褪黑素通过NRF2对氯胺酮诱导的尿路上皮损伤保护作用研究

Protective effect of melatonin on ketamine-induced urothelium injury via NRF2

目的在细胞水平探究褪黑素通过NRF2对氯胺酮相关性膀胱炎(KC)尿路上皮损伤的保护作用。方法首先利用Western blot验证NRF2在氯胺酮刺激后的SV-HUC-1细胞的表达水平变化;构建沉默NRF2的SV-HUC-1细胞株,利用流式细胞术检测氯胺酮共培养后的细胞凋亡率;利用CCK-8筛选褪黑素的工作浓度,将SV-HUC-1细胞分为对照组(CON),氯胺酮组(KET),褪黑素治疗组(KET+褪黑素)和褪黑素组,利用Western blot检测NRF2、HO-1蛋白表达丰度,利用实时荧光定量PCR检测IL-6 mRNA表达水平,并测定SOD水平变化情况。结果氯胺酮共培养后的SV-HUC-1细胞NRF2表达水平显著降低(P<0.001);成功构建沉默NRF2的稳定转染细胞株,si-NRF2组的NRF2表达水平低于si-NC组(P<0.05);si-NRF2+氯胺酮组[(14.717±0.601)%]和si-NC+氯胺酮组[(10.890±0.429)%]的细胞凋亡率显著高于si-NC组[(8.560±0.846)%](P<0.01)和si-NRF2组[(9.023±0.703)%](P<0.01);且siNRF2+氯胺酮组的凋亡率显著高于si-NC+氯胺酮组(P<0.001)。选取100μM为褪黑素的工作浓度。KET组的IL-6mRNA表达水平(1.415±0.286)显著高于CON组(1.024±0.230)(P<0.01);KET+褪黑素组(1.026±0.344)的IL-6mRNA表达水平显著低于KET组(P<0.01);KET+褪黑素组SV-HUC-1细胞的SOD(20.521±1.351)显著高于CON组(15.461±2.018)(P<0.001)和KET组(15.763±2.216)(P<0.0001);KET组SV-HUC-1细胞NRF2的表达丰度(0.428±0.101)低于CON组(1.500±0.081)(P<0.0001);KET+褪黑素组的NRF2表达丰度(0.804±0.100)高于KET组(P<0.01)。除此之外,KET+褪黑素的HO-1的表达丰度(1.419±0.098)高于CON组(0.352±0.168)(P<0.0001)和KET组(0.199±0.046)(P<0.0001)。结论褪黑素能够通过NRF2保护氯胺酮导致的尿路上皮损伤。

Objective To explore at the cellular level the protective effect of melatonin via NRF2on urothelium injury caused by ketamine cystitis(KC).Methods First,Western blot was used to verify the expression changes of NRF2in SV-HUC-1cells stimulated by ketamine.The SV-HUC-1cell line with silenced NRF2was constructed.Flow cytometry was employed to detect the apoptosis rate after the cells were co-cultured with ketamine.The working concentration of melatonin was screened using CCK-8.SV-HUC-1cells were divided into a control group(CON),a ketamine(KET)group,a melatonin treatment(KET+melatonin)group and a melatonin group.The abundance of NRF2and HO-1protein expressions was detected by Western blot;the IL-6 mRNA expression and SOD changes by real-time fluorescence quantitative PCR.Results There was a significant decrease in the expression of NRF2in SV-HUC-1cells co-cultured with ketamine(P<0.001).The stably transfected cell line with silenced NRF2was successfully constructed.The expression of NRF2in the si-NRF2group was lower than that in the si-NC group(P<0.05).The apoptosis rates in the siNRF2+ketamine group[(14.717±0.601)%]and si-NC+ketamine group[(10.890±0.429)%]were significantly higher than that in the si-NC group[(8.560±0.846)%](P<0.01)and that of the si-NRF2group[(9.023±0.703)%](P<0.01).The si-NRF2+ketamine group had significantly higher apoptosis rate than the siNC+ketamine group(P<0.001).100μM was selected as the working concentration of melatonin.The IL-6mRNA expression of the KET group(1.415±0.286)was significantly higher than that of the CON group(1.024±0.230)(P<0.01),while that of the KET+melatonin group(1.026±0.344)was significantly lower than that of the KET group(P<0.01).The SOD of SV-HUC-1cells in the KET+melatonin group(20.521±1.351)was significantly higher than that of the CON group(15.461±2.018)(P<0.001)and that of the KET group(15.763±2.216)(P<0.0001).The KET group had lower expression abundance of NRF2in SV-HUC-1cells(0.428±0.101)than the CON group(1.500±0.081)(P<0.0001).But the KET+melatonin group had higher abundance of NRF2(0.804±0.100)than the KET group(P<0.01).In addition,the expression abundance of HO-1in the KET+melatonin group(1.419±0.098)was higher than that in the CON group(0.352±0.168)(P<0.0001)as well as that in the KET group(0.199±0.046)(P<0.0001).Conclusion Melatonin can protect ketamine-induced urothelium injury via NRF2.