p38丝裂原活化蛋白激酶信号通路对高糖环境下HK-2细胞凋亡影响的研究
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摘要
目的探讨p38丝裂原活化蛋白激酶(p38MAPK)信号通路对高糖环境下HK-2细胞凋亡的影响。方法用p38 MAPK信号通路抑制剂作用于高糖环境下的HK-2细胞,MTT检测细胞活力,流式细胞术检测细胞凋亡情况,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法检测细胞活性氧簇(ROS)水平,Western blot检测p38MAPK、磷酸化的p38 MAPK(p-p38 MAPK)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)、含半胱氨酸的天冬氨酸蛋白水解酶3(caspase-3)蛋白水平,ELISA检测上清中肿瘤坏死因子α(TNF-α)水平。结果高糖作用后的HK-2细胞活力降低[(0. 79±0. 05)vs(0. 32±0. 02]),细胞凋亡率[(5. 35±0. 58)%vs(17. 42±1. 23)%]、细胞中ROS水平[(4. 62±0. 05)vs(20. 65±0. 21)]、细胞培养液上清中TNF-α水平[(25. 64±1. 36)vs(50. 71±1. 28)mg/L]均升高,cleaved caspase-3、caspase-3、p-p38 MAPK蛋白水平升高,与对照组HK-2细胞比较,差异有统计学意义(P<0. 05)。在细胞培养液中加入p38 MAPK信号通路抑制剂培养后,细胞凋亡率降低[(17. 42±1. 23)%vs(12. 65±1. 14)%vs(9. 17±0. 85)%vs(7. 25±0. 64)%],细胞活力升高[(0. 32±0. 02)vs(0. 41±0. 03)vs(0. 52±0. 04)vs(0. 68±0. 05)],细胞中ROS水平[(20. 65±0. 21)vs(16. 21±1. 23)vs(12. 04±1. 36)vs(9. 75±0. 09)]和细胞培养液上清中TNF-α水平[(50. 71±1. 28)vs(43. 23±2. 24)vs(36. 01±2. 17)vs(32. 84±1. 22)mg/L]均降低,细胞中cleaved caspase-3、caspase-3、p-p38 MAPK的蛋白水平降低,且p38 MAPK信号通路抑制剂浓度越高,作用越显著,与单纯高糖培养后的细胞比较,差异有统计学意义(P<0. 05)。结论抑制p38 MAPK信号通路可减弱高糖诱导的HK-2细胞凋亡。
Objective To investigate the effect of p38 MAPK signaling pathway on apoptosis of HK-2 cells in high glucose environment. Methods HK-2 cells were cultured by p38 MAPK signaling pathway inhibitors in high glucose environment. The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. ROS levels was detected by DCFH-DA staining. The protein levels of p38 MAPK,p-p38 MAPK,cleaved caspase-3,and caspase-3 were determined by western blot. The level of TNF-a in the culture supernatant was examined by ELISA. after high glucose treatment. Results The viability was decreased [(0. 79 ± 0. 05) vs(0. 32 ± 0. 02) ], the apoptotic rate was increased[(5. 35 ± 0. 58) %vs(17. 42±1. 23)%],the levels of ROS was elevated [(4. 62±0.05)vs(20. 65±0.21)),the level of TNF-a in the culture supernatant [(25. 64± 1. 36)vs(50. 71 ± 1. 28)mg/L] and the protein levels of cleaved caspase-3, caspase-3 and p-p38 MAPK were all significantly increased in HK-2 cells cultured with high glucose compared with HK-2 cells cultured with normal glucose(P<0.05). After adding p38 MAPK signaling pathway inhibitors, the apoptotic rate was decreased [(17. 42 ± 1. 23) % vs(12. 65 ± 1. 14)% vs(9. 17 ±0.85)% vs(7. 25 ±0.64)%],cell viability was increased[(0.32 ±0.02) vs(0.41 ±0.03)vs(0.52 ±0.04)vs(0.68±0.05)],ROS levels in the cells[(20.65±0.21)vs(16.21±1.23)vs(12.04±1.36)vs(9. 75 ± 0. 09) ] and the TNF-a content in the culture supernatant [(50. 71 ± 1. 28) vs(43. 23 ± 2. 24) vs(36. 01 ± 2. 17) vs(32. 84±1. 22) mg/L] were decreased, the protein levels of cleaved caspase-3, caspase-3 and p-p38 MAPK were also decreased. Moreover, the higher the concentration of p38 MAPK signaling pathway inhibitors was used, the more remarkable the effect was observed as compared with the cells cultured with high glucose(P<0.05). Conclusion Inhibition of p38 MAPK signaling pathway may attenuate the apoptosis of HK-2 cells induced by high glucose.
Objective To investigate the effect of p38 MAPK signaling pathway on apoptosis of HK-2 cells in high glucose environment. Methods HK-2 cells were cultured by p38 MAPK signaling pathway inhibitors in high glucose environment. The cell viability was measured by MTT assay. Apoptosis was analyzed by flow cytometry. ROS levels was detected by DCFH-DA staining. The protein levels of p38 MAPK,p-p38 MAPK,cleaved caspase-3,and caspase-3 were determined by western blot. The level of TNF-a in the culture supernatant was examined by ELISA. after high glucose treatment. Results The viability was decreased [(0. 79 ± 0. 05) vs(0. 32 ± 0. 02) ], the apoptotic rate was increased[(5. 35 ± 0. 58) %vs(17. 42±1. 23)%],the levels of ROS was elevated [(4. 62±0.05)vs(20. 65±0.21)),the level of TNF-a in the culture supernatant [(25. 64± 1. 36)vs(50. 71 ± 1. 28)mg/L] and the protein levels of cleaved caspase-3, caspase-3 and p-p38 MAPK were all significantly increased in HK-2 cells cultured with high glucose compared with HK-2 cells cultured with normal glucose(P<0.05). After adding p38 MAPK signaling pathway inhibitors, the apoptotic rate was decreased [(17. 42 ± 1. 23) % vs(12. 65 ± 1. 14)% vs(9. 17 ±0.85)% vs(7. 25 ±0.64)%],cell viability was increased[(0.32 ±0.02) vs(0.41 ±0.03)vs(0.52 ±0.04)vs(0.68±0.05)],ROS levels in the cells[(20.65±0.21)vs(16.21±1.23)vs(12.04±1.36)vs(9. 75 ± 0. 09) ] and the TNF-a content in the culture supernatant [(50. 71 ± 1. 28) vs(43. 23 ± 2. 24) vs(36. 01 ± 2. 17) vs(32. 84±1. 22) mg/L] were decreased, the protein levels of cleaved caspase-3, caspase-3 and p-p38 MAPK were also decreased. Moreover, the higher the concentration of p38 MAPK signaling pathway inhibitors was used, the more remarkable the effect was observed as compared with the cells cultured with high glucose(P<0.05). Conclusion Inhibition of p38 MAPK signaling pathway may attenuate the apoptosis of HK-2 cells induced by high glucose.
引文
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[14] Gao P, Wu X, Shui H, et al. Fluvastatin inhibits angiotensinⅡ-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway. Mol Biol Reports,2012, 39:4719-4725.
[15] Yu L,Gan X,Liu X,et al. Calcium oxalate crystals inducestight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway. Ren Fail,2017, 39:440-451.
[16]牛娟,刘培雯,黄杜娟,等.鞘内注射p38MAPK抑制剂SB203580对CCI大鼠脊髓背角多种嘌呤受体表达的影响.神经解剖学杂志,2017, 33:47-52.
[17] Zhang X, Liang D, Lian X, et al. Berberine activates Nrf2nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol3-kinase/Akt-dependent mechanism. Apoptosis, 2016, 21:721-736.
[18] Jiao X,Li Y, Zhang T,et al. Role of Sirtuin3 in high glucoseinduced apoptosis in renal tubular epithelial cells. Biochem Biophys Res Commun, 2016, 480:387-393.
[19]陈慧.PTS2诱导HeLa细胞凋亡及其分子机制的研究.南京:南京师范大学,2007.
[20] Oliveira LRD,Mombach JCM, Castellani G. A simple stochastic model for the feedback circuit between p16INK4a and p53 mediated by p38MAPK:Implications for senescence and apoptosis.Mol Biosystems,2015, 11:2955-2963.
[2] Gou R,Chen J,Sheng S,et al. KIM-1 mediates high glucoseinduced autophagy and apoptosis in renal tubular epithelial cells.Cell Physiol Biochem, 2016, 38:2479-2488.
[3]房艳宇,罗晓光,苏阳,等.探讨BV2细胞活化中是否存在p38MAPK及JAK2-STAT通路的磷酸化激活.中国免疫学杂志,2016, 32:629-632.
[4] Dai T, Natarajan R, Nast CC, et al. Glucose and diabetes:Effects on podocyte and glomerular p38MAPK, heat shock protein 25,and actin cytoskeleton. Kidney Int,2006,69:806-814.
[5]杨加培,孙浩,王丹丹,等.大黄酸通过MAPK信号转导通路诱导HK-2细胞凋亡.中国实验方剂学杂志,2015, 21:147-151.
[6]杨丽霞,姜良恩,王志程,等.中草药黄酮类化合物防治糖尿病肾病的实验研究进展.中国实验方剂学杂志,2015, 21:230-234.
[7] Chen GJ, Fei W, Pang XX, et al. UrotensinⅡinhibits autophagy in renal tubular epithelial cells and induces ECM production in early diabetic mice. J Diabetes Invest,2016,8:629-629.
[8] Yamashita T,Fujimiya M,Nagaishi K,et al. Fusion of bone marrow-derived cells with renal tubules contributes to renal dys-function in diabetic nephropathy. FASEB J,2012, 26:1559-1568.
[9] Wu G,Tu Y,Jia R. The influence of fasudil on the epithelialmesenchymal transdifferentiation of renal tubular epithelial cells from diabetic rats. Biomed Pharmacother, 2010, 64:124-129.
[10] Meier M, Nitschke M, Hocke C, et al. Insulin inhibits caspase-3 activity in human renal tubular epithelial cells via the PI3-kinase/Akt pathway. Cell Physiol Biochem, 2008, 21:279-286.
[11] Liu X, Ye F, Xiong H, et al. IL-beta Induces IL-6 production in retinal Muller cells predominantly through the activation of P38MAPK/NF-kappa B signaling pathway. Exp Cell Res, 2015,331:223-231.
[12] Xue J, Chen F, Wang J, et al. Emodin protects against concanavalin a-induced hepatitis in mice through inhibiting activation of the p38 MAPK-NF-κB signaling pathway. Cell Physiol Biochem,2015, 35:1557-1570.
[13]何丹,任朋亮,范雪娇,等.p38MAPK与疾病关系的研究进展.西部医学,2014, 26:259-262.
[14] Gao P, Wu X, Shui H, et al. Fluvastatin inhibits angiotensinⅡ-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway. Mol Biol Reports,2012, 39:4719-4725.
[15] Yu L,Gan X,Liu X,et al. Calcium oxalate crystals inducestight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway. Ren Fail,2017, 39:440-451.
[16]牛娟,刘培雯,黄杜娟,等.鞘内注射p38MAPK抑制剂SB203580对CCI大鼠脊髓背角多种嘌呤受体表达的影响.神经解剖学杂志,2017, 33:47-52.
[17] Zhang X, Liang D, Lian X, et al. Berberine activates Nrf2nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol3-kinase/Akt-dependent mechanism. Apoptosis, 2016, 21:721-736.
[18] Jiao X,Li Y, Zhang T,et al. Role of Sirtuin3 in high glucoseinduced apoptosis in renal tubular epithelial cells. Biochem Biophys Res Commun, 2016, 480:387-393.
[19]陈慧.PTS2诱导HeLa细胞凋亡及其分子机制的研究.南京:南京师范大学,2007.
[20] Oliveira LRD,Mombach JCM, Castellani G. A simple stochastic model for the feedback circuit between p16INK4a and p53 mediated by p38MAPK:Implications for senescence and apoptosis.Mol Biosystems,2015, 11:2955-2963.