Influence Of Effective Components Of Chuanxiong Rhizome And Peony Root On Platelet Activation And PKCβ1Expression During Vascular Endothelial Injury Induced By High Blood Glucose Fluctuations

According to recent studies, the prognosis of cardiovascular events in type2diabetes mellitus (T2DM) is equal to that found in patients with coronary heart diseases (CHD) not associated with T2DM. T2DM is hence upgraded from "risk factor" to "equivalent" of CHD. Diabetic vascular complications are the main causes of cripples and death of the diabetic patients. CHD is closely related to Blood-stasis Syndrome (BSS) according to Traditional Chinese Medicine (TCM) theory. The high blood viscosity syndrome and vascular complications associated with thrombosis of diabetes patients are the very concrete reflections of BSS in TCM. Our previous studies had investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with BSS by oligonucleotide microarray technique. Further studies conclusively showed that protein kinase C beta1(PKCβ1) played a key role in the regulation of CHD with BSS.High blood glucose is the main pathological factor of diabetes vascular lesions, which has two kinds of manifestations:fluctuant high blood glucose and constant high blood glucose. Compared with constant high blood glucose, fluctuant high blood glucose can lead to worse consequences on endothelial function and diabetes related chronic cardiovascular complications. Therefore, studies on the concrete mechanism and effective methods to prevent it have lately become a research focus in the field of diabetic cardiovascular disease prevention and treatment. PKC pathway is considered to be a key mechanism of diabetes vascular lesions. Then, whether PKCβ1, a key regulatory genes of CHD with BSS, plays an important role in diabetes vascular lesions (Diabetes with BSS), especially in vascular endothelial injury caused by high blood glucose fluctuation? BSS is strongly linked with platelet aggregation and activation, whether high blood glucose fluctuation has effect in platelet aggregation and activation, and whether PKCβ1is involved in?In our study, we explored the close relationships between high blood glucose fluctuations, endothelial injury, platelet aggregation and activation, and PKCβ1after inclusion of38T2DM patients. With "fluctuant high blood glucose cultured human umbilical vein endothelial cell (HUVEC) model" and "Human platelet-HUVEC experimental system", we observed the effects of effective components of Chuanxiong Rhizome and Peony Root on fluctuant high blood glucose induced endothelial injury and platelet aggregation, and explored the relative mechanisms and its correlation with PKCβ1. By introducing the concept of Glucose Index (GI), we established T2DM rat model with high blood glucose fluctuation and explored the effects of effective components of Chuanxiong Rhizome and Peony Root on endothelial function, platelet activation and aggregation, and PKCβ1expression of T2DM rats with high blood glucose fluctuations.This study contains two parts:literature review and experimental research.1Literature review:Including the following three reviews:"Diabetic angiopathy, Blood Stasis Syndrome and Activating Blood Circulation to Remove Stasis""Fluctuant high blood glucose, Endothelial injury and Platelet activation " and "Research advance on PKCβ1and Diabetic angiopathy"2Experimental research:Including the following five parts.Study I:Research on the relationships between blood glucose fluctuation, endothelial injury, platelet activation and PKCβ1in T2DM patients. Objective:To observe the correlations between blood glucose fluctuation, endothelial injury, platelet activation and PKCβ1in T2DM patients.Methods:Thirty-eight diagnosed type2diabetic patients with stable glucose-lowering treatment were enrolled. Seven point (preprandial and120-min postprandial for each meal and bedtime) capillary glucose was measured with an ultrasmart glucose meter. The mean amplitude of glycemic excursions (MAGE) were calculated. HbAlc was measured by ion-exchange high-performance liquid chromatography. Levels of E-selectin. Von Willebrand Factor (vWF) and PKCβ1were examined by ELISA. The peripheral blood platelet maximum aggregation rate and platelet membrane protein level of CD62p was determined by platelet fluorescent aggregometer and flow cytometry, respectively. The general indexes were measured and then the correlations of E-selectin, vWF, PKCβ1, platelet maximum aggregation rate, CD62p and MAGE were analyzed.Results:In simple correlation analysis, there were no significant correlations between age, body mass index, course, medical history and E-selectin, vWF, PKCpi, Platelet maximum aggregation rate, CD62p and MAGE. There were significant correlations between endothelial injury markers (E-selectin and vWF), Platelet maximum aggregation rate, CD62p and MAGE (P<0.01). There were certain correlations between PKCβ1and endothelial injury markers (E-selectin and vWF), platelet aggregation and activity (Platelet maximum aggregation rate and CD62p), MAGE and HbAlc (P<0.05or P<0.01). Multivariate regression analysis showed that PKCβ1increased linearly with increasing HbAlc and MAGE, increasing platelet maximum aggregation rate and CD62p, and increasing E-selectin and vWF.Conclusions:PKCβ1, the key regulatory genes of coronary artery disease with BSS, is closely related with endothelial injury and platelet aggregation and activity of T2DM patients with blood glucose fluctuation.Study Ⅱ:The PKCβ1expressions during vascular endothelial injury induced by high blood glucose fluctuations and the intervention of effective components of Chuanxiong Rhizome and Peony Root.Objective:To observe the expression of PKCβ1during vascular endothelial injury induced by high blood glucose fluctuations and the effect of effective components of Chuanxiong Rhizome and Peony Root.Methods:HUVECs were incubated for8days in media containing different glucose concentrations:5.56mmol/L (normal glucose, N),25mmol/L (constant high glucose, W), or a daily alternating5.56or25mmol/L glucose (fluctuant high glucose, B). LY333531(200nM) was pretreated on W group and B group, respectively. Meanwhile, B group were treated with tetramethylpyrazine hydrochloride (500μM), paeoniflorin (100μM), metformin hydrochloride (1mM) and aspirin enteric-coated tablets (1mM), respectively, which were called as TMP group, PAE group, MH group and ASA group. On the9th day, the morphological changes were observed by light and electron microscopy, cell viability and apoptosis were measured by cell counting kit and flow cytometry. The concentrations of TNF-a, PECAM-1, ROS and T-AOC in the cell culture supernatant were measured by ELISA method. The expression of PKCβ1in HUVECs was measured by Western blot.Results:1. Morphological results showed that both W group and B group could induce HUVECs injury obviously. The cells in these two groups showed swelling, apoptosis and necrosis obviously, especially in B group. After treated by LY333531or TMP, PAE, MH and ASA, the cell injury was alleviated markedly.2. The cell viability decreased and cell apoptosis rate (mainly late apoptosis) increased significantly in W group and B group compared with N group (P<0.01), especially in B group. The cell viability pretreated with LY333531increased significantly (P<0.05or P<0.01), while cell apoptosis rate significantly increased compared with B group (P<0.01). TMP, PAE, MH and ASA manifested the similar protective effects as LY333531, and PAE were the best of them.3. In comparison with N group, levels of TNF-a, PEC AM-1and ROS in W group and B group were all significantly increased (P<0.01). T-AOC in those two groups were significantly decreased (P<0.01). In comparison with W group, these indexes above in B group showed significantly increased or decreased further, respectively (P<0.05or P<0.01). Pretreated with LY333531, levels of TNF-a, PECAM-1and ROS were all significantly decreased (P<0.05or P<0.01), meanwhile T-AOC was significantly increased (P<0.05or P<0.01). TMP, PAE, MH and ASA showed the similar effect as LY333531pretreatment.4. In comparison with N group, the expression of PKCβ1in W group and B group were all significantly increased (P<0.05or P<0.01). In comparison with W group, B group significantly increased further (P<0.05or P<0.01). Pretreated with LY333531, the expression of PKCβ1was decreased significantly (P<0.05or P<0.01), just as treated with TMP, PAE, MH and ASA.Conclusions:High blood glucose can induce HUVEC injury and apoptosis obviously, which is closely related to PKCβ1over-expression. The effective components of Chuanxiong Rhizome and Peony Root can protect HUVEC suffering fluctuant high glucose obviously, and the protective mechanism of which is closely related to its effects of relieving vessel stress, alleviating inflammatory reaction and inhibitory effect on the expression of PKCβ1.Study III:The influence of high blood glucose fluctuations on platelet aggregation and the intervention of PKCβ1inhibitor and effective components of Chuanxiong Rhizome and Peony Root.Objective:To observe the expression of PKCβ1during vascular endothelial injury induced by high blood glucose fluctuations and the effect of effective components of Chuanxiong Rhizome and Peony Root.Methods:The first step, HUVECs were incubated for8days in media containing different glucose concentrations:5.56mmol/L (normal glucose, N),25mmol/L (constant high glucose, W), or a daily alternating5.56or25mmol/L glucose (fluctuant high glucose, B). On the9th day, the cell culture supernatant were collected and stored at-80℃for use. The second step,15healthy volunteers were enrolled and fasting blood were draw for preparation of platelet rich plasma (PRP) and platelet poor plasma (PPP). The third step, the cell culture supernatants of first step were added to PPP collected in second step for platelet maximum aggregation rate examination. There were nine groups in all:normal glucose group (N), constant high glucose group (W), constant high glucose+LY333531group (WL), fluctuant high glucose group (B), fluctuant high glucose+LY333531group (BL), fluctuant high glucose+TMP (TMP), fluctuant high glucose+PAE (PAE), fluctuant high glucose+MH (MH) and fluctuant high glucose+ASA (ASA).Results:In comparison with N group, the platelet maximum aggregation rate in W group and B group were all significantly increased (P<0.01). In comparison with W group, B group significantly increased further (P<0.01). Pretreated with LY333531, TMP, PAE, MH and ASA for15minutes can significantly decrease the platelet maximum aggregation rate induced by high glucose fluctuations (P<0.01).Conclusions:The cell culture supernatants induced by high blood glucose fluctuations can promote platelet aggregation obviously. The PKCβ1inhibitor and effective components of Chuanxiong Rhizome and Peony Root all have antagonistic effects on this. The anti-platelet aggregation effect of effective components of Chuanxiong Rhizome and Peony Root is closely related to its inhibitory effects on PKCβ1expression.Study Ⅳ:Establishment of T2DM rats model with high blood glucose fluctuation Objective:To establish T2DM rats model with high blood glucose fluctuation.Methods:Thirty Sprague Dawley rats were involved. The T2DM models were induced by intraperitoneal injection of a small dose of streptozotocin (STZ,30mg/kg) plus high fat and high caloric laboratory chow for4weeks. The rats were fed with the fixed time and fixed location (8:00-9:00,14:00-15:00,20:00-21:00). Three days after STZ injected, the fasting blood glucose and postprandial blood glucose were measured. The fasting blood glucose greater than11.1mmol/L and postprandial blood glucose not more than33.3mmol/L was selected for further study.. Six point (preprandial and120-min postprandial for each meal) caudal vein glucose was measured with an ultrasmart glucose meter and the process lasted for three days. Then, the mean amplitude of glycemic excursions (MAGE) and largest amplitude of glycemic excursions (LAGE) were calculated. Blood samples were obtained from abdominal aorta after treatment, and levels of fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), glucagon and insulin were measured, meanwhile insulin sensitive index (ISI) was calculated.Results:1. In comparison with N group, levels of body weight, serum FBG, TC and TG in high GI diet group and low GI diet group were all significantly increased (P<0.05or P<0.01), however, there were no significant difference between those two groups (P>0.05).2. In comparison with N group, levels of glucagon and insulin in high GI diet group and low GI diet group were all significantly increased (P<0.01), meanwhile ISI was decreased obviously (P<0.01). However, there were no significant difference between high GI diet group and low GI diet group (P>0.05). 3. In comparison with N group, levels of MAGE and LAGE in high GI diet group and low GI diet group were all significantly increased (P<0.01), and there were significant differences between high GI diet group and low GI diet group (P<0.05, P <0.01).Conclusions:Without impacting the levels of blood glucose, there appeared more obvious blood glucose fluctuation in high GI diet T2DM rats than low GI diet T2DM rats. We can establish T2DM rat model with high blood glucose fluctuation and relative constant high blood glucose by feeding high GI diet and low GI diet successfully.Study V:Influence of effective components of Chuanxiong Rhizome and Peony Root on platelet activation and PKCβ1expression during vascular endothelial injury induced by high blood glucose fluctuationsObjective:To observe the changes of platelet activation and PKCβ1expression during vascular endothelial injury induced by high blood glucose fluctuations and the intervention of effective components of Chuanxiong Rhizome and Peony Root.Methods:One hundred Sprague Dawley rats were involved.10rats were chosen as normal group (N). The other90rats were fed by fat and high caloric laboratory chow for4weeks. All the rats were fed with the fixed time and fixed location (8:00-9:00,14:00-15:00,20:00-21:00). Then T2DM models were induced by intraperitoneal injection of a small dose of streptozotocin (STZ,30mg/kg). Three days after STZ injected, the fasting blood glucose and postprandial blood glucose were measured. The fasting blood glucose greater than11.1mmol/L and postprandial blood glucose not more than33.3mmol/L were selected for further study, and there were62rats in all. Then the diabetic rats selected were divided into6groups according to average blood glucose level and body weight:11rats with low GI diet (W),11rats with high GI diet (B),10rats with high GI diet and MH gavage (MH),10rats with high GI diet and ASA gavage (ASA),10rats with high GI diet and TMP gavage (TMP),10rats with high GI diet and PAE gavage (PAE). Meanwhile,10rats was used as normal control group (N). Six weeks later, blood samples were obtained from abdominal aorta for biochemical detection, platelet protein extraction and flow cytometry. The histopathology of aorta was observed by H&E staining. The expression of PKCβ1in aorta and platelet were determined by Western blot.Results:1. Comparison of the aortic arteries histology among groups showed that there were more significant endothelial cells disorganization, injury and early atherosclerosis changes in both W group and B group than in N group, especially in B group. TMP, PAE, MH and ASA treatment manifested obvious protective effects on endothelial cells.2. In comparison with N group, levels of FBG, TG, TC all significantly increased in W group and B group (P<0.01), meanwhile body weight and INS were decreased obviously (P<0.01). However, there were no significant difference between W group and B group (P>0.05). TMP, PAE and MH treatment all significantly decreased the levels of FBG, TG and TC (P<0.05or P<0.01), meanwhile increased the INS level (P<0.01), except for ASA group.3. In comparison with N group, levels of vWF, E-selectin and ROS in W group and B group were all significantly increased (P<0.01), SOD and T-AOC in these two groups were significantly decreased (P<0.05or P<0.01). In comparison with W group, B group showed significantly increased or decreased further, respectively (P<0.05). After treatment with TMP, PAE, MH and ASA, levels of vWF, E-selectin and ROS were all significantly decreased (P<0.05or P<0.01), meanwhile levels of SOD and T-AOC were significantly increased (P<0.05or P<0.01).4. In comparison with N group, the platelet CD62p expression in W group and B group were all significantly increased (P<0.01). In comparison with W group, B group significantly increased further (P<0.05). The CD62p expression decreased obviously after TMP, PAE, MH and ASA treatment (P<0.05or P<0.01).5. In comparison with N group, the expression of PKCβ1in sera, aorta and platelet all significantly increased (P<0.05or P<0.01), and there were significant difference between W group and B group (P<0.05or P<0.01). TMP, PAE and MH treatment all significantly decreased the expression of PKCβ1in sera, aorta and platelet (P<0.05or P<0.01). ASA treatment decreased the expression of PKCβ1in aorta significantly (P<0.01), but had no effect on the expression of PKCβ1in sera and platelet (P>0.05).Conclusions:The effective components of Chuanxiong Rhizome and Peony Root can alleviate the vascular endothelial injury on T2DM rats with high glucose fluctuations obviously. The protective mechanism is closely related to their effects of relieving vessel stress, alleviating inflammatory reaction and inhibiting platelet hyperactivity and aggregation. And the most important effect was their inhibitory effect on the key regulatory genes of coronary artery disease with BSS.