| Objective: To explore whether curcumin(CUR)can inhibit chondrocyte apoptosis by regulating mi RNA-34 a / E2F1 / PITX1,to play the role of anti-obesity-related osteoarthritis(OA),so as to further clarify the pathogenesis of OA and explore the nutritional prevention and treatment measures of OA.Methods: 66 6-week-old male SD rats weighing 220-260 g were randomly divided into two groups: the ND group(12 rats)and the HFD group(54 rats).The ND group were fed with normal diet(10%)and the HFD group were fed with high-fat diet(60%).The rats in each group ate and drank freely at room temperature of 20-25 ? and relative humidity of 40-70%.Weight and food intake were measured every week for 28 weeks in total.At the end of 28 weeks,5 rats in each group were taken for gait analysis,and joints were taken for histopathological examination and Mankin score was calculated.After that,the remaining 7 rats in the general diet group were used as the control group(C)to continue to feed the general diet,and 49 rats in the high-fat diet group continued to feed the high-fat diet and were divided into seven groups: high-fat with non intervention group group(H),high-fat + curcumin in low-dose group(CL),high-fat +curcumin in high-dose group(CH),high-fat + curcumin in low-dose + agomir nomal control group(CLC),high-fat + curcumin in high-dose + agomir nomal control group(CHC),high-fat + curcumin in low-dose +agomir group(CLA),high fat + curcumin in high-dose + agomir group(CHA).Treatment route and dosage: the left and right hind limbs of rats in C,H,CL and CH groups were injected with sterile PBS(100 ?l);the left and right hind limbs of rats in CLC and CHC groups were injected with negative control(5 nmol)of 100 ?l sterile PBS;the left and right hind limbs of rats in CLA and CHA groups were injected with agomir(5 nmol)of 100 ?l sterile PBS respectively.One day later,group C and H were given 5% DMSO oil solution for intra-articular injection;group CL,CH,CLC,CHC,CLA and CHA were given CUR oil solution(12.5 mg/ml)dissolved in 5% DMSO for intra-articular injection.in 29-32 weeks,agomir was only injected once,CUR was injected once a week for 4 weeks.The low and high doses of CUR were 200 ?g/kg/w and 400 ?g/kg/w respectively(the dose was obtained according to the previous experiment).The volume of each rat was adjusted with the weight.At the end of the 32 nd week,the gait of the rats was analyzed;the subcutaneous and visceral fat was taken and the body fat ratio was calculated;the joints were fixed and embedded for histopathological detection;the apoptosis level of articular chondrocytes was detected by TUNEL kit;the expression level of miRNA-34 a was measured by qRT-PCR;the cartilage protein was extracted and the expression of E2F1 and PITX1 was measured by Western blot.Results:1?OA like changes in articular cartilage of obese rats induced by high fat diet.At the end of the 28 th week,the body weight and body fat ratio of rats in HFD group were higher than those in ND group(P<0.01,P<0.05);during the experiment,there was no significant difference in food intake between ND group and HFD group(P>0.05);The results of joint histology showed that the cartilage tissue in ND group was complete and uniform in thickness,the boundary between cartilage and bone was obvious,the cells on the surface of cartilage were arranged orderly,the surface of cartilage tissue was smooth and even,and the matrix staining was even.In HFD group,there were different thickness of cartilage,local thickening or local thinning,local damage of cartilage surface,unclear boundary between cartilage and bone,disordered arrangement of cells on the surface of cartilage,local cells clustering or sparse dispersion,uneven surface of cartilage tissue,uneven matrix staining;The Markin score of HFD group was significantly higher than that of ND group(P<0.05)?2?CUR treatment can significantly improve the OA like changes of cartilage tissue in high fat fed rats;compared with pure CUR,there was no significant difference in Markin score improvement between high and low dose CUR.At the end of the 32 nd week,the body weight and body fat ratio of 7 groups fed with high fat diet were significantly higher than those of the control group(P<0.01,P<0.05).There was no significant difference between the groups fed with high fat diet(P>0.05),and there was no significant difference between the groups fed with high fat diet(P>0.05)The surface of cartilage tissue was smooth and even.In group H,there were different thickness of cartilage,local thickening or local thinning,local damage on the surface of cartilage,unclear boundary between cartilage and bone,disordered arrangement of cells on the surface of cartilage,aggregation or sparse dispersion of local cells,uneven surface of cartilage and uneven matrix staining.In CL and CH group,the cartilage tissue was relatively complete,with occasional cartilage tissue damage,clear boundary between cartilage and bone,irregular arrangement of partial cartilage surface,occasional proliferation or reduction of chondrocytes,and relatively complete and smooth cartilage surface.In CLC,CHC and CHA groups,the boundary between cartilage and bone was slightly unclear,the arrangement of surface cells was disorder,the surface of cartilage tissue was uneven,and the matrix staining was uneven.In CLA group,there were local thickening or local thinning,local damage on the surface of cartilage,unclear boundary between cartilage and bone,aggregation or sparse dispersion of local cells,uneven surface of cartilage and uneven matrix staining;the Markin score in H group was significantly higher than that in C group(P<0.01).After CUR treatment,Markin scores of CL,CH group were significantly lower than H group(P<0.05).There was no significant difference in Markin scores between CL and CH groups(P>0.05).After the treatment of agomir,the Markin score of CLA group was the highest,and the score of CHA was similar to that of CLC and CHC group,but there was no significant difference among the four groups(P>0.05).3?The expression level of miRNA-34 a in the cartilage of rats with obesity-related OA induced by high fat increased significantly;CUR could reduce the expression level of miRNA-34a;low dose CUR did not significantly inhibit the increase of miRNA-34 a,but high dose CUR could inhibit the increase of miRNA-34 a.Compared with group C,the relative expression level of miRNA-34 a in group H was significantly higher(P<0.01);after CUR treatment,the relative expression level of miRNA-34 a was significantly lower(P<0.05);However,there was no significant difference in the relative expression level of miRNA-34 a between CL and CH group(P>0.05);compared with group CLC,the relative expression level of miRNA-34 a in group CLA was significantly higher(P<0.05);compared with group CHC,the relative expression level of miRNA-34 a in group CLA was no significant difference(P>0.05);compared with CLA group,the relative expression level of miRNA-34 a in CHA group decreased significantly(P<0.05).4?The expression level of E2F1 and PITX1 protein in cartilage of rats with obesity-related OA induced by high fat decreased significantly;CUR could enhance the expression of E2F1 and PITX1 protein;compared with CUR treatment alone,the effect of low-dose CUR on the expression of E2F1 and PITX1 was higher than that of high-dose CUR.The expression level of E2F1 and PITX1 in group H was significantly lower than that in group C(P<0.01)compared with H group,the expression level of E2F1 and PITX1 in CL group increased significantly(P<0.05);compared with H group and CL group,there was no significant difference in the expression level of E2F1 and PITX1 in CH group(P>0.05);compared with CLC group,the expression levels of E2F1 and PITX1 in CHC group were significantly lower(P<0.01);compared with CLC group,the expression levels of E2F1 and PITX1 in CLA group were significantly lower(P<0.05,P<0.01);compared with CLA group,the expression levels of PITX1 in CHA group were significantly lower(P<0.01).5?The apoptosis level of chondrocytes in rats with obesity-related OA induced by high fat increased significantly;CUR could inhibit the apoptosis of chondrocytes;compared with CUR alone,the inhibition effect of low-dose CUR was higher than that of high-dose CUR after the treatment of agomir and CUR.After TUNEL fluorescence staining,there were only a few apoptotic cells in group C;the apoptosis level of chondrocytes in group H was significantly higher than that in group C(P<0.01);the apoptosis level of chondrocytes in group CL and CH was significantly lower than that in group H(P<0.01,P<0.05),and there were a few apoptotic cells in group CH,and the expression level of apoptotic chondrocytes in group ch was significantly higher than that in group CL(P<0.05).Compared with CLC and CHC,there was no significant difference in the expression of apoptosis cells between CLA and CHA.Compared with CLA group,the expression level of apoptotic cells in CHA group was higher(P<0.05).Conclusion:1?High fat diet can induce obesity-related OA in rats,and CUR can delay the progression of OA.2?In the cartilage of rats with obesity-related OA,CUR may reduce the expression of miRNA-34 a,partly increase the expression of E2F1 and PITX1,then inhibit the apoptosis of chondrocytes. |
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