依托咪酯对缺氧复氧诱导心肌细胞炎性损伤及HMGB1/RAGE/NF-κB通路的影响

Effects of etomidate on inflammatory injury of cardiomyocytes induced by hypoxia/reoxygenation via the repression of HMGB1/RAGE/NF-κB pathway

目的:探讨麻醉诱导剂依托咪酯(Eto)在缺氧/复氧(H/R)后心肌细胞炎性损伤中的作用及其对高迁移率族蛋白B1/晚期糖基化终末产物受体/核因子κB(HMGB1/RAGE/NF-κB)通路的影响和机制。方法:随机将大鼠H9C2心肌细胞分成对照组、H/R组、Eto-L组(2 mg/L Eto预处理)、Eto-H组(4 mg/L Eto预处理)、正丁酸钠组(10 mmol/L HMGB1抑制剂正丁酸钠100μL)、Eto-H+正丁酸钠组;通过缺氧7 h后复氧2 h建立H/R心肌细胞模型,于造模前在Eto-L组、Eto-H组、正丁酸钠组及Eto-H+正丁酸钠组中分别添加相应药物处理48 h,而对照组、H/R组细胞常规培养;检测细胞上清液白介素1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白介素6(IL-6)水平及乳酸脱氢酶(LDH)漏出率;分别通过苏木精-伊红(HE)染色、MTT法、流式细胞仪、比色法观察H9C2细胞形态、活力、凋亡、半胱氨酸蛋白酶3(caspase-3)活性;Western blotting检测caspase-3及HMGB1/RAGE/NF-κB通路蛋白表达。结果:与对照组比较,H/R组细胞上清液中IL-1β、TNF-α及IL-6水平、LDH漏出率、细胞病理程度、凋亡率、caspase-3活性及其蛋白表达、p-NF-κB p65/NF-κB p65、RAGE、HMGB1表达水平增加,而细胞存活率降低(P<0.05);与H/R组比较,Eto-L组、Eto-H组、正丁酸钠组细胞上清液IL-1β、TNF-α及IL-6水平、LDH漏出率、细胞病理程度、凋亡率、caspase-3活性及其蛋白表达、p-NF-κB p65/NF-κB p65、RAGE、HMGB1表达水平减少,而细胞存活率增加(P<0.05),且Eto-L组、Eto-H组间上述指标比较,差异有统计学意义(P<0.05);与Eto-H组比较,Eto-H+正丁酸钠组细胞上清液IL-1β、TNF-α及IL-6水平、LDH漏出率、细胞病理程度、凋亡率、caspase-3活性及其蛋白表达、p-NF-κB p65/NF-κB p65、RAGE、HMGB1表达水平减少,而细胞存活率增加(P<0.05)。结论:Eto可以抑制H/R心肌细胞损伤,其机制可能是通过抑制HMGB1/RAGE/NF-κB通路和下调促炎性细胞因子的表达来实现。

Objective:To investigate the effects of etomidate(Eto) on the inflammatory damage of cardiomyocytes induced by hypoxia/reoxygenation(H/R) and the mechanism involved in the high mobility group protein B1/receptor for advanced glycation end product/nuclear factor kappa B(HMGB1/RAGE/NF-κB) pathway.Methods:Rat H9 C2 cardiomyocytes were randomly divided into control group,H/R group,Eto-L group(2 mg/L Eto pretreatment),Eto-H group(4 mg/L Eto pretreatment),sodium n-butyrate group(10 mmol/L HMGB1 inhibitor sodium n-butyrate 100 μL),and Eto-H+Sodium n-butyrate group.The H/R cardiomyocyte model was established by hypoxia for 7 hours and reoxygenation for 2 hours.Before modeling,the Eto-L group,Eto-H group,sodium n-butyrate group and Eto-H+sodium n-butyrate group were treated with corresponding drugs for 48 hours,while the control group and H/R group were cultured routinely.The levels of interleukin 1β(IL-1β),tumor necro-sis factor-α(TNF-α),interleukin 6(IL-6) and the leakage rate of lactate dehydrogenase(LDH) in the cell supernatant were detected.The morphology,viability,apoptosis,and caspase-3 activity of H9 C2 cells were examined by HE staining,MTT method,flow cytometry,and colorimetry,respectively.Western blotting was used to detect the expression of caspase3 and HMGB1/RAGE/NF-κB pathway proteins.Results:Compared with the control group,the levels of IL-1β,TNF-α and IL-6 in the cell supernatant,LDH leakage rate,cytopathological degree,apoptosis rate,caspase3 activity and its protein expression,the expression levels of p-NF-κB p65/NF-κB p65,RAGE and HMGB1 increased in H/R group,while the cell survival rate decreased(P<0.05);compared with H/R group,the levels of IL-1β,TNF-α and IL-6 in the cell supernatant,LDH leakage rate,cytopathological degree,apoptosis rate,caspase3 activity and its protein expression,the expression levels of p-NF-κB p65/NF-κB p65,RAGE andHMGB1 decreased in Eto-L group,Eto-H group,and sodium n-butyrate group,while the cell survival rate increased,and the difference in the above indicators between Eto-L group and Eto-H group was statistically significant(P<0.05);compared with Eto-H group,the levels of IL-1β,TNF-α and IL-6 in the cell supernatant,LDH leakage rate,cytopathological degree,apoptosis rate,caspase3 activity and its protein expression,the expression levels of p-NF-κB p65/NF-κB p65,RAGE and HMGB1 decreased in Eto-H + sodium n-butyrate group,while the cell survival rate increased(P<0.05).Conclusion:Eto may inhibit the injury of H/R cardiomyocytes by inhibiting the HMGB1/RAGE/NF-κB pathway and decreasing the production of inflammatory cytokines.