CAJ | 학술논문

目的：观察骨关节炎（OA）大鼠软骨自噬基因（Atg5、Atg7、Atg12）以及自噬相关蛋白mTOR、Beclin‐1、PI3K、Akt、LC3‐Ⅱ的表达，并分析其与IL‐4、IL‐10、IL‐1β、TNF‐α的相关性。方法20只大鼠随机分为正常组和模型组，每组各10只。模型组大鼠采用关节腔注射木瓜蛋白酶（Papain）和 L‐半胱氨酸（L‐cystine）方法制成骨关节炎大鼠模型。以光镜对大鼠软骨进行Mankin标准评分，以电镜观察OA大鼠软骨中自噬小体的形态及数目；采用荧光定量PCR检测软骨Atg5、Atg7、Atg12的表达，采用免疫印迹法（Western blot）检测软骨mTOR、Beclin‐1、PI3K、Akt、LC3‐Ⅱ的表达，采用ELISA法检测血清细胞因子IL‐4、IL‐10、IL‐1β、TNF‐α水平。结果与正常组大鼠比较，OA大鼠关节软骨Mankin评分明显升高（P＜0.01）；Atg5在软骨中降低（P＜0.01），并与IL‐4、IL‐10呈正相关（均 P＜0.01），与IL‐1β、TNF‐α呈负相关（P＜0.05或 P＜0.01）；Atg7在软骨中降低（P＜0.01）；mTOR在软骨中升高（P＜0.05），PI3K、Akt在软骨中均升高（均 P＜0.01），并与IL‐4、IL‐10呈负相关（ P＜0.05或 P＜0.01），与IL‐1β、T N F‐α呈正相关（ P＜0.05或 P＜0.01）；LC3‐Ⅱ、Beclin‐1在软骨中降低，与IL‐4、IL‐10呈正相关（ P＜0.05或 P＜0.01），与IL‐1β、T N F‐α呈负相关（ P＜0.05或P＜0.01）。结论 OA大鼠软骨在Mankin评分升高的同时，出现Atg5及Atg7表达的降低，PI3K／Akt‐mTOR通路上调，Beclin‐1下调，自噬受抑制，此过程可能参与OA软骨病变。
목적：관찰골관절염（OA）대서연골자서기인（Atg5、Atg7、Atg12）이급자서상관단백mTOR、Beclin‐1、PI3K、Akt、LC3‐Ⅱ적표체，병분석기여IL‐4、IL‐10、IL‐1β、TNF‐α적상관성。방법20지대서수궤분위정상조화모형조，매조각10지。모형조대서채용관절강주사목과단백매（Papain）화 L‐반광안산（L‐cystine）방법제성골관절염대서모형。이광경대대서연골진행Mankin표준평분，이전경관찰OA대서연골중자서소체적형태급수목；채용형광정량PCR검측연골Atg5、Atg7、Atg12적표체，채용면역인적법（Western blot）검측연골mTOR、Beclin‐1、PI3K、Akt、LC3‐Ⅱ적표체，채용ELISA법검측혈청세포인자IL‐4、IL‐10、IL‐1β、TNF‐α수평。결과여정상조대서비교，OA대서관절연골Mankin평분명현승고（P＜0.01）；Atg5재연골중강저（P＜0.01），병여IL‐4、IL‐10정정상관（균 P＜0.01），여IL‐1β、TNF‐α정부상관（P＜0.05혹 P＜0.01）；Atg7재연골중강저（P＜0.01）；mTOR재연골중승고（P＜0.05），PI3K、Akt재연골중균승고（균 P＜0.01），병여IL‐4、IL‐10정부상관（ P＜0.05혹 P＜0.01），여IL‐1β、T N F‐α정정상관（ P＜0.05혹 P＜0.01）；LC3‐Ⅱ、Beclin‐1재연골중강저，여IL‐4、IL‐10정정상관（ P＜0.05혹 P＜0.01），여IL‐1β、T N F‐α정부상관（ P＜0.05혹P＜0.01）。결론 OA대서연골재Mankin평분승고적동시，출현Atg5급Atg7표체적강저，PI3K／Akt‐mTOR통로상조，Beclin‐1하조，자서수억제，차과정가능삼여OA연골병변。
Objective To observe the expression of autophagy genes (Atg5 ,Atg7 ,Atg12)and autophagy related proteins , Beclin‐1 mTOR ,PI3K ,and Akt ,LC3‐Ⅱ in cartilage of OA rats ,and analyze its correlation with IL‐4 ,IL‐10 ,TNF‐α and IL‐1β.Methods Twenty rats were randomly divided into normal control group (NC)and model control group(MC)(n= 10 each group).In MC group ,osteoarthritis rat model was established by articular cavity injection of Papain and L‐cysteine.Cartilage of the rats was evaluated by Mankin standard score under light microscopy.Autophagosome form and number in OA rats cartilage were observed with electron microscope.Expression of Atg5 ,Atg7 and Atg12 in cartilage was detected with Real‐time PCR;Be‐clin‐1 ,mTOR ,PI3K ,LC3‐Ⅱ and Akt in cartilage were detected with Western blot.Serum cytokines IL‐4 ,IL‐10 ,TNF‐αand IL‐1βwere determined by ELISA.Results As compared with normal group ,Mankin score of cartilage of OA rats was increased significantly(P<0.01);Atg5 in cartilage was significantly reduced (P<0.01) ,and was positively correlated with IL‐4 and IL‐10(P<0.01) ,but was negatively correlated with TNF‐α and IL‐1β(P<0.05 or P<0.01);Atg7 in cartilage was significantly decreased(P<0.01);mTOR in cartilage was significantly increased(P<0.05);PI3K and Akt in cartilage were significantly in‐creased(both P<0.01) ,and were negatively correlated with IL‐4 and IL‐10(P<0.05 or P<0.01) ,but were positively correla‐ted with TNF‐αand IL‐1β(P<0.05 or P<0.01);LC3‐Ⅱ and Beclin‐1 were reduced in cartilage ,and positively correlated with IL‐4 and IL‐10(P<0.05 or P<0.01) .Conclusion At the same time with the increase of Mankin score in OA rats ,the expres‐sion of Atg5 and Atg7 is reduced ,expression of Atg12 increased ,PI3K/Akt‐mTOR pathway is up‐regulated while Beclin‐1 is down‐regulated ,and autophagy is suppressed ,which may participate in the pathological changes of OA rats cartilage.