly enriched in D-Arginine and D-ornithine metabolism,Glycerophospholipid metabolism,Phenylalanine metabolism,Pyrimidine metabolism,Aminoacyl-tRNA biosynthesis,Tyrosine metabolism,Arginine and proline metabolism.Enrichment analysis of KEGG differential metabolites in the negative model:SGT group vs.Model group differential metabolites were significantly enriched in Linoleic acid metabolism,Caffeine metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Vitamin B6 metabolism.Normal vs.Model group differential metabolites were significantly enriched in Citrate cycle(TCA cycle),Glyoxylate and dicarboxylate metabolism,alpha-Linolenic acid metabolism,Primary bile acid biosynthesis.(5)Most of the traditional Chinese medicines in GPTZF belong to liver meridian,spleen and stomach,48 potential targets of GPTZF are eo-expressed with KOA disease genes,and important targets are mainly enriched in 26 signaling pathways,thirteen of them are highly related to KOA.Conclusions:(1)The key target proteins of SGT were obtained by network pharmacology and analyzed by KEGG It was found that the treatment pathways of KOA mainly include immune,inflammation,apoptosis,glucose and lipid metabolism.(2)Med serum of SGT has an inhibitory effect on IL-1β-induced chondrocyte inflammation in rats.The mechanism is related to its regulation of NF-KB-related signal map.(3)SGT has an inhibitory effect on osteoarthritis in KOA rats models,and its mechanism may be related to regulating NF-KB-related signaling pathways.(4)Metabolomics analysis showed that SGT improved the rats KOA model through amino acid metabolism,energy metabolism and lipid metabolism.(5)KEGG analysis was used to obtain the key target proteins of the GPTZF through network pharmacological methods.It was found that the treatment of KOA mainly includes immune,inflammation,apoptosis,glucose and lipid metabolism,angiogenesis and cell autophagy.