| Squalene, belonging to the natural triterpene family, was reported that exerts protective effect on anti-cancer, resistance to "three hyper"(hypercholesterolemia, hypertension, hyperglycemia) and the regulation of immune due to its strong anti-oxidation function. It is called "the magic oil used in everything" in foreign country. The research aims at the protection mechanism of squalene on the high-glucose and H2O2models in pancreatic RIN-m5Fcell lines. The results were as follows:1. According to the data acquired from the protective effect of squalene to pancreatic β-cells, different concentrations of squalene intervene islet RIN-m5F cells grown with high glucose (25mmol/L) have shown that, compared with the control group, the apoptosis rate in high sugar model group increased by34.44%(P<0.01); Compared with model group, the average apoptosis rate in the concentration of25μg/ml,50μg/ml,75μg/ml of squalene treatment group were reduced by22.99%(P<0.01),6.67%(P<0.05) and6.90%, respectively. Squalene’anti-apoptotic effect is not remarkable, only in the concentration of25μg/ml are statistically significant difference. In group2, compared with the control group (DMSO), the apoptosis rate in high glucose model group raise72.55%(P<0.01), and squalene treatment group (50μg/ml) lowered7.98%. Compared with model group, squalene processing with high sugar concentration group were down by15.00%in apoptosis rate detecetion (P<0.05). The real-time fluorescent quantitative PCR analysis of apoptosis related genes, Bcl-2, Bax, Caspase-3, ATF-3, P38and JNK-1detected about the mRNA expression levels. Results showed that squalene significantly contributed to down-regulate pro-apoptotic gene ATF-3, JNK-1, P38(P<0.01). The protective effect of squalene to pancreatic β-cells might be related to ER stress-mediated apoptosis pathway to some extent.2. The result of insulinotropic effect of squalene on high-glucose induced islet β-cell found that,(1) In group1, compared with the control group, the insulin content in high sugar model reduced by11.53%(P<0.01), compared with model group, the average content of insulin in the squalene treatment groups increased by34.78%,48.14%and59.94%(P<0.01). With the enlargement of squalene concentration, the content of insulin increases in turn significancely.(2) In group2, compared with the control group (DMSO), the insulin levels in high glucose model group fell by7.76%(P<0.01), and squalene treatment group (50μg/ml) amplified21.78%(P<0.01). Compared with model group, squalene processing with high sugar concentration group increased by31.35%(P<0.01) in insulin detecetion. The real-time fluorescent quantitative PCR analysis of insulin secretion related genes, Sirtl, Ucp2and PLC-y deteced the mRNA expression levels. Results showed that squalene would significantly up-regulate Sirtl, down-regulate the relative quantitative expression of Ucp2(P <0.01). From the molecular level of squalene influence the amount of gene expression of insulin secretion related genes. Thus, squalene might play an crucial role in promoting islet (3RIN-m5F cell lines of insulin secretion.3. The induced model was explored by H2O2exposed with different concentrations and incubate for12h,18h,24h after treatment, respectively. The production of ROS were detected and the morphological struction after incubaton or hochst stain were observed to investigate the optimal conditions of oxidative stress model upon H2O2induced islet P-cell. The oxidative stress model show perfect situation at the concentration of250uMol/L after incubation of18h. Squalene intervene on the β3RIN-m5F cell lines induced by H2O2oxidative stress model analysis results show that compared with the control group, the average content of active oxygen increased by52.31%H2O2model group (P<0.01) insulin content decresed by22.06%(P<0.01), and apoptosis rate increased by318.66%(P <0.01); Compared with model group, the content of ROS level of25μg/ml of squalene treatment groups were reduced by21.40%(P<0.01), the insulin content increased by39.98%(P<0.01), the apoptosis rate were decreased by17.94%(P<0.05). The real-time fluorescent quantitative PCR analysis of insulin secretion related genes showed that squalene treatment group up-regulate Sirtl, PLC-y, down-regulate Ucp2, which activate insulin signal pathway and enhance secretion. Squalene could clear ROS which were induced in islet beta cells, but its anti-apoptotic effect is not obvious, this might be that non-Free-Radical caused apoptosis.Glucotoxicity and oxidative stress are both responsible for apoptosis and the impairment of glucose-stimulated insulin secretion in pancreatic β-cells. Squalene did exert the following effects on islet Rin-m5F cell lines in a dose-dependent manner:(a) it mounted the content of insulin secretion, and (b) it protected pancreatic β-cell against the damage caused by high-glucose and excess ROS.(c) it decreased intracellular ROS level, and (d) it prevented H2O2-induced oxidative injury. |
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