The Role Of MyD88-independent Pathway In The Production Of Inflammatory Cytokines Induced By Lycium Barbarum Polysaccharide In T2DM Mice

Objective:Chronic inflammation can cause damage of islet?cell,decrease of insulin secretion,interference of insulin signaling,and insulin resistance.It plays an important role in development of type 2 diabetes?T2DM?.Lycium barbarum polysaccharide?LBP?is one kind of main active ingredients of Lycium barbarum.Previous studies have shown that LBP can downgrade the level of blood glucose and reduce the level of inflammatory cytokins in patients with T2DM.However,its mechanism is still unclear.This study investigated the role of MyD88-independent pathway?TRIF/TRAM pathway?in the production of inflammatory cytokine induced with LBP in T2DM,and explored the anti-inflammatory and hypoglycemic mechanism of LBP.Methods:LBP was extracted with boiled water and alcohol and studied its characteristics.T2DM model was established with high-fat diet and intraperitoneal injection of streptozotocin at low dose?80 mg/kg?on MyD88^-/-mice?n=40?.Model mice were randomly divided into model group,metformin group?40 mg/kg?and low,medium and high LBP groups?20,40,80 mg/kg?and administrated for 3-month.Another 8 MyD88^-/-mice were used as control.Body weight and blood glucose were recorded during this time.After treatment,serum levels of interleukin-1??IL-1??,IL-6,IL-8,IL-10,tumor necrosis factor??TNF-??and transforming growth factor?1?TGF-?1?in MyD88^-/-mice were detected by ELISA.RT-PCR and Western blot were used to determine gene and protein expression of key molecules?TRAM,TRIF,RIP1 and TRAF6?in MyD88-independent pathway in peritoneal macrophages of MyD88^-/-mice.Results:1.Sugar content in LBP was determined by phenol-sulfuric acid method.The content of polysaccharide is 77.38%.Molecular weight of polysaccharides was determined by high performance liquid chromatography with gel chromatography.Contents of polysaccharides with molecular weight higher than 10kDa reached to 93.06%.Qualitative analyses of LBP by Fourier transform infrared spectroscopy and ultraviolet spectroscopy showed that the sample had typical polysaccharide structures and absorption of protein at 280 nm.Gas chromatography-mass spectrometry analysis showed that LBP was composed by arabinose0.31%,xylose 0.16%,mannose 41.68%,rhamnose 3.46%,glucose 53.83%and galactose0.56%.2.After treatment with LBP for 3 month,MyD88^-/-T2DM mice showed the same levels of body weight and blood glucose as the beginning.ELISA results showed that the levels of IL-8,TGF-?1 and IL-10 in the model group were significantly higher than those in control group.The levels of serum IL-1?and TGF-?1 in low and medium groups were significantly lower than those in the model and control groups and IL-10 of low LBP group was higher than control.RT-PCR results found that gene expressions of TRAM,TRIF,TRAF6 and RIP1expressed higher levels with metformin treatment and lower levels with various concentration of LBP.Western blot results showed that no statistically significant difference was found in protein expression between each group,except the expressions of TRIF and RIP1 in the high LBP group.Conclusion:It can't proved that LBP has effect on the production of inflammatory factors and the level of blood glucose via MyD88-independent pathway in the TLR/NF-?B pathway.