Effects Of High Blood Glucose Fluctuation On PI3k/Akt Signaling Pathway In Vascular Endothelial Cells And The Intervention Of PQS

The outcomes of cardiovascular events in diabetes are equal to those found in patients with coronary artery diseases (CAD) not associated with type 2 diabetes mellitus (T2DM). T2DM is hence upgraded from "risk factor" to "equivalent" of CAD. Vascular endothelial dysfunction plays a critical role in the occurrence and development of diabetic cardiovascular complications, which is also termed as one of the most important pathophysiologic basis of these diseases. There are two kinds of behaving form of hyperglycemia original. For the fluctuant high blood glucose can have more deleterious influences than constant high glucose on endothelial function and diabetes chronic cardiovascular complications, studies on the concrete mechanism and effective methods to prevent it have recently become a research focus in the field of diabetic cardiovascular disease. Panax quinquefolius saponin of stem and leaf (PQS) are the effective parts of American ginseng, which can regulate lipid metabolism, lower the blood glucose, improve insulin resistance and myocardial ischemia and so on simultaneously. Previous studies had shown that PQS could increase glucose transportation and accelerate glucose uptake, improve insulin resistance and increase insulin sensitivity of adipocytes by initiating PI3K/Akt signaling pathway. PI3K/Akt signaling pathway not noly plays extremely important roles in various processes of cell life including growth, apoptosis, survival and proliferation, but also in the initiation and progression of vascular diseases.In our study, we observe the influences of high blood glucose fluctuations on endothelial function and effects of PQS by establishing T2DM rats model and HUVECs model, and explore the exact mechanisms by measuring related factors.This study is divided into three parts:literature review, clinical study and experimental research.1 Literature Review:In this part, two reviews are reviewed:High blood glucose fluctuation and cardiovascular complications, PI3K/Akt signaling pathway and vascular endothelial dysfunction in diabetes mellitus.2 Animal experimental research:Influence of high blood glucose fluctuation on endothelial function and PI3K/Akt signaling pathway of type 2 diabetes mellitus rats and the interventional effects of PQSObjective:①To observe the influence of high blood glucose fluctuation on the endothelial function of type 2 diabetes mellitus (T2DM) rats.②To observe the effects of PQS on the endothelial function of type 2 diabetes mellitus rats with high blood glucose fluctuation, and explore the relative mechanisms and its correlation with PI3K/Akt signaling pathway.Methods:The T2DM models were induced by intraperitoneal injection of a small dose of streptozotocin (STZ,35mg/kg) plus high fat and high caloric laboratory chow. Then diabetic rats were divided into steady high blood glucose (SHG) group and fluctuant high blood glucose group(FHG) according to fasting blood glucose coefficient of variation(FBG-CV), then the FHG group rats were divided into six groups according to the levels of FBG-CV and fasting blood glucose:PQS 30mg/kg/d group(PQL), PQS 60mg/kg/d group(PQH), MH 150mg/kg/d group(MHL), MH 300mg/kg/d group (MHH), and a-LA 300mg/kg/d group (a-LA) and fluctuant high blood glucose control group (FHG). Meanwhile,10 rats with high fat and high caloric laboratory chow were used as hyperlipemia control group (HLP),10 rats without any treatment were used as normal control group (NOR). Eight weeks later, the aortic arteries tectology observation of morphological change, biochemical assay of superoxide dismutase (SOD) and malondialdehyde (MDA), ELISA assay of hepatocyte growth factor (HGF), AGEs, LOX-1, TNF-a, and sICAM-1, p-GSK-3β,p-Akt, GSK-3β, Akt expression by Western-blot, these were measured to explore effects and possible mechanism of high blood glucose fluctuation and PQS.Results:①In comparison with NOR group, the levels of plasma HGF and serum AGEs, LOX-1, NO, MDA, ET-1, TNF-αand sICAM-1 in SHG and FHG groups were all significantly increased (P<0.01),SOD was significantly decreased; in comparison with SHG group, plasma HGF and serum AGEs, LOX-1, NO, MDA, ET-1, TNF-αand sICAM-1 in FHG group were all significantly increased further (P<0.01 or P<0.05), meanwhile the SOD decreased further; in comparison with FHG group, the levels of plasma HGF and serum AGEs, LOX-1, NO, MDA, ET-1, TNF-α, sICAM-1 in PQS, MH and a-LA groups were all decreased significantly (P<0.01), meanwhile SOD in those two groups were all increased significantly (P<0.01).②Western-blot results showed p-GSK-3βand p-Akt phosphorylation of SHG and FHG groups were significantly increased than the NOR group (P<0.01), while the FHG group showed a further decrease compared with SHG group (P<0.01). The p-GSK-3βand p-Akt phosphorylation of PQL, PQH, MHL, MHH, a-LA group was increased significantly in contrast with FHG group (P<0.01).③Comparison of the aortic arteries histology among groups showed no significant differences either before or after treatment.Conclusions:①There is obvious vascular endothelial injury and endothelial dysfunction in T2DM rats, which is related to oxidative stress, inflammatory reaction, AGE, LOX-1 over-production, and decreased activity of PI3K/Akt signaling pathway. Glucose fluctuation can further exacerbate these.②PQS can alleviate the vascular endothelial injury on T2DM rats with high glucose fluctuations obviously. The protective mechanism of PQS is closely related to its effects of relieving vessel stress, adjusting glycolipid metabolism, alleviating inflammatory reaction and decreasing AGEs and LOX-1 production. And the PI3K/Akt signaling pathway activation plays an important role in above reactions.3 Experimental Cell ResearchStudy 1:Influence of high blood glucose fluctuation on HUVECs and PI3K/Akt signaling pathwayObjective:To observe the influence of fluctuant high glucose on human umbilical vein endothelial cells (HUVECs) and PI3K/Akt signaling pathway.Methods:HUVECs were incubated for 8 days in media containing different glucose concentrations:5.56 mmol/1 (normal glucose, NOR),25 mmol/1 (steady high glucose, SHG), or a daily alternating 5.56 or 25 mmol/1 glucose (fluctuant high glucose, FHG). LY294002 (20umol/L) and CsA (2umol/L) were pretreated on SHG and FHG groups respectively. On the 9th day, the morphological changes were observed by light and electron microscopy, cell viability was measured by MTT method, cell membrane damage was determined by lactate dehydrogenase (LDH) leakage, the NO, ET-1, TNF-a, sICAM-1 and LOX-1 concentrations in the cell culture supernatant were measured by ELISA method. The activities of SOD, NOS and the contents of MDA in the cell lysate were examined by enzyme method or spectrophotometry. HUVECs p-GSK-3β,p-Akt, GSK-3β,Akt expression were measured by Western-blot.Results:①Morphological results showed that both SHG and FHG could induce HUVECs injury obviously. The cell survival rates and cell viability in SHG and FHG groups decreased significantly compared with NOR group(P<0.01), while FHG group decreased further compared with SHG group (P<0.05). Cell survival rates and cell viability pretreated with LY294002 decreased significantly (P<0.01 or P<0.05). In contrast, pretreated with CsA significantly increased cell survival rates and cell viability (P<0.01).②In comparison with NOR group, the level of NO, MDA, ET-1, TNF-a, sICAM-1, LOX-1 in SHG and FHG groups were all significantly increased (P<0.01 or P<0.05), SOD in thoses two groups were significantly decreased(P<0.01 or P<0.05). In comparison with SHG group, FHG group were all significantly increased or decreased further respectively (P<0.01or P<0.05). Pretreated with LY294002, NO, MDA, ET-1, TNF-α, sICAM-1, LOX-1 were all significantly increased (P<0.01 or P<0.05), meanwhile SOD were significantly decreased (P<0.01 or P<0.05). In contrast, pretreated with CsA showed just the opposite (P<0.01).③In comparison with NOR group, the phosphorylation level of GSK-3(3,Akt in SHG and FHG groups were all significantly decreased(P<0.01 or P<0.05). In comparison with SHG group, FHG group significantly decreased further (P<0.01or P<0.05). Pretreated with LY294002, the phosphorylation level of GSK-3β,Akt were decreased significantly (P<0.01 or P<0.05). In contrast, pretreated with CsA showed just the opposite (P<0.01).Conclusions:High blood glucose can induce HUVECs dysfunction and injury obviously, which is related to oxidative stress, inflammatory reaction and LOX-1 over-production, decreased activityof PI3K/Akt signaling pathway and opening of MPTP. And glucose fluctuation can aggravate these futher.Study 2:The protective mechanism of PQS on endothelial injury and its correlation with PI3K/Akt signaling pathwayObjective:To observe the protection of PQS on HUVECs suffering fluctuant high glucose and investigate the relative mechanism including PI3K/Akt signaling pathway.Methods:HUVECs were all incubated for 8 days in fluctuant high glucose. They were treated with PQS 0.1mg/ml (PH), PQS 0.05mg/ml (PL), DS 0.05mg/ml (DS), MH 1mM (MH), a-LA 100umol/L (a-LA) respectively. Then LY294002 (20umol/L) were pretreated on the above five goups respectively, and they were PHL, PLL, DSL, MHL, a-LAL. On the 9th day, the morphological changes were observed and relative factors were measured as Study 1.Results:①Morphological results showed that PQL, PQH, DS, MH andα-LA all can alleviate HUVECs injury obviously. The cell survival rates and cell viability were all significantly increased compared with FHG group (P<0.01). Cell survival rates and cell viability pretreated with LY294002 decreased significantly compared with medication groups respectively (P<0.01) expect DSL goup.②In comparison with NOR group, the levels of NO, MDA, ET-1, TNF-α, sICAM-1, LOX-1 in PQL, PQH, DS, MH and a-LA groups were all significantly decreased(P<0.01 or P<0.05),meanwhile the level of SOD were increased significantly(P<0.01 or P<0.05). Pretreated with LY294002, NO, MDA, ET-1, TNF-α, sICAM-1, LOX-1 were all significantly increased (P<0.01 or P<0.05), SOD were significantly decreased(P<0.01 or P<0.05) expect DSL③In comparison with NOR group, the phosphorylation levels of GSK-3β, Akt in PQS, DS and MH groups were all increased significantly(P<0.01). Pretreated with LY294002, the phosphorylation levels of GSK-3β, Akt were all decreased significantly (P<0.01) expect DSL. Conclusions:②PQS can protect HUVECs suffering fluctuant high glucose obviously.②The protective mechanism of PQS is closely related to its effects of relieving vessel stress, alleviating inflammatory reaction and decreasing LOX-1 production. And upregulation of Akt and GSK-36 expressions plays a important role in above reactions.③An interesting founding in this study is that the protection of PQS is closely related to PI3K/Akt signaling pathway, however, the protection of DS, which is one of the important components of PQS, seems poorly related to PI3K/Akt signaling pathway.