[Action mechanism of Fangji Huangqi Decoction in treating knee osteoarthritis via regulating AMPK/mTOR pathway and activating autophagy based on network pharmacology and animal experiments].

Abstract

Combining network pharmacology and animal experiments,this study explored the mechanism of Fangji Huangqi Decoction in intervening knee osteoarthritis(KOA).Potential targets of Fangji Huangqi Decoction for the treatment of KOA were obtained through multiple databases,such as traditional Chinese medicine system pharmacology(TCMSP) and Gene Cards.A proteinprotein interaction(PPI) network was constructed,and GO function and KEGG pathway enrichment analysis was performed by using the bioinformatics resource database,namely the Database for Annotation,Visualization,and Integrated Discovery(DAVID).Molecular docking was conducted between key components and core targets.Rats were divided into a control group,a model group,and Fangji Huangqi Decoction groups with low and high doses.The KOA model was established by anterior cruciate ligament transection(ACLT).Cartilage and synovium were evaluated by hematoxylin-eosin(HE) staining and safranin O-fast green(SFG)staining.The levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA).The expressions of microtubule-associated protein 1 light chain 3β(LC3B),phosphorylated AMPactivated protein kinase(p-AMPK),and phosphorylated mammalian target of rapamycin(p-mTOR) were detected by immunohistochemistry(IHC).The expressions of collagen type Ⅱ alpha 1 chain(COL2A1),aggrecan(ACAN),matrix metalloproteinase 13(MMP13),p-AMPK,p-mTOR,autophagy receptor SQSTM1(p62),and LC3-Ⅱ/Ⅰ were detected by Western blot.Network pharmacology analysis identified 180 potential targets of Fangji Huangqi Decoction for the treatment of KOA,which were mainly enriched in AMPK and autophagy-related pathways.Animal experiments show that compared with model group,Lequesne MG scores,OARSI scores,and Mankin scores were significantly reduced in Fangji Huangqi Decoction group(P<0.01,P<0.001).ELISA results show that the levels of IL-1β,TNF-α,and IL-6 were significantly decreased in Fangji Huangqi Decoction group(P<0.001).IHC results show that the expressions of LC3B and p-AMPK are up-regulated,while the expression of p-mTOR was downregulated in Fangji Huangqi Decoction group(P<0.001).Western blot results show that Fangji Huangqi Decoction increases the expressions of COL2A1,ACAN,p-AMPK,and LC3-Ⅱ/Ⅰ,and decreases the levels of p-mTOR,MMP13,and p62(P<0.001),with a more obvious effect in the high-dose group.In conclusion,Fangji Huangqi Decoction may improve KOA by activating the AMPK/mTOR signaling pathway to regulate autophagy,alleviate inflammatory response,and promote cartilage matrix restoration.