Protective Effect Of Sequoyitol On Vascular Endothelium In Diabetic Rats And The Underlying Mechanisms

BACKGROUNDDiabetes mellitus is a group of metabolic diseases characterized by hyperglycemia as the basic pathological change, and accompanied by multiple organs damage. Diabetic vascular complications are the leading reason for disability and death in diabetes patients. A lot of studies have shown that oxidative stress is one of the most important mechanisms responsible for diabetic vascular complications.The family of SHC has three members:ShcA, ShcB and ShcC. The mammalian ShcA gene eneodes three isoforms:p46shcA, p52shcA and p66shcA. P46shcA and p52shcA are the products of alternative translation initiation sites within the same transcript. P66shcA is important in aging and in the pathogenesis of aging-associated diseases in mammals. Recently, it has been demonstrated that genetic ablation of p66shcA in the mouse was shown to reduce production of intracellular oxidants and consequently prolong life span by30%.Sequoyitol (also called5-O-methyl-myo-inositol), a new natural herbal compound, is extracted from Taxus Chinensis. In vitro, it has been demonstrated that sequoyitol competitively inhibits the activity of α-glucosidase and stimulates glucose uptake into adipocytes.In the present study, we investigated the protective effect of sequoyitol on vascular endothelium in diabetic rats and the underlying mechanisms.METHODSIn vivo study:type2diabetic rats (T2DM) were established by bonus injection of streptozotocin (STZ,35mg/kg) intraperitonealy and fed high-fat and high-glucose diet. Rats were divided into5groups: control, T2DM, T2DM plus Acarbose (20mg/kg), T2DM plus Sequoyitol (25mg/kg), T2DM plus Sequoyitol (50mg/kg). These animals were orally treated with drugs for4weeks. The oral glucose tolerance test (OGTT) were performed and the concentrations of fasting blood glucose (FBG) and insulin (FIns) were measured. The values of area under curve (AUC) and insulin activity index (IAI) and insulin secretion index (IS) were calculated to evaluate insulin resistance (IR). P66shcA mRNA expression in the isolated aorta was performed by real time PCR. Protein expression of p66shcA and phosphorylated p66shcA in the isolated aorta were detected by immunohistochemistry. The level of intracellular reactive oxygen species (ROS) was determined by using dihydroethidium (DHE) staining of aorta and the level of serum malondialdehyde (MDA). Vasodilator responses to acetylcholine in aortic rings were measured. In vitro study:after cultured with serum-free medium for24h, human umbilical vein endothelial cells (HUVECs) were pretreated with various concentrations (0.3,1,3μM) of sequoyitol for1h prior to exposure to glucose (30mM). Cell injury was evaluated using observation of lactic dehydrogenase (LDH) leakage. Cell apoptosis was determined using caspase-3activity. P66shcA mRNA expression was detected by real time PCR. Protein expression of p66shcA and phosphorylated p66shcA were detected by western blot. The level of intracellular ROS was determined by using DHE staining and the level of intracellular MDA.RESULTS1. The level of FBG, area under the curve of glucose, and HOME.IR were markedly increased while IAI and IS significantly decreased in diabetic rats. However, sequoyitol markedly decreased the level of FBG and increased insulin secretion while improving IS and HOME.IR, but had no effect on area under the curve of glucose or IAI.2. Both the mRNA and the protein expressions of p66shcA in the isolated aorta were up-regulated concomitantly with the increase of phosphorylated p66shcA protein level in diabetic rats. However, sequoyitol down-regulated the expression of p66shcA mRNA and protein accompanied with the decrease of protein level of phosphorylated p66shcA. 3. The levels of serum MDA and ROS in the isolated aorta were significantly increased in diabetic rats, which was attenuated by treatment with sequoyitol.4. Vasodilator responses to acetylcholine in aortic rings of diabetic rats were markedly decreased compared with control rats. However, sequoyitol markedly improved endothelium function.5. Treatment with glucose (30mM) for24h significantly increased the release of LDH as well as activity of caspase-3in HUVECs. However, sequoyitol significantly inhibited hyperglycemia-induced the release of LDH and caspase-3activity.6. Treatment with glucose (30mM) for24h significantly increased the mRNA and protein expressions of p66shcA concomitantly with the increase of phosphorylated p66shcA protein level in HUVECs, which was attenuated by pretreatment with sequoyitol in a concentration-dependent manner.7. Treatment with glucose (30mM) for24h significantly increased intracellular ROS as well as MDA in HUVECs. However, sequoyitol reduced the increase of ROS generation induced by hyperglycemia.CONCLUSION1. Sequoyitol reduced blood glucose and stimulated insulin secretion.2. Sequoyitol improved endothelial function in diabetic rats and the protective effect of sequoyitol was related to inactivation of p66shcA/ROS pathway.