Therapeutic effects of losartan and astragalus membranaceus on diabetic nephropathy

AIM: To observe the protective effects of losartan and astragalus membranace on the kidney of diabetic rats, and to study their possible mechanisms. METHODS: The diabetic rats were induced by a single intraperitoneal injection of streptozotocin. At the end of 12th week,changes in urinary albumin excretion, urinary β₂-MG excretion, Ccr,NO,ET-1 levels in blood, urinary and renal tissue were observed. Serum and urinary TGF-β₁ concentration,average volume of glomeruler,average thickness of glomerular basement membrane were also measured. RESULTS: In the treated diabetic rats, urinary albumin excretion, urinary β₂-MG excretion, Ccr, urinary and renal tissue NO, urinary TGF-β₁, average volume of glomeruler, average thickness of glomerular basement membrane decreased obviously as compared with diabetic untreated rats. These effects were enhanced when losartan was combined with astragalus membranace. CONCLUSION: Losartan or astragalus membranace reversed the injury of renal structure and function in STZ-induced diabetic rats. The protective effects were enhanced when losartan was combined with astragalus membranace. The decrease in NO,ET,TGF-β₁ concentration in renal tissue may be one of mechanisms for this action.     

Expression and probable role of extracellular signal-regulated protein kinases in renal fibrosis associated with diabetic in mice

AIM: To investigate the expression and probable role of extracellular signal-regulated protein kinase (ERK1/2) in renal fibrosis associated with diabetic in mice.METHODS: Male homozygous C57BL/6 mice were divided at random into control group (intraperitoneally injected with citrate buffer) and diabetes group (received 5 consecutive daily intraperitoneal injections of streptozotocin at dose of 50 mg·kg^-1·d^-1).All mice were followed up for 16 weeks.Diabetes was confirmed by serum glucose levels exceeding 16.7 mmol/L.Mice were killed at 0,4,8,12 and 16 weeks respectively after streptozotocin injection.The kidney tissues were obtained from diabetic and control mice.Serum glucose,kidney weight/body weight (KW/BW),24 h albumin excretion rate (UAE) and the serum creatinine (Scr) were measured.The kidney tissue was used for histological and morphometric studies of glomerular size,glomerular matrix expansion (PAS),and the expression of TGF-β₁,phosphorylated ERK1/2 and collagen Ⅲ by immunohistochemical staining.RESULTS: The serum level of glucose in streptozotocin -induced diabetic mice increased significantly.The kidney weight/body weight ratio,glomerular volume and glomerular matrix expansion in diabetic mice were obviously higher than those in control mice.Serum creatinine and 24 h albumin excretion rate in diabetic mice increased significantly compared with control mice.TGF-β₁,phosphorylated ERK1/2 and collagen Ⅲ levels were obviously increased in the kidney of diabetic mice compared with those in control mice (P<0.01).TGF-β₁ expression was positively related to the expression of phosphorylated ERK1/2.CONCLUSION: The overexpression of phosphorylated ERK1/2 in diabetic kidney may play an important role in the development of renal fibrosis associated with diabetic nephropathy in mice.     

Influence of irbesarten on renal hypertrophy in streptozotocin-induced diabetic rats LIU Bi-cheng,LUO Dong-dong,ZHANG Xiao-liang.

AIM: To investigate the influence of irbesartan (Irb), a new angiotensin II receptor 1 antagonist, on renal hypertrophy in streptozotocin (STZ)-induced diabetic rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: normal group (Group N, n=7), diabetic group (Group DN, n=6) and irbesartan treated group (Group DNI, n=7). In the experimental group, after the rats subjected to uninephrectomy, STZ was given by peritoneally injection at bolus dose of 50 mg/kg to induce diabetes. Blood glucose (BG), body weight (BW), urinary albumin excretion (Ualb), 24 hour proteinuria (24 h Upro) were measured at week 4, 8, 12, respectively. By the end of experiment at week 12, creatinine clearance (Ccr), kidney weight (KW), indicator of renal hypertrophy (KW/BW), renal total protein content (RTP), glomerular area (AG) and glomerular volume (VG) as well as glomerular basement membrane (GBM) were determined by semi-quantitative pathology technique. RESULTS: It was showed that there was no significant difference in BG between group DN and DNI, while Irb significantly reduced the increasing of Ualb, 24 h Upro in diabetic rats compared to control group (P＜0.01, respectively). Furthermore, Irb markedly inhibited the increasing of KW, KW/BW, RTP, A_G, V_G in diabetic rats (P＜0.05, P＜0.01, respectively). Of interest, Irb significantly prevented the increasing of GBM in diabetic rats. CONCLUSION: Irb exerts its early renal protective action by reducing proteinuria and inhibiting renal hypertrophy as well as the thickening of GBM.     

Role of renal cortex endothelin and NO in the development of proteinuria

AIM: To test the hypothesis that locally produced renal endothelin(ET) and nitric oxide(NO) play more important role in initiating proteinuria than circulating ET and NO. METHODS: (1) Rat nephrotic syndrome model was made by single injection of adriamycin, and the urinary protein excretion per 24 hours for every two days was measured. (2) Rat plasma and renal cortex were collected respectively 4 days, 8 days, 32 days and 56 days after the injection, and renal cortex homogenate was further prepared; ET of both plasma and renal cortex homogenate were determined by radioimmunoassay; NO of both plasma and renal homogenate were measured by the method of Griess. (3) The correlation between urinary protein and ET or NO concentration was analyzed respectively. RESULTS: (1) The urinary protein excretion of normal rat was 4-7 mg/d, that in nephrotic rat at the 4 time points was 5, 10, 241 and 201 mg/d, there was a significant increase of that at day 8, peaked at day 32, and less decrease at day 56. (2) The plasma ET in nephrotic rats of the 4 time points was 134, 150, 538 and 445 μg/L, respectively, which of day 8, day 32 and day 56 shown significant higher than that of normal control (126-129 μg/L). (3) The ET in renal cortex homogenate of nephrotic rat of the 4 time points was 364.6, 652.3, 1 526.8, 1 393.6 pg/g, all of that was much more higher than that of normal rat . (4) The plasma NO in nephrotic rat of the 4 time points was 40, 36, 8 and 11(μmol/L), there was considerable difference through day 8 to day 56 than control (42-46μmol/L, P＜0.05). (5) The NO in renal cortex homogenate of nephrotic rat was 80, 69, 8, 25 μmol/L that of normal rat was (114-124) μmol/L, there was significant difference between nephrotic rat and control from day 4 through day 56( P＜0.05). (6) The correlation coefficient of plasma ET and NO with separately urinary protein excretion shown statistic significance on only day 8 and day 56, while all of that of ET and NO in renal homogenate closely related with urinary protein from day 4 through to day 56. CONCLUSION: The ET and NO produced locally in renal play more important role in the development of proteinuria.     

Effects of piceatannol on rat kidney with diabetic nephropathy in early stage

AIM: To observe the effect of piceatannol on the kidney of diabetic nephropathy rats in early stage, and to explore the possible mechanisms.METHODS: The rats were randomly divided into 5 groups:control group, model group, low dose of piceatannol treatment group, medium dose of piceatannol treatment group and high dose of piceatannol treatment group. The rat model of diabetic nephropathy was induced accordingly, and the rats received 20 mg/kg, 40 mg/kg or 60 mg/kg of piceatannol by gavage once a day for 4 weeks. Blood glucose was detected by glucometer. The urea nitrogen and creatinine levels in the serum were measured by urease-glutamate dehydrogenase enzymatic and inosine acid oxidase methods, respectively, and 24 h urinary microalbumin was analyzed by immune transmission turbidimetry test. Moreover, the pathological changes of the kidney tissues were observed under microscope with HE staining. The protein expression of TGF-β1 and Smad 7 and the phosphorylation levels of Smad2 and Smad3 were determined by Western blot. RESULTS: Compared with model group, piceatannol treatment significantly decreased the levels of blood glucose, blood urea nitrogen and urinary microalbumin, but had no effects on serum creatinine. Furthermore, HE staining showed that the increased mesangial cells, matrix hyperplasia and degenerated epithelial cells in model group were markedly inhibited after piceatannol treatment. Additionally, piceatannol treatment also reduced the protein expression of TGF-β1 and Smad 7, and the phosphorylation levels of Smad2 and Smad3. CONCLUSION: Piceatannol attenuates pathological progression in the kidney of diabetic nephropathy rats in early stage, which may be through inhibiting TGF-β/Smad signaling pathway.     

A pilot study on mechanisms of hyperadiponectinemia in two rat models with different in vivo insulin sensitivity

AIM:To explore the possible mechanisms of hyperadiponectinemia in the insulin-resistant Zucker fatty rats (ZF) and insulin-sensitive nephrotic syndrome rats induced by puromycin aminonucleoside (PA).METHODS:8-week old Zucker rats were fed with standard rat chow for 4 weeks and Wistar rats were intraperitoneally injected single dose of PA 10 d before experiments.Blood samples and 24 h urines were collected for detecting the lipid profiles, adiponectin (ADPN), albumin and urine albumin excretion rate (UAER).A frequently sample intravenous glucose tolerance test (FSIVGTT) was conducted.The insulin sensitivity index (SI) and glucose effectiveness index (SG) were calculated according to Bergmans minimal model.Epididymal white adipose tissues were then extracted for weighting.Correlations between ADPN and other parameters were analyzed by Pearsons correlation.RESULTS:Both ZF rats and PA-injected rats showed significantly higher plasma ADPN levels compared with their own controls ［(7.44±1.23)mg/L vs (2.44±0.33)mg/L and (8.64±0.88)mg/L vs (2.95±0.46)mg/L, respectively, P<0.01］.SI and SG were markedly decreased and negatively correlated to ADPN in Zucker rats, but not changed and correlated to ADPN in Wistar rats.Hyperlipidemia was observed in either ZF rats or PA-injected rats and was positively correlated to ADPN levels.Two hyperadiponectinemic rats showed markedly higher UAER ［(64.8±14.2)mg/24 h vs (14.9±14.6)mg/24 h and (275.1±64.5)mg/24 h vs (15.4± 4.5)mg/24 h, respectively, P<0.01］ and positively correlated with ADPN.The ratio of white adipose tissue to body weight was higher in ZF rats than that in their control rats despite its disproportion with ADPN, while it was comparable between PA-injected and normal rats.The renal creatinine clearance rate was not altered in both strains of rats.CONCLUSION:Hyperadiponectinemia is a common property in ZF rats and PA-induced nephrotic syndrome rats, and it cant be interpreted by changes of in vivo insulin sensitivity.Markedly increased UAER in both rats is the key mechanism of hyperadiponectinemia.     

Effects of vasoconstrictor and endothelium-dependent relaxationagent on thoracic aortic rings of diabetes mice

AIM: To discuss the effects of vasoconstrictor and endothelium-dependent relaxation agent on the thoracic aortic rings in different stages of diabetes mellitus. METHODS: Streptozotocin (STZ, 40 mg/kg) was injected intraperitoneally to establish diabetic model in C57BL/6J mice. At the 17th, 22nd, 28th week, diabetic and age-matched control mice were sacrificed respectively and the effects of vasoconstrictors: phenylephrine (PE), 60 mmol/L KCl and endothelium-dependent relaxation agent respectively (ACh) were measured in two groups using thoracic aortic rings. RESULTS: The level of fasting plasma glucose concentration 2 weeks after STZ treatment increased higher (≥11.1 mol/L) in STZ-induced diabetic mice than that in age-matched controls, and maintained at this level in entire experiment course. On the contrary, the weight was decreased significantly. The responses of thoracic aortic rings in STZ-induced diabetic mice to PE were increased, unaltered and increased at the 17th, 22nd, 28th weeks, respectively compared to that in age-matched controls. The responses to 60 mmol/L KCl were also increased in all stages. While the responses to ACh were increased, unaltered and decreased at the 17th, 22nd, 28th weeks, respectively compared to that in age-matched controls. CONCLUSION: The responses of thoracic aortic rings to vasoconstrictor enhance in STZ-induced diabetic mice. However, the endothelial functions potentiate initially due to compensation, and then lower exhibiting endothelial damage.     

Effects of pulse high-volume hemofiltration on serum concentration of moxifloxacin in patients with continuous renal replacement therapy

AIM: To study the changes of the serum moxifloxacin concentration in the patients with continuous renal replacement therapy when applied for pulse high-volume hemofiltration (PHVHF). METHODS: The patients applied for PHVHF were intravenously given moxifloxacin at the dose of 400 mg in 60 min. Blood samples were collected from the arterial (input) and venous (output) lines of the extracorporeal circuit immediately at different time points after the beginning of infusion, and replacement fluid was taken at the corresponding time points. The serum concentration of moxifloxacin was measured by high-performance liquid chromatography (HPLC). The pharmacokinetics parameters were determined using DAS 2.1.1 software.RESULTS: Two-compartment model was used to achieve the best concentration-time relation of moxifloxacin. The levels of blood urea nitrogen (BUN), serum creatinine (SCr) and serum K⁺ were obviously decreased after application of PHVHF (P<0.05). The peak serum concentration (C_max) of moxifloxacin was (4.843±1.854) mg/L. The half-life (T_1/2) was (4.822±2.126) h and the peak time (T_max) was (1.31±0.59) h. The total volume of distribution (V_d) was (82.63±24.69) L. The total clearance (CL_tot) was (14.36±8.43) L/h. All the ratios of area under the concentration-time curve (AUC) to 90% minimal inhibitory concentration (MIC₉₀) were more than 100 when the values of MIC₉₀ were 0.25 mg/L, 0.12 mg/L and 0.03 mg/L. All the values of C_max/MIC₉₀ were more than 10 when the values of MIC₉₀ were 0.25 mg/L, 0.12 mg/L and 0.03 mg/L.CONCLUSION: Application of PHVHF obviously improves the renal functions and eliminates a part of moxifloxacin in the serum. However, the serum concentration of moxifloxacin remains at the effective level for controlling the pathogenic bacteria.     

The protective effects of angiotensin Ⅱ receptor blocker irbesartan on kidney function in diabetic rats

AIM: To investigate the effects and mechanisms of irbesartan, one of the angiotensin Ⅱreceptor blockers, on kidney function in diabetic rats. METHODS: Forty adult male Wistar rats were randomly divided into four groups: control group, diabetes group, irbesartan group and captopril group. At the end of 12 weeks, the rats were sacrificed. Urine volume, body weight, kidney weight/body weight, plasma, glucose, glycosylated hemoglobin (HbA₁c), urinary β₂-microglobulin (β₂-MG) excretion, urinary albumin excretion rate (UAR), creatinine clearance (Ccr) were measured. Nitric oxide (NO) and endothelin-1 (ET-1) levels in plasma, urinary and renal tissues were determined. RESULTS: Urine volume, kidney weight/body weight, plasma glucose, HbA1C, UAR, Ccr, urinary β₂-MG excretion, NO and ET-1 levels of urinary, blood and renal tissue in diabetic rats were significantly higher than those of normal controls ( P<0.01). UAR, Ccr, urinary β₂-MG excretion, ET-1 and NO levels of urinary and renal tissue in rats of irbesartan and captopril groups were significantly lower than those of DM rats ( P<0.01). There were positive relationships among the levels of plasma, urinary, renal tissue ET-1, NO and UAR, Ccr and urinary β₂-MG excretion. CONCLUSION: Irbesartan could prevent from the injury of renal function in STZ-induced diabetic rats. And it maybe one of the most importan mechanisms that irbesartan could reduce the NO and ET-1 levels in STZ-induced diabetic rats.     

Expression of CTGF and its role in the streptozotocin-induced diabetic rat kidney

AIM:To investigate the change of connective tissue growth factor(CTGF) expression and its role in streptozotocin-induced diabetic rat kidney. METHODS:Male SD rats were randomly divided into two groups: control(sham operated rats, group C，n=32) and diabetic rats (group DN，n=35). Rats in each group were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Body weight（BW）, blood glucose（BG）, 24-hour urine volumn（UV）, kidney weight, KW/BW,24-hour urinary albumin excretion (24Ualb), creatinine clearance (Ccr), kidney weight (KW), KW/BW, glomerular area (A_G), proximal tubular area (A_T) and the width of GBM、TBM at each time point were measured. Expression of CTGF and α-SMA were detected by immunostaining. RESULTS:There was a significant increase of 24 h Ualb, Ccr, KW/BW, A_G, V_G and the expression of CTGF in glomeruli and tubuli from week 1 onward in diabetic rats compared with those in group C (P<0.05, P<0.01, respectively), and an increasing expression of α-SMA mainly located in dilated tubuli from week 4 was found in group DN, which was more evident at week 8 accompanied by the decrease in A_T. Diabetic rats also had a significant increase in A_T from week 1 onward, which peaked at week 4. CONCLUSION:In the early stage of DN, the time-dependently upregulated CTGF might mediate the renal hypertrophy, which might be associated with the subsequent tubular epithelial-mesenchymal transdifferentiation (EMT) and renal fibrosis.     

Histochemical changes of skin in diabetic rats

AIM: To explore the histochemical changes of diabetic skin and the pathogenesis of impaired wound healing in diabetes. METHODS: 54 male Sprague-Dawley (SD) rats weighing 200-220 g were randomized into control and STZ-induced diabetic groups. The shaved skin specimens from the back of rats were collected in 4, 8 and 12 weeks post STZ-induction, respectively. Hematoxylin-eosin dye was used for histological examination. Meanwhile, the skin glucose contents were measured by Beckman’s autoanalyzer. Skin AGEs concentrations were assessed by detecting total fluorescence in tissue collagen and immunohistochemistry assay. RESULTS: The skin thickness in diabetic animals was decreased, with the features of multilayer epithelium structure disappeared in epidermis and collagen fibers atrophied, swollen and degenerated in dermis; The inflammatory responses in the dermis of diabetic animals were increased obviously. The results also revealed that skin glucose contents in diabetic rats ［(2.64±1.03）mg/g skin］ were 2-3 times higher than those in the controls ［(0.74±0.33)mg/g skin］ (P<0.01). The collagen fluorescence and AGEs positive expressions in diabetic skin enhanced significantly when compared with age-matched controls over the whole experimental course (P<0.05). CONCLUSIONS: A histochemical changes had already been occurred in diabetic skin before injury, which may be result from the local biochemical factors such as high concentrations of glucose and AGEs. These might be an important mechanism in the pathogenesis of impaired wound healing in diabetes.     

Protective effect of curcumin on myocardium in diabetic rats

AIM: To study the effects of curcumin (Cur) on diabetic cardiomyopathy (DCM) in rats. METHODS: Male Wistar rats (n=75) were divided into control group and diabetes model group, in which the rats were fed with high-fat diet and then intraperitoneally injected with streptozotocin (STZ, 40 mg/kg). Fasting blood glucose was measured 72 h and 1 week after STZ injection. The diabetic rats were diagnosed when sustained fasting blood glucose levels ≥ 11.6 mmol/L. The diabetic rats were randomly divided into DCM group, DCM+Cur 100 mg/kg group and DCM+Cur 200 mg/kg group. After treatment for 16 weeks, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) level were measured, and the level of cardiac troponin I (cTnI) in the serum was determined by enzyme-linked immunosorbent assay. The protein expression of protein kinase C (PKC) was detected by Western blotting. RESULTS: Curcumin significantly decreased the blood glucose level, increased the body weight, inhibited MDA content and up-regulated the GSH-Px activity in the diabetic rats. Furthermore, curcumin treatment inhibited the diabetes-induced protein expression of PKC. CONCLUSION: Curcumin may have a protective effect on diabetic cardiomyopathy by attenuating oxidative stress.     

Expression of cathepsin B and cystatin C and its potential role in diabetic rats

AIM: To observe the expressions of cathepsin B (CB) and cystatin C (CC) in different stage of diabetic rats and to investigates their potential roles.METHODS: Sixty rats were divided into diabetes mellitus group induced by intravenous injection of streptozotocin (55 mg/kg) and normal group injected with citrate buffer. Ten rats were sacrificed respectively at the end of fourth week, eighth week and sixteenth week in both groups. 24 h urine excretion was collected in rats before sacrifice. The blood and the kidney were also collected. The mRNA and protein expressions of CB and CC in kidney were detected by real time PCR and immunohistochemical staining, respectively.RESULTS: At the end of eighth week, the expression of Ccr, 24 h urinary protein excretion, CB, CC in diabetic rats increased significantly, compared to the results at the fourth week (P<0.01 or P<0.05). With the aggravation of diabetic nephropathy, the expressions of CC, colⅣ, FN and 24 h urinary protein excretion were up-regulated significantly (P<0.01 or P<0.05). The expression of CB in diabetic rats was up-regulated at eighth week significantly (P<0.01), whereas, at the end of sixteenth week it was down-regulated significantly (P<0.01). The 24 h urinary protein excretion, the expressions of colⅣ at protein and mRNA levels and FN were negatively correlated with CB (P<0.01).CONCLUSION: The unbalance of CB and CC exists in diabetic nephropathy renal tissue, which is likely to lead to the accumulation of extracellular matrix.     

Effect of extracts of ginkgo biloba leaves on activation of RBL-2H3 cells

AIM: To investigate the mechanism of extracts of ginkgo biloba leaves (EGB) on degranulation from mast cells. METHODS: Wistar rats were randomly divided into 6 groups, including normal group, control group, high dose of EGB group (30 mg/kg), moderate dose of EGB group (10 mg/kg), low dose of EGB group (3 mg/kg) and positive control medicine group (azelastine, 5 mg/kg), each group had 10 rats (half female and half male). The experiment of passive cutaneous anaphylaxis reaction (PCA) and colorimetry were used to determine the effect of EGB on degranulation of mast cells in vivo. For in vitro study, various doses of EGB (200 mg/L, 100 mg/L and 50 mg/L) were added to the culture medium of RBL-2H3 cells cultured with 200 μg/L of dinitrophenyl (DNP) specific IgE overnight. The azelastine (30 mg/L) was selected as the positive control. The antigen (DNP-human serum albumin, DNP-HSA, 20 μg/L)-induced release of degranulation was measured by enzymatic assay, histamine by EIA, leucotriene C4 (LTC4), interleukin-4(IL-4) and tumor necrosis factor-α (TNF-α) by ELISA, respectively. In addition, the effect of EGB on phosphorylation of Akt and p38 was observed by Western blotting. RESULTS: The results showed that treatments with high dose of EGB (30 mg/kg) and moderate dose of EGB (10 mg/kg) were followed by a decrease in PCA of rats. All doses of EGB (200 mg/L, 100 mg/L and 50 mg/L) obviously suppressed the degranulation from RBL-2H3 cells, whereas results in high dose group (200 mg/L) and moderate dose of EGB group (100 mg/L) indicated significantly inhibitory effect on β-hexosaminidase, histamine, LTC4, IL-4,TNF-α, phosphorylation of Akt and p38 of RBL-2H3 cells induced by antigen. CONCLUSION: EGB may suppress the anaphylactic reaction by inhibiting the action of mast cells. Akt and p38 at least in part contribute to this event.     

Study on the role of oxidative stress in the kidneys of diabetic rats

AIM: To investigate the changes of oxidative stress in the kidneys and their roles in nephropathy in diabetic rats. METHODS: Diabetic rats were induced by streptozotocin (STZ). 36 rats were divided into three groups randomly: (1) NC group, normal control rats; (2) DM group, diabetic rats received protamine zinc insulin (PZI) 2U-4U/2 d; (3) DT group, diabetic rats received PZI 9-12 U/kg body weigh/day. 12 weeks later, rats were killed, blood glucose, blood cholesterol, serum creatinine, blood urea nitrogen, HbA1c, urinary creatinine, and urinary protein for 24 h were measured. The activities of antioxidant enzymes in renal cortex, including total superoxide dismutase (TSOD), Cu-Zn superoxide dismutase (Cu-Zn SOD), Mn superoxide dismutase (Mn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and maleic dialdehyde (MDA) were measured by chromatometry. RT-PCR was performed to detect the expression of different antioxidant enzymes mRNA. RESULTS: For all the targets we measured, there was no significant difference between NC and DT groups. Compared with the other two groups, the levels of blood glucose, cholesterol, trigalloyl glycerol, HbA1c in DM group increased significantly. The activities of TSOD, Cu-Zn SOD and CAT decreased significantly. The activity of GSH-Px increased significantly. There was no significant difference among the activities of Mn SOD in all three groups. The level of MDA in DM group was much higher than that in NC or DT group. The relative expression levels of GSH-Px and Cu-Zn SOD mRNA in DM group were higher than those in other two groups, while the relative expression level of CAT decreased. Mn SOD mRNA was expressed without significant difference in all groups. Compared with NC or DT group, urinary protein in DM group increased significantly, while creatinine clearance rate decreased. CONCLUSIONS: Hyperglycemia affected the expression of antioxidant enzymes. Oxidative stress was caused by hyperglycemia in diabetic rats and may be an important factor in the etiology of diabetic nephropathy.     

The expression of heme oxygenase-1 in vascular smooth muscle cells induced by lipopolysaccharide

AIM:To study the alterations of heme oxygenase-1 mRNA in vascular smooth muscle cells(VSMC) induced by lipopolysaccharide(LPS) and the role of heme oxygenase(HO)/carbon monoxide(CO)pathway in the disorders of regulation of cardiovascular system by LPS. METHODS: LPS (final concentrations 10 mg/L,30 mg/L and 50 mg/L) was added in cultured VSMCs for 6 h respectively or 10 mg/L LPS for 9 h and 18 h. MDA content, LDH release and the rate of trypan blue uptake of VSMC were measured. HO-1 mRNA expression was examined by Northern Blot. RESULTS:VSMC HO-1 mRNA expression was increased gradually with the increasing of LPS concentration. When final concentration of LPS was 50 mg/L, the HO-1 mRNA expression of VSMC was increased by 176.7% compared with control. When LPS final concentration was 10 mg/L, the HO-1 mRNA expression increased gradually along with the culture time. When cultured for 18 h, the HO-1 mRNA expression of VSMC was increased by 195.6% compared with control. Only at LPS 50 mg/L for 6 h and 10 mg/L for 18 h, the rate of trypan blue uptake,MDA content and LDH release were significantly increased. CONCLUSION: LPS can induce the HO-1 mRNA expression of VSMC and that were dose-dependent and time-dependent. The inducible HO may play an important role in the pathogenesis of vascular system under LPS.     

巴氏蘑菇多糖对糖尿病小鼠脂代谢紊乱的干预作用

选择健康雄性昆明小鼠随机分组,建立糖尿病小鼠模型后分别灌胃不同剂量的巴氏蘑菇(Agaricus blazei)多糖(低、中和高剂量组分别灌胃50、100和200mg/kg/d),阳性对照组灌胃200mg/kg/d阿卡波糖,正常对照和模型组灌胃生理盐水,7周后考察巴氏蘑菇多糖对糖尿病小鼠脂代谢的影响。结果表明:与糖尿病模型组相比,多糖各剂量组和阳性对照组空腹血糖值和血清总胆固醇含量均显著下降;多糖中、高剂量组和阳性对照组的血清甘油三酯含量也显著下降;与模型组相比,附睾脂肪中阳性对照组的glut4、pi3k、akt1和akt2mRNA的表达量均显著升高,多糖组中glut4(低、中和高剂量组)、akt1(低、中和高剂量组)、akt2(中和高剂量组)和pi3k(高剂量组)mRNA的表达量显著升高。     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

Apoptosis in diabetic foot ulcers and human fibroblast cells treated with AGEs

AIM：To investigate cell apoptosis in diabetic foot ulcers and the effect of advanced glycosylation end products (AGEs) on apoptosis in human fibroblast cells. METHODS：Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited. The clinical and biochemical features were compared by statistics methods. Skin biopsies were obtained from foot. Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex (SABC) staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique was used to detect apoptosis of the skin tissues. Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose (changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose). After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3. Other cells were trypsinized, washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis. RESULTS：Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration. The protein level of cleaved caspase-3 was significantly higher in diabetic group, suggesting that apoptosis was increased in diabetic skin tissues. TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%±08%), which further confirmed that cell apoptosis was increased in diabetic foot tissues. In human fibroblasts, the levels of cleaved caspase-3 in normal group, sustained high glucose group, fluctuant high glucose group and AGEs group were 080±0.13, 1.22±0.18, 1.46±0.32 and 1.83±0.25, respectively. The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99%±0.24% and 6.83%±0.36%, respectively. Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group. CONCLUSION：Increased apoptosis in diabetic foot ulcers is one of the most important reasons for impaired wound healing. As compared to sustained high glucose and glucose fluctuations, AGEs induce greater apoptosis in human fibroblast cells.