Study On Mechanism Of Portulaca Oleracea L.Polysaccharides Improving Pancreaticβ-cell Insulin Resistance

Recently,increased attention has been paid to study on the resources of natural medicine for their wide sources, few side effects, cheap prices and relatively stable efficiency. Portulaca oleracea L. polysaccharide (POP) was an important active components extracted from the traditional Chinese herb Portulaca oleracea L.POP has been reported to be associated with beneficial effects such as inhibition activity of preadipocytes proliferation, antidiatics and antioxidant. Both domestic and international scholars have done many researches and report on the separation, purification, structure elucidation and biological activity (antioxidant, hypoglycemic lipid regulating) of POP. However, its hypoglycemic mechanism at the molecular level has not been elucidated. In this paper, the anti-diabetic mechanism of effect POP was analyzed on the following aspects based on previous researches:1. To establish the model of insulin resistance:taking pancreatic P cells (HIT-T15) as the subject investigated and different concentrations of palmitic acid or high-glucose hpalmitic acid as the inducer to carry out the detection of cell survivability by inducing respectively1h,4h,8h,24h,48h,72h. Finally, the results shows that the optimum inducing condition was as follows:the concentrations of palmitic acid and glucose were respectively0.5mol/ml and33.3mol/ml, and the processing time was24h.2. To detect the effect of POP on the viability of pancreatic β cell:The HIT-T15cells (5×104/ml) were seeded in96-well plates. After24hs’culture, they were divided into control group, model group of the insulin resistance, POP treatment group, and were cultivated in RPMI1640medium containing POP (0.01,0.1,1.0mg/ml). At different time points (2,4,8,24,48,72h), the methods of neutral tetrazolium blue (MTT)colorimetry, red uptake (NRU) and lactate dehydrogenase (LDH) were subsequently applied to carry out the detection of cell survivability. The results showed that POP can inhibit death of the fat or high-fat high-sugar-induced pancreatic (3-cell and can promote the proliferation of pancreatic β-cell.3. To detect the effects of POP on mRNA and protein expression of insulin signal transduction pathway of relevant molecular targets. The HIT-T15cells (1×105/ml) were seeded in culture plates. And after cultivating for24hs, they were divided into six groups:A. Control group, B. hpalmitic acid nduced insulin resistanc group, C. high-glucose hpalmitic acid induced insulin resistanc group, D. POP treatment high-fat (palmitic acid) induced insulin resistanc group, E. POP treatment high-glucose hpalmitic acid induced insulin resistanc group and F:POP group. After24hs’respective treatment with Portulaca oleracea L. polysaccharide (0.1mg/ml), and extracting proteins and RNA in each group of cells by using the techniques of Western bloting and reverse transcription-polymerase chain reaction (RT-PCR) to test the expression of target molecules (mRNA) and protein of phosphatidylinositol3-kinase (PI-3K), c-jun N-terminal kinase (JNK), pancreatic duodenal homeobox factor (PDX-1), PPARr, STAT-5B gene related to the signal transduction pathway of insulin resistance from the angle of the molecular level to bring to light the molecular mechanism of POP regulating pancreatic β cell insulin resistance itself. The results showed that high-fat and high-sugar high-fat processed HIT-T15cells can inhibit the expression of target molecules of IR、IRS-2、PI-3K、AKT、GLUT-1、GLUT-2related to the signal transduction pathway of insulin resistance and the expression of PDX-1、 PPARr、STAT-5B as well, and meanwhile, can activate the signaling pathway of c-jun N-terminal kinase (JNK). When treated with Portulaca oleracea L., the expression of target molecules and protein of IR、IRS-2、PI-3K、AKT、GLUT-1、GLUT-2、PDX-1、 PPARr、STAT-5B could be obviously up-regulated and the expression of protein of c-jun N-terminal kinase (JNK) pathway down-regulated.This paper, starting from establishing the model of insulin resistanc, explores at cellular and molecular levels the regulation of POP aimed at the expression of relevant molecular target mRNA and protein related to the signaling pathway of insulin, and makes a comprehensive analysis of the protection of pancreatic β cells by POP and the reduction of the regulation mechanisms of cellular and molecular of glycemic in order to discover new action targets of POP hypoglycemic for the purpose of laying a theoretic foundation for the development of innovative drugs with independent intellectual property rights.