N-乙酰半胱氨酸对G/GO诱导hRPE细胞氧化应激与凋亡的影响及其机制
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摘要
糖尿病性视网膜病变(DR)是糖尿病常见的严重并发症,是糖尿病患者致盲的重要原因。糖尿病慢性并发症发病机制的统一学说指出,慢性高血糖导致的氧化应激诱导的细胞凋亡是DR发病机制的中心环节,继而通过多种途径最终导致DR的发生,阻断这一环节便可以阻断DR发生的其他病理进程。因此有效的拮抗高糖导致的氧化应激是保护视网膜免受损伤继而延缓DR发生发展的关键。
本研究以葡萄糖/葡萄糖氧化酶(G/GO)诱导人视网膜色素上皮(hRPE)细胞及链脲佐菌素(STZ)诱发2型糖尿病大鼠为研究对象,通过体外细胞培养和体内动物实验研究两个层面,利用流式细胞术(FCM)、逆转录聚合酶链反应(RT-PCR)、免疫印迹法(Western blot)和免疫荧光双标染色等技术,从细胞、分子水平,凋亡途径及信号转导机制等方面研究发现,G/GO不仅导致hRPE细胞氧化应激损伤、活性氧簇和活性氮簇释放,而且还启动线粒体途径诱导hRPE细胞的凋亡,建立了氧化应激致hRPE细胞凋亡的细胞模型;N-乙酰半胱氨酸(NAC)通过调控线粒体凋亡途径相关基因Bcl-2、Bax、细胞色素C和Caspase-9的表达,激活PI-3K/Akt和NF-κB信号通路,对损伤的hRPE细胞及视网膜组织发挥了有效的保护作用。因此,我们的研究通过探讨早期DR发生、发展的可能原因,以及NAC防治DR的具体作用机制,为研究DR的发病机制及其治疗措施奠定了基础。
Diabetes mellitus (DM) is one of common diseases that threaten human health severily. It will lead to multisystem damage and progressive pathological changes of tissue and organs damage such as eye, kidney, nerve, heart and vessels. Diabetic retinopathy (DR) is one of the most common and severest micrangium complication.It is also the main reason that leads to blindness in developed countries now.Though the pathogenesy of DR is not clear, the persistent chronic hyperglycemia is the main factor of DR’s development.In recent years it is paying more and more attention to the effect of oxidative stress in the pathogenesy of DR.The enhancement of oxidative stress with high glucose causes releasing of reactive oxygen species (ROS), stimulate apoptosis and destroy retinal barricade. The apoptosis caused by chronic hyperglycemia-induced oxidative stress is the center piece in the pathogenesy of DR. Blocking the center piece will block the other pathological process of DR.
Depending on study objects of glucose/glucose oxidase (G/GO)-induced human retinal epithelial cells(hRPE) and streptozotocin(STZ)-induced rat model of type 2 diabetes, from two aspects of cell culture in vitro and animal studies in vivo ,by the technique of flow cytometry (FCM), reverse transcription-polymerase chain reaction, western blot and double-labelling technique of immunofluorescence and histochemistry staining, by the cell and molecular level ,apoptosis pathway and signal transduction mechanism , we discovered G/GO not only induced oxdative stress damage , release of ROS and RNS, but also induced hRPE apoptosis by priming mitochondrial pathway. Oxidative stress-induced cell apotosis model was established. N-acetylcysteine( NAC ) protects impaired hRPE cells and retinal tissues effectively through regulating the expressions of mitochondrial apoptosis pathway-induced related genes of Bcl-2,Bax and Caspase-9 and the signal pathways of PI-3K/Akt and NF-κB.
1.The effect of G/GO-induced apoptosis on hRPE
The hRPE cells was cultured in the 33mM high glucose with different concentrations of GO (5-20mU/mL).MTT chromatometry was used to detect the survival rate of hRPE. Annexin V-FITC/PI fluorescent staining was used to detect the apoptosis rate of cells.The shapes of hRPE cells were observed by optic phase microscope and the shapes of nucleus were observed by fluorescent microscope with the DAPI dyeing.The results manifested the survival rate of cells decreased to 81.4%±8.7%(P<0.05)and the apoptosis rate was 23.2% when hRPE cells were cultured with G/GO(33 mM G/15mU GO)for 6h. The cells shows typical apoptosis features:cellular membrane became rough, the cells shrinked and turned round,the refraction enhanced,tentacles of cells decreased obviously and the space between cells were increased;the nucleus were irregular and pyknoti, the chromatin gathered obviously and the nucleus of some cells turned to karyolobism or karyorrhexis.It indicates G/GO(33mM G/15mU)induces oxidative stress and apoptosis of hRPE cells.
2.The effect of NAC on G/GO-induced hRPE oxidative stress and apoptosis
By MTT chromatometry, Annexin V-FITC/PI fluorescent staining and DAPI dyeing, +NAC group inhibited toxicity of G/GO ,the survival rate of hRPE increased obviously, the number of apoptotic cells with positive staining was decreased, the shapes of cells and nucleus were regular, karyolobism and karyorrhexis seldom happened. The levels of ROS ,RNS and mitochondrial membrane potential (△Ψm)were detected by FCM with dyes of DHE、carboxy-H2-DCFDA、DHR123 and DiOC6(3).The content of MDA ,activity of SOD and GSH-px and Ca2+conveying ability of mitochondria were detected by spectrophotometer. The results manifested the mean fluorescence intensity(MFI), activity of SOD and GSH-px and the content of MDA were increased obviously( P < 0.05).However, those of +NAC group were lower than G/GO group.The number of cells in G/GO group with descending△Ψm increased from 1.44% to 42.14% and that of +NAC group decreased to 14.04%、6.56% and 5.78% respectively. The Ca2+conveying ability of mitochondria in G/GO group was lower than control group and that of +NAC group recovered to normal level. It indicates NAC inhibits accumulation of ROS and RNS in hRPE cells, protects mitochondria and cells against the oxidative damage and apoptosis induced by G/GO.
3.The mechanism of the protection of NAC against hRPE oxidative stress and apoptosis induced by G/GO
The mRNA levels of Bcl-2 and Bax were detected by RT-PCR.The protein levels of cytochrome C (Cyt C)and Caspase- 9, phosphorylation levels of PI-3K/Akt and NF-κB were detected by western blot.The results manifested with the prolonged action of G/GO(33mM G/15mU GO), the expression of Bax mRNA、Cyt C and Caspase- 9 proteins was increased and expression of Bcl-2 mRNA was decreased gradually ,which there was significant difference comparing with control group.NAC inhibited the change of the expression of Bax and Bcl-2 mRNA、Cyt C and Caspase- 9 proteins. The phosphorylation levels of Akt was decreased and that of NF-κB was increased in G/GO group.NAC group inhibited the change of the phosphorylation levels of Akt and NF-κB in (33mM G/15mU GO)G/GO group.There was no influence to the Akt protein and nonphosphorylated NF-κB in G/GO and NAC groups.It indicates NAC protects hRPE cells apoptosis and oxidative stress induced by G/GO through regulating the expressions of mitochondrial apoptosis pathway-induced related genes of Bcl-2,Bax,Cyt C and Caspase-9 and the signal pathways of PI-3K/Akt and NF-κB.
4.The initial study of NAC protection of the retina of rat model with type 2 diabetes
Establishment of experimental rat model of type 2 diabetes with streptozotocin and high-glucose-high-fat diet.There were 4 rats choosed randomly to be normal control group (NC). There were 20 rats divided into 4 groups, which were 1 month and 3 month diabetes mellitus groups(DM) and NAC-treated groups(NAC).NAC was dissolved in drinking water.The rats of NAC groups drank 1.4～1.5 g/kg NAC everyday.The pathomorphology of rat retina was studied by HE dyeing.The apoptosis of retinal cells was studied by TUNEL method. The expression of Bax ,Bcl-2 and Caspase- 9 proteins were studied by double-labelling technique of immunofluorescence and histochemistry staining.The results manifested there were apoptotic cells in inner and outer nuclear layer of DM 1m group. The apoptosis appeared in retinal ganglial cells, inner nuclear layer and retina, which were more than NC group and NAC group and different in apoptotic index significantly. The expression of Bcl-2 of DM 1m group was more than NC group, but that of DM 3m group decreased. The expression of Bax of DM 1m group was not significant and Bax was diffused in INL and ganglion cell layer (GCL). The expression of Bax of DM 3m group increased and extended. The expression of Caspase- 9 of DM 3m group increased maily in INL. It indicates apoptosis occurs in early DR.Through enhancement of expression of Bcl-2 , reduction of expression of Bax and inhibination of activity of Caspase- 9 in retinal neurocytes of STZ-induced rats, NAC inhibits apoptosis and relieves oxidative damage of retina in early DM.
Our research indicates G/GO induces oxidative stress and apoptosis through mitochondrial pathway in hRPE cells.NAC protects hRPE cells and retina of rats with diabetes by inhibitation of apoptotic signal pathways.Therefore, we investigate the possible reason of development in early DR and mechanism of prevention and cure of DR with NAC.It establishes the foundation on studying pathogenesy and therapeutic measure of DR.
本研究以葡萄糖/葡萄糖氧化酶(G/GO)诱导人视网膜色素上皮(hRPE)细胞及链脲佐菌素(STZ)诱发2型糖尿病大鼠为研究对象,通过体外细胞培养和体内动物实验研究两个层面,利用流式细胞术(FCM)、逆转录聚合酶链反应(RT-PCR)、免疫印迹法(Western blot)和免疫荧光双标染色等技术,从细胞、分子水平,凋亡途径及信号转导机制等方面研究发现,G/GO不仅导致hRPE细胞氧化应激损伤、活性氧簇和活性氮簇释放,而且还启动线粒体途径诱导hRPE细胞的凋亡,建立了氧化应激致hRPE细胞凋亡的细胞模型;N-乙酰半胱氨酸(NAC)通过调控线粒体凋亡途径相关基因Bcl-2、Bax、细胞色素C和Caspase-9的表达,激活PI-3K/Akt和NF-κB信号通路,对损伤的hRPE细胞及视网膜组织发挥了有效的保护作用。因此,我们的研究通过探讨早期DR发生、发展的可能原因,以及NAC防治DR的具体作用机制,为研究DR的发病机制及其治疗措施奠定了基础。
Diabetes mellitus (DM) is one of common diseases that threaten human health severily. It will lead to multisystem damage and progressive pathological changes of tissue and organs damage such as eye, kidney, nerve, heart and vessels. Diabetic retinopathy (DR) is one of the most common and severest micrangium complication.It is also the main reason that leads to blindness in developed countries now.Though the pathogenesy of DR is not clear, the persistent chronic hyperglycemia is the main factor of DR’s development.In recent years it is paying more and more attention to the effect of oxidative stress in the pathogenesy of DR.The enhancement of oxidative stress with high glucose causes releasing of reactive oxygen species (ROS), stimulate apoptosis and destroy retinal barricade. The apoptosis caused by chronic hyperglycemia-induced oxidative stress is the center piece in the pathogenesy of DR. Blocking the center piece will block the other pathological process of DR.
Depending on study objects of glucose/glucose oxidase (G/GO)-induced human retinal epithelial cells(hRPE) and streptozotocin(STZ)-induced rat model of type 2 diabetes, from two aspects of cell culture in vitro and animal studies in vivo ,by the technique of flow cytometry (FCM), reverse transcription-polymerase chain reaction, western blot and double-labelling technique of immunofluorescence and histochemistry staining, by the cell and molecular level ,apoptosis pathway and signal transduction mechanism , we discovered G/GO not only induced oxdative stress damage , release of ROS and RNS, but also induced hRPE apoptosis by priming mitochondrial pathway. Oxidative stress-induced cell apotosis model was established. N-acetylcysteine( NAC ) protects impaired hRPE cells and retinal tissues effectively through regulating the expressions of mitochondrial apoptosis pathway-induced related genes of Bcl-2,Bax and Caspase-9 and the signal pathways of PI-3K/Akt and NF-κB.
1.The effect of G/GO-induced apoptosis on hRPE
The hRPE cells was cultured in the 33mM high glucose with different concentrations of GO (5-20mU/mL).MTT chromatometry was used to detect the survival rate of hRPE. Annexin V-FITC/PI fluorescent staining was used to detect the apoptosis rate of cells.The shapes of hRPE cells were observed by optic phase microscope and the shapes of nucleus were observed by fluorescent microscope with the DAPI dyeing.The results manifested the survival rate of cells decreased to 81.4%±8.7%(P<0.05)and the apoptosis rate was 23.2% when hRPE cells were cultured with G/GO(33 mM G/15mU GO)for 6h. The cells shows typical apoptosis features:cellular membrane became rough, the cells shrinked and turned round,the refraction enhanced,tentacles of cells decreased obviously and the space between cells were increased;the nucleus were irregular and pyknoti, the chromatin gathered obviously and the nucleus of some cells turned to karyolobism or karyorrhexis.It indicates G/GO(33mM G/15mU)induces oxidative stress and apoptosis of hRPE cells.
2.The effect of NAC on G/GO-induced hRPE oxidative stress and apoptosis
By MTT chromatometry, Annexin V-FITC/PI fluorescent staining and DAPI dyeing, +NAC group inhibited toxicity of G/GO ,the survival rate of hRPE increased obviously, the number of apoptotic cells with positive staining was decreased, the shapes of cells and nucleus were regular, karyolobism and karyorrhexis seldom happened. The levels of ROS ,RNS and mitochondrial membrane potential (△Ψm)were detected by FCM with dyes of DHE、carboxy-H2-DCFDA、DHR123 and DiOC6(3).The content of MDA ,activity of SOD and GSH-px and Ca2+conveying ability of mitochondria were detected by spectrophotometer. The results manifested the mean fluorescence intensity(MFI), activity of SOD and GSH-px and the content of MDA were increased obviously( P < 0.05).However, those of +NAC group were lower than G/GO group.The number of cells in G/GO group with descending△Ψm increased from 1.44% to 42.14% and that of +NAC group decreased to 14.04%、6.56% and 5.78% respectively. The Ca2+conveying ability of mitochondria in G/GO group was lower than control group and that of +NAC group recovered to normal level. It indicates NAC inhibits accumulation of ROS and RNS in hRPE cells, protects mitochondria and cells against the oxidative damage and apoptosis induced by G/GO.
3.The mechanism of the protection of NAC against hRPE oxidative stress and apoptosis induced by G/GO
The mRNA levels of Bcl-2 and Bax were detected by RT-PCR.The protein levels of cytochrome C (Cyt C)and Caspase- 9, phosphorylation levels of PI-3K/Akt and NF-κB were detected by western blot.The results manifested with the prolonged action of G/GO(33mM G/15mU GO), the expression of Bax mRNA、Cyt C and Caspase- 9 proteins was increased and expression of Bcl-2 mRNA was decreased gradually ,which there was significant difference comparing with control group.NAC inhibited the change of the expression of Bax and Bcl-2 mRNA、Cyt C and Caspase- 9 proteins. The phosphorylation levels of Akt was decreased and that of NF-κB was increased in G/GO group.NAC group inhibited the change of the phosphorylation levels of Akt and NF-κB in (33mM G/15mU GO)G/GO group.There was no influence to the Akt protein and nonphosphorylated NF-κB in G/GO and NAC groups.It indicates NAC protects hRPE cells apoptosis and oxidative stress induced by G/GO through regulating the expressions of mitochondrial apoptosis pathway-induced related genes of Bcl-2,Bax,Cyt C and Caspase-9 and the signal pathways of PI-3K/Akt and NF-κB.
4.The initial study of NAC protection of the retina of rat model with type 2 diabetes
Establishment of experimental rat model of type 2 diabetes with streptozotocin and high-glucose-high-fat diet.There were 4 rats choosed randomly to be normal control group (NC). There were 20 rats divided into 4 groups, which were 1 month and 3 month diabetes mellitus groups(DM) and NAC-treated groups(NAC).NAC was dissolved in drinking water.The rats of NAC groups drank 1.4～1.5 g/kg NAC everyday.The pathomorphology of rat retina was studied by HE dyeing.The apoptosis of retinal cells was studied by TUNEL method. The expression of Bax ,Bcl-2 and Caspase- 9 proteins were studied by double-labelling technique of immunofluorescence and histochemistry staining.The results manifested there were apoptotic cells in inner and outer nuclear layer of DM 1m group. The apoptosis appeared in retinal ganglial cells, inner nuclear layer and retina, which were more than NC group and NAC group and different in apoptotic index significantly. The expression of Bcl-2 of DM 1m group was more than NC group, but that of DM 3m group decreased. The expression of Bax of DM 1m group was not significant and Bax was diffused in INL and ganglion cell layer (GCL). The expression of Bax of DM 3m group increased and extended. The expression of Caspase- 9 of DM 3m group increased maily in INL. It indicates apoptosis occurs in early DR.Through enhancement of expression of Bcl-2 , reduction of expression of Bax and inhibination of activity of Caspase- 9 in retinal neurocytes of STZ-induced rats, NAC inhibits apoptosis and relieves oxidative damage of retina in early DM.
Our research indicates G/GO induces oxidative stress and apoptosis through mitochondrial pathway in hRPE cells.NAC protects hRPE cells and retina of rats with diabetes by inhibitation of apoptotic signal pathways.Therefore, we investigate the possible reason of development in early DR and mechanism of prevention and cure of DR with NAC.It establishes the foundation on studying pathogenesy and therapeutic measure of DR.
引文
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