Effects of Free Anthraquinones Extract from the Rhubarb on Cell Proliferation and Accumulation of Extracellular Matrix in High Glucose Cultured-Mesangial Cells

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of free anthraquinones (FARs) extract, which was extracted from the rhubarb and purified by macroporous resin DM130 with gradient mixtures of ethanol/water as the lelution solvents, in high glucose-cultured glomerular mesangial cells (MCs). The cell proliferation was determined by CCK-8 assay, the levels of TGF-beta1, CTGF, ColIV and FN proteins in the supernatant of MCs were measured by ELISA assays, and the mRNA levels of these four genes were detected by RT-PCR. The results showed that the increased proliferation of MCs, the mRNA levels and protein expression of TGF-beta1, CTGF, ColIV and FN induced by high glucose were inhibited after the treatment with the FARs extract. This indicated that FARs extract could inhibit cell proliferation and the expression of main extracellular matrix induced by high glucose in MCs. The FARs extract exhibited potential values for prophylaxis and therapy of DN.

Xiaokening stimulates endothelial nitric oxide release in diabetic rats

<p><b>INTRODUCTION</b>Diabetes mellitus induces microangiopathic changes that lead to endothelial dysfunction. This study investigated the effect of Xiaokening, a type of Chinese compound medicine, on the mesenteric arteriolar endothelial cell function of diabetic rats and its underlying mechanism.</p><p><b>METHODS</b>Diabetes mellitus was induced in rat models via intraperitoneal injection of 60 mg/kg streptozotocin and observed over three weeks. Mesenteric arterioles, which were isolated in a cannulated and pressurised state, were incubated with intravascular injections of 1, 3 or 5 g/L Xiaokening for 24, 48 or 72 hours. The effects of Xiaokening on the release of nitric oxide (NO) on the mesenteric arterioles were detected under shear stress of 1, 10 and 20 dyn/cm(2). Biochemical methods were used to determine the activities of superoxide dismutase (SOD) and xanthine oxidase (XO). The expressions of endothelial NO synthase (eNOS), SOD and XO in the mesenteric arterioles were assessed using Western blot.</p><p><b>RESULTS</b>Compared to normal rat arterioles, less NO was released in the mesenteric arterioles of diabetic rats. Xiaokening was found to have a concentration- and time-dependent effect on NO release; when the shear stress was increased, there was a gradual increase in the release of NO. Compared to normal arterioles, the expression of eNOS in the mesenteric arterioles of diabetic rats was lower. Incubation with Xiaokening increased SOD activity and expression, and decreased XO activity and expression in the mesenteric arterioles of the diabetic rats.</p><p><b>CONCLUSION</b>Xiaokening was able to significantly increase NO release and improve the endothelial function of mesenteric arterioles through antioxidative mechanisms.</p>

Effect of sitagliptin on the expression of NO/iNOS in type 2 diabetic nephropathy / 中国生化药物杂志

Objective To investigate effect of sitagliptin ( SIT) on the expression of inducible nitric oxide synthase ( iNOS) and nitric oxide ( NO) in type 2 diabetic nephropathy.Methods 30 rats were randomly divided into normal group (NC group), diabetic nephropathy group (DN group) and sitagliptin treatment group (SIT group).The type 2 diabetes mellitus (T2DM) rats were induced by a high fat diet (HFD) plus repeated low dose streptozocin (STZ) injections.At the end of the 12th week in treatment,there were 6 rats in each group, the NO level was determined by Griess method.mRNA levels of iNOS RT-PCR was detect ed by.The expression of iNOS protein was detected by western blot and immunohistochemical method. ResuIts Compared with the NC group, the expression of iNOS and NO of DN group increased significantly.However, compared with DN group, the expression of iNOS and NO of SIT group decreased significantly.ConcIusion SIT can decrease the expression of iNOS and NO, which implies SIT may protect the type 2 diabetic kidney.

Effects of FAR on myocardial fibrosis of diabetic rats / 中国药理学通报

Aim To study the effects of Free Anthra-quinone from Rhubarb (FAR)on myocardial CTGF and collagen expression and interstitial fibrosis in dia-betic rats.Methods The male SD rats were randomly divided into normal group (CON),diabetic cardiomy-opathy group (DCM) and FAR treatment group (FAR).Streptozocin was intraperitoneally injected in-to the animals in the latter 2 groups to induce diabetic rat model.The model was expected to be stable for 2 weeks before the treatment.At the end of the 8th week in treatment,fasting plasma glucose and heart mass in-dex were measured.Masson staining was used to ob-serve the myocardial fibrosis.RT-PCR was used to de-tect the mRNA levels of CTGF,procollagen type Ⅰand collagen type Ⅲ.Immunohistochemical method was used to detect the content of CTGF.ELISA was used to detect the depositions of collagen type I and collagen type Ⅲ. Results Compared with CON group,fasting plasma glucose,heart mass index,the degree of myocardial fibrosis,and the expressions of CTGF,collagen type I and collagen type Ⅲ in left ven-tricular myocardial tissue of DCM group were signifi-cantly increased. However, compared with DCM group,fasting plasma glucose,heart mass index,the degree of myocardial fibrosis,and the expressions of CTGF,collagen type I and collagen type Ⅲ in left ven-tricular myocardial tissue of FAR-treated rats were sig-nificantly decreased.Conclusion FAR retards the process of myocardial fibrosis in diabetic rats by down-regulating the expression of CTGF,reducing the syn-thesis and depositions of collagen type I and collagen type Ⅲ.

Rhein and archen induce the apoptosis of mesangial cells in high glucose rats / 中国组织工程研究

BACKGROUND:Our previous study has already manifested that Chinese medicine Xiaokening can effectively prevent and treat the early diabetic nephropathy. OBJECTIVE:To compare the effect of rhein and archen on the apoptosis of mesangial cells of rats cultured with high glucose. METHODS:Mesangial cells were stimulated by different concentrations of rhein and archen (20, 40 and 80 μmol/L). Morphology of apoptotic cells was observed by hematoxylin-eosin staining. Karyon apoptosis was examined by fluorescence staining of DAPI. cellapoptosis rate was detected by flow cytometry. RESULTS AND CONCLUSION:The outcome of hematoxylin-eosin staining and DAPI staining indicated that the effect of rhein was much stronger than that of archen on the apoptosis of mesangial cells in the rats cultured with high glucose. Comparison of the apoptosis rate also indicated that the rhein had a stronger effect on the earlier and the later stage apoptotic rate of mesangial cells than archen. Both rhein and archen with different concentrations can induce apoptosis of mesangial cells, but the effect of rhein is much stronger.

Effect of Acupuncture on Leptin Receptor Gene Expression in Arcuate Nucleus of Type 2 Diabetes Mellitus Rats / 中国康复理论与实践

@#Objective To study the relationships among leptin, leptin receptor (OB-R) gene in arcuate nucleus and type 2 diabetes mellitus, and the effect of acupuncture on them. Methods Obese rats due to diet were injected in abdomen with small dosage streptozotocin to induce type 2 diabetes mellitus model, and were divided into acupuncture, glibenclamide and model group. Normal rats were in control. After treatment for 4 weeks, fasting blood sugar (FBS), fasting insulin (FINS), leptin, OB-R mRNA expression in arcuate nucleus were evaluated. Height, weight were measured and Lee's index were calculated before and after treatment. Results The FBS and FINS were higher in model group ［(15.79±1.87) mmol/L, (37.20±3.92) mIU/L］ than in control ［(5.16±0.13) mmol/L, (23.57±1.63) mIU/L］, but decreased in acupuncture group ［(5.46±0.20) mmol/L, (20.87±1.80) mIU/L］ and glibenclamide group ［(5.26±0.13) mmol/L, (28.10±1.86) mIU/L］. Leptin was lower in model ［(0.95±0.15) ng/ml］ than in control ［(1.32±0.15) ng/ml］, but was higher in glibenclamide group ［(1.83±0.23) ng/ml］ than in control. Weight and Lee's index decreased in model, acupuncture and glibenclamide groups after treatment. OB-R mRNA expression in arcuate nucleus in model wasn't different from control, acupuncture or glibenclamide group, nor between acupuncture and control, but was lower in glibenclamide group than in control. Conclusion Leptin decreased in type 2 diabetes mellitus rats when they became thin. Leptin resistance appears when treated with glibenclamide. Acupuncture may improve the OB-R mRNA expression in arcuate nucleus to some extent.

The Protective Effect of Rhodobryum giganteum (Schwaegr.) Par. on Umbilical Vascular Endothelial Cells and the Influence on Secretion of NO and NOS / 中国实验方剂学杂志

Objective: To study the effect and mechanism of Rhodobryum giganteum (Schwaegr.) Par. 's anti-atherosclerotic effect. Methods:Vascular endothelial ceils were cultivated and the H2O2 were used to induce the oxidative stress injury of human umbilical vascular endothelial cells (HUVEC). Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was added to cultivated HUVEC, and the activity of the cells was carefully determined by OD value with MTF method. The Griess Reagent was used to detect the NO concentration of different groups. At the same time, the NO fluorescence analysis probe, DAF-FM DA. (3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate), was used to determine the activity of NO synthase. Results:The most suitable stimulating concentration of H2O2 on HUVEC is 12.5 mmol· L-1. Water extract of Rhodobryum giganteum (Schwaegr.) Par. Was co-cultured with HUVEC damaged byH2O2 .The OD values indicate that 3.33,2.50 mg· mL-1 water extract groups increase activity of the cells significantly compared with the model group (P <0.05) ,and 3.33 mg·mL-1 is much better than 2.50 mg·mL-1 dose group. To determine the content of NO and NOS, the 2.50 mg·mL-1 dose has significant effect (P < 0.05). Conclusions: The H2O2 concentration of 12.5 mmol· L-1 could be used to establish the injured model of endothelial cell successfully. 2.50 and 3.33 mg·mL-1 water extract of Rhodobryum giganteum (Schwaegr.) Par. Have protective effect on ECV304 injured by H2O2. 2.50 mg· mL-1 water extract of Rhodobryurn giganteum (Schw aegr.) Par could increase the activity of NOS and promote the synthesis and secretion of NO.

Effect of Xiaokening on adjusting the proliferation of mesangial cells in rats under high glucose / 中国组织工程研究

BACKGROUND: Chinese medicines have better effects on preventing and treating diabetic nephropathy at early period, and the effects are caused by the diversity of the effective components of Chinese medicines which acts on the different targets of diabetic nephropathy.OBJECTIVE: To observe the effect of Xiaokening on the proliferation of mesangial cells under high glucose, and investigate the possible mechanism. DESIGN: A controlled observation.SETTING: Department of Endocrinology, the First Affiliated Hospital of Nanjing Medical University.MATERIALS: The experiments were completed in the Research Room of Cell Culture, Research Center of Endocrine Metabolism, the First Affiliated Hospital of Nanjing Medical University from July 2003 to May 2004. The rat mesangial cell lines HBZY-1 were bought from Chinese Center for Typical Culture Collection.METHODS: The mesangial cells were passaged and cultured according to the instructions. ① Grouping according to different concentration of stimulation: In the high glucose+Xiaokening group, Xiaokening of 6 concentrations were used, I.e., 20.0, 40.0, 60.0, 80.0, 120.0, 200.0 g/L. Meanwhile,normal glucose control group and high glucose control group were also set,the glucose concentration in the medium was 5.6 and 30 mmol/L respectively. The proliferations were observed after 72 hours. Grouping according to different time points of stimulation: There were normal glucose control group, high glucose control group and high glucose+Xiaokening group, the glucose concentration in the medium was 5.6, 30 and 30 mmol/L respectively, and the Xiaokening concentration was 60 g/L. The proliferations were observed at 24, 48 and 72 hours respectively in the three groups.② The dispensed cell suspension was placed into the 96-well plate, and 200 μL suspension for each well. The culture plate was placed in the inoculation box containing CO2 (0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant was deserted, cell solutions containing different drugs of different concentrations were added in each group, 200 μL for each well. The 96-well plate was then placed in the culture box containing CO2 (0.05 in volume fraction) and 100% humidity at 37 ℃ for inoculation.In the groups of different concentrations, 20 μL MTT (5 g/L) was added into the wells after 72 hours. In the groups of different time points, 20 μL MTT (5 g/L) was added into the wells at 24, 48 and 72 hours. Then the plates were inoculated at 37 ℃ for 4 hours. Under microscope, it was observed that MTT get into the cells, the supernatant was sucked away, then 150 μL dimathyl sulfoxide was added to dissolve methyl alcohol, and shaken to even with plate rocking bed for 30 s, and the absorbance (A) values were recorded with the microplate reader at the wavelength of 492 nm.MAIN OUTCOME MEASURES: The proliferations of mesangial cells (A value) were observed at different time points and in Xiaokening of different concentrations.RESULTS: ①Proliferation of mesangial cells at different time points: The A values in the high glucose control group at 24, 48 and 72 hours (0.685 ±0.010, 0.750±0.087, 0.659±0.018) were higher than those in the normal glucose control group (0.586±0.054, 0.598±0.040, 0.527±0.047, P ＜ 0.05). In both the high and normal glucose control groups, the A value at 48 hours was higher than that at 24 hours (P ＜ 0.05), but the A value at 72 hours was lower than that at 48 hours (P ＜ 0.05). The A value in the high glucose+Xiaokening group at 72 hours was 0.538±0.023, and it was lower than that in the high glucose control group (P ＜ 0.05). ② Proliferation of mesangial cells in Xiaokening of different concentrations: Xiaokening high er than 60.0 g/L could obviously restrain the excessive stimulation of high glucose to the mesangial cells, and the effect was in a concentration-de pendent manner, but too high concentration (120.0 g/L) would result in the abscission and death of cells, whereas no survived cells could be observed in extremely high concentration (200.0 g/L). CONCLUSION: Xiaokening could inhibit the proliferation of mesan gial cells stimulated by high glucose after 72 hours in a concentration dependent manner, but too high concentration would cause strong cytotoxicity.

Regulations of acupuncture on hepatic tissue IRS2 gene expression in type 2 diabetes rats / 中华中医药杂志

Objective:To study the relationship between hepatic tissue insulin receptor substrate 2(IRS2) gene and type 2 diabetes,and the regulation of acupuncture on IRS2 gene expression of hepatic tissue in type 2 diabetes mellitus(T2DM) rats.Methods:Obese rats due to diet were injected in abdomen with small dose streptozotocin(STZ) to induce T2DM.Then these T2DM rats were stochastically divided into acupuncture group,Glibenclamide group and model group.After 4 weeks treatment,fasting blood sugar(FBS) was examined with active blood sugar meter,fasting insulin(FINS) was examined by radioimmunoassav,hepatic tissue IRS2mRNA was evaluated in real-time RT PCR,and normal rats were in control.Results:Serum levels of FBS(15.79?1.87) and FINS(37.20?3.92) in model group were higher than those in normal group(5.16?0.13,23.57?1.63),serum levels of FBS(5.46?0.20) and FINS(20.87?1.80) in acupuncture group were obviously lower than those in model group,which were similar to normal group.FBS(5.26?0.13) reduced obviously and serum FINS(28.10?1.86) reduced in some degree in Glibenclamide group compared with model.Relative contents of IRS2mRNA in model group were 2.19 folds compared with that in normal group.And in Glyburide group it was 4.59 folds compared with that in normal group.In acupuncture group it was 4.04% of that in the normal group.Conclusion:Abnormal hepatic tissue IRS2 mRNA expression may be one of pathogenesis of T2DM.Acupuncture can obviously depress hepatic tissue IRS2 mRNA expression,but Glibenclamide can not.

Thoughts on ameliorating equipment and apparatus in the preparation room of the hospital / 医学研究生学报

Based on the requirements of Good Preparation Practice in Medical Institutions(GPP) and considering the actual conditions of the hospital,we decided upon a right way in the reconstruction of the hospital's preparation room and amelioration of the equipment and apparatus that constituted 30% of the investment.The results were reduced investment,higher efficiency,with perfect conformity to the GPP requirements.

Experimental study on treatment of simple obesity with acupuncture / 医学研究生学报

Objectives: Observe the effects of acupuncture on obese SD rats fed by self-prescribed high calorie diet. 　Methods: The model group were male SD rats fed by self-prescribed high calorie diet, while the control group were male SD rats fed by common roughage. After acupuncture, the changes of some indexes were observed such as eating and drinking amount,body weight,length, Lee index,fattiness in groin and circumrenal areas, TG,TC,BG levels in blood etc.　 Results: The obesity was defined as the average body weight of the model group was 20% heavier than the average body weight in the control group. And the obesity ratio was 56%. The indexes were changed in different degrees with statistic significance after one period of treatment.　 Conclusions: Over-nutrition is one of the causes of obesity. Our high calorie feed for obese rat is reasonable. Acupuncture has obvious therapeutic effects for simple obesity.

Studies on HPLC fingerprint of Herba Rhodobryi Rosei / 中草药

Objective To establish an HPLC fingerprint of Herba Rhodobryi Rosei.Methods The fingerprint chromatography has been determined by RP-HPLC.The analysis was carried out with Dikma ODS C18 column(250 mm?4.6 mm,5 ?m).The mobile phase were:0.5% HAcCN(A),0.5% HAcH2O(B);Elution method:0-50 min,A was 20%-55%;50-60 min,A was 55%-95%;60-85 min,A was 95%-100%;keeping 5 min.Flow-rate was 1.0 mL/min.Wavelength was 360 nm and temperature was 30 ℃.It was analysized with the Estimating System of Similarity of 2004A Version(the Country's Pharmacopeia Committee)on the Chinese Medicine Fingerprint Chromatography.Results The fingerprint chromatography of Herba Rhodobryi Rosei was estabilished.Conclusion The method can be used in quality control of Herba Rhodobryi Rosei with accuracy and better repeatability.