Li Zhiqiang, Zhu Shaihong, Huang Lihua, Shang Mingming, Yu Can, Zhu Hongwei, Han Duo, Huang Hui, Yu Xiao, Li Xia
Department of Hepatopancreatobiliary Surgery, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan, China.
Center for Medical Experiments, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan, China.
Biochem Biophys Res Commun. 2018 Feb 5;496(2):294-301. doi: 10.1016/j.bbrc.2018.01.037. Epub 2018 Jan 6.
This study aimed to explore the mechanism of impaired autophagy flux induced by exendin-4 and its role on cell apoptosis in pancreatic AR42J cells. The AR42J cells were treated with various concentration of exendin-4 for several time points to assess its cytotoxicity by MTT assay. Then the AR42J cells were treated by 10pM exendin-4 for 72 h, the cell death was analyzed by flow cytometry and caspase-3 level was examined by Western blot with or without the pretreatment of z-VAD-fmk to testify whether exendin-4 induces the cell apoptosis. The protein levels of LC3B, p62 and LAMP-2 were assessed by Western blot, the mRNA level of LAMP-2 was quantified by quantitative PCR in the absence or presence of LAMP-2 over-expression plasmid and the expression and activity of CatB and CatL were tested by ELISA or activity assay methods in AR42J cells treated by exendin-4. The normal rats and the diabetes-model rats by high-fat and high-sugar diet for two month then with streptozotocin intraperitoneally were subcutaneously injected with exendin-4 for 10 weeks to test the expression of LAMP-2 mRNA and protein in the pancreas. Cells pretreated with Bafilomycin A1 were detected for LC3B and p62 expressions by Western blot. Cells pretreated by 3-MA were used to assess whether 3-MA can protect from exendin-4 cytotoxicity. We found that exendin-4 can decrease the AR42J cell viability as well as increase the cell death and cleaved caspase-3 level, which all can be inhibited by z-VAD-fmk. Exendin-4 can downregulate the expression of LAMP-2 and then impair the autophagy flux to induce the accumulation of LC3B-II and p62, but cannot change the expression and activity of CatB and CatL. Bafilomycin A1 almostly have no impact on the change of LC3B and p62 protein levels induced by exendin-4. Both 3-MA and overexpressed LAMP-2 can reduce the cytotoxicity of exendin-4. Therefore, we considered the down-regulation of LAMP-2 which can impair the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes to induce the AR42J cell apoptosis as the potential mechanism of chronic pancreatitis induced by exendin-4.
本研究旨在探讨艾塞那肽-4诱导自噬通量受损的机制及其在胰腺AR42J细胞凋亡中的作用。用不同浓度的艾塞那肽-4处理AR42J细胞若干时间点，通过MTT法评估其细胞毒性。然后用10pM艾塞那肽-4处理AR42J细胞72小时，在有无z-VAD-fmk预处理的情况下，通过流式细胞术分析细胞死亡情况，并用蛋白质印迹法检测caspase-3水平，以证实艾塞那肽-4是否诱导细胞凋亡。通过蛋白质印迹法评估LC3B、p62和LAMP-2的蛋白质水平，在有无LAMP-2过表达质粒的情况下，通过定量PCR定量LAMP-2的mRNA水平，并通过ELISA或活性测定方法检测经艾塞那肽-4处理的AR42J细胞中组织蛋白酶B（CatB）和组织蛋白酶L（CatL）的表达和活性。将正常大鼠以及通过高脂高糖饮食两个月后腹腔注射链脲佐菌素建立的糖尿病模型大鼠皮下注射艾塞那肽-4 10周，以检测胰腺中LAMP-2 mRNA和蛋白质的表达。用巴弗洛霉素A1预处理的细胞通过蛋白质印迹法检测LC3B和p62的表达。用3-甲基腺嘌呤（3-MA）预处理的细胞用于评估3-MA是否能保护细胞免受艾塞那肽-4的细胞毒性作用。我们发现，艾塞那肽-4可降低AR42J细胞活力，增加细胞死亡和裂解的caspase-3水平，而z-VAD-fmk均可抑制这些作用。艾塞那肽-4可下调LAMP-2的表达，进而损害自噬通量，诱导LC3B-II和p62积累，但不改变CatB和CatL的表达及活性。巴弗洛霉素A1对艾塞那肽-4诱导的LC3B和p62蛋白质水平变化几乎没有影响。3-MA和过表达的LAMP-2均可降低艾塞那肽-4的细胞毒性。因此，我们认为LAMP-2下调可通过抑制自噬体与溶酶体融合损害自噬通量，从而诱导AR42J细胞凋亡，这是艾塞那肽-4诱导慢性胰腺炎的潜在机制。
