Oleanolic Acid Protects Against Osteoarthritis In Rats Via PI3K/AKT/mTOR Autophagic Pathway

Osteoarthritis(OA)is a chronic proliferative inflammatory disease of the articular bone system,also known as chronic osteoarthritis.It often manifests clinically as joint stiffness,pain and swelling,which seriously affects the quality of life of animals and causes economic losses.Veterinarians are mostly treated clinically by non-steroidal anti-inflammatory drugs(NSAIDs),but there are more side effects.Therefore,it is crucial to deeply study the pathogenic mechanism of OA and develop safe and effective OA drugs.Plant extracts are widely used in clinical practice because of their low price,low toxic side effects,and various pharmacological activities such as anti-inflammatory and antioxidant.Studies have shown that autophagy plays an important role in the treatment of OA,and moderate autophagy can maintain the normal physiological function of chondrocytes.PI3K/AKT/mTOR pathway is an important pathway regulating cellular autophagy in vivo,which is also involved in the occurrence and development of OA.Based on this,this experiment took PI3K/AKT/mTOR autophagy pathway as an entry point to explore the effects and molecular mechanisms of oleanolic acid(OLA)on OA rats.The experiment was divided into in vivo and in vitro experiments.In the in vivo experiment,a rat OA model was established by joint cavity injection of sodium iodoacetate(MIA),which was administered for 28 consecutive days.Behavioral tests,Von-Frey mechanical pain test,cold hypersensitivity test and knee joint swelling test were performed weekly.Serum samples were collected weekly,and the levels of inflammatory factors and MMPs in rat serum were measured by ELISA.At the end of the experiment,joint samples were collected from each group,and the morphological,X-ray imaging and histopathological observations and scores were performed on rat knee joints,respectively.i NOS and MMP13 protein expression in rat cartilage were detected by immunohistochemistry to assess the protective effect of OLA on cartilage in OA rats.In in vitro experiments,ATDC5 chondrocytes were identified by toluidine blue staining and immunofluorescence assay,and OLA,Chloroquine(CQ)and LY294002 intervention doses were screened by CCK-8 method.An inflammatory injury model was established by 10 ng/m L IL-1β.i NOS,COX2,MMP3,MMP13,Beclin1,P62,LC3II/LC3Ⅰ and PI3K/AKT/mTOR pathway-related protein levels were detected by immunoblotting.m RFP-GFP-LC3 was detected by MDC staining and transmission electron microscopy for autophagic vesicle number and morphology.adenovirus co-localization of autophagy lysosomes and autophagosomes,and RT-q PCR for Atg-related gene levels.A pathway blockage model was established using autophagy inhibitor CQ and PI3 K inhibitor LY294002 to detect autophagy and pathway-related protein expression.To clarify the mechanism by which OLA ameliorates cartilage degeneration in OA rats by regulating PI3K/AKT/mTOR autophagic pathway.The results showed that(1)In a rat OA model,OLA gavage reduced joint swelling,relieved pain,inhibited cartilage pathological changes,and decreased the expression of MMP13 and i NOS in cartilage.In addition,OLA intervention was able to reverse the levels of IL-1β,TNF-α,i NOS,COX-2,MMP3 and MMP13 in the serum of OA rats.(2)In the inflammatory injury model established by IL-1β,OLA treatment decreased the expression of i NOS,COX-2,MMP3,MMP13 and P62 in ATDC5 chondrocytes and promoted Beclin1 and LC3II/LC3 I protein expression.atg-related gene levels were significantly elevated and autophagic vesicle aggregation was significantly increased after OLA intervention.Transfection with m RFP-GFP-LC3 adenovirus observed a significant increase in the number of autophagosomes and autophagic lysosomes after OLA intervention.Autophagy-associated proteins were significantly inhibited by pretreatment of cells with CQ.Using LY294002 pretreatment cells,PI3K/AKT/mTOR pathway proteins were significantly inhibited and autophagy-associated protein expression was elevated,similar to the results of OLA intervention.It indicates that OLA promotes autophagy and protects OA rats by inhibiting PI3K/AKT/mTOR pathway.Conclusion:(1)OLA inhibited MIA-induced OA pain in rats,reduced serum levels of IL-1β,TNF-α,i NOS,COX-2,MMP3 and MMP13,improved cartilage pathological features and protected cartilage degeneration.(2)In an in vitro model of IL-1β-induced inflammatory injury,OLA promoted autophagy to exert anti-inflammatory and anti-matrix degradation effects through regulating the PI3K/AKT/mTOR pathway.