The Role Of Blood Stasis And Lipotoxicity In Glucose And Lipid Metabolic Disorder And The Therapeutic Effect Of Huoxue-Jiangtang Decoction

Objective1. Clinical research partIn this part, the objective is to investigate the correlation of blood stasis scores, macroangiopathy and free fatty acid spectrum and the differences of plasma metebolomic profiles between type 2 diabetic patients with blood stasis syndrome or not, so as to clarify the possible relationship of blood stasis and lipotoxicity in diabetes and the subsequent pathophysiological changes; and to provide a theoretical basis for TCM treating glucose and lipid disorder and related diseases.2. Experimental research partIn this part, the therapeutic effect and the molecular mechanism of Huoxue-Jiangtang decoction, a Chinese herb compound works to promote blood circulation to remove meridian obstruction and invigorate Qi nourish Yin, was studied, on insulin resistance, lipid disorder, islet 0 cell apoptosis, and pancreas proteome change in rats, and insulin secretion impairment in INS-1 cell. So as to investigate the effect and mechanism of Huoxue-Jiangtang decoction on lipotoxicity impairment from different aspects of pathophysiological changes involved in lipotoxicity.Methods1. Literature research partAccess to relevant information on type 2 diabetes in the past ten years, and then analyze the data, the TCM etiology and pathogenesis of type 2 diabetes, and the role of blood stasis in the progress of type 2 diabetes were summarized, meanwhile, the importance of lipotoxicity in type 2 diabetes was also systematically overviewed.2. Clinical research partTwenty-five type 2 diabetic patients with blood stasis syndrome were collected between June,2014 and December,2014 in our hospital. Meanwhile, another 25 diabetic patients without blood stasis syndrome were subjected to control group. The basic information, blood stasis scores, blood glucose and lipids were observed. Plasma interleukin 6 (IL-6), tumor necrosis factor a (TNF-a), and interleukin 18 (IL-18) were tested by Elisa. Macroangiopathy of patients in different groups were examined by color Doppler ultrasound. And the factors related to blood stasis scores were analyzed. Furthermore, gas chromatography-mass spectrography (GC-MS) was used to explore the difference of serum free fatty acid spectrum in different groups. Besides, the relationship of Free Fatty Acid Spectrum, Blood Stasis Scores and macroangiopathy was analyzed. Meanwhile, Nuclear magnetic resonance spectroscopy was applied to detect the plasma metabolite content in diabetic patients with blood stasis syndrome or not. The main factors contributed to the difference of metabolic component were analyzed by principal component analysis, so as to speculate the distinction of metabolic pathway in patients of two groups.3. Experimental research partAt first, the components in Huoxue-Jiangtang decoction were detected by Liquid chromatography mass spectrometry (LC-MS)Then, The rat model of insulin resistance (IR) and hyperlipidemia was established by feeding a high-fat-diet for 28 weeks. the rats were randomly divided into model group, Huoxue-Jiangtang decoction treated group and fenofibrate group. The corresponding therapy was given through gastric tube for rats in different treatment groups. Meanwhile, a control group was set in normal animals. Oral glucose tolerance test (OGTT), insulin releasing test (IRT), HOMA-IR, insulin sensitivity index (ISI), serum lipid level, and lipid metabolism related proteins were detected. Liver pathology was determined and liver triglyceride content was examined.After that, pancreatic pathology and TG content were examined, and islet β cell apoptosis was detected by Transmission electron microscopy and TUNEL methods, and apoptosis index was calculated. The protein expression of B cell lymphoma-2 (Bcl-2) and Bcl-2 related X protein (Bax) in pancreas was tested by western blot. pancreatic proteome was also examined with iTraq quantitative proteomics techniques, the differently expressed protein was discovered, and verified with immunohistochemical method.In the end, The cell model of lipotoxicity-induced insulin secretion disorder cell model was established by adding 0.25mmol/L palmitic acid to the culture medium. The model cells were divided into model group, high dose group (20% serum containing herb), middle dose group (15% serum containing herb), and low dose group (10% serum containing herb). The corresponding intervention was administered and a control group was also set. Insulin concentration in culture medium, cyclic adenosine monophosphate (cAMP) level in cells were determined by Elisa, and protein expression of GLP-1R, cAMP-regulated guanine nucleotide exchange factor (Epac-2), Protein Kinas B (PKA or Akt) and pAkt (Ser473) were examined by western blot after stimulation of glucose and GLP-1.Results1. Clinical research part(1) Compared with control group, the blood stasis scores, total cholesterol (TC), total triglyceride (TG), low density lipoprotein cholesterol (LDL-c), IL-6, TNF-α, IL-18, the discovery rate of carotid plaque and femoral artery plaque, endometrial thickness of carotid, carotid plaque area and femoral artery plaque area were significantly increased in 2 diabetic patients with blood stasis syndrome (P<0.05)(2) In correlation analysis, the positive correlation of age, duration of diabetes, endometrial thickness of carotid, carotid plaque area, femoral artery plaque area and the blood stasis scores were discovered (P<0.05)(3) In GC-MS examination, totally 21 fatty acids were found in serum, and total free fatty acid (TFFA), saturated fatty acid (SFA), lauric acid (C12:0), palmitic acid (16:0), stearicacid (C18:0), arachidonic acid (C20:4n6), docosanoic acid (22:0) and carnaubic acid (C24:0) were found to contribute the difference of FFA spectrum between two groups.(4) in Pearson or spearman correlation analysis, C12:0, C16:0, C18:0, C22:0, TFFA and SFA were positively correlated with blood stasis scores (P<0.05). However, only SFA entered the regression equation in the multiple linear regression analysis.(5)C12:0, C16:0, C18:0, C20:4n6, TFFA and SFA were positively, while linoleic acid (C18:3n3), Cis-5,8,11,14,17-eicosapentaenoic acid (C20:5n3) were negatively correlated with carotid plaque area (P<0.05), while C16:0 was positively, and C18:3n3, cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) and nervonicacid were negatively correlated with femoral artery plaque area (P<0.05).(6) Totally 47 metabolites were detected in the two groups. Metabolic components were distinct with partly overlapping.(7) Compared with the control group, acetone, acetoacetate, α-hydroxybutyrate,α-hydroxy isovaleric acid,β-hydroxybutyrate, 2-Oxoisocaproate,3-methyl-2-oxovalerate,3-hydroxy-isobutyric acid and glycine significantly increased (P<0.05), while fumarate, citrate, N-acetyl ornithine, pyroglutamate and methionine decreased (P<0.05) in diabetic patients with blood stasis syndrome. The other 33 metabolites in two groups were not significantly discriminated (P>0.05).2. Experimental research part(1) Chromocors and tanshinones were found in Huoxue-Jiangtang decoction by LC-MS analysis.(2) Effect of Huoxue-Jiangtang decoction on insulin resistance and protein expression of GLP-1R and GLUT-4 in liver and adipose tissue:In contrast to the control group, the OGTT and IRT were impaired, TC, TG, LDL-c, HOMA-IR, FFA, liver TG content were significantly increased while HDL-c and ISI were decreased in the rats of model group (P<0.05). At the same time, liver glucagnon-like peptide-1 receptor (GLP-1R), glucose transpoter 4 (GLUT-4) and adipose GLUT-4 protein expression were decreased (P<0.05). While adipose GLP-1R protein expression was increased (P<0.01) in model group. Compared with model group, the OGTT, IRT, serum concentration of TG, TC, LDL-c, FFA in Huoxue-Jiangtang decoction treated group were decreased. Meanwhile, GLUT-4 protein in liver and adipose tissue, GLP-1R protein expression in liver tissues were increased while liver lipid accumulation and TG content were decreased in in the rats of Huoxue-Jiangtang decoction treated group (P<0.05). However, the expression of GLP-1R protein in adipose tissue were not changed in treatment group compared with model group.(3) Effect of Huoxue-Jiangtang decoction on β cell apoptosis:In contrast to the control group, the TG content and protein expression of Bax in pancreas tissue were increased (P<0.01), and pancreas fat deposition and early β cell apoptosis were found in model group, Meanwhile, β cell apoptosis index was increased while protein expression of Bcl-2 was decreased in model group (P<0.01). Compared with model group, the TG content and protein expression of Bax were decreased in pancreas (P<0.05), pancreas fat deposition and cell apoptosis index was decreased in Huoxue-Jiangtang decoction treated group (P<0.05). besides, the protein expression of Bcl-2 was increased in Huoxue-Jiangtang decoction treated group (P<0.01). Correlation analysis revealed that β cell apoptosis index was positively correlated with pancreas TG content.(4) Effect of Huoxue-Jiangtang decoction on pancreas proteome:In contrast to the control group,41 proteins were up-regulated and 24 were down regulated in pancreatic tissue of model group. However, after the treatment with Huoxue-Jiangtang decoction,28 proteins were up-regulated and 26 proteins down regulated, in which the modulation of 18 proteins was conformity (up/down regulated in model group and down/up regulated in Huoxue-Jiangtang decoction treated group at the same time).12 proteins were up-regulated in model group vs. control, while down-regulated in Huoxue-Jiangtang decoction treated group vs. model group, and 6 proteins were down-regulated in model group vs. control, while up-regulated in Huoxue-Jiangtang decoction treated group vs. modes group. After screening and verifying by immunohistochemical method, protein expression of Enoyl-CoA, Hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh), sterol carrier protein 2 (Scp-2), Fatty Acid Transporter (FATP-1) were increased in model group while decreased in Huoxue-Jiangtang decoction treated group.(5) Effect of Huoxue-Jiangtang decoction on β cell insulin secretion:20% serum containing Huoxue-Jiangtang decoction was proved to be the proper concentration for cell culture by MTT. In contrast to control group, insulin releasing, cAMP concentration, protein expression of GLP-1R, Epac-2, Akt, pAkt (Ser473) were all decreased in model group (P<0.05). Compared with model group, insulin secretion, and cAMP content were increased in all the three treatment groups (P<0.01). In addition, in high dose and middle dose groups, protein expression of GLP-1R, Epac-2 and Akt were up-regulated (P<0.01), while pAkt (Ser473) expression was increased in high dose group (P<0.01) However, the expression of pAkt (Ser473) in middle dose group, the expression of GLP-1R、Epac-2、Akt、pAkt (Ser473) in low dose group were not significantly changed in contrast to model group (P>0.05). Compared with high dose group, cAMP content, expression of GLP-1R, Epac-2, Akt, and pAkt (Ser473) were down-regulated in low dose group (P<0.05)Conclusion1. Clinical research partSerum FFA spectrum was significantly different in type 2 diabetic patients with blood stasis syndrome or not, and long chain SFA contributed most to this variation. Serum FFA spectrum was correlated with blood stasis scores and diabetic macroangiopathy, at the same time, patients with blood stasis manifested more serious disorder in lipid and energy metabolism. Indicating that lipotoxicity and the existence of blood stasis are closely correlated with each other in type 2 diabetes.2. Experimental research partMulti-target and mechanism of Huoxue-Jiangtang decoction treatment on lipotoxicity were revealed. In other words, Huoxue-Jiangtang decoction was effective in dealing with different pathophysiological changes in impairment caused by lipotoxicity such as insulin resistance, hyperlipidemia,β cell apoptosis, insulin secretion disorder etc. the specific mechanisms are as follows:(1) The mechanism of Huoxue-Jiangtang decoction in treating insulin resistance and hyperlipidemia in rats may be related to inducing the expression of GLUT-4 protein in liver and adipose tissue as well as increasing the expression of GLP-1R protein in liver.(2) The mechanism of Huoxue-Jiangtang decoction in preventing β cell apoptosis in rats may be related to the regulation of protein expression of Bcl-2 and Bax in pancreas.(3) Pancreatic proteome was changed after long-term high-fat-diet and Huoxue-Jiangtang decoction treatment. The screened protein mainly participated in fatty acid oxidation and transportation.(4) serum containing Huoxue-Jiangtang decoction was effective in treating lipotoxicity-induced insulin secretion disorder in INS-1 cell, and the mechanism may be related to increased cAMP content and protein expression of GLP-1R, Epac-2, Akt and pAkt (Ser473)...