乙酰唑胺对IL-1β诱导的大鼠关节软骨细胞凋亡的影响及机制研究  

Effect of acetazolamide on IL-1β-induced apoptosis in rat articular chondrocytes and its mechanism

作　　者：陈伟娜 李春梅 朱仲珍 蔡莉[2] 李荣[1] 

机构地区：[1]安徽医科大学药学院,合肥230032 [2]安徽医科大学病理学教研室,合肥230032

出　　处：《安徽医科大学学报》2018年第5期700-706,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81102273);高校优秀青年人才支持计划重点项目(编号:gxyq ZD2016045)

摘　　要：目的探讨水通道蛋白1(AQP1)抑制剂乙酰唑胺(AZ)对IL-1β诱导的大鼠关节软骨细胞凋亡的影响及机制。方法分离培养大鼠关节软骨细胞主要采用胰酶-胶原酶法,IL-1β诱导软骨细胞凋亡,AZ体外处理IL-1β刺激的关节软骨细胞,MTT法检测细胞增殖,hoechst 33258染色、Annexin V-FITC/PI双染和rhodamine-123染色检测细胞凋亡,Western blot法检测AQP1、Bcl-2、Bax、Caspase3、IκBα、pIκBα、NF-κB p65和p-NF-κB p65的蛋白表达。结果 AZ(12.5、25、50μmol/L)剂量依赖性地提高IL-1β诱导的软骨细胞增殖;AZ可以抑制IL-1β诱导的大鼠关节软骨细胞凋亡,表现为减少细胞凋亡形态学改变、降低软骨细胞凋亡率和提高软骨细胞线粒体膜电位;与正常组比较,IL-1β组AQP1蛋白表达增加,Bcl-2蛋白表达降低,Bax和Caspase3蛋白表达升高,IκB蛋白表达下降,p-IκBα和p-NF-κB p65蛋白表达增加;与IL-1β组比较,AZ抑制AQP1表达,提高Bcl-2表达,降低Bax和Caspase3表达,抑制IκBα的降解和磷酸化,并减少p-NF-κB p65蛋白表达。结论 AZ体外能抑制IL-1β诱导的关节软骨细胞凋亡。该项结果可能与下调Caspase3和Bax(均为促凋亡基因)表达、上调Bcl-2(抗凋亡基因)表达及抑制NF-κB炎症通路活化有关。Objective To investigate the effects and mechanisms of acetazolamide（ AZ）,an aquaporin 1（ AQP1）inhibitor,on IL-1β-induced apoptosis in rat articular chondrocytes. Methods Articular chondrocytes were isolated and cultured by trypsin and collagenase digestion method. IL-1β was used to induce chondrocytes apoptosis in vitro.Different doses of AZ were added to IL-1β-treated chondrocytes. The cell proliferation was detected by MTT assay.The cell apoptosis was evaluated by hoechst 33258 staining,flow cytometry analysis and rhodamine-123 staining.Protein levels of AQP1,Bcl-2,Bax,Caspase3,NF-κB p65,p-NF-κB p65,IκBα and p-IκBα were assayed by Western blot. Results AZ（ 12. 5,25,50 μmol/L） increased the proliferation of IL-1β-induced chondrocytes with a dose-dependent manner. AZ could inhibit the IL-1β-induced rat articular chondrocyte apoptosis,manifested by reducing apoptosis morphological changes,decreasing the rate of chondrocyte apoptosis and increasing mitochondrial membrane potential. Compared with normal group,increased AQP1 protein level,reduced Bcl-2 protein level and increased protein levels of Bax and Caspase3 were observed in IL-1β-induced group. In addition,IκB expression in IL-1β-induced group was lower than normal group,whereas p-IκBα and p-NF-κB p65 protein levels of IL-1β-induced group were higher than normal group. Compared with IL-1β-induced group,AZ decreased AQP1 expression,increased Bcl-2 expression and down-regulated the expressions of Bax and Caspase3. Moreover,AZ inhibited the degradation and phosphorylation of IκBα and reduced p-NF-κB p65 protein level. Conclusion AQP1 inhibition by AZ can decrease chondrocytes apoptosis induced by IL-1β,which might be related to up-regulating Bax and Caspase3（ all pro-apoptotic genes）,down-regulating Bcl-2（ anti-apoptotic gene）,and inhibiting the activation of NF-κB inflammatory pathway.

关 键 词：乙酰唑胺 水通道蛋白1 类风湿性关节炎 关节软骨细胞 凋亡 NF-ΚB通路 

分 类 号：R593.22[医药卫生—内科学]