The Protective Effects And Mechanisms Of Tongluo Recipe On The Endothelial Cell Of The Myocardium And Aorta In Diabetic And Obese Rats

Background and objectives:Nowadays obesity and diabetes have become the main diseases that affect human health,and diabetic complications have been the major cause leading to high morbility and mortality of cardiovascular diseases.Dysfunction of endothelial cells attributed to chronic hyperglycemia related oxidative stress is a major factor leading to diabetic macroanglopathy and microanglopathy,and obesity related chronic mild inflammation which leads to insulin resistance is the initial factors contributing to diabetic vascular complications.Osteopontin(OPN) is a multifunctional extracelluar matrix protein and also a kind of cell secretory protein,proinflammation factor and adhesion molecular.It plays a great role in mediating cell aggregation,adhesion, migration,mineralization and reconstruction of bone,immunoregulation,signal transduction and immunity to infectious diseases.Injuries,reparation and remodeling of blood vessles all could stimulate expression of OPN.Hyperglycemia could stimulate expression of OPN in smooth muscle cells.Expression of OPN is up-regulated in smooth muscle cells from diabetic rats and diabetic patients' tunica media of arteries.Oxidative stress induced by hyperglycemia could activate PKC,increase expression of OPN, down-regnlate expression of iNOS,decrease NO,thus affect endothelial functions. Recently OPN has been found to be a pre-inflammation factor.It is highly expressed in fat tissues,could mediate infiltration of macrophage into fat tissue,and is associated with the development of insulin resistance.OPN also has an important role in diabetic anglopathy[4].This study explored the possible mechanisms of Tongluo recipe's protective effects on endothelial functions by observing the pathological changes of myocardium and aorta of obese and experimental diabetic rats,measuring plasma OPN, CRP of obese and experimental diabetic rats and mRNA of iNOS and expression of OPN protein in myocardium and aorta of obese and experimental diabetic rats,so that provide new target and theoretical evidence of diabetic complications.Materials and methods:We studied the type 1 diabetic rats,obese and experimental type 2 diabetic rats.A number of method including HE dying,electronmicroscope, enzyme-labeled immunosorbent assay,immunohistochemistry,westernblot and RT-PCR were used.The lesions caused by oxidative stress and lipotoxicity,changes of adhere factors and the pre-inflammation factor OPN and the protective effects of TLR on myocardial cells and endothelia of aorta.We also compared the effects of TLR with antioxidants including a-lipoic acid,atorvastatin to further determine the protective effects of TLR and its possible mechanisms.Results:1) The effects of TLR on oxidative stress of obese and diabetic rats:â‘ Plasma oxidative stress parameters:SOD,GSH,T-AOC,CAT in type 1 diabetic rats significantly reduced compared with normal controls and MDA significantly increased. SOD,GSH,T-AOC,CAT in type 1 diabetes+TLR and type 1DM+ALA increased compared with normal controls,but there were no significant difference.MDA in type 2 diabetic rats was higher than normal controls and SOD significantly reduced.SOD in type 2 diabetes +ALA,type 2 diabetes+TLR were lower than normal controls,but the difference has no significance.MDA in type 2 diabetes +ALA,type 2 diabetes+TLR was lower than DM,but higher than NC.The difference had no significance.â‘¡Serum CRP: CRP of obese rats was significantly higher than that of normal controls.CRP of obese+TLR was lower than that of obese rats and the difference was significant.There was no significant difference among obese+atorvastatin,obese+ALA and obese+TLR.CRP of type 2 diabetic rats was significantly higher than that of normal controls.There was no significant difference among type 2 DM+atorvastatin,type 2 DM +ALA and type 2 DM +TLR.CRP of the three groups was lower than that of type 2 diabetic rats;â‘¢Changes of blood lipids:TG of obese rats was higher than that of normal controls.TG of obese+TLR increased at the fourth and eighth week,but remained lower than normal controls.At the 12^th week it decreased significantly.TG of type 2 diabetic rats was higher than that of normal controls.TG of type 2 DM+TLR reduced from the fourth and it was lower than diabetic rats by the end of the 12^th week.The TG of type 2 DM remained higher than normal controls.The TG of type DM+TLR was lower than that of type 2DM+atorvastatin and it reduced significantly compared with that of type 2 DM+AL.TC:At the 4^th week TC of obese rats was higher than that of normal controls.TC of obese+TLR also increased at the fourth and eighth week,but remained lower than obese rats.At the 12^th week it decreased.TC of type 2 diabetic rats was significantly higher than that of normal controls. The peak appeared at the 4^th week and it began to reduce at the 8^th and 12^th week.TC of type 2 DM+TLR reduced from the fourth and it was lower than diabetic rats by the end of the 12^th week.However the TC of type 2 DM remained higher than normal controls.The TC of type DM+atorvastatin reduced more than that of type 2DM+TLR,and the TC of type 2 DM+ALA did not reduce so much as that of type 2 DM+atorvastatin and type 2DM+TLR.HDL:HDL of obese rats was lower than that of normal controls.HDL of obese+TLR was higher than that of obese rats.HDL of type 2 diabetic rats was significantly lower than that of normal controls.TC of type 2 DM+TLR increased significantly compared with diabetic rats,but it was lower than diabetic rats.However the HDL of type 2 DM remained higher than normal controls.There was no significant difference between type 2 DM+TLR and type 2 DM+atorvastatin but the difference between type 2 DM+TLR and type 2 DM+ALA had statistical significance.LDL:LDL of obese rats was higher than that of normal controls.LDL of obese+TLR was higher than that of normal controls but lower than that of obese rats.LDL of type 2 diabetic rats was significantly higher than that of normal controls.The LDL of type 2 DM+TLR was significantly lower than that of type 2 DM,but it was higher than normal controls Reduction of LDL in type 2 DM+TLR was less than that of type 2 DM+atorvastatin but more than type 2 DM+ALA.â‘£iNOS:Serum iNOS of obese rats was lower than that of normal controls.The iNOS of type 2 DM+TLR was significantly higher than that of type 2 DM,but it was still lower than normal controls.There was no significant difference in iNOS between type 2 DM+TLR and type 2 DM+atorvastatin but the difference between type 2 DM+TLR and type 2 DM+ALA had statistical significance;⑤24-hour urine proteins:There was no significant difference on the 24-hour urine proteins between obese rats and normal controls and there also had no statistical difference among obese+TLR, obese+ALA and obese+atorvastatin.24-hour urine proteins of type 2 diabetic rats were significantly higher than those of normal controls.The peak appeared at the 4^th week and began to reduce at the 8^th and 12^th week.24-hour urine proteins of type 2 DM+TLR began to reduce from the 4^th week and were significantly lower than those of type 2 diabetic rats at the 8^th and 12^th week,but remained higher than those of normal controls.The reduction of type 2 DM+atorvastatin was more than that of type 2 DM +TLR,and reduction of type 2 DM +TLR was more than that of type 2 DM+ALA.2)histological changes of myocardia and aorta of diabetic rats:â‘ HE dying:normal myocardia had the morphology of short cylinder.The nucleus was oval-shape and in the middle of the cytoplasma.Myofibrilla could be seen in the cytoplasma.The outline of cell was clear.Mitochondria of diabetic rats were swollen and some of the cristae were broken.Vacuolization could be seen in parts of the mitochondria.The lesions of type 2 DM+TLR were significantly milder than those of type 2 DM.There was no significant difference between type 2 DM+TLR and type 2 DM+atorvastatin.The normal endothelia of intima of aorta pectoralis and smooth cells of tunica media were in regular array and complete.There was no significant difference between obese rats and normal controls.At the 12^th week Endothelial cells of diabetic rats were in irregular array and the smooth cells of tunica media proliferated.The lesions of type 2 DM+TLR were significantly milder than those of type 2 DM.There was no significant difference between type 2 DM+ALA and type 2 DM+atorvastatin.â‘¡electronmicroscope:In NC,tochondria of myocardial cells were complete.Crista mitochondriales were in parallel array.Myofilament was seen clearly. Myocomma was in good order and intercalary disc was normal.The structures and numbers of mitochondria were normal.At the 12^th week myofilament of myocardial cells of type 2 diabetic rats were twisted and the mitochondria were swollen.Some of the cristae were broken.Vacuolization could be seen in parts of the mitochondria.The lesions of type 2 DM+TLR were significantly milder than those of type 2 DM.There was no significant difference between type 2 DM+TLR and type 2 DM+atorvastatin.The normal endothelial cells were thin and flat.The nucleus was fusiform..At the 12^th week the mitochondria of endothelia cells of aorta pectoralis of type 2 diabetic rats were well-stacked and slightly swollen.The mitochondria cristae were clear.No smooth cells penetrated into theca interna. Smooth cells of tunica media proliferated and the gap between myocardial tissues widened. The sarcomere was vague.The mitochondria of endothelia cells of aorta pectoralis of type 2 DM+TLR were well-stacked and slightly swollen.The mitochondria cristae were clear. No smooth cells penetrated into theca interna.The gap between myocardial tissues was normal and the sarcomere was clear.3) Expression of iNOS mRNA:The expression of iNOS mRNA of myocardial cells and aorta of obese rats were not different from that of normal controls.The expression of iNOS mRNA of myocardial cells and aorta of type 2 diabetic rats were significantly lower than that of normal controls.The expression of iNOS mRNA of myocardial cells and aorta of type 2 DM+TLR were not different than that of normal controls and there was no significant difference between type 2 DM+TLR and type 2 DM+atorvastatin.4) Expression of PKCβ₂ and OPN:The expression of PKCβ₂ of myocardial cells of obese rats increased compared with normal controls.The expression of PKCβ₂ of myocardial cells of obese+TLR was lower than that of obese rats.There was no significant difference between obese+TLR and obese+atorvastatin.The expression of PKCβ₂ of myocardial cells of type 2 diabtic rats increased significantly compared with normal controls.The expression of PKCβ₂ of myocardial cells of type 2 DM+TLR was higher than that of normal controls but lower than that of type 2 diabetic rats.The expression of PKCβ₂ of myocardial cells of type 2 DM+ALA and type 2 DM+atorvastatin was higher than that of normal controls and type 2 DM+TLR but lower than that of type 2 diabetic rats.There was no significant difference between the two groups.â‘¡OPN:The OPN of obese rats increased compared with normal controls.The OPN of obese+TLR was lower than that of type 2 diabetic rats but higher than that of normal controls.There was no significant difference between obese+ALA and obese+atorvastatin.However the OPN in the above two groups was higher than that of obese+TLR.The expression of OPN of type 2 diabtic rats increased significantly compared with normal controls.The expression of OPN of type 2 DM+TLR was significantly lower than that of type 2 diabetic rats.There was no significant difference between type 2 DM+ALA and type 2 DM+atorvastatin.Conclusions:1) As the increase of insulin level in the obese rats,the oxidative stress parameters were characterized by the decreasing of the antioxdiative parameters and the increasing of oxidative parameters.There were no obvious pathological changes of the myocardial cells and endothelial cells.The plasma OPN was higher than that of normal controls,2) The antioxidative parameters in diabetic rats continued to decrease further.The oxidative stress parameters in type 1 diabetic rats were significantly higher than those of obese or diabetic rats.The changes of serum CRP,NO and 24-hour urine proteins reflected the dysfunction of endothelia cells of type 2 diabetic rats.The pathological changes of diabetic rats further affirmed the impairment of endothelia function.The expressions of PKCβ₂ in myocardial cells and OPN in myocardial cells and aorta of type 2 diabetic rats were significant higher than those of normal controls.3) The intervention of TLR decreased the 24-hour urine proteins,CRP and oxidative stress parameters and increased the antioxidative parameters and NO.It also improved the lesions of myocardial cells and endothelial cells of rats and decreased the expression of PKCβ₂ of myocardial cells,serum OPN and expression of OPN in myocardial cells and aorta.It could be one of the mechanisms of the TLR that down-regulating the expression of OPN,enhancing the activity of iNOS and increasing the synthesis of NO by inhibition the oxidative stress, decrease of the CRP and 24-h urine proteins,inhibition of expression of PKCβ₂.Part 1 Effects of Tongluo recipe on oxidative stress of obese and diabetic ratsTo investigate the oxidative stress lesions in diabetic and obese rats and the effects of TLR,compare the effects of TLR with those of antioxidants ALA,atorvastatin and explore the possible mechanisms of TLR protective effects on endothelia cells.The results shows:It could be one of the mechanisms of the TLR that down-regulating the oxidative stress lesions,increasing the synthesis of NO by inhibition the oxidative stress,decrease of the CRP and 24-h urine proteins.It maybe the one of the mechanisms protecting the function of endothelium.Part 2 Ultrastructural changes of myocardium and aorta ofdiabetic ratsTo observe the histological and ultrastructural changes of myocardium and aorta of obese and experimental type 2 diabetic rats and the protective effects of TLR.The results shows:The changes of myocardial cells and endothelial cells of aorta of obese rats were not significant.The pathological changes of myocardial cells and endothelial cells of aorta of type 1 diabetic rats were more significant than those of type 2 diabetic rats.Intervention of TLR could improve the pathological changes of changes of myocardial cells and endothelial cells of aorta of type 1 and 2diabetic rats.Compared with atorvastatin there was no significant difference and TLR had protective effects on myocardial cells and endothelial cells of aorta.Part 3 Expression of iNOS mRNA in myocardium and aorta of obese and experimental type 2 diabetic rats and intervention effects of TLRTo investigate the expression of iNOS mRNA in myocardium and aorta of obese and experimental type 2 diabetic rats and intervention effects of TLR.The results shows:The expression of iNOS mRNA of myocardial cells and endothelial cells of aorta of obese rats did not significantly differ from that of normal controls.The expression of iNOS mRNA of myocardial cells and endothelial cells of aorta of type 2 diabetic rats was significantly lower than that of normal controls.The intervention of TLR could increase expression of iNOS mRNA of myocardial cells and endothelial cells of aorta and the synthesis of NO,thus protected the endothelia cells.This kind of effects was not significantly different from those of atorvastatin.Part 4 Expression of protein OPN in myocardium and aorta of obese and experimental type 2 diabetic rats and the intervention effects of TLRTo investigate the expression of protein OPN in myocardium and aorta of obese and experimental type 2 diabetic rats and the intervention effects of TLR.The results shows:TLR could decrease plasma OPN of diabetic and obese rats and expression of protein PKCβ₂ of myocardial cells of diabetic rats.The effects of TLR were not significantly different from those of atorvastatin and ALA.TLR could also decrease expression of protein OPN in myocardium and aorta of obese and experimental type 2 diabetic rats.The effects were stronger than those of atorvastation and ALA.To protect the function of endothelium.