Study On The Hypoglycemic Mechanism Of Mulberry Leaf Water Extract On T2DM Mic

The pathogenesis of diabetes are complex and diverse,and the recent data revealed that the concentration of inflammatory cytokines like IL-1β,IL-6 and TNF-α is associated with type 2 diabetes(T2DM)and insulin resistance(IR).It has been reported that type2 diabetes is connected with autoimmune system,and the levels of TLR2 and TLR4 of the patients of type 2 diabetes are much higher than that of the controls,indicating that the signaling pathways of TLR2 and TLR4 are activated and that type 2 diabetes,the chronic inflammatory autoimmune disease,closely relates with TLRs.Mulberry leaves are planted in Asia,Africa,the Americas and Oceania and other parts of the world,especially in Asia.Mulberry leaves are used to treat wind-heat common cold and governance hyperactivity dry cough,possessing the effects of moistening lung,clearing liver,and improving vision.In the late of sixteenth century,Comprehensive Outline of the Materia Medica(Bencao Gangmu)by Li Shizhen says that the mulberry tea is able to treat diabetes.Correspondingly,the modern pharmacological studies show that the mulberry can lower the blood glucose,the main active ingredients of which are total polysaccharides and deoxynojirimycin(DNJ).The mechanisms focus on the inhibition of α-glucosidase and the protecton of β-cell from apoptosis.The previous experiment revealed that mulberry leaves could down-regulate the expression of TLR2 and TLR4 in the pancreas of KKAy mice.This research will further discuss the anti-diabetic mechanism from TLRs and insulin signaling pathway,which hopes to provide a novel idea and strategy for developing the mulberry leaves into the supplementary drugs for anti-diabetes.The experiment was divided into two sections.For the first section,it aimed to investigate the effects of water extracts of mulberry leaves on hyperglycemia and inflammatory cytokines in T2DM mice,being fed with high-fat and high-sugar diet for 10 weeks and low-dose streptozotocin(STZ)for three times.The successful T2D-model mice were divided into five groups,namely,the model group,the metformin group,the water extract of Mulberry leaves,namely low-dosage(2g/kg),middle dosage(4g/kg)and high-dosage(8g/kg),and the normal mice were divided into the normal control group.The medicinal interference lasted for 10 weeks,at the same time,the control group and model group were administered the drinking water of animals.The body weight and the blood glucose were measured each week during the administration.The Oral Glucose Tolerance Test(OGTT)and the 24-hour intake of diet and water were measured in the week of 6th,8th and 10th.After the 10-week administration,the insulin,TNF-α,IL-1β and IL-6 in the serum were tested by ELISA,and total cholesterol and triglyceride by Microplate Reader.The results showed that the mulberry leaves could significantly decrease the fasting blood glucose(p<0.05)compared with the models.There was no obvious change in the body weight in the six groups,though it appeared the increasing trend in the metformin group,the group of Mulberry leaves.The 24-hour intake of water in the group of Mulberry leaves was much lower than that of the models(p<0.01).The area under curve(AUC)of OGTT of Mulberry leaves was critically smaller(p<0.05,or p<0.01)than that of the models in the week of 6th,8th and 10th.The serum concentrations of insulin in the Mulberry leaves were much higher(p<0.01)than that of the models.The serum concentrations of TNF-α and triglyceride in the Mulberry leaves were much less(p<0.01)than that of the models.These results can be induced that the Mulberry leaves could significantly improve the impaired glucose tolerance,lower the levels of inflammatory cytokines,increase the secretion of insulin and ameliorate the diabetes,and that it also appears good hypolipidemic effect.For the second section,it explored the mechanism of hypoglycemia of mulberry leaves.Firstly,real-time-PCR was applied to investigate the expression of TLR1-9,InsR,IRS1 and GLUT4 mRNA in the liver,muscle and fat.It is hoped to inspect the effect of gene levels of TLRs and insulin pathways on insulin sensitive tissues upon the water extracts of mulberry leaves.The results showed that significant decrease in TLR7,TLR9 mRNA in the liver and TLR2 of the group of Mulberry leaves was observed compared with the models in the tissue of muscle(p<0.01).The Mulberry leaves could notably increase the expression of InsR and IRS1 mRNA(p<0.05,or p<0.01),which was prominently higher than that of the models.Western blot was applied to investigate protein expressions of IKK,TRAF6,NF-κB p65,MyD88 and TLRs in the liver and muscle,and protein expression of PI3K p85,InsR,phospho IRS1,IRS1,and GLUT4 in the fat tissue.It is hoped to inspect the effect of protein levels of TLRs and insulin pathways on insulin sensitive tissues upon the water extracts of mulberry leaves.Mulberry leaves could dramatically down-regulate the protein expression of TLR2,TRAF6,NF-κB p65 and MyD88(p<0.01)in the muscle of T2DM mice.Compared with models,the Mulberry leaves could significantly increase the protein expression of InsR and IRS1(p<0.05,or p<0.01).The protein expression of phospho IRS 1 in the fat tissue of T2DM in the mulberry leaves was much lower(p<0.01)than that of the models.The mulberry leaves possibly play the role in improving diabetes through MyD88-dependent signaling pathways,inhibiting the expression of TLR2,MyD88,TRAF6 and NF-κB in the muscle of T2DM mice,increasing the expression of IRS1 and InsR,and decreasing the expression of phospho IRS1 to improve IR and inflammation.To sum up,Mulberry leaves could dramatically decrease the concentration of TNF-α and IL-6,the expression of TLR2,MyD88,TRAF6,NF-κB,and phospho IRS1,and increase the expression of IRS l and InsR,suggesting that Mulberry leaves could play the role in inhibiting the proinflammatory state via TLRs and improve the insulin signaling pathway to treat diabetes.