TRPV4活化调控炎性小体Nod样受体家族3在小鼠膀胱上皮细胞损伤中作用和机制研究
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摘要
目的探讨瞬时受体电位离子通道香草素受体亚家族4(transient receptor potential vanilloid receptor 4, TRPV4)通道活化在小鼠原代培养的膀胱上皮细胞损伤中的作用及其分子机制。方法小鼠1只,原代培养膀胱上皮细胞,并分为对照组、刺激组、β-羟基丁酸(β-hydroxybutyrate, BHB)预处理组和细胞因子释放抑制药物3(cytokine release inhibitory drug 3, MCC950)预处理组,刺激组采用TRPV4激动剂GSK1016790A 100μmol/L进行刺激;BHB预处理组和MCC950预处理组分别给予10 mmol/L BHB和50μmol/L MCC950后采用TRPV4激动剂GSK1016790A进行刺激;对照组正常培养。4组细胞采用Calcein-AM/PI双染色法观察活细胞数和死细胞数;采用乳酸脱氢酶(lactate dehydrogenase, LDH)细胞毒实验检测细胞LDH水平;采用Western blot法检测刺激组GSK1016790A刺激前及刺激3、6、9 h时细胞Nod样受体家族含pyrin结构域蛋白3(Nod-like receptor family pyrin domain-containing protein 3, NLRP3)及其下游活化产物胱冬肽酶-1(caspase-1 p10)相对表达量。结果刺激组Calcein-AM染色的活细胞数[(46.80±15.51)个]较对照组[(1 242.20±143.79)个]、BHB预处理组[(1 001.20±115.41)个]和MCC950预处理组[(1 144.00±138.62)个]减少(P<0.05),BHB预处理组、MCC950预处理组少于对照组(P<0.05),BHB预处理组与MCC950预处理组比较差异无统计学意义(P>0.05);刺激组PI染色的死细胞数[(1 111.60±127.49)个]较对照组[(33.80±8.41)个]、BHB预处理组[(251.80±48.67)个]和MCC950预处理组[(265.20±31.57)个]增多(P<0.05),BHB预处理组、MCC950预处理组多于对照组(P<0.05),BHB预处理组与MCC950预处理组比较差异无统计学意义(P>0.05);刺激组LDH水平[(85.27±7.18)u/L]高于对照组[(22.47±2.94)u/L]、BHB预处理组[(35.74±4.32)u/L]和MCC950预处理组[(37.09±4.09)u/L](P<0.05),BHB预处理组、MCC950预处理组高于对照组(P<0.05),BHB预处理组与MCC950预处理组比较差异无统计学意义(P>0.05);刺激组GSK1016790A刺激3、6、9 h时细胞中NLRP3(0.68±0.07、0.86±0.08、0.79±0.09)、caspase-1 p10表达量(0.68±0.06、0.51±0.07、0.47±0.05)均高于刺激前(0.36±0.05、0.24±0.06)(P<0.05)。结论 TRPV4可通过活化炎性小体NLRP3导致小鼠膀胱上皮细胞损伤,调控TRPV4-NLRP3可能为膀胱过度活动症治疗提供新靶点。
Objective To investigate the role of transient receptor potential vanilloid receptor 4(TRPV4) activation in primary cultured bladder epithelial cell injury in a mouse and the possible mechanism. Methods The primarily cultured bladder epithelial cells in a mouse were randomly divided into control group, GSK1016790 A group, β-hydroxybutyrate(BHB) pre-treatment group and cytokine release inhibitory drug 3(MCC950) pre-treatment group. GSK1016790 A group was exposed to 100 μmol GSK1016790 A. BHB pre-treatment group and MCC950 pre-treatment group were pretreated with 10 mmol/L BHB and 50 μmol/L MCC950 for 30 min, followed by stimulation with GSK1016790 A. Control group was routinely cultured. Calcein-AM/PI double staining was used to detect the alive and died cell counts, lactate dehydrogenase(LDH) Assay Kit was used to detect LDH level, and Western blot technique was used to detect the relative expressions of Nod-like receptor family pyrin domain-containing protein 3(NLRP3) and caspase-1 p10 before and after GSK1016790 A stimulation for 3, 6 and 9 h. Results The alive cell count was significantly less in GSK1016790 A group(46.80±15.51) than that in control group(1 242.20±143.79), BHB pre-treatment group(1 001.20±115.41) and MCC950 pre-treatment group(1 144.00±138.62)(P<0.05),less in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The died cell count was significantly more in GSK1016790 Agroup(1 111.60±127.49)than that in control group(33.80±8.41),BHB pre-treatment group(251.80±48.67)and MCC950 pre-treatment group(265.20±31.57)(P<0.05),more in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The content of LDH was significantly higher in GSK1016790 Agroup((85.27±7.18)u/L)than that in control group((22.47±2.94)u/L),BHB pre-treatment group((35.74±4.32)u/L)and MCC950 pre-treatment group((37.09±4.09)u/L)(P<0.05),higher in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The contents of NLRP3(0.68±0.07,0.86±0.08,0.79±0.09)and the expressions of caspase-1 p10(0.68±0.06,0.51±0.07,0.47±0.05)after stimulation for 3,6 and 9 hwere significantly higher than those before stimulation(0.36±0.05,0.24±0.06)(P<0.05).Conclusion TRPV4 induces bladder epithelial cell injury by activating NLRP3 in the mouse.To regulate TRPV4/NLRP3 signaling pathway would provide a novel target for the treatment of overactive bladder.
Objective To investigate the role of transient receptor potential vanilloid receptor 4(TRPV4) activation in primary cultured bladder epithelial cell injury in a mouse and the possible mechanism. Methods The primarily cultured bladder epithelial cells in a mouse were randomly divided into control group, GSK1016790 A group, β-hydroxybutyrate(BHB) pre-treatment group and cytokine release inhibitory drug 3(MCC950) pre-treatment group. GSK1016790 A group was exposed to 100 μmol GSK1016790 A. BHB pre-treatment group and MCC950 pre-treatment group were pretreated with 10 mmol/L BHB and 50 μmol/L MCC950 for 30 min, followed by stimulation with GSK1016790 A. Control group was routinely cultured. Calcein-AM/PI double staining was used to detect the alive and died cell counts, lactate dehydrogenase(LDH) Assay Kit was used to detect LDH level, and Western blot technique was used to detect the relative expressions of Nod-like receptor family pyrin domain-containing protein 3(NLRP3) and caspase-1 p10 before and after GSK1016790 A stimulation for 3, 6 and 9 h. Results The alive cell count was significantly less in GSK1016790 A group(46.80±15.51) than that in control group(1 242.20±143.79), BHB pre-treatment group(1 001.20±115.41) and MCC950 pre-treatment group(1 144.00±138.62)(P<0.05),less in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The died cell count was significantly more in GSK1016790 Agroup(1 111.60±127.49)than that in control group(33.80±8.41),BHB pre-treatment group(251.80±48.67)and MCC950 pre-treatment group(265.20±31.57)(P<0.05),more in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The content of LDH was significantly higher in GSK1016790 Agroup((85.27±7.18)u/L)than that in control group((22.47±2.94)u/L),BHB pre-treatment group((35.74±4.32)u/L)and MCC950 pre-treatment group((37.09±4.09)u/L)(P<0.05),higher in BHB pre-treatment group and MCC950 pre-treatment group than that in control group(P<0.05),and showed no significant difference between BHB pre-treatment group and MCC950 pre-treatment group(P>0.05).The contents of NLRP3(0.68±0.07,0.86±0.08,0.79±0.09)and the expressions of caspase-1 p10(0.68±0.06,0.51±0.07,0.47±0.05)after stimulation for 3,6 and 9 hwere significantly higher than those before stimulation(0.36±0.05,0.24±0.06)(P<0.05).Conclusion TRPV4 induces bladder epithelial cell injury by activating NLRP3 in the mouse.To regulate TRPV4/NLRP3 signaling pathway would provide a novel target for the treatment of overactive bladder.
引文
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[10] VILAPLANA J,LECHA M,TRULLAS C,et al.Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding[J].J Clin Invest,2007,117(11):3453-3462.
[11] BAROJA-MAZO A,MARTIN-SANCHEZ F,GOMEZ A I,et al.The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response[J].Nat Immunol,2014,15(8):738-748.
[12] 祁会丽,姚丽芬.细胞焦亡激活机制及相关疾病研究进展[J].中华实用诊断与治疗杂志,2016,30(5):417-419.
[13] 乔菲,郝蕾,晁旭.细胞焦亡与癌症关系的研究进展[J].中华实用诊断与治疗杂志,2018,32(11):98-100.
[14] JO E K,KIM J K,SHIN D M,et al.Molecular mechanisms regulating NLRP3 inflammasome activation[J].Cell Mol Immunol,2016,13(2):148-159.
[2] 李传章.TRP离子通道家族与膀胱功能相关性的研究进展[J].山东医药,2013,53(30):92-95.
[3] EVERAERTS W,NILIUS B,OWSIANIK G.The vanilloid transient receptors potential channel TRPV4:from structure to disease[J].Prog Biophys Mol Biol,2010,103(1):2-17.
[4] JANCZY J R,CIRACI C,HAASKEN S,et al.Immune complexes inhibit IL-1 secretion and inflammasome activation[J].J Immunol,2014,193(10):5190-5198.
[5] HUGHES F M,HILL H M,WOOD C M,et al.The NLRP3 inflammasome mediates inflammation produced by bladder outlet obstruction[J].J Urol,2016,195(5):1598-1605.
[6] ZHANG X L,YAO J,GAO K,et al.AMPK suppresses connexin 43 expression in the bladder and ameliorates voiding dysfunction in cyclophosphamide-induced mouse cystitis[J].Sci Rep,2016,6:19708.
[7] 陈潇雨,屈颖伟,王锁刚,等.药物联合应用治疗前列腺增生合并膀胱过度活动症临床疗效分析[J].医药论坛杂志,2016,37(5):4-6.
[8] 马煜.索利那新联合多沙唑嗪治疗前列腺增生症的临床观察[J].医药论坛杂志,2012,33(5):21-22.
[9] NILIUS B,APPENDINO G,OWSIANIK G.The transient receptor potential channel TRPA1:from gene to pathophysiology[J].Pflügers Archiv Eur J Physiol,2012,464(5):425-458.
[10] VILAPLANA J,LECHA M,TRULLAS C,et al.Deletion of the transient receptor potential cation channel TRPV4 impairs murine bladder voiding[J].J Clin Invest,2007,117(11):3453-3462.
[11] BAROJA-MAZO A,MARTIN-SANCHEZ F,GOMEZ A I,et al.The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response[J].Nat Immunol,2014,15(8):738-748.
[12] 祁会丽,姚丽芬.细胞焦亡激活机制及相关疾病研究进展[J].中华实用诊断与治疗杂志,2016,30(5):417-419.
[13] 乔菲,郝蕾,晁旭.细胞焦亡与癌症关系的研究进展[J].中华实用诊断与治疗杂志,2018,32(11):98-100.
[14] JO E K,KIM J K,SHIN D M,et al.Molecular mechanisms regulating NLRP3 inflammasome activation[J].Cell Mol Immunol,2016,13(2):148-159.