二仙汤对过氧化氢诱导的人卵巢颗粒细胞氧化损伤的保护作用
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摘要
目的探讨二仙汤对过氧化氢(H_2O_2)诱导人卵巢颗粒细胞(COV434)氧化损伤的保护作用及其可能机制。方法体外培养COV434细胞,0.5 mmol/L H_2O_2诱导COV434氧化损伤。将细胞分为空白组、模型组(H_2O_2 0.5 mmol/L)、二仙汤水提液低、中、高剂量组(0.5、1、5 mg/mL+H_2O_2 0.5 mmol/L)。MTT分析细胞存活率,DCFH-DA荧光探针检测细胞内活性氧族(ROS)水平,微量酶标法测定细胞培养液中乳酸脱氢酶(LDH)的释放。Annexin V-FITC/PI双染和PI染色,分别检测细胞凋亡和周期。Western blot检测HO-1、NQO-1、P-JNK、P-44/42、P-p38、Bax、Bcl-2、Caspase-9蛋白表达。结果与空白组比较,模型组细胞存活率下降,ROS及LDH水平增加,S期百分率、早期凋亡、晚期凋亡及总凋亡比例显著增高(P<0.01),Bax、Caspase-9及P-JNK蛋白表达升高(P<0.05)。与模型组比较,二仙汤水提液低、中、高剂量组LDH、S期百分率降低,G2/M期百分率升高(P<0.05),二仙汤水提液中、高剂量组细胞存活率升高,ROS释放量、早期凋亡及总凋亡比例降低(P<0.05,P<0.01),二仙汤水提液低、中剂量组P-44/42蛋白表达升高(P<0.05)。结论二仙汤在一定剂量范围内对H_2O_2诱导的COV434细胞损伤具有明显的保护作用,其保护效应可能与清除ROS,减轻DNA氧化损伤,调控MAPK-Nrf2信号通路及线粒体通路介导的细胞凋亡有关。
Objective To evaluate the protective effects and mechanisms of Erxian Decoction(EXD) on oxidative injury of human ovarian granulosa cells(COV434)induced by H_2O_2. Methods The model of oxidative injury was established by supplement with H_2O_2(0.5 mmol/L). COV434 cells were divided into control group, model group(H_2O_2 0.5 mmol/L), the low,medium and high dose group of EXD(EXD 0.5, 1, 5 mg/mL + H_2O_2 0.5 mmol/L). Cell growth activity was evaluated by MTT assay. Intracellular reactive oxygen species(ROS) production was measured by DCFH-DA.The activity of lactic dehydrogenase(LDH) were determined by microenzyme labeling.The apoptosis and cell cycle of COV434 were detected using Annexin V-FITC/PI. The involvement of HO-1, NQO-1, P-JNK, P-44/42, P-p38, Bax, Bcl-2, Caspase-9 in the oxidative stress-mediated signaling pathway was explored using Western blot analysis. Results Compared with the control group, H_2O_2 significantly decreased cell viability which was accompanied by an increase in ROS and LDH production, and the percentage of s-phase, early apoptosis, late apoptosis and total apoptosis increased significantly(P<0.01). Meanwhile,the levels of Bax, Caspase-9, P-JNK protein were increased(P<0.05). Compared with the model group, the LDH production and the percentage of S phase in the low, medium and high dose group of EXD were decreased, while the percentage of G2/M phase increased(P<0.05). The medium and high dose group of EXD increased viability of COV434 and decreased the elevated ROS production and early apoptosis and total apoptosis(P<0.05, P<0.01). Furthermore, the low and medium dose group of EXD significantly enhanced the expression of P-44/42(P<0.05). Conclusion Pretreatment with EXD alleviate H_2O_2-induced apoptosis in COV434 cells through both eliminate ROS, relieve oxidative DNA damage and regulating the MAPK-Nrf2 signaling pathway and mitochondrial pathway mediated apoptosis.
Objective To evaluate the protective effects and mechanisms of Erxian Decoction(EXD) on oxidative injury of human ovarian granulosa cells(COV434)induced by H_2O_2. Methods The model of oxidative injury was established by supplement with H_2O_2(0.5 mmol/L). COV434 cells were divided into control group, model group(H_2O_2 0.5 mmol/L), the low,medium and high dose group of EXD(EXD 0.5, 1, 5 mg/mL + H_2O_2 0.5 mmol/L). Cell growth activity was evaluated by MTT assay. Intracellular reactive oxygen species(ROS) production was measured by DCFH-DA.The activity of lactic dehydrogenase(LDH) were determined by microenzyme labeling.The apoptosis and cell cycle of COV434 were detected using Annexin V-FITC/PI. The involvement of HO-1, NQO-1, P-JNK, P-44/42, P-p38, Bax, Bcl-2, Caspase-9 in the oxidative stress-mediated signaling pathway was explored using Western blot analysis. Results Compared with the control group, H_2O_2 significantly decreased cell viability which was accompanied by an increase in ROS and LDH production, and the percentage of s-phase, early apoptosis, late apoptosis and total apoptosis increased significantly(P<0.01). Meanwhile,the levels of Bax, Caspase-9, P-JNK protein were increased(P<0.05). Compared with the model group, the LDH production and the percentage of S phase in the low, medium and high dose group of EXD were decreased, while the percentage of G2/M phase increased(P<0.05). The medium and high dose group of EXD increased viability of COV434 and decreased the elevated ROS production and early apoptosis and total apoptosis(P<0.05, P<0.01). Furthermore, the low and medium dose group of EXD significantly enhanced the expression of P-44/42(P<0.05). Conclusion Pretreatment with EXD alleviate H_2O_2-induced apoptosis in COV434 cells through both eliminate ROS, relieve oxidative DNA damage and regulating the MAPK-Nrf2 signaling pathway and mitochondrial pathway mediated apoptosis.
引文
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[6] Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis[J]. Free Radic Biol Med, 2010, 48(6): 749-762.
[7] 刘永胜, 赵丽慧. 二仙汤的研究及临床应用[J]. 光明中医, 2010, 25(4): 741-742.
[8] Zhong LL, Tong Y, Tang GW, et al. A randomized, double-blind, controlled trial of a Chinese herbal formula (Er-Xian decoction) for menopausal symptoms in Hong Kong premenopausal women[J]. Menopause, 2013, 20(7): 767-776.
[9] 缪正来主编. 实用方剂学辞典[M]. 南京: 江苏科技出版社, 1989: 4.
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[11] 沈小瑜, 方肇勤, 张伯讷, 等. 二仙汤及其拆方对老年大鼠部分抗氧化酶活性及其基因表达水平的影响[J]. 中国中西医结合杂志, 1995, 15(11): 672-674.
[12] Tsai-Turton M, Luong BT, Tan Y, et al. Cyclophosphamide-induced apoptosis in COV434 human granulosa cells involves oxidative stress and glutathione depletion[J]. Toxicol Sci, 2007, 98(1): 216-230.
[13] 刘建, 聂广宁, 杨洪艳. 过氧化氢诱导人卵巢颗粒细胞氧化应激模型的建立[J]. 广东医学, 2017, 38(7): 986-989.
[14] Jancar N, Kopitar AN, Ihan A, et al. Effect of apoptosis and reactive oxygen species production in human granulosa cells on oocyte fertilization and blastocyst development[J]. J Assist Reprod Genet, 2007, 24(2): 91-97.
[15] 王长海, 张仲海, 马静. 二仙汤治疗骨质疏松症50例[J]. 陕西中医, 1998, 19(5): 205.
[16] 艾浩, 张海英, 张玉立, 等. 二仙汤对顺铂损伤后小鼠卵巢功能调节作用研究[J]. 天津中医药, 2013, 30(5): 298-300.
[17] 刘波, 陈明, 李姗姗, 等. 二仙汤对去卵巢大鼠骨质疏松的影响[J]. 中国骨质疏松杂志, 2014, 20(2): 129-132.
[18] Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J]. Cell Res, 2002, 12(1): 9-18.
[19] Aggeli IK, Gaitanaki C, Beis I. Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells[J]. Cell Signal, 2006, 18(10): 1801-1812.
[20] Liu AL, Wang XW, Liu AH. JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons[J]. Exp Toxicol Pathol, 2009 61(2): 137-143.
[21] Park EJ, Kim YM, Park SW, et al. Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLP-induced septic mice[J]. Food Chem Toxicol, 2013, 55: 386-395.
[22] Sun Z, Huang Z, Zhang DD. Phosphorylation of Nrf2 at multiple sites by map kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response[J]. PLoS One, 2009, 4(8): e6588.
[23] Liu X, Zhang J, Wang S, et al. Astragaloside Ⅳ attenuates the H2O2-induced apoptosis of neuronal cells by inhibiting α-synuclein expression via the p38 MAPK pathway[J]. Int J Mol Med, 2017, 40(6): 1772-1780.
[24] Huang Y, Ma T, Ye Z, et al. Carbon monoxide (CO) inhibits hydrogen peroxide (H2O2)-induced oxidative stress and the activation of NF-κB signaling in lens epithelial cells[J]. Exp Eye Res, 2017, 166: 29-39.
[25] Heo J, Lee BR, Koh JW. Protective effects of epigallocatechin gallate after UV irradiation of cultured human lens epithelial cells[J]. Korean J Ophthalmol, 2008, 22(3): 183-186.
[2] Devine PJ, Perreault SD, Luderer U. Roles of reactive oxygen species and antioxidants in ovarian toxicity[J]. Biol Reprod, 2012, 86(2): 27.
[3] Karuputhula NB, Chattopadhyay R, Chakravarty B, et al.Oxidative status in granulosa cells of infertile women undergoing IVF[J]. Syst Biol Reprod Med, 2013, 59(2): 91-98
[4] Tsai-Turton M, Luderer U. Opposing effects of glutathione depletion and follicle-stimulating hormone on reactive oxygen species and apoptosis in cultured preovulatory rat follicles[J]. Endocrinology, 2006, 147(3): 1224-1236.
[5] Kumar M, Pathak D, Venkatesh S, et al. Chromosomal abnormalities & oxidative stress in women with premature ovarian failure (POF)[J]. Indian J Med Res, 2012, 135(1): 92-97.
[6] Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis[J]. Free Radic Biol Med, 2010, 48(6): 749-762.
[7] 刘永胜, 赵丽慧. 二仙汤的研究及临床应用[J]. 光明中医, 2010, 25(4): 741-742.
[8] Zhong LL, Tong Y, Tang GW, et al. A randomized, double-blind, controlled trial of a Chinese herbal formula (Er-Xian decoction) for menopausal symptoms in Hong Kong premenopausal women[J]. Menopause, 2013, 20(7): 767-776.
[9] 缪正来主编. 实用方剂学辞典[M]. 南京: 江苏科技出版社, 1989: 4.
[10] 沈小珩, 方肇勤, 吴敦序, 等. 二仙汤及其拆方对老年大鼠自由基代谢作用的实验研究[J]. 上海中医药杂志, 1996, 30(3): 40-42.
[11] 沈小瑜, 方肇勤, 张伯讷, 等. 二仙汤及其拆方对老年大鼠部分抗氧化酶活性及其基因表达水平的影响[J]. 中国中西医结合杂志, 1995, 15(11): 672-674.
[12] Tsai-Turton M, Luong BT, Tan Y, et al. Cyclophosphamide-induced apoptosis in COV434 human granulosa cells involves oxidative stress and glutathione depletion[J]. Toxicol Sci, 2007, 98(1): 216-230.
[13] 刘建, 聂广宁, 杨洪艳. 过氧化氢诱导人卵巢颗粒细胞氧化应激模型的建立[J]. 广东医学, 2017, 38(7): 986-989.
[14] Jancar N, Kopitar AN, Ihan A, et al. Effect of apoptosis and reactive oxygen species production in human granulosa cells on oocyte fertilization and blastocyst development[J]. J Assist Reprod Genet, 2007, 24(2): 91-97.
[15] 王长海, 张仲海, 马静. 二仙汤治疗骨质疏松症50例[J]. 陕西中医, 1998, 19(5): 205.
[16] 艾浩, 张海英, 张玉立, 等. 二仙汤对顺铂损伤后小鼠卵巢功能调节作用研究[J]. 天津中医药, 2013, 30(5): 298-300.
[17] 刘波, 陈明, 李姗姗, 等. 二仙汤对去卵巢大鼠骨质疏松的影响[J]. 中国骨质疏松杂志, 2014, 20(2): 129-132.
[18] Zhang W, Liu HT. MAPK signal pathways in the regulation of cell proliferation in mammalian cells[J]. Cell Res, 2002, 12(1): 9-18.
[19] Aggeli IK, Gaitanaki C, Beis I. Involvement of JNKs and p38-MAPK/MSK1 pathways in H2O2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells[J]. Cell Signal, 2006, 18(10): 1801-1812.
[20] Liu AL, Wang XW, Liu AH. JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons[J]. Exp Toxicol Pathol, 2009 61(2): 137-143.
[21] Park EJ, Kim YM, Park SW, et al. Induction of HO-1 through p38 MAPK/Nrf2 signaling pathway by ethanol extract of Inula helenium L. reduces inflammation in LPS-activated RAW 264.7 cells and CLP-induced septic mice[J]. Food Chem Toxicol, 2013, 55: 386-395.
[22] Sun Z, Huang Z, Zhang DD. Phosphorylation of Nrf2 at multiple sites by map kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response[J]. PLoS One, 2009, 4(8): e6588.
[23] Liu X, Zhang J, Wang S, et al. Astragaloside Ⅳ attenuates the H2O2-induced apoptosis of neuronal cells by inhibiting α-synuclein expression via the p38 MAPK pathway[J]. Int J Mol Med, 2017, 40(6): 1772-1780.
[24] Huang Y, Ma T, Ye Z, et al. Carbon monoxide (CO) inhibits hydrogen peroxide (H2O2)-induced oxidative stress and the activation of NF-κB signaling in lens epithelial cells[J]. Exp Eye Res, 2017, 166: 29-39.
[25] Heo J, Lee BR, Koh JW. Protective effects of epigallocatechin gallate after UV irradiation of cultured human lens epithelial cells[J]. Korean J Ophthalmol, 2008, 22(3): 183-186.