Role of STAT3 in the regulation of autophagy in the glomerular mesangial cells of diabetic nephropathy

Objective To observe the changes of STAT3 signaling transduction pathway and autophagy activity in human glomerular mesangial cells cultured in high glucose, as well as the effect of STAT3 on autophagy, exploring whether SAT3 further influence extracellular matrix proteins type IV collagen secretion through the regulation of autophagy. Methods Culture human renal mesangial cells under different conditions, STAT3 pathway was inhibited with specific blocking agent S3I-201 and siRNA respectively. The experiment was divided into: (1) Control group: normal glucose concentration; (2) High glucose group: divided into 12 h, 24 h, 48 h, 72 h incubation group. (3) High glucose+S3I-201 group: pretreated cells with 30 μmol/L S3I-201 (Selleck S1155) for 1 h, then incubation with high glucose for another 24 hours. (4) High glucose+STAT3-siRNA group: siRNA transfection firstly, then incubation with high glucose for 24 hours. (5) High glucose+S3I-201+3-MA group: pretreated cells with 2 mmol/L 3-MA (Selleck S2767) and 30 μmol/L S3I-201 for 1 h, then incubation with high glucose for another 24 hours. Western blot was employed to detect the protein of STAT3, p-STAT3 and autophagy related protein LC3, p62 expressions. The changes of autophagosome quantity was observed with transmission electron microscope. The extracellular matrix protein collagen IV expression was measured with ELISA. Results Compared with the control group, glomerular mesangial cells cultured with high glucose for 24h, the expressions of STAT3 and p-STAT3 increased (P＜0.01), while the expression of autophagy related proteins LC3II/LC3I decreased. The expression of p62 increased and the number of autophagosome reduced under transmission electron microscope, which all indicated the decrease of autophagy activity (P＜0.05). Blocking STAT3 signaling pathway with S3I-201 and STAT3-siRNA respectively, compared with high glucose group, LC3II/LC3I was up-regulated and p62 was down-regulated, and the number of autophagosome was increased significantly, which all indicated the increase of autophagy activity (P＜0.05). Extracellular matrix proteins collagen IV expression of cells cultured with high glucose was higher than the control group (P＜0.05), and the application of S3I-201 blocking STAT3 pathway caused type IV collagen expression to decrease (P＜0.05). The application of the autophagy inhibitor 3-MA could convert the result and lead to an increase of type IV collagen expression (P＜0.01). Conclusions High glucose could active STAT3 signaling pathway of human renal mesangial cell and increase STAT3, p-STAT3 expression. High glucose could inhibit autophagy activity of human renal mesangial cells. Inhibition of STAT3 pathway activation may reduce the inhibitory effect of high glucose on autophagy of human renal mesangial cells. High glucose leads to an increase of type IV collagen secretion of human glomerular mesangial cells. The activation of STAT3 pathway may increase type IV collagen secretion through negative regulation of autophagy, which eventually leads to diabetic nephropathy.     

细胞因子信号传导抑制蛋白1抑制高糖诱导的肾小球系膜细胞单核细胞趋化蛋白1的表达

目的 探讨细胞因子信号传导抑制蛋白1(SOCS-1)对高糖状态下肾小球系膜细胞单核细胞趋化蛋白1（MCP-1）表达的影响。 方法 体外培养人肾小球系膜细胞,应用脂质体2000分别转染pCR3.1-SOCS-1表达质粒和pCR3.1 空质粒载体,G418筛选阳性克隆。分别采用低糖（5.5 mmol/L）、高糖（30 mmol/L）、低糖+甘露醇（24.5 mmol/L甘露醇）和JAK-STAT信号通路抑制剂AG490 （10 μmol/L）进行刺激。Western印迹检测系膜细胞SOCS-1、信号转导和转录活化因子1、3（STAT1、STAT3）及其磷酸化蛋白（p-STAT1、p-STAT3）的表达。ELISA法和放免法测定细胞上清液中MCP-1、FN和Ⅳ型胶原的含量。RT-PCR法检测SOCS-1和MCP-1 mRNA的表达。 结果 高糖刺激系膜细胞SOCS-1蛋白和mRNA表达呈时间依赖性变化, 4 h表达达到峰值,然后逐渐减低,24 h达基线水平。与低糖组相比,高糖组系膜细胞STAT1和STAT3磷酸化水平显著上调（P < 0.01）; MCP-1 mRNA水平表达显著上调[（0.39±0.05）比（0.16±0.02）,P < 0.01];上清液中MCP-1[（459±67）比（241±19） ng/L]、FN[（5.84±0.61）比（3.41±0.31） mg/L]和Ⅳ型胶原[（16.45±2.30）比（9.56±1.52） μg/L] 含量均显著增加（均P < 0.01）。与空载体对照组相比,SOCS-1过表达组系膜细胞STAT1和STAT3的磷酸化水平显著下降（P < 0.05）;MCP-1 mRNA表达下调[（0.34±0.04）比（0.42±0.05）,P < 0.05]; 上清液中MCP-1[(387±47）比（463±56） ng/L]、 FN[（4.61±0.57）比（5.76±0.74） mg/L]和Ⅳ型胶原[(13.4±2.32)比（17.1±2.57） μg/L] 含量显著减少（均P < 0.05）。与高糖组相比,AG490组系膜细胞MCP-1 mRNA（0.31±0.04）表达显著下调;上清液中MCP-1[（361±53） ng/L]、FN[（5.46±0.71）mg/L]和Ⅳ型胶原[（15.2±1.97） μg/L]含量均减少。 结论 SOCS-1过表达抑制高糖状态下肾小球系膜细胞MCP-1及细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

Prostaglandin E2 receptor subtype 3-siRNA reduces the mesangial cell damage induced by TGF-β1through inhibiting MAPK pathway in mice

Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 (EP3) on transforming growth factor β1 (TGF-β1)-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into: (1)control group; (2)TGF-β1 (10 μg/L) group; (3)NC-siRNA plus TGF-β1 (10 μg/L) group; (4) EP3-siRNA group; (5)EP3-siRNA plus TGF-β1 (10 μg/L). Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP in cell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin (FN), connective tissue growth factor (CTGF), cyclooxygenase-2 (COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) were detected by real-time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was decected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased (P＜0.05), the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated (all P＜0.05). Compared with TGF-β1 group, the cell proliferation in EP3-siRNA plus TGF-β1 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated (all P＜0.05), the phosphorylation of ERK1/2, p38 MAPK were also declined (P＜0.05). Conclusion EP3-siRNA may reduce TGF-β1-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.     

苦参素对人肾小球系膜细胞磷酸化信号传导和转录激活因子3及其蛋白抑制剂表达的影响

目的 观察苦参素对脂多糖（LPS）诱导下人肾小球系膜细胞（HMC）增殖时磷酸化信号传导和转录激活因子3（p-STAT3）、活化的信号传导和转录激活因子3蛋白抑制剂（PIAS3）蛋白和mRNA表达的影响,并探讨其相互关系。 方法 体外培养HMC,并分为对照组、LPS模型组及苦参素干预组。培养12、24、48 h时以MTT法检测HMC的增殖情况;ELISA法检测细胞上清Ⅳ型胶原蛋白（ColⅣ）含量;同时收集同时间点细胞,采用Western印迹法检测p-STAT3和PIAS3的蛋白表达;实时荧光定量PCR法检测STAT3和PIAS3的mRNA表达。 结果 LPS组细胞增殖较对照组显著加快（P < 0.01）,ColⅣ的表达显著高于对照组 （P < 0.01）。苦参素干预后细胞增殖和ColⅣ的表达显著低于模型组（P < 0.01）。LPS诱导 12 h时细胞p-STAT3表达开始上调,各时间点p-STAT3表达量显著高于对照组（P < 0.01）。苦参素干预后,与同时间点LPS模型组相比显著下调（P < 0.01）。LPS诱导下PIAS3各时间点表达量显著下调（P < 0.01）。苦参素干预后,与同时间点LPS模型组相比均显著上调（P < 0.01）。 结论 苦参素对LPS诱导HMC增殖过程中p-STAT3蛋白及mRNA表达有下调作用,对PIAS3有上调作用。苦参素可能影响HMC增殖过程中JAK-STAT信号传导通路。     

Impact of TLR4-siRNA transfection on the expression of TLR4/MyD88 Signaling in rat tubular epithelial cells under high glucose condition

Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection. Methods Three TLR4-siRNA sequences were designed and synthesized. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of TLR4 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for forward experiments. After transfection for 24 h, cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ(AngⅡ) for 12 h, 24 h; cells without stimulation were as normal control. Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression. ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h. Results TLR4/MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or AngⅡ co-incubated NRK-52E(P＜0.01), the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P＜0.01). TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P＜0.01), MCP-1 and IL-6 production decreased remarkably compared with high glucose or AngⅡ co-stimulated group(P＜0.01). Conclusions High glucose can lead to the activation of TLR4/MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E, AngⅡ further augments these effects. The effect can be blocked efficiently by specific siRNA gene silence. TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.     

IL-6/STAT3通路在冷缺血-再灌注损伤后胆管上皮细胞再生中的意义

目的 探讨白介素6/信号转导及转录激活因子3(IL-6/STAT3)通路在冷缺血-再灌注损伤诱导的胆管上皮细胞(BEC)再生过程中的意义.方法 将大鼠随机分为4组:对照组、CP 1 h组、CP12 h组(供肝分别冷保存1、12 h后行原位肝移植术)和anti-IL-6组(CP12 h组术前1 h给予抗IL-6中和抗体0.5 ms/kg,术后每日给予相同剂量直至观察结束).术后1、3、7、14 d,分别采用ELISA法、免疫组织化学法测定肝组织IL-6浓度及BEC再生情况;实时荧光定量PER法、Western blot法检测各组BEC内IL-6 mRNA以及磷酸化STAT3(p-STAT3)、细胞周期蛋白(cyclin)D1表达水平,测定血清碱性磷酸酶(ALP)、谷氨酰转肽酶(GGT)浓度,并进行肝脏组织学检查.结果 与对照组相比,CP1 h组术后IL-6、p-STAT3及cyclin D1表达水平略增加,血清GGT、ALP仅有轻度、短暂的升高,BEC损伤及再生均不明显.CP 12 h组术后BEC损伤严重,血清GGT、AIP明显升高,至术后14 d恢复正常,肝组织及BEC内IL-6含量增加、BEC内p-STA13和cyclin D1蛋白表达上调,BEC再生明显.经anti-IL-6中和抗体处理后,CP 12 h组IL-6以及p-STAT3和cyelin D1表达降低,BEG再生明显抑制,术后14 d仍可见细胞损伤及ALP、GGT的升高.结论 IL-6/STAT3信号转导通路可能参与缺血-再灌注损伤后胆管上皮细胞的再生过程,对肝移植术后的胆道功能恢复有利.     

JAK2-STAT3 pathway regulates the expression of complement factor B in autosomal dominant polycystic kidney disease

Objective To investigate the role of JAK2-STAT3 pathway in the expression of complement factor B (CFB) in autosomal dominant polycystic kidney disease (ADPKD). Methods Renal tissue samples of patients with ADPKD after nephrectomy were collected. Normal renal tissue samples as control were taken from patients after radical nephrectomy. Renal tissue samples of Han: SPRD Cy/+ rats (ADPKD model) and wild-type Han: SPRD +/+ rats were also collected at 4, 8, 16 week. Han:SPRD Cy/+ rat renal tubular epithelial cells (16 w) were primarily cultured in vitro, then stimulated with the JAK2 inhibitor (WP1066) and STAT3 inhibitor (pyrimethamine) for 24 h respectively. Western blotting was used to detect the expression of p-JAK2, JAK2, p-STAT3, STAT3, CFB protein. Results Compared with control group, the protein expressions of p-JAK2, p-STAT3, STAT3, CFB significantly increased in the renal tissue of ADPKD patients (all P＜0.05). The protein expressions of p-JAK2, JAK2, p-STAT3, STAT3 and CFB also significantly increased in the renal tissue of Cy/+ rats compared with wild-type rats (all P＜0.01). When the Cy/+ renal tubular epithelial cells were treated with WP1066, the expressions of p-JAK2, p-STAT3, CFB were suppressed (P＜0.05) and the degree of inhibition was correlated with the WP1066 dose. Pyrimethamine inhibited the protein expressions of p-STAT3 and CFB in the tubular epithelial cells of Cy/+ rats (all P＜0.05) and the degree of inhibition was correlated with the pyrimethamine dose. Conclusions The JAK2-STAT3 pathway is abnormally activated in ADPKD and increases the protein expression of CFB. CFB protein level is correlated with the progress of ADPKD, suggesting that it may take part in the growth and development of ADPKD vesicles.     

Effect of heat shock protein 47 on epithelial-mesenchymal transdifferentiation induced by transforming growth factor β1 in the renal tubular epithelial cells

Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells. Methods HK-2 cells were exposed to TGF-β1 (0, 2.5, 5, 10 μg/L) for different time (0, 12, 24, 48 h). The expression of HSP47 was examined by Western blotting. Then HK-2 cells were exposed to 10 μg/L TGF-β1, the expressions of vimentin, zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR. Furthermore, the expressions of p-Smad3 and Smad3 were examined by Western blotting. HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1. Then the expressions of vimentin, ZO-1 were detected by Western blotting and real-time PCR, meanwhile Western blotting for HSP47, p-Smad3 and Smad3. Results Stimulating HK-2 with TGF-β1resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P＜0.05). Meanwhile, TGF-β1up-regulated the protein and mRNA expression of vimentin (P＜0.05), and down-regulated the protein and mRNA expression of ZO-1 (P＜0.05), all in time-dependent manner. Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3, which was peaked at 30 min, slightly decreased at 1 h, and then increased again between 24 and 48 h (P＜0.05). Compared to the TGF-β1group, inhibition of HSP47 expression in HK-2 up-regulated the protein and mRNA expression of ZO-1, down-regulated the protein and mRNA expression ofvimentin (P＜0.05) and down-regulated the ratio of p-Smad3/Smad3. HSP47 siRNA negative control had no significant effect on the expressions of ZO-1, vimentin and p-Smad3/Smad3 (P＞0.05). Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.     

过氧化物酶体增殖物激活受体α激动剂非诺贝特抑制高糖条件下的肾脏系膜细胞增殖

目的探讨非诺贝特（Fenofibrate）对高糖条件下大鼠肾脏系膜细胞（mesangial cells,MCs）HBZY-1增殖的影响及其作用机制。方法将对数生长期大鼠肾脏系膜细胞分成正常糖组（NG组）、高糖组（HG组）和非诺贝特组（FN组）。NG组细胞体系常规培养,HG组细胞体系加入40mmol／L葡萄糖,FN组细胞培养体系中加入40mmol／L葡萄糖和非诺贝特100μmol／L。各组细胞培养48h后,利用MTT方法检测细胞增殖;realtime-PCR（实时定量PCR）检测各组细胞PPAR-α、JAK2和STAT3基因表达;Westernblotting检测细胞中过氧化物酶体增殖物激活受体Q激动剂（peroxisome proliferators activedreceptor-α,PPAR-α）、p-PPAR-a、JAK2、STAT3、p-JAK2和p-STAT3蛋白的变化。结果与NG组比较,HG组系膜细胞增殖率明显增高（P〈0．05）,JAK2与STAT3mRNA表达水平无统计学差异（P〉0．05）,PPAR-α、JAK2和STAT3蛋白无明显变化（P〉0．05）,但p-PPAR-α、p-JAK2、p-STAT3蛋白明显增加（P〈0．05）;与HG组比较,FN组系膜细胞增值率和JAK2、p-sTAT3蛋白表达均明显下降（P〈0．05）,PPAR-α、JAK2与STAT3mRNA和JAK2与STAT3mRNA表达均无统计学差异,但p-PPAR-α蛋白表达进一步增加（P〈0．05）。结论PPAR-α激动剂非诺贝特能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制JAK2／STAT3信号激活。     

STAT3及其磷酸化STAT3和E钙黏蛋白在结直肠癌组织中的表达及意义

目的探讨结直肠癌组织中STAT3及其磷酸化STAT3（P-STAT3）和E钙黏蛋白（E—cadherin）表达的相关性及其与肿瘤浸润转移的关系。方法用免疫组织化学Elivision^TM plus法检测江西赣南医学院第一附属医院病理科存档的50例结直肠癌组织及相应癌旁组织中STAT3、P-STAT3及上皮细胞间质化（EMT）相关蛋白E—cadherin的表达,并分析其与结直肠癌临床病理特征的关系。结果50例结直肠癌组织中STAT3和p-STAT3及E—cadherin的阳性表达率分别为72％（36／50）、76％（38／50）和26％（13／50）,相应癌旁组织中的表达则分别为24％（12／50）和26％（13／50）及68％（34／50）,结直肠癌组织STAT3和p-STAT3的表达明显高于相应癌旁组织,而E—cadherin的表达则明显低于癌旁组织（均P〈0．05）。STAT3、P-STAT3及E．cadherin表达与肿瘤浸润深度、分化程度、肿瘤大小、淋巴结转移及TNM分期有关（均P〈0．05）。STAT3和P—STAT3蛋白在结直肠癌中的表达与E-cadherin呈显著负相关（均P〈0．05）。结论STAT3和p-STAT3可能通过对E—cadherin的抑制作用进而引起EMT现象,从而导致结直肠肿瘤的发生和进展。     

NF-κB抑制剂对前列腺癌PC-3细胞STAT3核转位的影响

目的:观察IL-6刺激后的前列腺癌PC-3细胞STAT3和NF-κB的表达情况;验证NF-κB抑制剂咖啡酸苯乙基酯(CAPE)对PC-3细胞IL-6和STAT3表达的影响。方法:20 ng/ml IL-6分别作用于PC-3细胞0、5、10、20、30、45 min后,Western印迹和实时荧光定量PCR检测STAT3和NF-κB蛋白和mRNA水平的表达差异;流式细胞技术检测细胞周期。采用TNF-α或TNF-α联合CAPE作用于PC-3细胞,收集培养液上清,ELISA检测IL-6的表达;同时用Western印迹检测p-STAT3的表达。结果:IL-6刺激PC-3细胞后,p-STAT3蛋白的表达明显上调,细胞增殖指数明显增高。TNF-α作用于PC-3细胞后,培养液中IL-6的表达上调,同时p-STAT3蛋白的表达亦上调(P<0.05)。CAPE联合TNF-α作用于PC-3细胞后,培养液中IL-6的表达及p-STAT3蛋白的表达均明显低于TNF-α作用后的表达水平(P<0.05)。结论:CAPE能抑制TNF-α引起的IL-6的分泌,从而抑制IL-6引起的STAT3核转位;通过CAPE抑制NF-κB表达,继而影响STAT3等相关细胞信号传导途径,可能成为前列腺癌治疗的一条新途径。     

Triptolide inhibits the tubulointerstitial fibrosis in rats of unilateral ureteral obstruction by up-regulating Ski expression

Objective To investigate the effects of triptolide on tubulointerstitial fibrosis in rats kidneys with the unilateral ureteral obstruction (UUO) by examining the expression of collagen type Ι (Col-Ι), Ski, Smad3, TGF-β1. Methods Sixty male SD rats were divided into three groups: Sham operation group (Sham group), UUO group and triptolide (0.2 mg•kg-1•d-1) treatment group. The levels of blood urea nitrogen (BUN), serum creatinine (Scr), pathological changes were measured. Col-Ι, Ski and Smad3 expressions were assessed by immunohistochemistry. Protein and mRNA expressions of Ski, Smad3, TGF-β1 were assessed by Western blotting and real-time PCR. Results Compared with Sham group, Scr and BUN increased significantly in UUO group (P＜0.05). Interstitial fibrosis was prominent and renal interstitial injury score increased significantly in UUO group (P＜0.05). The expressions of Col-Ι and Smad3 were increased in UUO group (P＜0.05). Compared with Sham group, the protein expressions of TGF-β1 and Smad3 were increased, the Ski protein was decreased in UUO group (P＜0.05). In triptolide group, the morphological changes were notably reduced (P＜0.05). Comparison with UUO group, triptolide could increase the protein and mRNA expressions of Ski significantly, and decreased the protein and mRNA expressions of Smad3 and TGF-β1 (P＜0.05). Conclusion Triptolide can reduce the tubulointerstitial fibrosis by up-regulating Ski, and down-regulating TGF-β1 and Smad3.     

Effects of silibinin on expression of integrin linked kinase,transforming growth factor β1 and α-smooth muscle actin in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of silibinin on the expression of integrin linked kinase (ILK), transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose(1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 12, 24, 48, 72 hours, high glucose (2.5%) for 24 hours after silibinin (5, 10, 20 mg/L) preincubate for 2 hours. ILK and α-SMA mRNA were detected by real-time PCR. ILK protein was detected by Western blotting. TGF-β1 protein in supernatants was detected by ELISA. Results Compared with the control group, the expresssion of ILK, TGF-β1 and α-SAM was significantly increased in groups stimulated by high glucose (all P＜0.05). Silibinin could significantly decrease the expression of ILK, TGF-β1 and α-SMA induced by high glucose (all P＜0.05). Conclusions High glucose can up-regulate the expression of ILK, TGF-β1 and α-SMA. Silibinin can reverse these changes.     

Effects of metformin on expression of AMP - activated protein kinase in rat glomerular mesangial cells

ObjectiveTo observe the effects of metformin on expression of Adenosine 5’- monophosphate (AMP)-activated protein kinase (AMPK), nuclear factor-κB (NF-κB) and transforming growth factor β1 (TGF - β1) in cultured rat glomerular mesangial cells (MCs), and explore its reno - protective mechanisms. Methods MCs were cultured in the medium with normal glucose (group NG, 5.6 mmol/L), high glucose (group HG, 25mmol/L) and different concentrations of metformin (group M1, M2, M3). After 48 h exposure, the supernatants and MCs were collected. The expression of NF-κB and TGF-β1 mRNA was analyzed by real time-PCR. Total-AMPK, phospho-Thr-172 AMPK (p-AMPK), NF -κB p65 and TGF-β1 were visualized by Western blot. ResultsThe real time-PCR and Western blot result showed MCs could express AMPK, NF-κB and TGF-β1 mRNA and protein. After stimulated by HG, the levels of intracellular NF - κB and TGF - β1 expressions were significantly increased compared with group NG (P＜0.05); The levels of NF-κB and TGF-β1 were significantly decreased in group M1, M2 and group M3 compared with group HG in a dose-dependent manner. After stimulated by HG, the level of intracellular p-AMPK were down-regulated compared with group NG(all P＜0.05); The expression of p-AMPK increased with the rising of metformin concentration, presenting the opposite trend (P＜0.05), while the level of total-AMPK protein was unchanged with exposure to HG or different concentrations of metformin(P＞0.05). ConclusionMetformin can suppress the expression of NF- κB and TGF-β1 of glomerular MCs induced by HG via AMPK activation, which may partly contribute to its reno-protection.     

靶向STAT3的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响

目的 构建大鼠STAT3基因的shRNA慢病毒表达载体,并观察其对大鼠血管平滑肌细胞增殖和凋亡的影响.方法 针对STAT3基因的不同部位设计4对shRNA的寡核苷酸片段,克隆到慢病毒载体PLKO.1中,构建靶向STAT3基因的慢病毒载体PLK0.1-STAT3-shRNA,检测并筛选最佳抑制效率的shRNA干扰载体.并将其转染大鼠血管平滑肌细胞,用噻唑蓝法和流式细胞仪检测沉默STAT3基因后对血管平滑肌细胞增殖和凋亡能力的影响.结果　靶向STAT3慢病毒表达载体构建成功.转染PLKO.1-STAT3-shRNA后,STAT3蛋白表达明显下降,其中以PLKO.1-STAT3-S1最为明显,达到90%以上;转染PLKO.1-STAT3-S1的细胞增殖能力(A值=0.25±0.05)明显低于未转染组(A值=0.62±0.12)和阴性对照组细胞(A值=0.59±0.11)(P＜0.05);而早期细胞凋亡率(26.9±2.8)%和晚期细胞凋亡率(9.5±1.6)%均明显高于未转染组和阴性对照组(P＜0.01).结论　成功构建并筛选最佳抑制效率的靶向STAT3慢病毒表达载体PLKO.1-STAT3-S1,该载体能有效抑制大鼠血管平滑肌细胞增殖,并促进细胞凋亡.

Abstract：

Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying STAT3 gene in rats, and to investigate its effects on proliferation and apoptosis of vascular smooth muscle cells by silencing STAT3. Methods Four oligonucleotides targeting STAT3 gene were synthesized and cloned into lentivirus vector PLKO. 1. The shRNA lentiviral vector with best transfection efficiency was detected and identified, which was transfected into vascular smooth muscle cells in rats, and its effects on proliferation and apoptosis of vascular smooth muscle cells were measured by MTT and flow cytometry after silencing STAT3. Results The recombinant lentivirus vector PLKO. 1-STAT3-shRNA was constructed successfully. PLKO. 1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically, especially PLKO. 1-STAT3-S1, whose transfection efficiency was more than 90%. The proliferation capacity of vascular smooth muscle cells transfected with PLKO. 1-STAT3-S1 (A value =0. 25 ±0. 05 ) was significantly lower than no-transfected group (A value =0. 62 ±0. 12) and negative control group (A value =0. 59 ±0. 11 )(P ＜ 0. 05). Meantime the early apoptosis rate (26. 9 ± 2. 8 ) % and late apoptosis rate (9. 5 ± 1.6 ) % in PLKO. 1-STAT3-shRNA-transfected group were significantly higher than in no-transfected group and negative control group (P ＜ 0. 01 ). Conclusion The recombinant lentivirus shRNA vector targeting STAT3,PLKO. 1-STAT3-S1, with best transfection efficiency, is constructed successfully. PLKO. 1-STAT3-S1 can inhibit the proliferation of vascular smooth muscle cells, and promote the cell apoptosis. This study lays the foundation for further studying on targeting treatment of vascular restenosis.     

JAK2信号通路介导人恶性黑素瘤A375细胞的自噬和凋亡机制研究

目的:探讨JAK2/STAT3信号通路介导人恶性黑素瘤A375细胞的自噬和凋亡活性,为黑素瘤的发生机制和潜在干预靶点提供理论依据。方法:人恶性黑素瘤A375细胞经复苏和传代后随机分为对照组和JAK2抑制剂干预组,比较两组细胞培养12h、24h、48h和72h的增殖率(采用MTT定量法)及凋亡率(采用流式细胞术),培养72h的细胞JAK2、p-JAK2、STAT3、LC3B和p-STAT3蛋白相对表达量(采用Western blot法),检测IL-6和TNF-α水平(采用ELISA法),检测Caspase-3和Bax/Bcl-2 mRNA相对表达量[采用反转录PCR(RT-PCR)法]。结果:两组培养12h、24h、48h和72h的细胞增殖率比较,干预组各时间点均明显小于对照组,而凋亡率明显大于对照组,差异有统计学意义(P<0.05)。干预组培养72h的细胞p-JAK2和p-STAT3蛋白相对表达量、IL-6和TNF-α水平明显低于对照组,但LC3B蛋白、Caspase-3和Bax/Bcl-2 mRNA相对表达量均明显高于对照组,差异有统计学意义(P<0.05)。结论:JAK2/STAT3信号通路异常激活可能是人黑素瘤A375细胞恶性增殖的重要通路之一,靶向干预JAK2/STAT3信号通路可以促进细胞自噬和凋亡活性上调,有望成为临床干预的重要靶点。     

白细胞介素6促胆囊癌细胞增殖、侵袭与JAK/STAT3信号通路的关系

目的:探讨炎症因子白细胞介素6(IL-6)对胆囊癌细胞生物学行为的影响及其与JAK/STAT3信号通路的关系.方法:将胆囊癌细胞株GBC-SD用IL-6或IL-6+AG490(JAK/STAT3通路抑制剂)作用后,分别用MTT法检测增殖情况;Transwell法检测侵袭能力;明胶酶谱法检测基质金属蛋白酶9(MMP-9)活性;Western blot法检测磷酸化STAT3(p-STAT3)和血管内皮生长因子(VEGF)蛋白的表达.结果:IL-6(10,50,100 ng/mL)作用后,GBC-SD细胞增殖呈浓度依赖性增加(均P＜0.05),AG490能取消IL-6对细胞增殖的促进作用.IL-6作用GBC-SD细胞后,细胞侵袭能力及MMP-9的分泌明显增加(均P＜0.05),细胞p-STAT3和VEGF的表达明显上调,加入AG490后,以上作用均被取消.结论:IL-6能促进胆囊癌细胞的增殖通和侵袭,其作用可能与其活化JAK/STAT3信号通路,从而上调下游的MMP-9和VEGF的表达有关.