Cav1.2在顺铂诱导C57BL/6J小鼠耳蜗螺旋神经元凋亡中的作用

Role of Cav1.2 in cisplatin induced apoptosis of cochlear spiral ganglion neurons in C57BL/6J mice

目的:探究顺铂(CDDP)诱导C57BL/6J小鼠耳蜗螺旋神经元(SGNs)凋亡过程中Cav1.2的作用及其可能的机制。方法:动物实验:选取8周龄雄性C57BL/6J小鼠分为以下两组(10只/组):生理盐水组(Control组)和顺铂给药组(Cisplatin组)。Control组每天腹腔注射生理盐水,Cisplatin组每周期前4 d以3 mg/kg的剂量进行顺铂腹腔注射,后10 d每日注射生理盐水,重复三个周期。给药结束后,听性脑干反应(ABR)检测小鼠听力阈值变化;小鼠内眦采血,并断颈取耳蜗,超氧化物歧化酶(SOD)以及丙二醛(MDA)试剂盒检测血清及耳蜗组织的SOD活性和MDA含量;免疫印迹法(Western blot)检测耳蜗组织相关凋亡蛋白表达;苏木精-伊红HE染色观察小鼠耳蜗螺旋神经节形态学变化;TUNEL染色观察小鼠耳蜗SGNs凋亡情况;免疫荧光观察耳蜗SGNs上Cav1.2的分布和表达。细胞实验:原代培养SGNs,根据CCK8选择顺铂5μmol/L干预12 h并随机分为:对照组(Control)、溶剂组(DMSO)、Cav1.2阻断剂组(N)、顺铂组(Cisplatin)、顺铂与Cav1.2阻断剂共同孵育组(Cisplatin+N)。Western blot检测Cav1.2蛋白表达;Hoechst33342染色观察各组SGNs凋亡情况,流式细胞术检测各组SGNs凋亡率,Western blot检测相关凋亡蛋白的表达,CA探针检测细胞内钙离子浓度变化,线粒体膜电位检测试剂盒(JC-1)检测膜电位变化,线粒体超氧化物指示剂(MitoSOX-red)检测线粒体释放ROS情况。结果:动物实验:与Control组相比,Cisplatin组小鼠听力阈值升高(P<0.01),血清及耳蜗组织MDA含量、耳蜗组织凋亡蛋白Cleaved-caspase-3、Bax蛋白水平和TUNEL阳性率、Cav1.2蛋白表达水平等均明显升高(P<0.05,P<0.01);血清及耳蜗组织SOD活性、耳蜗组织抗凋亡蛋白Bcl-2蛋白水平和SGCs密度均明显降低(P<0.05,P<0.01)。细胞实验:与Control组相比,Cisplatin组的Cav1.2表达、细胞凋亡率、Cleaved-caspase-3、Bax蛋白水平、细胞内钙离子浓度以及ROS释放均明显增加(P<0.05,P<0.01);而细胞的Bcl-2蛋白水平和线粒体膜电位则明显降低(P<0.01);Cav1.2阻断剂可部分逆转上述改变(P<0.05)。结论:顺铂可能通过上调Cav1.2促进钙内流,进而使线粒体ROS增多,引起SGNs氧化应激损伤从而诱导线粒体途径的细胞凋亡。

Objective:To investigate the role of Cav1.2 and its possible mechanism in the apoptosis of cochlear spiral ganglion neurons(SGNs)induced by cisplatin(CDDP)in C57 BL/6 J mice.Methods:Animal experiment:8-week-old male C57 BL/6 J mice were randomly divided into the following two groups(10 mice/group):normal saline group(Control group)and Cisplatin group(Cisplatin group).The Control group received daily intraperitoneal injections of normal saline,Cisplatin group was injected with cisplatin intraperitoneally at a dose of 3 mg/kg at the first 4 days of each cycle,and normal saline was injected daily at the last 10 days,repeat for 3 cycles.After administration,auditory threshold was detected by auditory brainstem response(ABR).Blood samples were collected from inner canthus of mice,and cochlea was cut off from neck.SOD and MDA kits were used to detect SOD activity and MDA content in serum and cochlea tissues.The expressions of apoptosis proteins in cochlear tissues were detected by Western blot.Morphological changes of spiral ganglion in mouse cochlea were observed by hematoxylin-eosin(HE)staining.TUNEL staining was used to observe the apoptosis of SGNs in cochlea of mice.The distribution and expression of Cav1.2 in SGNs of cochlea were observed by immunofluorescence.Cell experiment:Primary cultured SGNs were randomly divided into:control group(Control),solvent group(DMSO),Cav1.2 blocker group(N),cisplatin group,cisplatin and Cav1.2 blocker co-incubation group(Cisplatin+N).5μmol/L cisplatin was selected to treat SGNs based on the results of CCK8.Western blot was used to detect the protein expressions of Cav1.2.and apoptotic proteins.Hoechst33342 staining was used to observe the apoptosis of each group.Flow cytometry was used to detect the apoptosis rate of each group.Mitochondrial superoxide indicator(MitoSOX-Red)was used to detect the ROS release of mitochondria.Results:Animal experiments:Compared to the Control group,the hearing threshold was increased in Cisplatin group(P<0.01),the content of MDA in serum and cochlea tissues,apoptosis protein Cleaved caspase-3,Bax protein level,TUNEL positive rate,Cav1.2 protein expression level were increased significantly(P<0.05,P<0.01);the activity of SOD in serum and cochlear tissue,anti-apoptotic protein bcl-2 protein level and SGCs density in cochlear tissue were decreased significantly(P<0.05,P<0.01).Cell tests:Compared with the Control group,the expression of Cav1.2,apoptosis rate,Cleaved caspase-3,Bax protein level,intracellular calcium ion concentration,and ROS release were increased significantly only in Cisplatin group(P<0.05,P<0.01).The levels of bcl-2 protein and mitochondrial membrane potential were decreased significantly(P<0.01).Cav1.2 blockers could partially reverse the above changes(P<0.05).Conclusion:Cisplatin may increase intracellular Caconcentration through up-regulation of Cav1.2,and then damage mitochondria,causing oxidative stress injury of SGNs and inducing neuronal apoptosis.