Study On The Protective Effect And Mechanism Of Isoorient On Diabetic Macroangiopathy In Type 2 Diabetic Mice

Objective:The incidence rate of diabetes mellitus(DM)gets higher year by year within the scope of world,but the majority are type 2 diabetes(T2DM).Diabetic macroangiopathy is one of the most common complications in diabetes mellitus,which is the main cause of death and disability in diabetes.The exact etiology and mechanism of diabetic macroangiopathy is not clear yet presently.It is considered that the AGEs-RAGE-NF-κB pathway is closely related to the activation of the chronic inflammatory pathways,which is a key pathogenesis of diabetic macroangiopathy.The rapeutic methods of diabetic macroangiopathy included blood glucose levels,lipids and blood pressure.There are no effective evaluation methods to meet the needs of the patientsyet.Chinese medicine plays a important role and significance in the treatment of diabetes.With in-depth study,we found that the main monomer of caluabba Isoorientin(ISO)has a great value in the treatment of diabetes complications,but the mechanism of action still unclear.Therefore,based on inflammation and AGEs/RAGE/NF-k B-mediated pathways,the present study constructed the model of type 2 diabetes mellitus in mice(C57BL/6J)successfully,we tried to explore the molecular mechanism of ISO in treating diabetic macroangiopathy,which may provides a theoretical basis for Physicians in Research and Treatment about Diabetes.Methods:To build the model of T2 DM macroangiopathy in mice,we randomized the 50 male C57BL/6J mice into two groups.One is normal diet(ND)group,9 in this group,which we fed with normal diet for 4 weeks.The other one is molding group wich fed with high-fat diet(HFD)for 4 weeks.There is no limit for the usage of water.Type 2 diabetes mellitus mouse model was established by injecting 30 mg.kg-1 streptozotocin(STZ)once a week for 5 weeks.We monitered the body weight and the fasting blood-glucose level every week.If more than two blood glucose levels above 16.7mmol/L were defined as successful T2 DM model.T2 DM model were continued feeding with high fat diet for 8weeks which were defined as successful T2 DM macroangiopathy model.We randomized the T2 DM macroangiopathy model into two groups: model group and ISO-treated group,then the ISO-treated group was divided into three subgroup according to the dose of ISO:low-dose group(10mg.kg-1),middle-dose group(20mg.kg-1)and high-dose group(40mg.kg-1).There 10 mice were included in the model group,9 in each ISO treatment group.While the normal group rats and the model control group rats were injected with equal amount of 0.5% carboxymethyl cellulose.The total intervening time were 8 weeks.We detected the blood glucose levels,serum lipids such as TC,TG,HDL-C,LDL-C and so on.Aorta pathology morphology were observed by 1ight microscopy.RAGEs and VCAM-1 were measured by immuno histo chemical method.AGEs was measured by ELISA.RTQ-PCR was to detect the m RNA expression of RAGE、IL-6、NF-κB p65、TNF-α、VCAM-1.Results:1.The general condition and mental state of the ISO-treated group were significantly improved compared with the model group.After 4 and 8 weeks of treatment,compared to the model group,the interventional group of ISO were improved in body weight(P<0.01),and there were no significant difference between the groups(P>0.05).2.As to glucose metabolism,compared to the ND group,the T2 DM model group were increased in FBG(P<0.01);while compared to the T2 DM model group,the middle and low interventional group were reduced in FBG(P<0.01).3.As to blood lipid,compared to the ND group,the T2 DM group were significantly increased in TC,TG,LDL-C(P<0.01)and decreased significantly in HDL-C(P<0.01);compared to the T2 DM model group,the interventional group of ISO were significantly decreased in TC,TG,LDL-C(P<0.01)and increased significantly in HDL-C(P<0.01),there were no significant difference among the interventional group in TC,LDL-C,HDL-C(P>0.01),TG decreased significantly with the increasing concentration of ISO(10-40 u M)(P<0.05).4.As to the morphology of blood vessels,there were no pathological changes in normal group,the inner wall of vascular lumen was smooth,continuous integrity,no fracture,and the smooth muscle cells were arranged orderly and regular.While the T2 DM model group had showed that the wall of blood vessels were swelling,inner membrane became coarse,the vascular smooth muscle were broken,vascular smooth muscle cells were random and disorganized and some were by fibrous tissue.The three interventional groups of ISO had showed significant effect on delaying pathology changes of blood vessels,this phenomenon was most obvious in high-dose group of ISO.5.As to the level of AGEs,compared to the ND group,the T2 DM model group were significantly increased(P<0.01),while the interventional group were reduced significantly(P<0.01).6.As to RAGE 、 VCAM-1 protein expression : immunohis to chemical resu Its showed:compared to the ND group,RAGE、VCAM-1 increased significantly in the T2 DM model group,which were presented brown granule sediments,deeper color,and higher level of MOD value(P<0.01).Compared to the T2 DM model group,RAGE 、VCAM-1 in interventional reduced significantly with a lighter color and lower MOD values(P<0.01).And its effect is concentration dependent.7.The result of RT-q PCR showed:Compared to normal group,RAGE、VCAM-1、NF-κB P65 、TNF-α、IL-6 m RNA expression in the T2 DM group were significantly increased(P<0.01).Compared to the T2 DM model group,the interventional groups of ISO reduced significantly(P<0.01).Conclusion:ISO has a significant protective effect on diabetic macroangiopathy mice,through inhibiting the expression of AGEs/RAGE/NF-κB pathway and reducing the inflammatory reaction.