Clinical Effect of Jinlida and Tongxinluo on Type 2 Diabetic Kidney Disease Under Vessel Collateral Theory / 中国实验方剂学杂志

Objective: To observe the clinical efficacy of Jinlida (JLD) granules and Tongxinluo (TXL) capsules on type 2 diabetic kidney disease (DKD) under the guidance of vessel collateral theory. Method: A total of 120 patients with type 2 DKD, were randomly divided into 2 groups:the normal control group (60 cases) and the treatment group (60 cases). The patients in normal control group were treated with dietary control and hyperglycemia control. Based on treatment in control group, patients in treatment group were additionally treated with JLD granules (1 bag, tid), and TXL capsules (4 capsules, tid). The treatment was lasted for 12 weeks. The clinical efficacy, traditional Chinese medicine(TCM) syndrome scores were observed and compared before and after treatment. At the same time, the levels of glucose metabolism indexes including fasting blood glucose (FBG),postprandial 2 h plasma glucose (2 hPG), glycosylated hemoglobin (HbA1c) and the insulin resistance (IR); the levels of lipid metabolism indexes including total cholesterol(TC),triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and the high-density lipoprotein cholesterol (HDL-C); the levels of renal function indexes including urinary albumin excretion rate (UAER) and serum creatinine (SCr); as well as nailfold microcirculation were detected and compared. Result: ①The total effective rate was 80.0% in treatment group, significantly higher than 61.67% in the normal control group (PPPPPβ2-MG in treatment group was significantly obvious than that in the control group (PPConclusion: Tongxinluo combined with Jinlida can improve the clinical symptoms of patients with type 2 diabetic nephropathy and reduce urinary trace albumin, and its mechanism may be related to lowering glucose, regulating lipid metabolism and improving microcirculation.

Effect of Tongxinluo Treated Conditioned Medium of Cardiac Microvascular Endothelial Cells on Rats' Embryo Cardiomyocyte Apoptosis / 中国循环杂志

Objective: To explore the effect of impaired cardiac microvascular endothelial cells on rat's embryo cardiomyocyte apoptosis and the impact of Tongxinluo in such process. Methods: 3 kinds conditioned medium of cardiac microvascular endothelial cells were prepared. ① normal rat's cardiac microvascular endothelial cell (RCMECs) conditioned medium, ②homocysteine induced impaired RCMECs conditioned medium and ③ Tongxinluo treated RCMECs conditioned medium. Rat's embryo H9c2 cardiomyocytes were divided into 4 groups:① Control group, cells were cultured in DMEM without serum, ② N-CM group, cells were cultured in RCMECs conditioned medium, ③ H-CM group, cells were cultured in homocysteine induced impaired RCMECs conditioned medium and ④ T-CM group, cells were cultured in Tongxinluo treated RCMECs conditioned medium. Cell mitochondrial membrane potential (MMP) was detected by JC-1 agent, ROS level was examined by flow cytometry, cardiomyocytes apoptosis was measured by live cell imaging system, expression levels of cytochrome C (Cyt-C), cysteinyl aspartate specific proteinase-3 (Caspase-3), Bcl-2 associated X protein (Bax) and B-cell leukemia/lymphoma 2 protein (Bcl-2) were detected by Western blot analysis. Results: MMP, expression levels of ROS, Cyt-C and cardiomyocyte apoptosis were similar between Control group and N-CM group, P>0.05. Compared with N-CM group, H-CM group had decreased MMP, increased ROS and cardiomyocyte apoptosis, up-regulated expressions of Cyt-C, Caspase-3 and Bax, all P<0.01. Compared with H-CM group, T-CM group had increased MMP, decreased ROS and cardiomyocyte apoptosis, down regulated expressions of Cyt-C, Caspase-3 and Bax, all P<0.05; while up-regulated expression of Bcl-2, P<0.01. Conclusion: Conditioned medium of impaired cardiac microvascular endothelial cells may induce cardiomyocyte apoptosis and such process could be inhibited by Tongxinluo.

Downregulated transient outward potassium channel protein Kv4.2 and Kv4.3 expression in PVN contributes to sympathoexcitation in rats with chronic heart failure / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the transient outward potassium channel protein expression in paraventricular nucleus (PVN) and its contribution to renal sympathetic nerve activity (RSNA) in rats with chronic heart failure (CHF). METHODS:A rat model of CHF was prepared by acute myocardial infarction that was induced by ligation of the left ante -rior descending coronary artery .Four weeks after heart failure , echocardiogram was applied to identify the CHF model and plasma norepinephrine (NE), serum NH2-terminal pro-brain natriuretic peptide (NT-proBNP) were detected by ELISA. The expression of ransient outward potassium channel proteins Kv 4.2 and Kv4.3 at mRNA and protein levels was deter-mined by real-time PCR and Western blot .The mean arterial pressure ( MAP) , heart rate ( HR) and RSNA were measured in anesthetized rats with PVN microinjection of potassium channel blockers 4-AP.RESULTS:In CHF group , the rat car-diac function and Kv4.2 and Kv4.3 expression in PVN were obviously lower while plasma NE and serum NT-proBNP were obviously higher than those in sham group .Microinjection of 4-AP into PVN induced an increase in MAP , HR and RSNA in both sham and CHF rats , while the CHF rats exhibited smaller responses to 4-AP than sham-operated rats .CONCLU-SION:Downregulation of Kv4.2 and Kv4.3 expression in the PVN may be a potential mechanism for sympathoexciation in the rats with chronic heart failure .

Influence of Guishaotongluo on angiogenesis of adventitial vasa vasorum and oxidative stress in early stage of atherosclerosis / 中国药理学通报

Aim To observe the effect of Guishao-tongluo ( GSTL ) on the angiogenesis of vasa vasorum and oxidative stress in the early stage of atherosclero-sis. Methods The rabbits ( n =84 ) were randomly divided into 7 groups (n=12):control group,high-fat group, adventitial injury group, GSTL high(GH)and medium ( GM ) dose group, atorvastain group ( ATO ) , and Tongxinluo group ( TXL ) . The normal group was fed with common foodstuffs, and high-fat foodstuffs for the high-fat group to establish an early model of hyper-lipidemia, and all the other groups were fed with high-fat diet combined with carotid artery cannula to build early atherosclerosis carotid artery injury rabbit mod-els. The GSTL high and medium dose was given Guishaotongluo ultrafine powder 4. 16,2. 08 g·kg-1 · d-1 respectively. The atorvastain group and Tongxinluo group were given suspension of atorvastain solution 2. 5 mg·kg-1 ·d-1 , Tongxinluo supermicro powder 0. 6 g ·kg-1 ·d-1 . All groups were treated with gastric per-fusion for 4 weeks. Biochemical method was applied to detect blood lipid change. HE staining was used to ob-serve the pathological morphology of intima-media. Aactivity of serum superoxide dismutase( SOD) ,malon-dialdehyde ( MDA ) content and the total antioxidant capacity ( T-AOC ) in artery serum were detected. NADPH subunits p22phox mRNA, gp91phox mRNA in carotid arteries were located and semi-quantitated by fluorescence in situ hybridization. The expression of VEGF, VEGFR-2 in the carotid artery adventitia was detected by Western blot. Results Compared with normal group,the contents of TC,TG and LDL-C were significantly increased, and VEGF, VEGFR-2 protein levels were remarkly increased in high-fat and adventi-tial injury group. The carotid artery injuries,the degree of angiogenesis of vasa vasorum and NADPH subunits p22phox, gp91phox mRNA in adventitia tissue of the GH,GM, ATO and TXL group were milder in varying degrees compared with those of the vasa injury group. Also the activity of SOD,T-AOC increased,while MDA content,VEGF,VEGFR-2 protein levels were remarkly decreased ( P < 0. 5 or P < 0. 01 ) . Conclusions GSTL can inbibit adventitial neovascularization in the early stage of atherosclerosis, and its mechanism might be related to the increase of total antioxidant capacity of the vascular system and adventitia tissue.

Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection.</p><p><b>METHODS</b>Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot.</p><p><b>RESULTS</b>Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.</p>

Effect of Tongxinluo on Homocysteine-induced Rat’s Cardiac Micro Vascular Endothelial Cell Injury and the Oxidative Stress Mechanism / 中国循环杂志

Objective: To observe the effect of Tongxinluo (TXL) on homocysteine-induced rat’s cardiac micro vascular endothelial cell (RCMECs) injury and to study the oxidative stress mechanism. Methods: Primary RCMECs were cultured with tissue explants process, cell morphology was observed by inverted microscope and the cell was identiifed by CD31 immunolfuorescence method. RCMEC injury model was established by Homocysteine (Hcy) induction and the cells were divided into 5 groups: Control group, with normal cells, Hcy group, the cells were treated by Hcy at 10 mmol/L, Low-dose TXL group, Hcy treated cells were cultured with TXL at 100 mg/L, Middle-dose TXL (200 mg/L) group and high-dose TXL (400 mg/L) group. Cell survival rates were detected, supernatant levels of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined, intracellular protein expressions of reactive oxygen species (ROS) and endothelial nitric oxide synthase (eNOS) were detected and mRNA expression of endothelin-1 (ET-1) was measured in different groups respectively. Results: Compared with Control group, Hcy group showed decreased cell survival rate (74.61 ± 2.88)% vs (100.00 ± 2.07)%, increased supernatant level of MDA (4.10 ± 0.18) nmol/ml vs (1.92 ± 0.10) nmol/ml, reduced SOD activity (7.55 ± 0.71) U/ml vs (20.77 ± 0.68) U/ml, elevated ROS level(38.17 ± 10.28) % vs (19.83 ± 2.97) %, up-regulated mRNA expression of ET-1 and down-regulated protein expression of eNOS. Compared with Hcy group, the above indexes were improved in each TXL group at different levels. Conclusion: TXL could decrease Hcy induced RCMECs injury, such protection was conducted by reducing the oxidative stress mechanism in cells.

Effect of Dredging Collateral Drug on ox-LDL Injured THP-1 Cells Adhesion to Human Umbilical Vein Endothelial Cells in vitro / 中国循环杂志

Objective: To observe the effect of oxidative-low density lipoprotein (ox-LDL) injured human leukemia mononuclear cells (THP-1) adhesion to human umbilical vein endothelial cells (HUVECs)in vitrowith the intervening function of dredging collateral drug, tongxinluo (TXL) and ginsenoside (Rb1). Methods: Cell injury was induced by ox-LDL treatment. The cells were divided into 4 groups:①Normal control group,②Injury model group, the cells were cultured by ox-LDL,③TXL group, the cells were cultured with both ox-LDL and TXL,④Rb1 group. HUVEC viability was measured by MTS assay, adherence rate of THP-1 cells to HUVECs was tested by vital cell staining. The contents of monocyte chemoat-tractant protein (MCP-1), soluble vascular cell adhesion molecule (sVCAM-1), soluble inter vascular cell adhesion molecule-1 (sICAM-1) and E-selectin in HUVEC conditioned medium were detected by ELISA; protein expressions of CCR2, VLA4 and Mac-1 in THP-1 cells were examined by Western blot analysis. Results: Compared with Control group, HUVEC viability was decreased in Injury model group (100 ±1.31) % vs(75.57 ± 1.02) %, while increased in both TXL and Rb1 groups (99.25 ± 1.40) % and (99.48 ± 2.15) %; Injury model group showed elevated adherence rate of THP-1 cells to HUVECs, while the adherence rates were reduced in both TXL and Rb1 groups. Compared with Injury model group, TXL group and Rb1 group showed decreased levels of MCP-1, sVCAM-1, sICAM-1 and E-selectin in HUVEC conditioned medium; decreased protein expressions of CCR2, VLA4 and Mac-1 in THP-1 cells. Conclusion: TXL and Rb1 could protect HUVECs, reduce ox-LDL injury induced vascular endothelial cell adhesion and decrease relevant receptor expression in monocytes; therefore, inhibit injured monocytes adherence to vascular endothelial cells.

A parental case control study on the association between reduced folate carrier gene polymorphism and neural tube defects / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the association between reduced folate carrier gene (RFC1 A80G) polymorphism and the risk for child with neural tube defects (NTDs), and to provide epidemiological evidence for the existence of NTDs genetic marker.</p><p><b>METHODS</b>RFC1 (A80G) genotypes were detected using RFLP-PCR for blood DNA of 104 families with NTDs-affected children and 100 control families with no history of child-affected birth defects. Case-control study and transmission/disequilibrium test(TDT) for the RFC1 genotype of NTDs pedigree were carried out.</p><p><b>RESULTS</b>The G allele frequency of children with NTDs was higher than that of controls when compared to A allele( OR = 1. 64, 95% CI :1.08-2.49). The offspring of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (OR = 2.56, 95% CI: 1.04-6.36) in our study population. There was evidence of association between G allele and the risk of parent having a child with NTDs (OR = 1.56, 95% CI: 1.07-2.28) in the TDT analysis.</p><p><b>CONCLUSION</b>Our findings indicated that there was potential association between offspring RFC1 GG genotype and the risk of NTDs, and the G allele was a possible susceptible gene marker for an increased NTDs risk in the Chinese population.</p>