硫化氢对顺铂诱导近端肾小管上皮细胞凋亡的干预研究
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摘要
目的探讨硫化氢H_2S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。NaH(0.5S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。
Objective To explore the intervention of hydrogen sulfide(H_2S) 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 concentrationdependent manner, there was significant difference in the OD value between the 20 mg/L DDP group and the control group(P < 0.05). Adding H_2S(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 H_2S 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 Na HS 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 concentrationdependent manner.
Objective To explore the intervention of hydrogen sulfide(H_2S) 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 concentrationdependent manner, there was significant difference in the OD value between the 20 mg/L DDP group and the control group(P < 0.05). Adding H_2S(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 H_2S 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 Na HS 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 concentrationdependent manner.
引文
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[17]MISHRA P K,TYAGI N,SEN U,et al.H2S ameliorates oxidative and proteolytic stresses and protects the heart against adverse remodeling in chronic heart failure[J].Am J Physiol Heart Circ Physiol,2010,298(2):H451-H456.
[18]HU L F,LU M,TIONG C X,et al.Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models[J].Aging Cell,2010,9(2):135-146.
[19]LU M,ZHAO F F,TANG J J,et al.The neuroprotection of hydrogen sulfide against MPTP-induced dopaminergic neuron degeneration involves uncoupling protein 2 rather than ATP-sensitive potassium channels[J].Antioxid Redox Signal,2012,17(6):849-859.
[20]PAN T T,CHEN Y Q,BIAN J S.All in the timing:a comparison between the cardioprotection induced by H2S preconditioning and post-infarction treatment[J].Eur J Pharmacol,2009,616(1-3):160-165.
[21]PAN T T,FENG Z N,LEE S W,et al.Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes[J].J Mol Cell Cardiol,2006,40(1):119-130.
[22]PREDMORE B L,LEFER D J,GOJON G.Hydrogen sulfide in biochemistry and medicine[J].Antioxid Redox Signal,2012,17(1):119-140.
[2]KELLAND L.The resurgence of platinum-based cancer chemotherapy[J].Nat Rev Cancer,2007,7(8):573-584.
[3]BARABAS K,MILNER R,LURIE D,et al.Cisplatin:a review of toxicities and therapeutic applications[J].Vet Comp Oncol,2008,6(1):1-18.
[4]RYBAK L P.Mechanisms of cisplatin ototoxicity and progress in otoprotection[J].Curr Opin Otolaryngol Head Neck Surg,2007,15(5):364-369.
[5]MCWHINNEY S R,GOLDBERG R M,MCLEOD H L.Platinum neurotoxicity pharmacogenetics[J].Mol Cancer Ther,2009,8(1):10-16.
[6]ARANY I,SAFIRSTEIN R L.Cisplatin nephrotoxicity[J].Semin Nephrol,2003,23(5):460-464.
[7]SASTRY J,KELLIE S J.Severe neurotoxicity,ototoxicity and nephrotoxicity following high-dose cisplatin and amifostine[J].Pediatr Hematol Oncol,2005,22(5):441-445.
[8]SIDDIK Z H.Cisplatin:mode of cytotoxic action and molecular basis of resistance[J].Oncogene,2003,22(47):7265-7279.
[9]PABLA N,DONG Z.Cisplatin nephrotoxicity:mechanisms and renoprotective strategies[J].Kidney Int,2008,73(9):994-1007.
[10]KELLAND L.The resurgence of platinum-based cancer chemotherapy[J].Nat Rev Cancer,2007,7(8):573-584.
[11]BARABAS K,MILNER R,LURIE D,et al.Cisplatin:a review of toxicities and therapeutic applications[J].Vet Comp Oncol,2008,6(1):1-18.
[12]MCWHINNEY S R,GOLDBERG R M,MCLEOD H L.Platinum neurotoxicity pharmacogenetics[J].Mol Cancer Ther,2009,8(1):10-16.
[13]RYBAK L P.Mechanisms of cisplatin ototoxicity and progress in otoprotection[J].Curr Opin Otolaryngol Head Neck Surg,2007,15(5):364-369.
[14]AKCAY A,TURKMEN K,LEE D,et al.Update on the diagnosis and management of acute kidney injury[J].Int J Nephrol Renovasc Dis,2010,3:129-140.
[15]KOTSYUBA E P.Cystathionine beta-synthase in the central nervous system of the shore crab Hemigrapsus sanguineus[J].Dokl Biol Sci,2011,437:94-96.
[16]C H E RTO K V M,K O T S I U B A A E,K O T S I U B A E P.Cystathionine beta-synthase in the structural elements of the human brain and spinal cord[J].Tsitologiia,2011,53(8):665-670.
[17]MISHRA P K,TYAGI N,SEN U,et al.H2S ameliorates oxidative and proteolytic stresses and protects the heart against adverse remodeling in chronic heart failure[J].Am J Physiol Heart Circ Physiol,2010,298(2):H451-H456.
[18]HU L F,LU M,TIONG C X,et al.Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models[J].Aging Cell,2010,9(2):135-146.
[19]LU M,ZHAO F F,TANG J J,et al.The neuroprotection of hydrogen sulfide against MPTP-induced dopaminergic neuron degeneration involves uncoupling protein 2 rather than ATP-sensitive potassium channels[J].Antioxid Redox Signal,2012,17(6):849-859.
[20]PAN T T,CHEN Y Q,BIAN J S.All in the timing:a comparison between the cardioprotection induced by H2S preconditioning and post-infarction treatment[J].Eur J Pharmacol,2009,616(1-3):160-165.
[21]PAN T T,FENG Z N,LEE S W,et al.Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes[J].J Mol Cell Cardiol,2006,40(1):119-130.
[22]PREDMORE B L,LEFER D J,GOJON G.Hydrogen sulfide in biochemistry and medicine[J].Antioxid Redox Signal,2012,17(1):119-140.