罗格列酮抑制糖基化终产物诱导心肌成纤维细胞增殖和结缔组织生长因子及Smad的表达

Objective To investigate the effects of rosiglitazone on the proliferation,connective tissue growth factor and Smad expression in cultured cardiac fibroblasts induced by advanced glycosylation end-products (AGEs).Methods After being treated with various amounts of rosiglitazone,the cultured neonatal rat cardiac fibroblasts were incubated with AGEs.The status of cardiac fibroblasts proliferation and cell cycle were detected by 3-(4,5-dimethyhhiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTI) assay and flow cytometry.Furthermore,ELISA technique was applied to identify the level of TGF-β1.The protein expressions of CTGF and Smad in cardiac fibroblasts of neonatal SD rats were detected with Western blotting.Results The exposure of cardiac fibroblasts to AGEs at doses of 0-200 mg/L induced a dose-dependent increase in cell proliferation.At the concentration of rosiglitazooe (0.1,1,and 10 μmol/L),the cell proliferation was reduced compared with 200 mg/L AGEs group by O.823±0.072,0.785±0.060,0.601±0.081 vs 0.981±0.049,respectively (P < 0.05).The increased levels of TGF-β1 in supematants of cultured cardiac fibroblasts stimulated by AGEs were inhibited by rosiglitazone at the concentrations of 0.1,1,10μmol/L by 257.77±9.09,230.29±6.56,200.84±10.26 vs 300.68±8.56,respectively (vs 200 mg/L AGEs,P<0.01).Western blot indicated that pretreatment with rosiglitazone (0.1,1,and 10 μmol/L) inhibited CTGF protein production in a dose-dependent by 0.769±0.108,0.590±0.095,0.534±0.115 vs 1.021±0.113,respectively (vs 200 mg/L AGEs,P<0.01).It was also demonstrated that pretreatment with rosiglitazone (1 and 10 μmol/L) inhibited Smad2 protein production by 0.424±0.059,0.396±O.080 vs 0.572±0.073,respectively (vs 200 mg/L AGEs,P < 0.05 or P < 0.01).Meanwhile pretreatment with rosiglitazone (1 and 10 μmol/L) inhibited Smad4 protein production by 0.580±0.063,0.556±0.051 vs 0.672±0.059,respectively (vs 200 mg/L AGEs,P < 0.05 or P < 0.01).Conclusions The findings suggest that AGEs promote the proliferation of cardiac fibroblasts and stimulate the protein production of Smad and CTGF of cardiac fibroblasts.Rosiglitazone inhibits the above reaction.These results indicate that CTGF/Smad pathway may play an important role in the protective effect of rosiglitazone on myocardial fibrosis.     

Upregulation of miR-29b contributes to the anti-fibrotic effect of macrophage migration inhibitory factor in diabetic myocardial fibrosis

Background Macrophage migration inhibitory factor(MIF) possesses proinflammatory function when secreted from the cells, and it also exhibits antioxidant properties based on its intrinsic oxidoreductase activity.However, the role of MIF in cardiac fibrosis is not well known. In the present study, the effect of MIF on fibrosis-associated gene expression and the underlying mechanism were examined. Methods The collagen content in mouse myocardium was detected by Masson staining. Expressions of MIF and fibrosis-associated Col1a1, Col3a1 and α-SMA in mouse myocardium or mouse cardiac fibroblasts were detected by quantitative real-time PCR and Western blot assay, respectively. Mature miR-29b expressions in mouse myocardium and cardiac fibroblasts were determined by real-time PCR. Smad3 activation in MIF-treated cardiac fibroblasts was also detected by Western blot assay. Results Compared with the db / m control mice, the collagen content was significantly increased in the myocardium of diabetic db / db mice. MIF was up-regulated, but miR-29b was down-regulated in the diabetic myocardium. Quantitative real-time PCR and Western blot assay showed that MIF could inhibit fibrosis-associated Col1a1, Col3a1 and α-SMA expressions in mouse cardiac fibroblasts.Smad3 activation was inhibited, but miR-29b was up-regulated in MIF-treated cardiac fibroblasts. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA mRNA and 1protein expressions in cardiac fibroblasts. Conclusions MIF possesses the anti-fibrosis activity through inhibiting Smad3activation and through up-regulating miR-29b expression, and miR-29b can inhibit fibrosis-associated Col1a1,Col3a1 and α-SMA expressions in cardiac fibroblasts.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ, α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemiccal staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARy at mRNA and protein level markedly increased in HSCs of 10 umol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t=5.542, P<0.01), α-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0,01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (X2=16.682, P<0,01). CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ, decreases α-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

Effects of the tyrosine protein kinase inhibitor genistein on the proliferation,activation of cultured rat hepatic stellate cells

AIM：Hepatic stellate cell(HSC)plays a pivotal role in liver fibrosis and is considered as the therapeutic target for the treatment of hepatic fibrosis,Tyrosine protein kinase plays an important role in the proliferation,activation of HSC.The purpose of the study is to investigate the effects of the tyosine protein kinase inhibitor genistein on the proliferation and activation of cultured rat HSC.METHODS:Rat HSC were isolated from Wistar rats by in situ perfusion of collagenase and pronase and single-step density Nycodenz gradient,Culture-activated HSC were serum-starved and incubated with10^-9to10^-5mol/L concentration of genistein for 24,48or 72h,In PDGF-induced HSC proliferation,HSC were stimulated with10μg&#183;L^-1PDGF-BBfo15min,and thentreated with genistein for the same time.Cell proliferation was measured by MПassay and based on flow cytometric analysis of cell cycle.The a-smooth muscle actin(α-SMA）expression in HSC was studied with confocallaser microscopy and flow cytometry.c-fos,c-jun and cyclinD1expression in HSCwas also detected by flow cytometry.RESULTS:Genistein inhibited basal and PDGF-induced proliferation of HSCat the concentration of 10^-8to10^-5mol/L,and treatment with10^-7mol/L concentration of genistein for 48h inhibited the HSCproliferation significantly(the inhibition rate was 70.3%,P&lt;0.05).Immunofluorescence detected by confocal laser microscopy and flow cytometry showed that treatment with10^-7mol/L genistein for48h suppressed the expression of α-SMA significantly in HSC(the specific fluorescence intensity were60.2&#177;21.5vs35.3&#177;11.6and12.8&#177;10.4vs9.54&#177;6.39,respectively,bothP&lt;0.05).The intensity of c-fos,c-jun and cyclinD1 expression of HSCs treated with 10^-7mol/L genistein for 48h was also significantly decreased compared with the controls.CONCLUSION;Genistein influences proliferation of HSC,suppresses the expression of α-SMA in HSC and tinhibits the intensity of c-fos,c-jun and cyclinD1 expression of HCSs,Genistein has therapeutic potential against liver fibrosis.     

Role of transforming growth factor-beta signaling pathway in pathogenesis in pathogenesis of benign biliary stricture

AIM:To characterize the expression of members of the transforming growth factor-beta(TGF-β)/Smad/connective tissue growth factor(CTGF)signaling pathway in the tissue of benign biliary stricture,and to investigate the effect of TGF-β signaling pathway in the pathogenesis of benign biliary stricture.METHODS:Paraffin embedded materials from 23 cases of benign biliary stricture were analyzed for members of the TGF-β/Smad/CTGF signaling pathway.TGF-β1,TβRⅠ,T13RⅡ,Smad4,Smad7 and CTGF protein were detected by immunohistochemical strepto-advidinbiotin complex method,and CTGF mRNA was evaluated by hybridization in situ,while 6 cases of normal bile duct served as controls.The percentages of positive cells were counted.The correlation between TGF-β1,Smad4 and CTGF was analyzed.RESULTS:The positive expression ratios of TGF-β1,TβRⅠ,T13RⅡ,Smad4,CTGF and CTGF mRNA in 23 cases with benign biliary stricture were 91.3%,82.6%,87.0%,78.3%,82.6% and 65.2%,respectively,significantly higher than that in 6 cases of normal bile duct respectively(vs 33.3%,16.7%,50.0%,33.3%,50.0%,16.7%,respectively,P＜0.05).The positive expression ratio of Smad7 in cases with benign biliary stricture was 70.0%,higher than that in normal bile duct,but this difierence is not statistically significant 70.0% vs 50%,P＞0.05).There was a positive correlation between positive expression of TGF-β1,Smad4 and CTGF in cases with benign biliary stricture.CONCLUSION:The high expression of TGF-β/Smad/CTGF signaling pathway plays an important role in the pathogenesis of benign biliary stricture.     

非小细胞肺癌患者呼出气冷凝液中p53蛋白检测的临床意义

Objective To study the clinical significance of the detection of p53 protein in exhaled breath condensate (EBC) of patients with non-small cell lung cancer (NSCLC). Methods EBC and plasma of 98 patients with NSCLC were collected,p53 protein expression in EBC and plasma was detected by enzyme-linked immunosorbent assay,and the data were compared with those of 98 healthy controls. p53 protein expression in cancer tissue of 98 patients with NSCLC was detected by immunohistochemistry. p53 protein expression in EBC and plasma and positive expression rate of p53 protein in cancer tissue were compared among patients with different lung cancer type,stage,histologic type,tumor size,and lymph node metastasis,smoking history. The specificity and sensitivity of diagnosis of p53 protein in patients with NSCLC were analyzed by ROC curve. Results ① The level of p53 protein in EBC of patients with NSCLC was significantly higher than that in healthy control group [(233.99±7.91) ng/L vs ( 130. 26 ± 4. 73) ng/L,P ＜0. 01]. The level of p53 protein in serum of patients with NSCI.C was significantly higher than that in healthy control group [(292. 58 ± 8. 79) ng/L vs (141. 66±3. 33) ng/L,P ＜0. 01]. ② The level of p53 protein in EBC of patients with central lung cancer was higher than that in patients with peripheral lung cancer [(248. 22 ± 8. 58) ng/L vs (215. 78 ± 6.61) ng/L,P＜0. 01]. ③The level of p53 protein in EBC of patients with positive immunostaining group was higher than that in negative group [(249.77 ± 8.07) ng/L vs (216.86 ± 7.44) ng/L,P ＜ 0. 05]. ④The level of p53 protein in serum of smokers was significantly higher than that in non-smokers [(310.18 ± 9.04) ng/L vs (254. 55 ± 6. 91) ng/L,P ＜0. 01]. ⑤The positive expression rate of p53 protein in cancer tissue was 47. 96% (47/98). ⑥The sensitivity and specificity of diagnosis of p53 protein were 95. 90% and 90. 04% in plasma,and those were 92. 90% and 79. 59% in EBC. The cut off values of p53 protein were respectively 175. 68 ng/L and 166. 26 ng/L in EBC and serum. Conclusions The detection of p53 protein in EBC of patients with NSCLC is helpful for the diagnosis of lung cancer.     

过表达肌浆网钙ATP酶治疗实验性慢性缺血性心力衰竭

Objective Chronic myocardial ischemia (CMI) has become the most importat cause of heart failure (HF) all over the world. The aim of the current study was to investigate the effects of Sarcoendoplasmic reticulum calcium ATPaee 2a (SERCA2a) gene transfer on cardiac function and endoplasmic reticulum stress (ERS) associated myocardial apoptosis in a minipig HF animal model induced by CMI. Methods HF was induced in minipigs by implantation of ameroid constrictor in the initial segment of left anterior descending (LAD) branch of coronary artery. After confirmation of myocardial perfusion defects and cardiac function impairment by myocardial perfusion imaging and echocardiography, animals were divided into 4 groups (n =4 each): HF group, HF + enhanced green fluorescent protein (EGFP) group,HF + SERCA2a group, and shamed animals as control group. A total amount of 1×1012 v.g. Of rAAV1EGFP or rAAV1-SERCA2a were injected intramyocardially to each animal of HF + EGFP and HF +SERCA2a groups. Sixty days after gene transfer, protein level and activity of SERCA2a were examined,cardiac functions and changes of serum inflammatory and neuro-hormonal factors were determined. Apoptotic index of the ischemic myocardium, protein levels of ER stress marker glucose regulated protein 78 ( GRP 78) and ER stress specific apoptotic marker caspase-12 were also assayed. Results At the study end,echocardiographic and hemodynamic measurements indicated a significant improvement of both cardiac systolic and diastolic function in HF + SERCA2a group compared with HF/HF + EGFP groups [LVEF (60.2±8.6)%vs (44.2±7.1)% and (46.8±6.7)%, Ev/Ay 1.28±0.24 vs 0.77 ±0.17 and 0.80±0.21, +dp/dtmax(2713.9 ±434.0) mm Hg/s ( 1 mm Hg =0.133 kPa) vs (1892.3 ±434.2) mm Hg/s and (1931.2±397.4)mm Hg/s, -dp/dtmax (1422.1±334.4) mm Hg/s vs (848.3±308.3) mm Hg/s and (849.5±278.3)mm Hg/s, P＜0.05], along with increase in both SERCA2a protein level (1.13±0.26 vs 0.73 ±0.17 and 0.64±0.18, P＜0.05) and activity [(16.2±5.5) IU/ml vs (7.9±3.1) IU/ml and (7.5 ±2.8)IU/ml, P ＜0.05] compared with HF/HF + EGFP groups. Serum concentrations of inflammatory factor tumor necrotic factor α [(382.3±114.4) ng/L vs (732.3±201.4) ng/L and (689.8±192 5) ng/L, P＜0. 05], neural-hormonal factors brain natriuretic peptide [(142.6±45.3) ng/L vs (422.3±113.6) ng/L and(393.7 ±103.3)ng/L, P＜0.01], endothelin-1 [(111.4 ±37.5)ng/L vs (193.5 ±54.3)ng/L and (201.0±72.1)ng/L,P＜0.05] and angiotensin Ⅱ[(189.7±65.2)μg/L vs (538.3 ± 135.2) μg/L and ( 525.5±144.1)μg/L, P＜0.01] were also significantly decreased in HF + SERCA2a group compared with HF/HF + EGFP groups. The apoptotic index [(12.71±4.11)% vs(23.22±7.23) % and (24.31±6.38)%, P＜0.05], protein levels of GRP78 (1.27±0.33 vs 3.23±1.14 and 4.18±1.13, P＜0.05)and protein level ratios of cleaved caspase-12 to total caspase-12[(4.62±1.93)% vs (9.71±2.70)% and (10.14±2.81)%, P＜0.05] were also significantly reduced in the ischemic myocardium of HF+SERCA2a group compared with the HF/HF + EGFP groups. Conclusion Overexpression of SERCA2a significantly improved cardiac systolic and diastolic function in this HF model partly through attenuation of ER stress related myocardial apoptosis, suggesting its therapeutic potential for CM1 related heart failure.     

Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture

AIM： To characterize the expression of members of the transforming growth factor-beta （TGF-β）/Smad/ connective tissue growth factor （CTGF） signaling pathway in the tissue of benign biliary stricture, and to investigate the effect of TGF-β signaling pathway in the pathogenesis of benign biliary stricture. METHODS： Paraffin embedded materials from 23 cases of benign biliary stricture were analyzed for members of the TGF-β/Smad/CTGF signaling pathway. TGF-β1, TβRⅠ, TβRⅡ, Smad4, Smad7 and CTGF protein were detected by immunohistochemical strepto-advidinbiotin complex method, and CTGF mRNA was evaluated by hybridization in situ, while 6 cases of normal bile duct served as controls. The percentages of positive cells were counted. The correlation between TGF-β1, Smad4 and CTGF was analyzed. RESULTS： The positive expression ratios of TGF-β1, TβR Ⅰ , TβR Ⅱ, Smad4, CTGF and CTGF mRNA in 23 cases with benign biliary stricture were 91.3%, 82.6%, 87.0%, 78.3%, 82.6% and 65.2%, respectively, significantly higher than that in 6 cases of normal bile duct respectively （vs 33.3%, 16.7%, 50.0%, 33.3%, 50.0%, 16.7%, respectively, P 〈 0.05）. The positive expression ratio of Smad7 in cases with benign biliary stricture was 70.0%, higher than that in normal bile duct, but this difference is not statistically significant 70.0% vs 50%, P 〉 0.05）. There was a positive correlation between positive expression of TGF-β1, Smad4 and CTGF in cases with benign biliary stricture. CONCLUSION： The high expression of TGF-β/Smad/ CTGF signaling pathway plays an important role in the pathogenesis of benign biliary stricture.     

Effects of cholesterol on proliferation and functional protein expression in rabbit bile duct fibroblasts

AIM:To investigate the effect of cholesterol (Ch) on the growth and functional protein expression of rabbit bile duct fibroblasts.METHODS:The cultured bile duct fibroblasts were divided randomly into two groups:the control group and the experiment group (fibroblasts were incubated respectively with 0.6g/L Ch for 12, 24, 36 and 48 h).The growth and DNA synthesis of bile duct fibroblasts were measured by the means of ^3H-TdR incorporation.The total protein content of fibroblast was measured by BSA protein assay reagent kit,then the expression of α-actin was analyzed semiquantitatively by Western blot.RESULTS:After treatment with 0.6g/L Ch for 12, 24,36 and 48h, the values of ^3H-TdR incorporation of bile duct fibroblasts were respectively 3.1&#177;0.39, 3.8&#177;0.37,4.6&#177;0.48 and 5.2&#177;0.56mBq/cell, and the values of the corresponding control groups were 3.0&#177;0.33, 3.2&#177;0.39,3.7&#177;0.49 and 4.3&#177;0.43mBq/cell. After comparing the values of experiment groups and their corresponding control groups,it was found that the 3H-TdR incorporation of bile duct fibroblasts after treatment with 0.6g/L Ch for 24, 36 and 48h were significantly increased (P&lt;0.05, P&lt;0.01, P&lt;0.01),while the ^3H-TdR incorporation of 12-h group was not different statistically from its control group.Ch had no obvious effect on the total protein content of fibroblasts.After incubated with 0.6g/L Ch for 12, 24, 36 and 48h,the total protein content of each experiment group was not altered markedly compared with its corresponding control group.The values of experiment groups were 0.246&#177;0.051,0.280&#177;0.049, 0.263&#177;0.044 and 0.275&#177;0.056ng/cell, and those of corresponding control groups were 0.253&#177;0.048,0.270&#177;0.042,0.258&#177;0.050 and 0.270&#177;0.045 ng/cell.Western blot analysis revealed that the α-actin expression in fibroblasts affected by Ch for 12 and 24 h was not markedly changed compared with their corresponding control groups (P&gt;0.05),the values of total gray scale of 12- and 24-h groups were 1 748&#177;185 and 1 756&#177;173,respectively. But after stimulation with Ch for 36 h, the total gray scale of fibroblasts (1923&#177;204) was significantly higher than that of control group (1734&#177;197).When the time of Ch treatment was lengthened to 48h, the α-actin expression was markedly elevated, the total gray scale was 2189&#177;231 (P&lt;0.01 vs control group).CONCLUSION:Moderately concentrated Ch can promote the proliferation of bile duct fibroblasts at early stage, With the prolongation of Ch treatment, the α-actin expression of fibroblasts was also increased,but the hypertrophy of fibroblasts was not observed,