Protective Effects Of GABA On RIN-m5f-cells Is And Its Functional Mechanism

Objective: Oxidative stress (OS) is considered as one of major risk factors inoccurrence and development of diabetes. GABA is a main inhibitory neurotransmitter in theCNS. Study confirmed that pancreatic β cells secrete a large amount of GABA, whichsignificantly maintains the redox homeostasis and insulin secretion function of pancreatic βcells, but the functional mechanism is not clear. In this study, oxidative injury was inducedby high glucose (G) and H2O2on RIN-m5f cells, in order to investigate the anti-oxidativeeffect of GABA on islet β cells and its functional mechanisms.Methods: RIN-m5f cells were exposed to11mM,22mM,44mM G for48h, or to10μM,50μM,100μM H2O2for1hour to create oxidative injury model. GABA interventionexperiment contained following groups: control group, injury group (G/H2O2), GABA group(G/H2O2+GABA), lipoic acid group (G/H2O2+100μM LA). DCFH-DA was used to detectROS level; cellular redox status and insulin secretion were measured by related kits; cellviability was determined by MTT assay; mitochondrial membrane potential was detected byflow cytometry; relative genes levels were analyzed by RT-PCR; Western blot assay wereutilized to examine the protein expression of GSK-3β and Nrf2, and phosphorylation level ofp-GSK-3β (Ser9) and p-GSK-3β (Tyr216).Results: As the concentration of G and H2O2increased, intracellular ROS and MDAlevels were significantly rised(P<0.05), GAD1mRNA level was significantly reduced(P<0.01), and cells showed different degree of oxidative injury(P<0.05). The treatment of100μM and200μM GABA significantly decreased intracellular ROS and MDA levels(P<0.05), improved T-AOC, GSH-Px, CAT, SOD activity (P<0.05), up-regulatedanti-oxidative and anti-apoptosis genes mRNA levels, such as Nrf2, NQO1, HO-1, Bcl-2(P<0.05); increased mitochondrial membrane potential and cell viability (P<0.05).Furthermore, GABA significantly increased insulin secretion and its relative genes (INS-2,Pdx-1, MafA, GK, GLUT2, Gabra2) mRNA levels (P<0.05); Western blot analysis showed,in oxidative injured (44mM G and100μM H2O2) cells, GABA significantly inhibitedGSK-3β protein leve (P<0.05), increased p-GSK-3β (Ser9) and Nrf2levels (P<0.05), but hadno influence on p-GSK-3β (Tyr216)(P>0.05).Conclusions: This study found that GABA significantly improved anti-oxidative ability,cells viability and insulin secretion of oxidative injured (G and H2O2) RIN-m5f cell, exertedanti-apoptosis function. GABA regulate intracellular GSK-3β/Nrf2pathway relative genesmRNA levels, significantly increased nuclear/cytoplasmic ratio of Nrf2, improved cellanti-oxidative ability, and its functional mechamism is supposed to connected withup-regulating p-GSK-3β (Ser9) level and down-regulating GSK-3β protein level. And theanti-oxidative regulating function of GABA showed concentration-dependent effect, whichmeans as oxidative injury increases, the requirement of GABA increases.