瞬时感受器电位离子通道香草素受体4在睾丸缺血再灌注损伤中对GC-1细胞增殖及凋亡的作用及其机制

Effect of transient receptor potential vanilloid 4 on proliferation and apoptosis of GC-1 cells in testicular ischemia-reperfusion injury and mechanism

目的探讨瞬时感受器电位离子通道香草素受体4(TRPV4)在睾丸缺血再灌注损伤(IRI)中对GC-1细胞增殖和凋亡的作用及其机制。方法建立睾丸GC-1细胞缺氧复氧模型,采用蛋白质印迹法(Western blot)检测不同复氧损伤时间点TRPV4的表达变化;分别采用噻唑蓝(MTT)实验、流式细胞术检测转染TRPV4对GC-1细胞增殖和凋亡的影响;采用Western blot法检测睾丸组织中转染TRPV4对GC-1细胞中半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和细胞色素C(Cyt-C)表达的影响,组间比较采用t检验,多组间比较采用单因素方差分析。结果对照组及缺氧复氧组(0、6、12、24、48和72 h)TRPV4的蛋白表达水平分别为0.19±0.02、0.35±0.03、0.42±0.04、0.46±0.04、0.62±0.05、0.54±0.05、0.45±0.04。缺氧复氧组TRPV4表达显著高于未缺氧GC-1细胞的对照组,并在复氧24 h达到峰值(F=6.898,P<0.05),差异有统计学意义;缺氧复氧组中细胞增殖水平明显低于对照组(52.32±4.58比100.00±7.63,t=-9.280,P<0.05),差异有统计学意义,细胞凋亡水平高于对照组(15.60±1.72比4.08±0.87,t=10.352,P<0.05),差异有统计学意义;过表达TRPV4组中细胞增殖水平低于其对照组(23.65±3.98比51.35±4.67,t=-7.820,P<0.05),差异有统计学意义,细胞凋亡水平高于其对照组(26.93±2.15比14.62±1.68),t=7.814,P<0.05),差异有统计学意义;沉默TRPV4组中细胞增殖水平高于其对照组(72.49±6.21比53.18±5.14,t=4.150,P<0.05),差异有统计学意义,细胞凋亡水平低于其对照组(9.71±1.25比15.07±1.64,t=-4.502,P<0.05),差异有统计学意义。缺氧复氧组中Caspase-3和Cyt-C表达水平高于对照组(0.70±0.06比0.20±0.02,t=13.693,P<0.05;0.74±0.07比0.26±0.03,t=10.917,P<0.05),差异有统计学意义;过表达TRPV4组中Caspase-3和Cyt-C表达水平高于其对照组(1.25±0.11比0.69±0.07,t=7.439,P<0.05;1.38±0.14比0.72±0.07,t=7.303,P<0.05),差异有统计学意义;沉默TRPV4组中Caspase-3和Cyt-C表达水平低于其对照组(0.46±0.05比0.68±0.06,t=-4.879,P<0.05;0.45±0.05比0.72±0.06,t=-5.988,P<0.05),差异有统计学意义。结论TRPV4在GC-1细胞中高表达,其可能通过改变GC-1细胞的增殖和凋亡能力,从而影响睾丸IRI的发生发展。

Objective To investigate the effect of transient receptor potential vanilloid 4(TRPV4)on the proliferation and apoptosis of GC-1 cells in testicular ischemia-reperfusion injury(IRI)and the underlying mechanism.Methods The hypoxia reoxygenation model of testicular GC-1 cells was established.Western blotting was used to detect the protein expression of TRPV4 at different reoxygenation injury time points.Methyl thiazolyl tetrazolium(MTT)assay and flow cytometry were used to measure the effect of TRPV4 transfection on the proliferation and apoptosis of GC-1 cells.Western blotting was used to examine the protein expression level of Caspase-3 and cytoc hrome C(Cyt-C)after TRPV4 transfection.T test was used for comparison between groups,and one-way analysis of variance(ANOVA)was used for comparison among multiple groups.Results The protein expression level of TRPV4 in the normoxia group and hypoxia reoxygenation group(0,6,12,24,48 and 72 h)was(0.19±0.02,0.35±0.03,0.42±0.04,0.46±0.04,0.62±0.05,0.54±0.05,0.45±0.04),respectively.The expression of TRPV4 in the hypoxia reoxygenation group was significantly higher than that in the control group,and reached the peak at hypoxia 3 h/reoxygenation 24 h(F=6.898,P<0.05).The level of cell proliferation in the hypoxia reoxygenation group was significantly lower than that in the control group(52.32±4.58 vs.100.00±7.63,t=-9.280,P<0.05),and the level of apoptosis in the hypoxia reoxygenation group was higher than that in the control group(15.60±1.72 vs.4.08±0.87,t=10.352,P<0.05).The level of cell proliferation in TRPV4 overexpression group was lower than that in control group(23.65±3.98 vs.51.35±4.67,t=-7.820,P<0.05),and the level of apoptosis in TRPV4 overexpression group was higher than that in control group(26.93±2.15 vs.14.62±1.68,t=7.814,P<0.05).The level of cell proliferation in TRPV4 silencing group was higher than that in control group(72.49±6.21 vs.53.18±5.14,t=4.150,P<0.05),and the level of apoptosis was lower than that in control group(9.71±1.25 vs.15.07±1.64,t=-4.502,P<0.05).The expression levels of Caspase-3 and Cyt-C in hypoxia reoxygenation group were higher than those in control group(0.70±0.06 vs.0.20±0.02,t=13.693,P<0.05;0.74±0.07 vs.0.26±0.03,t=10.917,P<0.05).The expression levels of Caspase-3 and Cyt-C in TRPV4 overexpression group were higher than those in control group(1.25±0.11 vs.0.69±0.07,t=7.439,P<0.05;1.38±0.14 vs.0.72±0.07,t=7.303,P<0.05).The expression levels of Caspase-3 and Cyt-C in TRPV4 group were lower than those in control group(0.46±0.05 vs.0.68±0.06,t=-4.879,P<0.05;0.45±0.05 vs.0.72±0.06,t=-5.988,P<0.05).Conclusion TRPV4 is highly expressed in GC-1 cells,which may change proliferation and apoptosis of GC-1 cells,thus affecting the development of testicular IRI.