Yang Zhenkun, Wu Shuoxiong, Zhao Jingjing, Wang Zuoyu, Yao Min, Lu Peihua, Dong Wenyan, Sun Jie
Center of Clinical Research, Wuxi People's Hospital of Nanjing Medical University, Wuxi 214023, China.
Department of Anesthesiology, Wuxi Children's Hospital, Wuxi 214023, China.
Ann Palliat Med. 2020 Jan;9(1):90-97. doi: 10.21037/apm.2019.11.31.
Pancreatic beta cell damage induced by glucose toxicity is an important factor in type 2 diabetes mellitus (T2DM). It has become evident that endoplasmic reticulum stress (ERS)-induced apoptosis was contributed to beta cell dysfunction and insulin resistance. Our previous work showed that emulsified isoflurane (EIso) could alleviate ERS in lung reperfusion injury. This study aimed to elucidate whether EIso could alleviate apoptosis induced by glucose in rat islet RIN-m5F beta cells via inhibiting ERS.
RIN-m5F cells were divided into five groups: the control group; the 0.1G group, cultured in 0.1M glucose for 24 h; the 0.3G group, cultured in 0.3M glucose for 24 h; the 0.3G + 57E group, cultured in 0.3M glucose with 57 µM EIso for 24 h, and the 0.3G + 76E group, cultured in 0.3M glucose with 76 µM EIso for 24 h. First, cell proliferation was measured by MTT assay, and the level of insulin secretion was measured with enzyme-linked immunosorbent assay (ELISA) kit. Second, the expression of B cell leukemia/lymphoma 2 (Bcl-2) associated X (Bax) and Bcl-2 were detected by Western blotting. The level of caspase-3 activity was assessed by colorimetric method. Finally, the ERS marker CHOP and GRP78 expression were detected by Western blotting. The levels of activating transcription factor-6 (ATF6), X-box-binding protein 1 (Xbp1), and eukaryotic translation initiation factor-2α (eIF2α) mRNA were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) after being treated with EIso for 24 h.
We found that exposure to high glucose reduced RIN-m5F cell viability, stimulated the secretion of insulin, activated caspase-3, improved the expression of Bax, and down-regulated Bcl-2. EIso improved the survival and protected the function of RIN-m5F. Compared to the 0.3G group, treatment with EIso inhibited the activity of caspase-3, and decreased the expression of Bax. The expression of CHOP and GRP78 were significantly suppressed by EIso at 24 h in a dose-dependent manner. The level of ATF6, Xbp1, and eIF2α mRNA of RIN-m5F were enhanced by high glucose, but only eIF2α mRNA was significantly decreased by EIso treatment.
The present study suggests that high glucose induces rat islet beta cell RIN-m5F apoptosis and aggravates the function of beta cells. EIso protects beta cells against high glucose through the ERS-dependent apoptotic pathway and might serve as a potential therapy for diabetes.
葡萄糖毒性诱导的胰腺β细胞损伤是2型糖尿病（T2DM）的一个重要因素。内质网应激（ERS）诱导的细胞凋亡导致β细胞功能障碍和胰岛素抵抗，这一点已变得很明显。我们之前的研究表明，乳化异氟烷（EIso）可减轻肺再灌注损伤中的ERS。本研究旨在阐明EIso是否能通过抑制ERS减轻葡萄糖诱导的大鼠胰岛RIN-m5Fβ细胞凋亡。
将RIN-m5F细胞分为五组：对照组；0.1G组，在0.1M葡萄糖中培养24小时；0.3G组，在0.3M葡萄糖中培养24小时；0.3G + 57E组，在含57μM EIso的0.3M葡萄糖中培养24小时；0.3G + 76E组，在含76μM EIso的0.3M葡萄糖中培养24小时。首先，通过MTT法检测细胞增殖，并用酶联免疫吸附测定（ELISA）试剂盒检测胰岛素分泌水平。其次，通过蛋白质印迹法检测B细胞淋巴瘤/白血病-2（Bcl-2）相关X蛋白（Bax）和Bcl-2的表达。采用比色法评估半胱天冬酶-3的活性水平。最后，通过蛋白质印迹法检测ERS标志物CHOP和GRP78的表达。用EIso处理24小时后，通过定量实时聚合酶链反应（qRT-PCR）评估活化转录因子6（ATF6）、X盒结合蛋白1（Xbp1）和真核翻译起始因子2α（eIF2α）的mRNA水平。
我们发现，暴露于高糖环境会降低RIN-m5F细胞活力，刺激胰岛素分泌，激活半胱天冬酶-3，提高Bax的表达，并下调Bcl-2。EIso可提高RIN-m5F细胞的存活率并保护其功能。与0.3G组相比，EIso处理可抑制半胱天冬酶-3的活性，并降低Bax的表达。EIso在24小时时以剂量依赖的方式显著抑制CHOP和GRP78的表达。高糖可提高RIN-m5F细胞中ATF6、Xbp1和eIF2α的mRNA水平，但EIso处理仅使eIF2α的mRNA水平显著降低。
本研究表明，高糖诱导大鼠胰岛β细胞RIN-m5F凋亡并加重β细胞功能障碍。EIso通过ERS依赖性凋亡途径保护β细胞免受高糖损伤，可能成为糖尿病的一种潜在治疗方法。
