胡琦兰,张明山,王银银,韦映丽,陶玲,张敏,沈祥春
HU Qilan,ZHANG Mingshan,WANG Yinyin,WEI Yingli,TAO Ling,ZHANG Min,SHEN Xiangchun

• 1:省部共建药用植物功效与利用国家重点实验室&贵州医科大学
• 2:贵州医科大学生理学教研室
• 3:天然药物资源优效利用重点实验室&贵州医科大学药学院

摘要(Abstract)：

目的 探讨天麻素(GAS)抑制Keap1/Nrf2-泛素-蛋白酶体信号对高糖诱导原代大鼠心肌细胞损伤的作用及其机制。方法 利用40 mmol/L高糖制作原代大鼠心肌细胞尿病心肌细胞损伤模型，将细胞分为正常对照组(25 mmol/L葡萄糖)、模型组、GAS低剂量(0.75μmol/L)治疗组、GAS高剂量(1.5μmol/L)治疗组及阳性药(二甲双胍，0.2 mmol/L)治疗组；48 h后取出细胞；利用吉姆萨染色法观察细胞形态变化，乳酸脱氢酶(LDH)试剂盒检测细胞LDH外漏率，蛋白免疫印迹法检测血心钠肽(ANP)、脑钠肽(BNP)、核因子E2相关因子2(Nrf2)、Keap1及血红素氧合酶1(HO-1)的表达，免疫荧光法检测Nrf2;为进一步研究GAS对蛋白酶体的作用，引入蛋白酶体抑制剂MG132,并将细胞分为正常对照组、模型组、MG132治疗组(1.0μmol/L)、GAS治疗组(1.5μmol/L)及MG132与GAS联合治疗组(1.0μmol/L MG132+1.5μmol/L GAS),利用蛋白免疫印迹法检测细胞中Nrf2,Keap1及泛素-蛋白酶体相关蛋白泛素激活酶(UBE1)、人26S蛋白酶体非ATP酶调节亚基11(PSMD11)、人蛋白酶体亚基α7(PSMA7)的表达，免疫荧光法检测Nrf2,免疫沉淀法检测泛素化Nrf2的表达。结果 GAS可改善高糖诱导原代大鼠心肌细胞形态损伤、降低LDH外漏率(P<0.05);模型组细胞中ANP,BNP,Keap1,UBE1、PSMD11,PSMA7与泛素化的Nrf2的表达增加(P<0.05),Nrf2与HO-1的表达降低(P<0.05);给予不同浓度的GAS与阳性药二甲双胍后、可部分逆转上述蛋白的表达(P<0.05);GAS还可在一定程度上改善Nrf2的入核情况，同时GAS与MG132均有效下调模型组细胞中UBE1、PSMD11、PSMA7、Keap1与泛素化的Nrf2的表达(P<0.05),并增加Nrf2的表达(P<0.05),但GAS与MG132联用与单独使用GAS相比，差异无统计学意义(P>0.05)。结论 GAS可以改善高糖诱导的原代大鼠心肌细胞损伤，其机制可能是通过抑制Keap1介导Nrf2的泛素化、同时减少Nrf2的降解而抑制蛋白酶体活性。
Objective To explore the amelioration effect and molecular mechanism of gastrodin on high glucose-induced primary neonatal rat cardiomyocytes. Methods The primary rat myocardial cell injury model of urinary disease was established by using 40 mmol/L high glucose. The experimental groups were as follows: Control group(25 mmol/L D-glucose), Model group(40 mmol/L D-glucose), and Low dose gastrodin treatment group(40 mmol/L D-glucose+0.75 μmol/L), High dose gastrodin treatment group(40 mmol/L D-glucose+1.5 μmol/L), Metformin treatment group(40 mmol/L D-glucose+0.2 mmol/L). The cells were removed After 48 h and giemsa staining was used to observe the morphological changes of cells. Lactate dehydrogenase(LDH) leakage rate was measured by LDH kit. Western blot was used to detect the expression of atrial natriuretic peptide(ANP), B-type natriuretic Peptide(BNP), nuclear factor-E2-related factor 2(Nrf2), Keap1, heme oxygenase-1(HO-1), and ubiquitin-proteasome system-related proteins UBE1, PSMD11, PSMA7 on cells. Nrf2 was detected by immunofluorescence. To further explore the effect of GAS on Proteasome, MG132, a Proteasome inhibitor, was introduced for experiments. And the cells were divided into Control group, Model group, MG132 treatment group(40 mmol/L D-glucose+1.0 μmol/L), GAS treatment group(40 mmol/L D-glucose+1.5 μmol/L), MG132 and GAS combined treatment group(40 mmol/L D-glucose+1.0 μmol/L MG132+1.5 μmol/L GAS). Western blot was used to detect the expression of Nrf2, Keap1, and ubiquitin-proteasome system-related proteins UBE1, PSMD11, PSMA7 on cells. The expression of ubiquitinated-Nrf2 was detected by immunoprecipitation. Results GAS could improve the morphological damage of primary rat cardiomyocytes induced by high glucose and reduce the LDH leakage rate(P<0.05). In the model group, the expressions of ANP, BNP, Keap1, UBE1, PSMD11, PSMA7, and ubiquitinated Nrf2 were increased(P<0.05), while the expressions of Nrf2 and HO-1 were decreased(P<0.05). The expression of the above proteins could be partially reversed by different concentrations of GAS and metformin(P<0.05). GAS could also improve the nucleation of Nrf2 to some extent. At the same time, both GAS and MG132 effectively down-regulated the expression of UBE1, PSMD11, PSMA7, Keap1, and ubiquitinated Nrf2 in the model group cells(P<0.05), and increased the expression of Nrf2(P<0.05). There was no significant difference(P>0.05). Conclusion GAS can ameliorate the injury of primary neonatal rat cardiomyocytes induced by high glucose and its mechanism may be achieved through inhibiting Keap1 mediated Nrf2 ubiquitination and proteasome activity to reduce Nrf2 degradation.

关键词(KeyWords)： 天麻素;原代大鼠心肌细胞;血心钠肽;脑钠肽;核因子E2相关因子2;泛素蛋白酶体系统;高糖
primary neonatal rat cardiomyocytes;gastrodin;heart blood sodium peptide;brain natriuretic peptide;nuclear factor-e2-related factor 2;ubiquitin-proteasome system;high glucose

Abstract:

Keywords:

基金项目(Foundation): 国家自然基金(8217142504);; 贵州省自然科学基金(黔科合中引地[2023]003)

作者(Author): 胡琦兰,张明山,王银银,韦映丽,陶玲,张敏,沈祥春
HU Qilan,ZHANG Mingshan,WANG Yinyin,WEI Yingli,TAO Ling,ZHANG Min,SHEN Xiangchun

DOI: 10.19367/j.cnki.2096-8388.2024.07.001

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