硫化氢对顺铂诱导近端肾小管 上皮细胞凋亡的干预研究

Intervention of hydrogen sulfide on apoptosis of proximal renal tubular epithelial cells induced by cisplatin

摘要:

目的 探讨硫化氢H2S 对顺铂（DDP）诱导近端肾小管上皮细胞凋亡的干预作用。方法 研究分 为阴性对照组、DDP 组、DDP+ 硫氢化钠NaHS 组。DDP 组：DDP 浓度为0、1、2、5、10、20 和40 mg/L ； DDP+NaHS 组：NaHS 浓度分别为0、0.2、0.4、0.5、0.8、1.0 和2.0 mmol/L，加DDP（20 mg/L）共同作用。噻唑蓝 （MTT）实验：DDP 组、DDP+NaHS 组与人近端肾小管上皮细胞株（HK-2 细胞）共同培养，24 h 后测光密度（OD） 值。NaHS（0.5 和1.0 mmol/L）加DDP（20 mg/L）与HK-2 细胞共同培养24 h。4，6- 联脒-2- 苯基吲哚（DAPI）， Annexin V-FITC/PI 染色通过显微镜观察各组细胞的形态学改变。Annexin V-FITC/PI 流式细胞术检测各组细胞 凋亡率。结果 MTT 实验：0、1、2、5、10 和20 mg/L DDP 浓度的OD 值分别为（0.270±0.064）、（0.229±0.022）、 （0.198±0.024）、（0.189±0.069）、（0.188±0.030） 和（0.165±0.012），OD 值随DDP 浓度上升逐渐下降， 在 20 mg/L 低于阴性对照组（P <0.05）。20 mg/L DDP 加浓度分别为0.2、0.4、0.5、0.8 和1.0 mmol/L NaHS 的OD 值分别为（0.173±0.051）、（0.186±0.023）、（0.259±0.050）、（0.263±0.033） 和（0.268±0.098）， 以 上与浓度为20 mg/L DDP 的OD 值（0.153±0.017）比较，差异有统计学意义（P <0.05）。DAPI、Annexin VFITC/ PI 染色荧光显微镜显示，对照组HK-2 细胞平均凋亡细胞数为（4.230±1.015），20 mg/L DDP 影响下 为（35.020±6.079），两者差异有统计学意义（P <0.05），DDP 组高于阴性对照组。DDP+NaHS 组在0.5 和 1.0 mmol/L 的平均凋亡细胞分别为（22.550±2.912） 和（9.780±2.063），差异有统计学意义（P <0.05）， DDP+NaHS 组低于DDP 组。Annexin V-FITC/PI 流式细胞术检测，阴性对照组HK-2 细胞凋亡率（5.167± 0.612）%，在20 mg/L DDP 影响下，为（31.598±1.014）%，细胞凋亡率增加（P <0.05）。合用NaHS 凋亡减 少，在0.5 和1.0 mmol/L 的凋亡率为（18.375±2.239）% 和（11.636±1.233）%，差异有统计学意义（P <0.05）， DDP+NaHS 组低于DDP 组。结论 NaHS 对DDP 导致的近端肾小管上皮细胞的凋亡有保护作用。

Abstract:

Objective To explore the intervention of hydrogen sulfide (H2S) on apoptosis of proximal renal tubular epithelial cells induced by cisplatin (DPP). Methods The HK-2 cells were randomly assigned into a control group, DPP treated groups (0, 1, 2, 5, 10, 20 and 40 mg/L) and DDP (20 mg/L)+NaHS (0, 0.2, 0.4, 0.5, 0.8, 1.0 and 2.0 mmol/L) treated groups. After the HK-2 cells were co-cultured with DPP or DPP+NaHS for 24 h, MTT assay was used to measure the OD value. Using 4’,6-diamidino-2-phenylindole (DAPI) and Annexin V-FITC/PI double staining the morphological changes of each group were observed under fluorescence microscope, and flow cytometry was used to detect the apoptosis rate of each group. Results According to MTT, the OD value was (0.270 ± 0.064), (0.229 ± 0.022), (0.198 ± 0.024), (0.189 ± 0.069), (0.188 ± 0.030) and (0.165 ± 0.012) respectively in the DDP groups with different concentrations (0, 1, 2, 5, 10, and 20 mg/L), DDP accelerated HK-2 cell apoptosis in a concentration-dependent manner, there was significant difference in the OD value between the 20 mg/L DDP group and the control group (P < 0.05). Adding H2S (0.2, 0.4, 0.5, 0.8, and 1.0 mmol/L) and DPP (20 mg/L) into the cells, the OD was (0.173 ± 0.051), (0.186 ± 0.023), (0.259 ± 0.050), (0.263 ± 0.033) and (0.268 ± 0.098) respectively, with significant increases compared to (0.153 ± 0.017) after adding 20 mg/L DDP alone (P < 0.05), the protective effect of H2S was also in a concentration-dependent manner. Through the DAPI, Annexin V-FITC/PI staining, under the effect of 20 mg/L DDP, the average number of apoptotic cells in visual field of HK-2 cells was (35.020 ± 6.079), while that in the normal negative control group was (4.230 ± 1.015), there was statistical significance between them (P < 0.05). Adding 0.5 and 1.0 mmol/L of NaHS could ameliorate apoptosis, the average number of apoptotic cells in visual field was (22.550 ± 2.912) and (9.780 ± 2.063), which were significantly lower than that of the DDP group (P < 0.05). Flow cytometry revealed that 20 mg/L DDP incubated with HK-2 cells for 24 h led to apoptosis with the rate of (31.598 ± 1.014)% which was significantly higher than (5.167 ± 0.612)% of the normal negative control group (P < 0.05). However, adding NaHS (0.5 and 1.0 mmol/L) could reduce apoptosis, the apoptosis rate were (18.375 ± 2.239)%, (11.636 ± 1.233)%, which were statistically lower than that of the DDP group (P < 0.05). Conclusions The apoptosis of human renal tubular epithelial cells induced by cisplatin can be protected by hydrogen sulfide in a concentration-dependent manner.