The Effect Of LPS On The Autophagy And Apoptosis In INS-1Rat Insulinoma Cells

Research backgrounds：Compared with nondiabetic, there are significant differencesin beta cell number and function in type2diabetes, characterized by decrease in thenumber and decline in the function. The patients with type2diabetes have asignificantly higher mortality rates in beta cells, most of cell via apoptosis pathway,however, autophagy also contribute to the loss of beta cell mass.This shows thatautophagy involved in the beta cells failure in some degree. Autophagy is aevolutionarily conserved machinery for degradation and recycling of variouscytoplasmic components such as long-lived proteins and organelles. It plays animportant role in maintain the cell homeostasis, through satisfied their own needs ofmetabolism and some organelles’ updation. In some conditions, such as lack ofnutrition, autophagy can reallocate nutrients from unnecessary processes to morepivotal ones required for survival, so autophagy is also very important for maintainingcell survival. However, autophagy is also thought to be a programmed cell deathmechanism same as apoptosis, which in some cases can lead to cell death. Theinternal environment is different between diabetics and nondiabetic, such as highsugar levels, high levels of blood lipids, high blood viscosity and low-grade chronicinflammation in diabetics. In vitro studies, autophagy can be induced bystreptozotocin, free fatty acid, high sugar etc in beta cell, however inflammatoryfactor which assumed to be closely related with diabetes occurrence and development has not been reported. This article aims to study the effects and the underlyingmechanism of the inflammatory reaction inducer lipopolysaccharide (LPS) onautophagy and apoptosis in INS-1rat insulinoma cells, so as to offer new clues to theprevention or treatment of type2diabetes.Objective: To investigate the effects of lipopolysaccharide (LPS) on autophagy andapoptosis of rat insulinoma INS-1cells and the underlying mechanisms.Methods：（1）INS-1cells at log growth phase were treated with0.1,1.0or10.0mg/LLPS for24h, then cell morphological characteristics were observed by microscope,sulforhodamine(SRB) assay was carried out to evaluate the effect of LPS on cellviability, and reactive oxygen species (ROS) were detected bydichlorofluorescein-diacetate (DCFH-DA) fluoro-probe. The autophagy markermicrotubule-associated protein1light chain3(LC3-Ⅱ) on the membrane ofautophagosomes and the apoptosis marker cleaved poly ADP ribose polymerase(PARP) were detected by Western blotting analysis.（2） The autophagy essentialgene Atg7was silenced via siRNA-mediated RNAi method, then cells were treatedwith1.0mg/L LPS for another24h. Atg7, LC3-Ⅱ and cleaved PARP were detectedby Western blotting analysis.（3）INS-1cells were treated with400μmol/LN-acetylcysteine (NAC, a scavenger of ROS). And1h later cells were treated with10.0mg/L LPS for another24h. LC3-Ⅱ and cleaved PARP were detected withWestern blotting methods. Differences between groups were calculated with analysisof variance and t test.Results （1） After treated with0.1,1or10mg/L LPS for24h, morphologicalchanges including cell shrinkage, cell size reduction and turn round were observed.Compared with the control group（0.755±0.030）, the viabilities significantlydecreased in0.1,1and10mg/L LPS groups (0.658±0.042,0.658±0.015,0.634±0.029,F=30.98, P<0.01）. Compared with the control group （0.447±0.012）, the proteinlevels of LC3-Ⅱ significantly increased in0.1,1and10mg/L LPS groups(0.992±0.030,0.949±0.020,0.982±0.013respectively, F=525.79, P<0.01）. Comparedwith the control group (0.055±0.001), the levels of cleaved PARP significantlyincreased in0.1,1and10mg/L LPS groups (0.313±0.007,0.390±0.011,0.500±0.033,F=327.093, P<0.01）. A remarkable increase of ROS was detected in INS-1cells treated with10mg/L LPS for24h.（2） Compared with control, the protein levels ofAtg7, LC3-Ⅱ and cleaved PARP significantly decreased in Atg7siRNA transfectiongroup (t=156.069,123.154,103.246, P<0.01).（3） Compared with LPS10.0mg/Lgroup，the protein level of LC3-Ⅱ and cleaved PARP significantly decreased in LPS10.0mg/L+NAC group（t=66.37,26.84, P<0.01）.Conclusion LPS induces autophagy and apoptosis in INS-1cells, and autophagypromotes apoptosis herein. Reactive oxygen species are involved in autophagy andapoptosis induced by LPS in INS-1cells.