Curcumin Promotes Autophagy Via Inhibiting Akt/mTOR Signaling Pathway By Regulating MiR-34a To Improve The Effect Of High-fat Diet-induced Osteoarthritis In Obese Rats

Objective: To investigate whether curcumin improve obesity-related osteoarthritis(Osteoarthritis,OA)via promoting autophagy by regulating miR-34a/Akt/m TOR,playing the role of improving the condition of obesity-related osteoarthritis(Osteoarthritis,OA)by protecting articular cartilage,so that we could explore the pathogenesis of OA and provide experimental basis for nutritional control methods.Methods: Sixty-six SD rats with body weight of 240 g±20 g(6 weeks of age)were divided into normal diet(ND)group and high-fat diet(HFD)group,there is no difference in body weight between the two groups.Rats in ND group fed with maintenance feed(10% kcal from fat),HFD group was fed homemade high-fat feed(60% kcal from fat).All rats were weighed body weight and recorded food intake weekly.At the 28 th week,5 rats in each group were randomly selected for gait analysis.Then these rats were sacrificed,and the hind limbs were fixed,decalcified,and embedded for histopathological examination,followed by Safranin O-fast green staining and HE staining.After that,the ND group renamed to C(control)group,continued to be fed with the maintenance food.The high-fat group was randomly divided into 7 groups: pure high-fat(H)group,high-dose curcumin(HH),low-dose curcumin(HL),high-dose curcumin plus NC(HH-NC),low-dose curcumin plus NC(HL-NC),low-dose curcumin plus agomir(HL-Ago),high-dose curcumin plus agomir(HH-Ago),n=7 in each group.At the beginning of the 29 th week,the hind limb joints of the HL-NC and HH-NC groups were injected into negative control NC dissolved in sterile PBS.The hind limbs of the HL-Ago group and HH-Ago group were injected with agomir dissolved in sterile PBS.Group rats were injected with sterile PBS into the hind limb joints,and the injection volume of each joint was 100 μl.One day later,the hind limb joints of groups C and H were injected with DMSO.The remaining six groups were injected with corresponding volumes of curcumin solution dissolved in DMSO at two doses,low and high,respectively.The injection volumes of the solutions corresponding to low and high doses were 16μl./kg and 32 μl/kg(obtained according to previous experiments).Curcumin or DMSO was injected every week,until the 32 nd week,a total of 4 injections.At the end of the 32 nd week,the rats were subjected to gait analysis.Fats of each animal were taken to calculate body fat ratio after sacrifice.Joints were taken for histopathological examination,safranine O-fast green staining and HE staining were performed.Immunofluorescence was used to detect the levels of articular chondrocytes Beclin1,LC3 B,p62,p-Akt,and p-m TOR.Cartilage RNA was extracted,and the relative expression level of miRNA-34 a was determined by q RT-PCR.Results: 1.High-fat diet increased the body weight and body fat of rats,and induced OA-like changes.Compared to the ND group,the body weight of the HFD group rised(P<0.01),and the body fat ratio of the HFD group also increased(P<0.05).Safranin O-fast green staining and HE staining were used.Joints of the HFD group rats appeared OA-like changes,and Mankin score increased,which was statistically significant compared with the ND group(P<0.01).2.Curcumin treatment improved the performance of osteoarthritis in high-fat diet rats,and the improvement effect was exerted by reducing the level of miR-34 a.The 7 groups of rats fed a high-fat diet had significantly higher body weight and higher body fat ratio than the control group.Curcumin treatment improved OA-like lesions,and the Mankin score was lower than that of group H(P <0.05).Compared to the HL-NC group,the histological damage of HL-Ago was more serious,but the Mankin score was not statistically different.Compared to the C group,the expression of miR-34 a in articular cartilage of group H was significantly up-regulated(P<0.01),and curcumin treatment reduced its expression(P<0.05).Compared to the HL-NC group,the expression of miR-34 a in the HL-Ago group was significantly increased(P <0.05),and the expression of miR-34 a in the HH-Ago group was significantly lower than that in the HL-Ago group(P <0.05).3.Autophagy decreased in cartilage tissue of rats with high-fat diet,curcumin restored autophagy levels by inhibiting miR-34 a.Compared to the C group,the expression of Beclin1 and LC3 B in group H decreased,and the expression of p62 increased.Curcumin treatment up-regulated the expression level of Beclin1,LC3 B,and decreased the expression level of p62.Compared to the HL-NC group,Beclin1 and LC3 B expression in the HL-Ago group decreased,and p62 expression increased.Compared to the HH-NC group,Beclin1 and LC3 B expression in the HH-Ago group decreased.Compared to the HL-Ago group,HH-Ago group expressed increased LC3B(P <0.05)and decreased p62(P <0.01).4.In the cartilage of high-fat diet rats,miR-34 a inhibited autophagy by promoting Akt/m TOR,and curcumin reduced the expression of p-Akt and p-m TOR by inhibiting miR-34 a.Compared to the C group,the expression of p-Akt and p-m TOR in articular cartilage of H group was significantly increased(P<0.01,P <0.01),and curcumin decreased their expression(P <0.01).Compared with the HL-NC group,the expression of p-Akt and p-m TOR in the HL-Ago group increased(P <0.01,P <0.05);compared with the HH-NC group,the expression of p-Akt in the HH-Ago group decreased(P <0.01).Compared to the HL-Ago group,the expressions of p-Akt in HH-Ago group declined(P<0.05).And the level of p-m TOR in the HL-Ago group was higher than that in the HH-Ago group(P<0.05).Conclusion:1.HFD leads to obesity-related OA-like lesions in rats,and curcumin can protect the cartilage of OA.2.In the cartilage tissue of obese OA rats,curcumin promotes autophagy by reducing the expression of miR-34 a.3.Mi R-34 a may inhibit autophagy by activating Akt/m TOR pathway,and curcumin can resume cartilage autophagy by inhibiting miR-34 a.