医用臭氧对大鼠骨关节炎软骨细胞中PPARγ及自噬水平表达的影响  

Effects of medical ozone on the expression of PPARγ and autophagy in chondrocytes of osteoarthritis in rats

作　　者：孙盼盼 赵旭[2] 林小雯[1] 傅志俭[1] SUN Panpan;ZHAO Xu;LIN Xiaowen;FU Zhijian(Department of Pain Management,Shandong Provincial Hospital Affiliated to Shandong University,Jinan 250021,Shandong,China;Department of Anesthesia,Shandong Provincial Hospital Affiliated to Shandong University,Jinan 250021,Shandong,China)

机构地区：[1]山东大学附属省立医院疼痛科,山东济南250021 [2]山东大学附属省立医院麻醉科,山东济南250021

出　　处：《山东大学学报（医学版）》2020年第6期14-21,共8页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81271346,81771199)。

摘　　要：目的探讨医用臭氧对大鼠骨性关节炎软骨细胞中过氧化物酶体增殖物激活受体γ(PPARγ)及自噬相关蛋白表达的影响,及PPARγ在臭氧诱导的软骨细胞自噬中的作用。方法选取出生3 d以内的Wistar大鼠10只,获取四肢关节软骨进行原代软骨细胞的分离培养,并通过甲苯胺蓝染色及Ⅱ型胶原蛋白免疫荧光进行软骨细胞的鉴定。通过10 ng/mL白介素-1β(IL-1β)刺激24 h构建骨关节炎软骨细胞模型。造模后采用不同浓度臭氧处理30 min,采用CCK-8法检测不同浓度臭氧对软骨细胞活力的影响,选出最佳臭氧浓度。造模后加入PPARγ特异性抑制剂GW9662处理12 h,再给予30μg/mL臭氧处理。将原代软骨细胞随机分为对照组、模型组、模型臭氧组、正常臭氧组、模型臭氧+抑制剂组及模型抑制剂组。采用Western blotting法检测PPARγ蛋白及自噬相关蛋白LC3Ⅱ、P62及Beclin-1的表达。采用免疫荧光技术检测软骨细胞的自噬相关蛋白LC3B及P62的表达。结果经甲苯胺蓝染色及Ⅱ型胶原蛋白免疫荧光鉴定,分离培养的细胞为软骨细胞。不同浓度臭氧处理后,30μg/mL臭氧处理后骨性关节炎软骨细胞的活力得到改善(P<0.05),50、70μg/mL臭氧明显抑制骨性关节炎软骨细胞的活力(P<0.05)。30μg/mL臭氧提高了骨性关节炎软骨细胞中PPARγ及自噬相关蛋白LC3Ⅱ及Beclin-1的表达水平,抑制了P62蛋白的表达(P<0.05)。PPARγ特异性抑制剂GW9662(20 mmol/mL)抑制臭氧对骨性关节炎软骨细胞自噬相关蛋白LC3Ⅱ、Beclin-1上调作用,并可逆转臭氧对P62的抑制作用。结论30μg/mL医用臭氧可以促进骨性关节炎软骨细胞自噬,PPARγ激活在臭氧诱导骨性关节炎软骨细胞的自噬中起到促进作用。Objective To investigate the effects of ozone(O3)on the expressions of peroxisome proliferators-activated receptorγ(PPARγ)and autophagy-related proteins in rat osteoarthritis chondrocytes and the role of PPARγin ozone-induced autophagy of chondrocytes.Methods Wistar rats within 3 days of birth were chosen and the articular cartilage of extremities were obtained for the isolation and culture of primary chondrocytes.The chondrocytes were identified with toluidine blue staining and type II collagen immunofluorescence.The cells were stimulated with 10 ng/mL interleukin-1β(IL-1β)for 24 h to construct osteoarthritis chondrocytes models.After that,osteoarthritis chondrocytes were treated with different concentrations of ozone for 30 min,the effects on cell activity were detected by CCK-8 assay and the optimal O3 concentration of 30μg/mL was selected.After IL-1βtreatment,PPARγ-specific inhibitor GW9662 was added to the osteoarthritis chondrocytes and treated for 12 h,which were then treated with 30μg/mL O3.The primary chondrocytes were randomly divided into control group,IL-1βgroup,IL-1β+O3 group,control+O3 group,IL-1β+O3+GW9662 group and IL-1β+GW9662 group.The expressions of PPARγand autophagy-related proteins including LC3Ⅱ,P62 and Beclin-1 were detected with Western blotting.The expressions of LC3 B and P62 were detected with immunofluorescence staining.Results Toluidine blue staining and type II collagen immunofluorescence identified the cultured cells were chondrocytes.The cell activity was improved with 30μg/mL ozone(P<0.05),and decreased with 50 and 70μg/mL ozone(P<0.05).The 30μg/mL ozone increased the expressions of PPARγ,LC3Ⅱand Beclin-1,while decreased the level of P62(P<0.05).GW9662(20 mmol/mL)inhibited the up-regulation effect of ozone on the levels of LC3Ⅱand Beclin-1,and reversed the inhibition of ozone on P62.Conclusion Ozone at 30μg/mL can promote autophagy of osteoarthritis chondrocytes,and activation of PPARγcan promote ozone-induced autophagy in osteoarthritis chondrocytes.

关 键 词：臭氧 白介素-1Β 骨性关节炎 过氧化物酶体增殖物激活受体Γ 自噬 

分 类 号：R575.1[医药卫生—消化系统]