The Protective Mechanism Of Morin On Rat Pancreatic β-cells

Polyphenols widely distributed in fruits,vegetables and other foods have attracted extensive attention due to their multiple biological activities such as anti-inflammatory and antioxidant.The active precise effects and intrinsic mechanism of polyphenols have become study focuses.Islet is the important tissue to maintain blood glucose balance,so protecting pancreatic cells from oxidative stress and inflammatory damages posseses distinguished significance to our body health.As a dietary polyphenol,morin is abundant in dietary vegetables and fruits.Numerous studies found morin have strong antioxidant and anti-inflammatory activity.Therefore,the purpose of this study is to clarify the protective effect and the intrinsic mechanism of morin on pancreatic β cells,and to show safe and effective dietary polyphenols for improving blood glucose balance in the body.This study used saturated fatty acid(FFA),streptozocin(STZ)and lipopolysaccharide(LPS)treatment to mimic the environment of stress,to determine the protective mechanism of morin on pancreatic β cells,and analyze the main antioxidant and anti-inflammatory regulation genes in transcription level,protein expression and enzyme activity.The content mainly has the following three parts:(1)DPPH assay showed the morin at a concentration of 50 μM scavenged 50%DPPH radical.MTT results showed that morin below 500 μM had no toxic effect on the viability of RINm5 F cells.FFA was added to RINm5 F cells to mimic a cellular environment with high level free fatty acids to carry out further study on the protective effects of morin on the pancreatic cells.MTT assay found the IC50 of FFA was 0.5 mM.Pretreatment with 400 μM Morin can effectively improve cell survival rate and protect cells from apoptosis.PI staining further proved that morin can inhibit FFA induced apoptosis via flow cytometric analysis.The administration of morin increased the activity of FFA-induced intracellular antioxidant enzymes GPx and SOD in high fat-induced cells.(2)The antioxidant activity of morin was further examined in the pancreatic cells treated with STZ.MTT assay was used to determine the optimal concentration of morin on STZ treated cells.Effects of intracellular morin on STZ-induced ROS levelswere determined by confocal laser microscopy and flow cytometry.Use real-time quantitative PCR,protein experession and CAT detection kit to measure the effects of morin on CAT transcription,protein expression and enzyme activity.SiRNA were used to verify the internal mechanism of morin on pancreatic β cells protection.The results showed that 25 μM morin markedly improved the viability of RINm5 F cells exposured to 6 mM STZ.Morin effectively protect RINm5 F through suppressing ROS production and upregulating CAT expression.(3)The protective effect of morin against LPS challenge was investigated.The anti-inflammatory ability of morin was demonstrated through MTT assay and RT-PCR.MTT assay demonstrated the IC50 of LPS was 1.75 mg/mL,while 100 μM morin can effectively improve cell activity and protect cells from death.The gene expression analysis showed LPS treatment significantly increased the expressions of TLR4,NLRP3,ASC and TNF-α,while morin can markedly suppress gene expression associated with NLRP3 pathway and prevent pancreatic β cells against LPS induced damages.In summary,this study demonstrated morin played protective effects against the pancreatic β-cell damages and elucidated its mechanism: morin improved the antioxidant enzyme activity in FFA treated pancreatic β-cells.Morin exerted high antioxidant through increasing the CAT expression in STZ treated oxidative stress,and showed anti-inflammatory activity in LPS challenge by suppressing NLRP3 pathway associated gene expression.Our findings provided the necessary theoretical evidence for the special population to protect their health through dietary adjustment.