Study On The Mechanism Of Polysaccharide From Portulaca Oleracea L.Regulating Na~+ Channel In Islet ? Cells

Insulin is the only hypoglycemic hormone,and insulin resistance or impaired insulin secretion leads to the development of type 2 diabetes.While the intracellular network for regulation of insulin secretion is complex and multifactorial,such as hormones,nonglouse nutrients and ion channels.Increased extracellular glucose,and glucose enters the cell through the GLUT.Afterwards,the glucose is catabolized via glycolysis,tricarboxylic acid cycle and oxidative phosphorylation,the ATP/ADP ratio increases,while the ATP-sensitive potassium ion(K_ATP)Channel closure,membrane depolarization,T-type Ca²⁺channels and voltage-gated Na⁺channels?VGSCs?open,causing rapid depolarization of membranes,activation of L-type Ca²⁺channels,and insulin release.VGSC have not been studied as exhaustively as other channels in?cells.However,it is well known that modulators of K_ATPTP channels and Ca²⁺channels can influence insulin secretion,and several reports suggesting novel roles of Na⁺channels in?cells have appeared.Polysaccharide,which is a major active component isolated from Portulaca oleracea L.?POP?,has also received extensive study.Previous researches on the mechanism of anti-diabetic effect of POP generally focused on antioxidation and signal transduction.However,the study on the regulation of insulin secretion by POP by regulating the activity of VGSC on INS-1 cells remains unknown.In this work,the molecular mechanism of POP based on VGSC to regulate insulin secretion was investigated in INS-1 cells using whole-cell patch-clamp techniques,MTT,fluorescence imaging experiment and enzyme-linked immunosorbent assay?ELISA?and molecular biological method.1.The protective effect of POP on INS-1 cells was studied by MTT assay.The results showed that INS-1 cells could increase the survival rate when the concentration of POP was 0.01-0.5 mg/mL.And when the concentration of POP was more than 0.5 mg/mL,POP inhibited the survival of INS-1 cells.2.The effect of POP on intracellular Ca²⁺concentration,mitochondrial membrane potential and cell membrane potential were studied by fluorescence imaging.The results showed that POP increased Ca²⁺concentration,improved mitochondrial membrane potential and depolarized cell membrane.The results showed that POP promoted the synthesis of intracellular ATP by ATP assay kit.The effect of POP on insulin synthesis and secretion was investigated by enzyme linked immunosorbent assay.The results showed that POP promoted the synthesis and secretion of insulin.3.The impact of POP on VGSC is studied using whole-cell patch clamp technology under the voltage clamp conditions.The results showed that POP could partially inhibit voltage-gated sodium channel current(I_Na)in a concentration-dependent manner,inhibited the steady-state activation of I_Naa peak current,and shifted the I_Naa steady-state inactivation curve to a more negative potential.Compared with the control,the recovery time of I_Naa was prolonged and the attenuation of I_Naa was increased.4.The effect of POP on Nav1.3 and Nav1.7 was studied by Western blotting.The results showed that POP increased the expression of Nav1.3 and decreased the expression of Nav1.7.In conclusion,these results suggest that POP may protect INS-1 cells by partially inhibiting VGSC activity by promoting the exchange of information between mitochondria and membrane potential,thereby increasing the concentration of Ca²⁺and promoting the synthesis of ATP.Furthermore,the insulin synthesis and secretion was promoted by up-regulating Nav1.3 levels and down-regulating Nav1.7 levels in INS-1 cells.