WNK4激酶通过溶酶体途径抑制BK通道表达

目的 研究WNK4激酶对BK通道的调节作用及机制.方法 将BK和WNK4野生型(WNK4-WT)或CD4(对照)质粒DNA共同转染进Cos-7细胞中,采用免疫染色-共聚焦激光显微镜、化学发光法、Western印迹法检测BK在细胞上的分布、细胞膜表面蛋白和总蛋白的表达;并使用质子泵抑制剂bafilomycin A1( Baf A1)阻断溶酶体降解检测BK蛋白表达水平的减少是否由于其蛋白降解增多所致.结果 免疫染色-共聚焦激光显微镜发现,与对照组相比,WNK4-WT组BK在细胞膜表面的分布明显减少.化学发光法检测结果显示,对照组BK的细胞膜表面蛋白表达水平为299.9±18.6,WNK4-WT组中其细胞膜表面蛋白表达水平为148.4±13.7,比对照组显著下降(P＜0.01).Western印迹结果提示,WNK4-WT组BK的总蛋白表达水平比对照组明显减少.和对照组(100％)相比,WNK4-WT显著减少BK的总蛋白水平(42.3％±15.2％,P＜0.01),而Baf A1则逆转WNK4-WT对BK蛋白的抑制作用(82.2％±12.1％,P＜0.05).结论 WNK4激酶能同时抑制BK在Cos-7细胞膜表面蛋白和总蛋白的表达水平;WNK4激酶抑制BK通道蛋白的表达是通过增加其在溶酶体内的降解所致的.     

7,8-dihydroxyflavone protects human renal proximal tubular cells from hypoxia injury via inhibiting endoplasmic reticulum stress

Objective To observe the effects of 7,8-dihydroxyflavone (7,8-DHF) on hypoxia induced endoplasmic reticulum stress (ERS) in human proximal tubular epithelial cells (HK-2). Methods The mRNA level of ERS associated biomarkers was evaluated by RT-PCR assay in cell hypoxia damaged model. And HK-2 cells were pretreated with different concentrations of 7,8-DHF through CCK-8 assay; meanwhile CCAAT/enhancer-binding protein homologous protein (CHOP), Cyr61, Akt and p-Akt were determined by western blotting assay. Moreover, HK-2 cells were pretreated by LY294002, a kind of PI3K/Akt inhibitor, to inhibit the PI3K/Akt signaling, and its effects on protein level induced by 7,8-DHF was detected. HK-2 cells was then over-expressed Cyr61 and exposed to hypoxia Apoptosis rate and CHOP expression were determined. Results Compared to hypoxia group (P＜0.01), Hypoxia for 12h was effective in inducing ERS (P＜0.01), while pretreatment with 7,8-DHF (100 μmol/L) increased cell proliferation significantly . The protein expressions of Cyr61 and p-Akt in H+7,8-DHF group were higher, but the level of CHOP was decreased (P＜0.05). With LY294002 pretreated, the expression of Cyr61, p-Akt was down-regulated (all P＜0.05) while the expression of CHOP was up-regulated (P＜0.05). In comparison to empty plasmid group, when cells were transfected with over-expression of Cyr61 plasmid and exposed to hypoxia, the number of apoptotic tubular cells was decreased (P＜0.01). And over-expression of Cyr61 significantly reduced CHOP expression compared with the empty plasmid group (P＜0.01). Conclusion Pretreatment of 7,8-DHF could protect cells from hypoxia injury and inhibit ERS, which may involve the Akt-Cyr61 signaling pathway.     

S期激酶相关蛋白2调控大鼠系膜细胞增殖

目的 探讨S期激酶相关蛋白2（Skp2）表达变化对系膜细胞增殖的影响。 方法 设计合成大鼠Skp2 siRNA和对照siRNA、pIRES-GFP-Skp2质粒和pIRES-GFP质粒。采用脂质体转染法进行细胞转染。半定量PCR和Western印迹法检测Skp2的mRNA、蛋白表达。原代培养的大鼠系膜细胞以每孔3000个接种于96孔板,分为以下6组：（1）无血清组;（2）20％胎牛血清（FCS）组;（3）10％FCS+pIRES-GFP质粒转染组;（4）10％FCS+pIRES-GFP-Skp2质粒转染组;（5）20％FCS+对照siRNA组;（6）20％FCS+Skp2 siRNA组。四甲基偶氮唑盐（MTT）法检测细胞相对活力和相对细胞数;BrdU标记法检测S期细胞;流式细胞仪检测细胞周期。 结果 半定量PCR法结果显示,与阴性对照siRNA组相比,Skp2 siRNA转染后Skp2 mRNA表达显著下调。Western印迹结果显示,与pIRES-GFP质粒转染组相比,pIRES-GFP-Skp2质粒转染后Skp2蛋白表达显著上调。MTT、BrdU和细胞周期分析显示,与相应对照组相比,Skp2质粒转染后相对细胞数增加（A值：0.419±0.088 比 0.305±0.036,P ＜ 0.01）、S期细胞数增多（BrdU 阳性细胞：0.21±0.04比0.15±0.03,P ＜ 0.01;S期细胞数：20.18±0.64比14.33±0.37,P ＜ 0.01）;Skp2 siRNA转染后相对细胞数减少（A值：0.328±0.069比0.482±0.133,P ＜ 0.01）;S期细胞数减少（BrdU 阳性细胞：0.17±0.01比0.24±0.00,P ＜ 0.01;S期细胞数：16.52±0.75比23.81±1.25,P ＜ 0.01）。 结论 Skp2过表达促进系膜细胞增殖,下调Skp2表达可抑制系膜细胞增殖。     

二氮嗪预先给药对大鼠心肌微血管内皮细胞缺氧复氧时PI3K mRNA和Akt mRNA表达的影响

目的 评价二氮嗪预先给药对大鼠心肌微血管内皮细胞缺氧复氧时磷脂酰肌醇-3激酶(PI3K) mRNA和蛋白质丝氨酸苏氨酸激酶(Akt) mRNA表达的影响.方法 培养SD大鼠心肌微血管内皮细胞,以1×106个/ml密度接种于96孔培养板(100μl/孔)或培养皿(2 ml/皿),采用随机数字表法,将其随机分为4组(n=25).正常对照组(C组)不作任何处理;缺氧复氧组(H/R组)、二氮嗪预先给药组(DZ组)、二氮嗪预先给药+线粒体ATP敏感性钾通道阻断剂5-羟葵酸组(DZ+ 5-HD组)均进行缺氧2h,复氧2h.DZ组和DZ+ 5-HD组在缺氧前2h分别加入100 μmol/L二氮嗪、100μnol/L二氮嗪+ 100 μmol/L 5-羟葵酸.于复氧2h时测定细胞活力、细胞凋亡率、PI3K mRNA和Akt mRNA表达.结果 与C组比较,H/R组细胞活力降低,细胞凋亡率升高,PI3K mRNA和Akt mRNA表达上调(P＜0.05或0.01);与H/R组比较,DZ组细胞活力升高,细胞凋亡率降低,PI3K mRNA和Akt mRNA表达上调(P ＜0.05或0.01);5-羟葵酸可逆转二氮嗪预先给药导致的上述改变(P＜0.05或0.01).结论 二氮嗪预先给药减轻大鼠心肌微血管内皮细胞缺氧复氧损伤的机制与激活线粒体ATP敏感性钾通道,促进PI3K和Akt基因的转录有关.     

小分子RNA干扰靶向巨噬细胞移动抑制因子对大肠癌影响的实验研究

目的 分析靶向巨噬细胞移动抑制因子的小分子干扰RNA(MIFsiRNA)对大肠癌生长与荷瘤小鼠生存质量的影响,并探讨其作用机制.方法 利用盲肠造疝原位接种瘤块法建立大肠癌动物模型.将成功建模后的BALB/C小鼠随机分为3组,每组10只,每周2次分别给予DEPC水、MIFsiRNA(0.15 nmol/g)和非特异性siRNA(0.15 nmol/g)瘤内注射,每天称量各组小鼠的饮水、饮食量和体重,每周测量1次肿瘤体积.4周后处死小鼠,称取肿瘤重量,用ELISA法和免疫组织化学法检测小鼠血清和组织中的MIF水平,分光光度法检测肿瘤组织中Caspase-3蛋白的表达,TUNEL法检测肿瘤组织中凋亡细胞数.结果 MIFsiRNA干预组小鼠血清中MIF表达比其他两组低[(22±6) ng/ml)比(32±8)ng/ml、(33±8) ng/ml,P＜0.01],组织中MIF阳性细胞数比其他两组少[(85±20)/500个比(423±23)/500个、(442±31)/500个,P＜0.01];干预后第3周与第4周MIFsiRNA干预组小鼠肿瘤体积均明显小于其他两组(P＜0.01).处死小鼠后肿瘤重量也明显轻于其他两组[(1.93±0.21)g比(4.40±0.30)g、(5.25±0.44)g,P＜0.01];建模后15～31 d饮水量较其他两组多(P＜0.01),建模后8～31 d饮食量也较其他两组多(P＜0.01),3组小鼠体重变化之间相比差异无统计学意义(P＞0.05);MIFsiRNA干预组小鼠肿瘤组织中Caspase-3蛋白表达比其他两组高[(0.74±0.06) μg比(0.57±0.08)μg、(0.56±0.02)μg,P＜0.01],凋亡细胞数较其他两组多[(12±2)/100个比0、0,P＜0.01].结论 敲低MIF基因表达可以抑制大肠癌的生长,提高荷瘤小鼠的生存质量,其可能的作用机制是激活Caspase-3促进细胞凋亡.

Abstract：

Objective To analyze the effect of siRNA targeting MIF( MIFsiRNA) on the growth of colorectal cancer xenografts and the life quality of tumor-bearing mice.Methods BALB/C mouse model carring colorectal cancer was established.Thirty mice were divided into three groups randomly and managed respectively with intratumor injection of DEPC water, MIFsiRNA(0.15 nmol/g) and non-specific siRNA (0.15 nmol/g), respectively twice a week for consecutively 4 weeks.Drinking water, fodder consumed and body weight was recorded daily, and tumor volume was measured once a week.Mice were sacrificed after four weeks.ELISA and immunohistochemistry were used to detect the expression of MIF in serum and in tumor tissues.Spectrophotometric detection was used to detect caspase-3 protein.TUNEL was used to detect apoptotic cells.Results MIF expression in serum in MIFsiRNA group was lower than the other two groups [(22 ± 6) ng/ml vs (32 ± 8) ng/ml and (33 ± 8) ng/ml, P ＜ 0.01]; MIF expression in tissues was less than the other two groups [(85 ± 20) /500 vs.(423 ± 23) /500 and (442 ± 31) /500, P ＜ 0.01]; Tumor was smaller than the other two groups at third and fourth week (P ＜ 0.01) ; Tumor weight was significantly less than the other two groups [(1.93 ±0.21) g vs (4.40 ±0.30) g and (5.25 ±0.44) g, P＜0.01]; Mice in MIFsiRNA group were healthier than the other two groups as judged by water and fodder consumption (P ＜ 0.01 ) , while weight change was not significantly different among the three groups ( P ＞ 0.05 ).Caspase-3 protein in tissues was higher than the other two groups [(0.74 ±0.06) μg vs (0.57 ±0.08) μg and (0.56 ±0.02) μg, P ＜0.01]; Apoptosis cells in tissues were higher than the other two groups [(12 ± 2)/ 100 个vs 0 and 0, P ＜ 0.01].Conclusions Knockdowning MIF gene expression inhibits the growth of colorectal cancer xenografts and improves life quality of tumor-bearing mice, possibly by a mechanism in which MIFsiRNA activates caspase-3 promoting cell apoptosis.     

Active vitamin D reduces macrophage infiltration by TREM-1 in renal tissue of diabetic nephropathy rats

Objective To investigate the effects of active vitamin D (VD) on the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in renal tissue of diabetic nephropathies (DN) rats and to explore the impact of TREM-1 on adhesion and migration capacity of macrophage. Methods DN rat models were established by streptozotocin. Rats were randomly distributed into four groups: control (NC) group, VD group, DN group and DN+VD group (DN rats with 0.1 μg?kg-1?d-1 calcitriol by gavages). Rats were sacrificed respectively at 8 weeks and 12 weeks after treatment. Pathological changes in kidney tissue were detected and the expressions of CD68 and TREM-1 were acquired by immunohistochemistry stain and Western blotting. In vitro, RAW264.7 cells were divided into NC group, VD group, high glucose (HG) group and HG+VD group. In HG+VD group rats were treated by high glucose with 10-8 mol/L 1,25(OH)2D3. TREM-1 expression was measured by immunohistochemistry stain and Western blotting, and the ability of macrophage in migration and adhesion was evaluated by Transwell migration assay and adhesion assay. TREM-1 siRNA was transferred to silence TREM-1 expression, while plasmid of TREM-1 was transferred for high expression. Their ability of adhesion and migration in macrophage and the effect of 1,25(OH)2D3 were examined. Results (1) Compared with the NC group, the expressions of CD68 and TREM-1 were increased in DN group (P＜0.05), whereas markedly decreased in DN+VD group (P＜0.05). (2) The number of adhesion and migration cells, and the expression of TREM-1 protein in macrophage were obviously increased in HG group as compared with those in NC group (all P＜0.05); whereas above changes were markedly decreased in HG+VD group than those in HG group (P＜0.05). (3) The number of adhesion and migrated macrophage was reduced after TREM-1 siRNA intervention (all P＜0.05). VD could significantly decrease the effect of high glucose on adhesion and migrated macrophages after TREM-1 siRNA (all P＜0.05). (4) Adhesion and migration of macrophage were increased via TREM-1 overexpression (all P＜0.05), but the effects of VD on high glucose-induced adhesion and migration of macrophage were disappeared. Conclusions VD can suppress the adhesion and migration of macrophage via reducing the expression of TREM-1, and inhibit infiltration of macrophage in renal tissue of DN rats.     

Smad3沉默对IGFBPrP1诱导肝星状细胞分泌细胞外基质作用的影响

目的 明确胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein related protein1,IGFBPrP1)是否通过Smad3信号通路影响肝星状细胞分泌细胞外基质.方法 (1)化学合成2对针对Smad3基因的siRNAs(siRNA1,siRNA2),转染肝星状细胞株(HSC-T6).采用实时定量PCR和Western blot法筛选抑制效率较高的siRNA用于干扰实验;(2)将肝星状细胞株(HSC-T6)分为4组:阴性对照组、siRNA-Smad3转染组、siRNA-Smad3+IGFBPrP1组和IGFBPrP1组.将筛选的抑制效率较高的siRNA转染HSC-T6细胞株,田Western b1ot检测各组Smad3、纤维连接蛋白及Ⅰ型胶原的表达.结果 (1)siRNA2-Smad3对Smad3的抑制效率较高;(2)与阴性对照组相比,siRNA-Smad3转染组Smad3蛋白的表达显著下降(P＜0.01).与IGFBPrP1组相比,siRNA-Smad3+IGFBPrP1组纤维连接蛋白和Ⅰ型胶原蛋白的表达均显著降低(P＜0.01).结论 IGFBPrP1影响肝星状细胞分泌细胞外基质的机制之一是通过Smad3信号通路来实现的.

Abstract：

Objective To identify the effect of IGFBPrP1 on the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway. Methods (1)Two pairs of chemically synthesized siRNAs (siRNA1, siRNA2) targeting Smad3 were transfected into HSC-T6 cells,real-time PCR and Western blot were used to evaluate the silence efficiency, and the better siRNA was used. (2)HSC-T6 cells were divided into four groups: Negative control group, siRNA-Smad3 transfection group, siRNA-Smad3+IGFBPrP1 group and IGFBPrP1 group. The better siRNA was chosen to transfect into HSC-T6 cells. The protein expressions of Smad3, fibronectin and Collagen Ⅰ were evaluated by Western blot. Results (1)siRNA2-Smad3 inhibited Smad3 gene expression stronger than another siRNA. (2)After transfection of siRNA2-Smad3, the protein expression of Smad3 was significantly decreased compared to the negative control group(P＜0.01). The protein expression of fibronectin and Collagen Ⅰ in IGFBPrP1 stimulating HSCs treated with siRNA2-Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2-Smad3 (P ＜0. 01 ).Conclusion IGFBPrP1 induces the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway.     

WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling

With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca²⁺-activated K⁺ (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the α subunit of BK (HEK-BKα cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKα channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKα protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKα expression and decreased ERK1/2 phosphorylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKα expression. Similarly, pretreatment of HEK-BKα cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKα expression in a dose-dependent manner. Knockdown of WNK1 expression also increased the ubiquitination of BKα channels. Notably, mice fed a high-K⁺ diet for 10 days had significantly higher renal protein expression levels of BKα and WNK1 and lower levels of ERK1/2 phosphorylation compared with mice fed a normal-K⁺ diet. These data suggest that WNK1 enhances BK channel function by reducing ERK1/2 signaling-mediated lysosomal degradation of the channel.     

WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.     

Effect of HIF1α-KIM1 pathway on extracellular matrix degradation in human tubular epithelial cells under high glucose

Objective To investigate the effect of hypoxia inducible factor 1α (HIF1α)-kidney injury molecule 1 (KIM1) pathway on extracellular matrix degradation in human tubular epithelial cells under high glucose, and to explore the possible mechanism of this pathway participated in renal interstitial fibrosis of DN. Methods The human tubular epithelial cells (HK2) were cultured in vitro and divided into the following groups: (1)Normal control Group (D-glucose 5.6 mmol/L); (2) Mannitol group (D-glucose 5.6 mmol/L+D-mannitol 24.4 mmol/L); (3) High glucose group (D-glucose 30 mmol/L); (4) Control siRNA group; (5) HIF1α siRNA group; (6) KIM1 siRNA group. The corresponding indexes were measured at 12th, 24th and 36th hours. Western blotting, immunofluorescence and qRT-PCR were used to examine the expression of HIF1α, KIM1, matrix metalloproteinase-9 (MMP9), tissue inhibitor of metalloproteinases-1 (TIMP1), fibronectin (FN) and type I collage (COL-I) in protein and mRNA. Results Compared with the control group, the protein and mRNA expression of HIF1α, KIM1, TIMP1, FN and COL-I in the high glucose group were increased in a time-dependent manner (P＜0.01), and MMP9 was decreased in time-dependent manner (P＜0.01). Compared with the high glucose group, the protein and mRNA expression of HIF1α, KIM1, TIMP1, FN and COL-I in HIF1α siRNA group was decreased (P＜0.01), and MMP9 was increased (P＜0.01); However, the protein and mRNA expressions of KIM1, TIMP1, FN and COL-I in KIM1 siRNA group was decreased (P＜0.01), MMP9 was increased (P＜0.01), and the change of HIF1α was of no significance. Conclusions Down-regulation of HIF1α can significantly inhibit the expression of KIM1 in HK2 and decrease the expression of extracellular matrix, and down-regulation of KIM1 can also decrease the extracellular matrix under high glucose, which suggests that HIF1α may regulate the expression of KIM1 in human tubular epithelial cells under high glucose condition, and this pathway may participate in renal interstitial fibrosis of DN.     

钠-氢交换蛋白1小干扰RNA对抗霉素A诱导人肾小管上皮细胞缺血再灌注损伤的保护机制

目的 通过构建钠-氢交换蛋白1（NHE-1）小干扰RNA (siRNA)抑制肾小管上皮细胞NHE-1的表达，探讨其对细胞缺血再灌注损伤的保护作用机制。 方法 根据人NHE-1全长基因序列设计并合成NHE-1-siRNA，转染人肾小管上皮细胞系（HKC），以无关siRNA转染组作为对照。利用10 μmol/L 抗霉素A诱导细胞来模拟缺血缺氧环境。用RT-PCR、Western印迹法检测NHE-1的表达。用BCECF/AM、Fluo-3/AM和SBFI-AM标记HKC，通过激光共聚焦显微镜分别检测其细胞内pHi、Ca2+和Na+浓度的变化。用Hoechst 33342染色技术及Annexin V/PI 染色结合流式细胞仪技术检测细胞凋亡情况。利用荧光探针JC-1检测细胞线粒体膜电位的变化。 结果 特异性NHE-1-siRNA能有效抑制HKC的NHE-1表达，与无关siRNA转染组相比，NHE-1-siRNA转染组NHE-1 mRNA和蛋白表达水平明显下调(均P < 0.05)。抗霉素A刺激后，2组细胞NHE-1 mRNA及蛋白表达水平显著上调，而NHE-1-siRNA转染组低于无关siRNA转染组；同时NHE-1-siRNA转染组细胞凋亡率（8.9%±2.9%）明显低于抗霉素A处理组（18.8%±3.2%）和抗霉素A+无关siRNA转染组（17.4%±3.6%）（均P < 0.05）；NHE-1-siRNA转染组线粒体膜电位明显增高；与无关siRNA转染组相比较，NHE-1-siRNA转染组经抗霉素A处理后，细胞内钠离子、氢离子及钙离子增高的幅度较低（P < 0.05）。 结论 NHE-1-siRNA可抑制肾小管上皮细胞内NHE-1的表达，对抗霉素A诱导肾小管上皮细胞的缺血再灌性损伤有一定的保护作用。其机制可能通过抑制NHE-1表达，延缓细胞内Na+的积聚，减轻细胞内钙离子的超载，抑制损伤细胞的线粒体膜电位下降，减少细胞的凋亡。     

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.     

鞘内注射P300 siRNA对神经病理性痛大鼠的镇痛效果

目的 评价鞘内注射p300小干扰RNA(p300siRNA)对神经病理性痛大鼠的镇痛效果.方法 取鞘内置管成功的雄性SD大鼠96只,采用坐骨神经慢性压迫性损伤法(CCI)建立神经病理性痛模型,随机分为4组(n=24):假手术+生理盐水组(S组)、CCI+生理盐水组(CCI组)、CCI+转染试剂组(V组)和CCI+p300siRNA组(P组).于术后3～6 d时鞘内注射生理盐水、转染试剂或p300siRNA(siRNA 4μg溶于转染试剂)各20μl,2次/d,10μl/次,连续4 d.于术前1 d(基础状态)、术后1、3、5、7、9、11、14 d时测定机械痛阈和热痛阈;于术后3、7、14 d时取腰段脊髓,测定p300蛋白及其mRNA、乙酰化组蛋白H3(Ac-H3)的表达.结果 与基础值比较,CCI组、V组和P组术后各时点机械痛阈和热痛阈降低(P＜0.05).与S组比较,其余各组机械痛阈和热痛阈降低,p300蛋白及其mRNA和Ac-H3蛋白表达上调(P＜0.05).与CCI组比较,P组机械痛阈和热痛阈升高,p300蛋白及其mRNA和Ac-H3蛋白表达下调(P＜0.05).结论 鞘内注射p300 siRNA可减轻大鼠神经病理性痛,其机制与抑制脊髓p300和Ac-H3蛋白的表达有关.     

吡格列酮对高糖诱导大鼠腹膜间皮细胞合成细胞外基质的作用及机制

目的　观察过氧化物酶体增殖物活化受体γ配体吡格列酮对高糖作用下大鼠腹膜间皮细胞（RPMC）合成细胞外基质的作用以及调节机制。 方法 胰蛋白酶消化法分离培养RPMC。随机分为正常对照组、高糖组（2.5%葡萄糖）、吡格列酮干预组（10 μmol/L、20 μmol/L+2.5%葡萄糖）、二硫氨基甲酸吡咯烷干预组（PDTC,NF-κB抑制剂,25 μmol/L、50 μmol/L+2.5%葡萄糖）、姜黄素干预组[活化蛋白1（AP-1）抑制剂,15 μmol/L、30 μmol/L+2.5%葡萄糖]。RT-PCR方法检测纤连蛋白（FN）、Ⅰ型胶原（COLⅠ）、纤溶酶原激活抑制因子1（PAI-1）、c-fos、c-jun mRNA表达。ELISA方法检测细胞上清液中FN、COLⅠ和PAI-1蛋白水平。Western印迹方法检测IκBα、磷酸化IκBα（p-IκBα）、NF-κBp65、磷酸化NF-κBp65 （p-p65）蛋白表达。 结果 常规培养的腹膜间皮细胞表达基础量的FN、COLⅠ和PAI-1,高糖显著上调其蛋白及mRNA表达（P < 0.01）。吡格列酮预处理后,高糖诱导的FN、COLⅠ和PAI-1蛋白及mRNA表达显著低于高糖组（P < 0.01）。高糖作用后,磷酸化IκBα和NF-κBp65水平显著增高,c-fos、c-jun mRNA表达增加,与对照组差异有统计学意义（P < 0.01）。PDTC预处理后,高糖诱导RPMC的FN和PAI-1蛋白水平降低（P < 0.01）,COLⅠ蛋白表达无明显变化。AP-1抑制剂姜黄素预处理后,高糖诱导的RPMC FN、COLⅠ和PAI-1蛋白水平均显著降低,与对照组差异有统计学意义（P < 0.01）。吡格列酮抑制高糖条件下磷酸化IκBα和NF-κB p65水平,抑制c-fos、c-jun mRNA表达（P < 0.05或P < 0.01）。 结论 NF-κB和AP-1信号通路参与高糖条件下RPMC的FN、COLⅠ和PAI-1表达的调节。吡格列酮通过NF-κB 和AP-1途径下调高糖诱导的RPMC的FN、 COLⅠ和PAI-1的表达,从而发挥抗纤维化作用。     

重组pEGFP-N1-H2Bl质粒载体诱导心脏移植免疫耐受

目的 探讨H2-Bl基因诱导小鼠心脏移植免疫耐受的机制和效果.方法 建立小鼠颈部心脏异位移植模型,供体心脏经主动脉根部灌注H2-Bl质粒真核表达载体后进行心脏移植.实验分4组:对照组、环孢素A(CsA)组、H2-Bl质粒转染组、H2-Bl质粒转染+CsA组.各组于术后1、3和7天各动态枪测供心病理改变,免疫组织化学方法测供心CD40表达情况,流式细胞仪检测供心血清中Th1/Th2细胞因子变化,记录移植心脏存活时间.结果 对照组移植后排斥反应最重,其余组与之比较均有所减轻,以H2-Bl质粒转染+CsA组排斥反应最轻.免疫组化显示术后7天时H2-Bl质粒转染组CD40与对照组相比差异有统计学意义(P＜0.05),CsA组、H2-Bl+CsA组CD40与对照组相比差异有统计学意义(P＜0.01).H2-Bl+CsA组Th2细胞因子表达较其余组增加而Th1细胞因子则减少,到术后7天时各组Th细胞因子与对照组相比差异均有统计学意义(P＜0.05).与对照组相比,其余各组小鼠供心存活天数均延长(P＜0.05).结论 H2-Bl基因干预可一定程度诱导移植心脏免疫耐受,延长同种异体小鼠颈部移植心脏存活时间.     

PRS-CTGF-siRNA对TGF-β1诱导的人腹膜间皮细胞细胞外基质及VEGF表达的影响

目的 观察pRetro-Super(PRS)反转录病毒载体介导的表达人结缔组织生长因子(CTGF)小分子干扰RNA(siRNA)对体外培养的人腹膜间皮细胞(HPMC) 细胞外基质和血管内皮生长因子(VEGF)表达的影响。 方法 根据siRNA靶序列要求及PRS反转录病毒载体特点分别设计4 对寡核苷酸，构建表达人CTGF基因siRNA 的PRS-CTGF-siRNA1～4重组反转录病毒载体。以脂质体2000将重组反转录病毒载体转染PT67包装细胞，继而感染HPMC。采用RT-PCR法检测mRNA表达及Western印迹法检测蛋白质表达。 结果 5 μg/L外源性转化生长因子β1（TGF-β1）刺激可诱导HPMC表达CTGF、纤连蛋白（FN）、I型胶原（Col I）、层粘连蛋白（LN）和VEGF明显增高； 而PRS-CTGF-siRNA 1～4组与TGF-β1刺激组比较，HPMC细胞内CTGF、FN、Col I、LN mRNA和蛋白表达和VEGF mRNA表达明显较低（P < 0.01），各干扰组对CTGF mRNA抑制率分别为 69.3％、22.2%、27.4%和38.8%，其中以PRS-CTGF-siRNA 1组最为明显；同时PRS-CTGF-siRNA 1组相对于TGF-β1刺激组，VEGF蛋白表达也明显较低（P < 0.01）；而PRS空载体组与TGF-β1刺激组比较，差异无统计学意义（P ＞ 0.05）。 结论 PRS-CTGF-siRNA重组反转录病毒载体可明显抑制TGF-β1诱导的细胞外基质及VEGF表达的增加。     

机械牵张对A549细胞穿透素-3表达的影响

目的 探讨械牵张对肺泡上皮腺癌细胞A549(A549细胞)穿透素-3(PTX-3)表达的影响.方法 体外培养人A549细胞,将A549细胞接种于包被胶原基底膜的培养板上,待细胞贴壁并融合50%后,随机分为正常对照组(Ⅰ组)、假机械牵张组(Ⅱ组)、机械牵张组(Ⅲ组)、siRNA对照组(Ⅳ组)和siRNA+机械牵张组(V组).Ⅳ组仅转染PTX-3 siRNA;Ⅴ组转染PTX-3 siRNA后24 h,进行周期性机械牵张4 h.机械牵张结束后24 h时测定PTX-3 mRNA、PTX-3蛋白水平及细胞凋亡情况.结果 与Ⅰ组和Ⅱ组比较,Ⅲ组PTX-3 mRNA和PIX-3蛋白水平上调,Ⅳ组和Ⅴ组上述两指标下调(P＜0.05或0.01);与Ⅲ组比较,Ⅴ组上述两指标下调(P＜0.01).Ⅲ组和Ⅴ组细胞凋亡率高于Ⅱ组(P＜0.01);Ⅴ组细胞凋亡率低于Ⅲ组(P＜0.01).结论 机械牵张可上调A549细胞PTX-3 mRNA表达.     

Regulation of melatonin on Toll-like receptor 4 signaling in diabetic db/db mice kidneys

Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys. Methods The 48 10-week-old male db/db mice were randomly divided into db/db group, db/db+MT 50 μg/kg group, db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group, each consisting of 12 mice. These mice received i.p. injections of MT These mice received i.p. injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution, given every day]. Alternatively, 12 db/m mice served as the control group. db/m and db/db group were injected i.p. with the same volume of PBS/DMSO solution. The animals were sacrificed after 12 weeks of dosage administration. Blood glucose (BG), body weight (BW), kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined; Kidney pathological lesions were evaluated by renal pathological staining. Immunohistochemistry of renal TLR4, NF-κB p65, and ED-1 was performed to determine the immunoreactivity. Western blotting was used to detect the expression of renal TLR4, myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF-3) and NF-κB p65, while the mRNA expressions of renal tumor necrosis factor -α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were much higher in db/db mice group (P＜0.01), while KW in db/db+MT (100, 200 μg/kg) groups and UAER level in db/db+MT (50, 100, 200 μg/kg) groups were distinctly decreased compared with those in db/db group (P＜0.01). In week 12 db/db mice, the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P＜0.01). The above kidney histopathologic lesions were distinctly ameliorated by 50, 100, 200 μg/kg MT (P＜0.05). Immunohistochemistry intensity of renal TLR4, NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P＜0.01), and that were remarkably decreased in db/db+MT (50, 100, 200 μg/kg) mice compared with db/db mice (P＜0.05). Western blotting showed that the protein expression of renal TLR4, MyD88, TRIF, IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P＜0.05) and weaker in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.05). Futhermore, the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P＜0.01) and lower in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.01). Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.     

CCN3 inhibits deposition of extracellular matrix of glomerular mesangial cells via promoting the expression of microRNA-29a

Objective To investigate the effects of nephroblastoma over-expressed protein (CCN3) on the formation of extracellular matrix (ECM) induced by transforming growth factor -β1 (TGF-β1) in human mesangial cells (HMCs) and its underlying signal transduction mechanism related with microRNA-29(miRNA-29). Methods HMCs were pretreated with different doses of exogenous CCN3 (5 μg/L, 50 μg/L and 500 μg/L) or transfected with pcDNA3.1(+)-CCN3 before exposed to TGF-β1(2 μg/L), to observe the expression of fibronectin (FN), type Ⅰ collagen (COLⅠ) and miRNA-29a, b and c. The mimics or inhibitor of the miRNA-29a were transfected into HMCs to analyze whether miRNA-29a affect CCN3. The expressions of FN mRNA, COLⅠmRNA and miRNA-29 family were detected by real time PCR. The protein expressions of FN and COLⅠ were detected by Western blotting and cell immunofluorescence. Results (1) Compared with the normal control group, the expressions of FN and COLⅠ were up-regulated in TGF-β1 group, while the expressions of miRNA-29a, b, c were down-regulated in TGF-β1 group (all P＜0.05). (2) Compared with the TGF-β1 group, the expressions of FN and COLⅠ were decreased when pretreated with the different doses of exogenous of CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05). Meanwhile, the expression of miRNA-29a was significantly increased when pretreated with 50 μg/L and 500 μg/L CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05); whereas miRNA-29b and c had no statistical difference (all P＞0.05). (3) Compared with TGF-β1+CCN3 group, the expressions of FN and COLⅠ were decreased in CCN3+TGF-β1+miRNA-29a mimics group (all P＜0.05), whereas the expressions of FN and COLⅠ in CCN3+TGF-β1+miRNA-29a inhibitors group were increased (all P＜0.05). Conclusions CCN3 reduces the TGF-β1-induced production of ECM by the up-regulation of miRNA-29a.     

丹参酮ⅡA对兔纤维环细胞能量代谢的保护作用

[目的]考察丹参酮ⅡA(TSⅡA)对白细胞介素-1β(IL-1β)诱导的兔纤维环细胞能量代谢障碍的保护作用.[方法]藻酸盐串珠立体培养兔纤维环细胞,将细胞分为7组,在培养过程中加入不同浓度的药物:A组为空白对照不加入药物,B组加入4 μg/ml TSⅡA,C组加入10ng/ml IL-1β,D～G组在给予10 ng/mlIL-1β同时分别加入0.5、1、2和4 μg/ml TSⅡA.于培养3 d后行Na+-K+-ATP酶活性检测、,琥珀酸脱氢酶活性检测、MTT法细胞增殖情况检测以及细胞凋亡的流式细胞仪检测.[结果]G组Na+-K+-ATP酶活性(3.23±0.28U/mgprot)较C组(1.118±0.15U/mgprot)明显增高(P<0.01),与A组接近(3.57±0.15 U/mgprot)(P>0.05).G组琥珀酸脱氢酶活性(12.48±0.97U/mgprot)较c组(3.03±0.60 U/mgprot)明显增高(P<0.01),与A组接近(14.24±1.56 U/mgprot)(P>0.05).G组MTT试验吸光度(0.77±0.06)较C组(0.31±0.07)明显增高(P＜0.01),随着TSⅡA浓度的升高,D～G组吸光度随着TsIIA上升而上升.G组细胞死亡细胞比例和凋亡细胞比例分别为21.08±1.46%和8.99±0.33%,均显著低c组(43.11±2.7,P<0.01和11.71±0.32,P<0.01).[结论]TSIIA能够减轻IL-1β对纤维环细胞能量代谢的抑制作用,从而改善纤维环细胞的增殖、死亡及凋亡.