镉对大鼠肾小管上皮细胞的毒性作用及NAC的保护效应
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摘要
镉是环境中常见的重金属污染物,由于工业生产中镉使用量的增加及相关工业废物带来的污染加重,环境中的镉含量呈快速上升趋势,给公众健康带来的危害引起了广泛关注。肾脏是慢性镉毒性损伤的靶器官和蓄积部位,国内外研究者从职业性暴露、环境污染及动物试验多方面对镉所致肾毒性机理进行了广泛研究。本研究以SD大鼠为试验动物,通过体外培养细胞的方法探讨了镉对大鼠肾小管上皮细胞的毒性损伤效应及N-乙酰半胱氨酸(NAC)的保护效应,为进一步认识镉的肾毒性作用提供了理论依据。
本试验采用机械筛网结合酶消化法建立大鼠原代肾小管上皮细胞培养模型,在传一代细胞增殖活性最强时间段进行镉(2.5μmol/L、5μmol/L)染毒。主要进行以下试验:①cck-8还原法测定不同组合的镉在不同染毒时间(3、6、12、24h)对rPTCs存活率的影响;②测定镉染毒12h对rPTCs凋亡率、坏死率、乳酸脱氢酶释放率、细胞内GSH水平及凋亡形态学的影响,同时添加NAC,观察其对镉所致细胞毒性损伤的保护效果;③测定镉染毒12h对rPTCs膜ATP酶(Ca2+-ATPase、Na+/K+-ATPase)活性、胞内pH、线粒体膜电位、活性氧及钙离子水平的影响。
结果表明:①5μmol/LCd组从6h开始、2.5μmol/LCd组从12h开始,其细胞存活率显著低于对照组(P<0.05或P<0.01),且存活率降低幅度与染毒剂量、染毒时间呈正相关;②染毒12h,各染毒组细胞凋亡率、坏死率、乳酸脱氢酶释放率均极显著高于对照组(P<0.01);染毒组细胞表现核皱缩、呈新月形、染色质致密浓染、核碎裂等典型凋亡特征;NAC对镉所致细胞凋亡有显著保护效应,但对细胞坏死率和乳酸脱氢酶释放率无明显影响;③与对照组比较,染毒组GSH含量均极显著降低(P<0.01),NAC有显著保护效应;④染毒各组细胞内活性氧和钙离子水平均极显著高于对照组(P<0.01),线粒体膜电位水平、胞内pH、Ca2+-ATPase与Na+/K+-ATPase活性均极显著低于对照组(P<0.01)。
上述试验结果可以得出以下结论:①镉暴露对rPTCs的毒性损伤呈浓度依赖性和时间依赖性;②细胞凋亡与细胞坏死是镉所致rPTCs死亡的2种死亡类型,其中凋亡性死亡在低剂量镉所致细胞损伤过程中发挥主导作用。氧化应激在镉染毒所致细胞凋亡型死亡过程中发挥重要作用,抗氧化剂NAC对镉所致的肾小管上皮细胞毒性损伤有显著保护效应;③镉暴露导致rPTCs线粒体膜电位降低而促进细胞凋亡,同时细胞内酸化、钙离子超载、氧化还原平衡状态失调等一系列细胞内环境稳态失衡促进了细胞凋亡。
Cadmium (Cd) is now recognized to be one of most important heavy metal contaminants in the environment. Due to its increased industrial uses and environmental pollution with the related waste products, concentrations of cadmium is increasing rapidly in the environment in recent years. Adverse health effects caused by cadmium have provoked a significant public health concern. The kidney is the target organ and the primary accumulation site of chronic cadmium exposure. The nephrotoxicity induced by cadmium has been extensively studied and widely reported in occupationally and environmentally exposed human subjects, as well as in various experimental models. In this study, the toxic effects of cadmium on the kidney of Sprague-Dawley (SD) rats were investigated in vitro, which will offer some theoretic evidences for further exploring the mechanism in nephrotoxicity of cadmium.
In studies, the primary cultures of rat proximal tubular cells (rPTCs) were cultured by mechanical grinding, filtering and chemical digestive methods. The first passage was used to perform the experimental design when it was in its highest cell viability.
Effects of cadmium (2.5μmol/L, 5μmol/L) on the rPTCs were investigated in the following assays.①Effects of different doses of lead and/or cadmium on the survival rates in rPTCs for a time range of 3, 6, 12 and 24h were detected by using the cck-8 reduction method.②Effects of cadmium on the apoptotic rates, necrotic rates, LDH release, contents of GSH and apoptotic morphological changes in rPTCs over a 12h period were investigated. In addition, the protective effect of N-acetyl-L-cysteine (NAC) against cadmium induced cellular damage was investigated.③Activities of Ca2+-ATPase and Na+/K+-ATPase, intracellular pH, levels of mitochondrial membrane potential (ΔΨ), reactive oxygen species (ROS) and intracellular [Ca2+]i in rPTCs were detected after exposed to lead and/or cadmium for 12h.
The results are as follows:①The cell survival rates in the cadmium groups were significantly lower than those of control groups since these cells were exposed to high-dose (5μmol/L) and low-dose (2.5μmol/L) groups for six and twelve hours, respectively (P<0.05 or P<0.01). Furthermore, the degree of decrease in the cell survival rate was positively correlated with the dose and the exposure time.②After exposure to cadmium for 12h, the apoptotic rates, necrotic rates, LDH release in these exposed groups were significantly higher than those in the control group (P<0.01). After a 12h exposure time, it showed typical apoptotic features in the cadmium groups, i.e., nuclear chromatin condensed and fragmented chromatin was characterized by a scattered, drop-like structure. Apoptosis induced by cadmium can be efficiently prevented by NAC, but the necrotic rates and LDH release were not affected by NAC.③Compared with the control group, activities of the GSH level in the exposed groups decreased significantly (P<0.01), and it can be efficiently prevented by NAC.④After exposed to cadmium for 12h, intracellular ROS and [Ca2+] in rPTCs increased significantly (P<0.01), while the mitochondrialΔΨ, intracellular pH, activities of Ca2+-ATPase and Na+/K+-ATPase decreased significantly (P<0.01). Based on these results, the conclusions are as follows:①cadmium exposure induced cellular death in rPTCs, depending on both the concentration and the exposure time.②Cellular death induced by cadmium is medicated by two mechanisms, necrotic and apoptotic. The apoptotic mechanism played a chief role in the cellular death induced by cadmium at these doses. Moreover, oxidative stress could be implicated in the apoptotic mechanism mediated by cadmium. In addition, the cellular damage induced by lead and/or cadmium can be significantly prevented by NAC.③Depletion of mitochondrialΔΨand a disorder of intracellular homeostasis, i.e. intracellular acidification, calcium overload, disturbance in the prooxidant–antioxidant balance, promoted the development of apoptosis in rPTCs.
本试验采用机械筛网结合酶消化法建立大鼠原代肾小管上皮细胞培养模型,在传一代细胞增殖活性最强时间段进行镉(2.5μmol/L、5μmol/L)染毒。主要进行以下试验:①cck-8还原法测定不同组合的镉在不同染毒时间(3、6、12、24h)对rPTCs存活率的影响;②测定镉染毒12h对rPTCs凋亡率、坏死率、乳酸脱氢酶释放率、细胞内GSH水平及凋亡形态学的影响,同时添加NAC,观察其对镉所致细胞毒性损伤的保护效果;③测定镉染毒12h对rPTCs膜ATP酶(Ca2+-ATPase、Na+/K+-ATPase)活性、胞内pH、线粒体膜电位、活性氧及钙离子水平的影响。
结果表明:①5μmol/LCd组从6h开始、2.5μmol/LCd组从12h开始,其细胞存活率显著低于对照组(P<0.05或P<0.01),且存活率降低幅度与染毒剂量、染毒时间呈正相关;②染毒12h,各染毒组细胞凋亡率、坏死率、乳酸脱氢酶释放率均极显著高于对照组(P<0.01);染毒组细胞表现核皱缩、呈新月形、染色质致密浓染、核碎裂等典型凋亡特征;NAC对镉所致细胞凋亡有显著保护效应,但对细胞坏死率和乳酸脱氢酶释放率无明显影响;③与对照组比较,染毒组GSH含量均极显著降低(P<0.01),NAC有显著保护效应;④染毒各组细胞内活性氧和钙离子水平均极显著高于对照组(P<0.01),线粒体膜电位水平、胞内pH、Ca2+-ATPase与Na+/K+-ATPase活性均极显著低于对照组(P<0.01)。
上述试验结果可以得出以下结论:①镉暴露对rPTCs的毒性损伤呈浓度依赖性和时间依赖性;②细胞凋亡与细胞坏死是镉所致rPTCs死亡的2种死亡类型,其中凋亡性死亡在低剂量镉所致细胞损伤过程中发挥主导作用。氧化应激在镉染毒所致细胞凋亡型死亡过程中发挥重要作用,抗氧化剂NAC对镉所致的肾小管上皮细胞毒性损伤有显著保护效应;③镉暴露导致rPTCs线粒体膜电位降低而促进细胞凋亡,同时细胞内酸化、钙离子超载、氧化还原平衡状态失调等一系列细胞内环境稳态失衡促进了细胞凋亡。
Cadmium (Cd) is now recognized to be one of most important heavy metal contaminants in the environment. Due to its increased industrial uses and environmental pollution with the related waste products, concentrations of cadmium is increasing rapidly in the environment in recent years. Adverse health effects caused by cadmium have provoked a significant public health concern. The kidney is the target organ and the primary accumulation site of chronic cadmium exposure. The nephrotoxicity induced by cadmium has been extensively studied and widely reported in occupationally and environmentally exposed human subjects, as well as in various experimental models. In this study, the toxic effects of cadmium on the kidney of Sprague-Dawley (SD) rats were investigated in vitro, which will offer some theoretic evidences for further exploring the mechanism in nephrotoxicity of cadmium.
In studies, the primary cultures of rat proximal tubular cells (rPTCs) were cultured by mechanical grinding, filtering and chemical digestive methods. The first passage was used to perform the experimental design when it was in its highest cell viability.
Effects of cadmium (2.5μmol/L, 5μmol/L) on the rPTCs were investigated in the following assays.①Effects of different doses of lead and/or cadmium on the survival rates in rPTCs for a time range of 3, 6, 12 and 24h were detected by using the cck-8 reduction method.②Effects of cadmium on the apoptotic rates, necrotic rates, LDH release, contents of GSH and apoptotic morphological changes in rPTCs over a 12h period were investigated. In addition, the protective effect of N-acetyl-L-cysteine (NAC) against cadmium induced cellular damage was investigated.③Activities of Ca2+-ATPase and Na+/K+-ATPase, intracellular pH, levels of mitochondrial membrane potential (ΔΨ), reactive oxygen species (ROS) and intracellular [Ca2+]i in rPTCs were detected after exposed to lead and/or cadmium for 12h.
The results are as follows:①The cell survival rates in the cadmium groups were significantly lower than those of control groups since these cells were exposed to high-dose (5μmol/L) and low-dose (2.5μmol/L) groups for six and twelve hours, respectively (P<0.05 or P<0.01). Furthermore, the degree of decrease in the cell survival rate was positively correlated with the dose and the exposure time.②After exposure to cadmium for 12h, the apoptotic rates, necrotic rates, LDH release in these exposed groups were significantly higher than those in the control group (P<0.01). After a 12h exposure time, it showed typical apoptotic features in the cadmium groups, i.e., nuclear chromatin condensed and fragmented chromatin was characterized by a scattered, drop-like structure. Apoptosis induced by cadmium can be efficiently prevented by NAC, but the necrotic rates and LDH release were not affected by NAC.③Compared with the control group, activities of the GSH level in the exposed groups decreased significantly (P<0.01), and it can be efficiently prevented by NAC.④After exposed to cadmium for 12h, intracellular ROS and [Ca2+] in rPTCs increased significantly (P<0.01), while the mitochondrialΔΨ, intracellular pH, activities of Ca2+-ATPase and Na+/K+-ATPase decreased significantly (P<0.01). Based on these results, the conclusions are as follows:①cadmium exposure induced cellular death in rPTCs, depending on both the concentration and the exposure time.②Cellular death induced by cadmium is medicated by two mechanisms, necrotic and apoptotic. The apoptotic mechanism played a chief role in the cellular death induced by cadmium at these doses. Moreover, oxidative stress could be implicated in the apoptotic mechanism mediated by cadmium. In addition, the cellular damage induced by lead and/or cadmium can be significantly prevented by NAC.③Depletion of mitochondrialΔΨand a disorder of intracellular homeostasis, i.e. intracellular acidification, calcium overload, disturbance in the prooxidant–antioxidant balance, promoted the development of apoptosis in rPTCs.
引文
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