| Objective To investigate the mechanism of icariin-mediated PI3K-Akt-mTOR pathway regulating autophagy of articular chondrocytes on osteoarthritis(OA).Methods(1)Vitro experiment:Rat OA model was established and chondrocytes were isolated,cultured and the optimal drug concentration was determined.Chondrocytes were divided into 6 groups:control group(cultured in blank medium),rapamycin group(cocultured with 100nM rapamycin),3-methyladenine group(co-cultured with 12mM 3methyladenine)and 40,60,80μM icariin group(co-cultured with 40,60,80μM icariin respectively).The viability of chondrocytes was evaluated by CCK-8 assay.AO-PI staining and flow cytometry were used to observe the apoptosis of chondrocytes.Real-time fluorescence quantitative PCR and Western blotting were used to detect the expression of autophagy-related genes in chondrocytes.(2)Vivo experiment:Rats were divided into 6 groups:sham operation group,OA group,rapamycin group and icariin low,medium and high dose groups,6 rats in each group.Sham operation group and OA group were intraperitoneally injected with normal saline 10ml/kg once a day.Rapamycin group intraperitoneal injection of rapamycin lmg/kg/times,2 times a week.icariin low,medium and high dose groups were intraperitoneally injected with icariin 20,40,80 mg/kg/time,once a day.After 4 weeks of continuous intervention treatment,the rats were sacrificed,and Micro-CT scan of knee joint and safranin O staining,immunohistochemical detection and immunofluorescence determination of cartilage tissue were performed.Results(1)The fluorescence expression intensity of type II collagen in chondrocytes of OA group was significantly lower than that of sham group,indicating that OA model was success-fully established.In this experiment,100M of rapamycin,12mM of 3-methyladenine and 40μM,60μM,80μM of icariin were finally determined as the optimal drug concentration for cell experiments.(2)Compared with the control group,the apoptosis rate of 3-methyladenine group increased,the apoptosis rate of rapamycin group and icariin group decreased,and decreased with the increase of icariin concentration.In addition,compared with the control group,the apoptosis rate of 3-methyladenine group increased from 20.25%to 37.56%,while the apoptosis rate of rapamycin group and 40,60,80μM icariin group decreased to 11.76%and 16.12%,12.75%,11.04%,respectively.(3)Compared with the control group,ATG7,LC3 and LC3-II were significantly inhibited in the 3-methyladenine group,and significantly increased in the rapamycin group and 60μM and 80μM icariin groups(P<0.05,P<0.01).(4)Compared with the control group,the levels of PI3K,p-AKT1,p-mTOR,p-p70S6Kp-AK T1/AKT1,p-mTOR/mTOR,p70S6K/p-p70S6K in chondrocytes were significantly increased in 3-methyladenine group(P<0.01),and significantly decreased in rapamycin group and 80μM icariin group(P<0.01).(5)Micro-CT showed that compared with the OA group,the pathological changes of articular cartilage and articular bone in the rapamycin group and the icariin group were significantly improved,and improved in a dose-dependent manner.Safranin O staining showed that compared with OA group,rapamycin group significantly improved cartilage degeneration and fibrous layer loss,and icariin group also induced this cartilage improvement ability in a dose-dependent manner.The OARSI scores of rapamycin group and icariin groups were also significantly decreased(P<0.01).(6)Compared with sham operation group,the expression of Beclin-1,ATG7 and LC3-Ⅱ/LC3-I in OA group was significantly decreased,while that in rapamycin group and icariin group was significantly increased(P<0.01),and that in icariin group was increased in a dose-dependent manner.The protein levels of PI3K,p-AKT1,p-mTOR and p-p70S6K in OA group were significantly higher than those in sham group,but were significantly inhibited in rapamycin and icariin groups(P<0.01),and decreased in a dose-dependent manner.Conclusion The mechanism of protects chondrocytes by icariin may be to inhibit PI3K-AKTmTOR signaling pathway,activate chondrocyte autophagy,and reduce chondrocyte apoptosis rate. |
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