Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.     

Ⅱ型肺泡上皮细胞间质转分化中的Notch-1/ Twist-1信号通路机制*

目的:通过研究Notch-1/Twist-1信号通路参与Ⅱ型肺泡上皮细胞间质转分化(EMT)作用,为阐明肺纤维化(PF)发病机制提供理论依据。方法:动物实验设对照组和博莱霉素(BLM)组(n=15)。气管注射BLM(7 500 U/kg)诱导肺纤维化大鼠模型,造模后28 d取左肺下叶固定于10%福尔马林中,进行HE、Masson及转化生长因子-β1(TGF-β1)免疫组化染色。体外培养Ⅱ型肺泡上皮细胞(RLE-6TN),细胞实验设对照(Control)组、TGF-β1(5 ng/ml)组、TGF-β1+ Notch-1 siRNA阴性对照组(NC siRNA, 100 pmol/L)和TGF-β1+Notch-1 siRNA干扰组(Notch-1 siRNA, 100 pmol/L),每组设9个复孔。细胞先用NC siRNA或Notch-1 siRNA预处理24 h,再用TGF-β1处理48 h。检测肺组织和(或)II型肺泡上皮细胞内TGF-β1、I型胶原(collagen I)、III型胶原(collagen III)、E-钙粘蛋白(E-Cadherin)、紧密连接蛋白-1(ZO-1)、波形蛋白(Vimentin)、N-钙粘蛋白(N-Cadherin)、Notch-1、Notch-1胞内域(NICD)、Hes-1和Twist-1 mRNA和(或)蛋白表达。结果:动物实验结果显示,与对照组相比,BLM组肺泡萎缩、塌陷并发生融合,肺泡间隔明显增宽,可见大量炎性细胞的浸润。肺间质胶原纤维沉积明显增多,collagen I和collagen III的表达明显增加(P＜0.01);另外,肺BLM组织中E-cadherin和ZO-1表达明显下降而Vimentin和N-cadherin的表达明显增加(P＜0.01);同时,BLM组肺组织TGF-β1、Notch-1、NICD、Hes-1和Twist-1的表达也明显上调(P＜ 0.01)。细胞实验结果显示,与Control相比,TGF-β1组Notch-1、NICD、Hes-1、Twist-1、collagen I和collagen III的表达明显升高,同时E-Cadherin和ZO-1的表达明显降低而Vimentin和N-cadherin的表达明显升高(P＜0.01)。与TGF-β1组相比,Notch-1 siRNA能够明显降低TGF-β1诱导Notch-1、NICD、Hes-1和Twist-1的表达(P＜0.05或P＜ 0.01),同时E-Cadherin和ZO-1的表达明显升高而Vimentin和N-cadherin的表达明显降低(P＜0.05或P＜0.01)。另外Notch-1 siRNA还能够明显降低TGF-β1诱导的collagen I和collagen III的表达(P＜0.05或P＜0.01)。结论:Notch-1/Twist-1信号通路参与了Ⅱ型肺泡上皮细胞EMT,可能参与了肺纤维化的发生发展。     

冠状动脉结扎致心肌纤维化大鼠模型的建立

目的建立大鼠心肌纤维化（myocardial fibrosis,MF）模型,探讨其病变规律,为临床防治MF研究提供实验动物模型。方法 100只雄性Wistar大鼠随机分为模型组（92只）和伪手术组（8只）,模型组进行心脏冠状动脉结扎（coronary artery ligation,CAL）,手术后第7、14、21、28、35、42、49、56天分别处死;留取心脏标本,HE染色和Masson染色观察心肌组织基本结构,定量测定心脏组织羟脯氨酸含量、心肌胶原和转化生长因子β1（transfor-ming growth factor,TGF-β1）的表达。另设立伪手术组作为对照。结果与伪手术组组相比,模型组大鼠手术7 d后心肌组织炎性反应即已严重,心肌细胞断裂,心肌胶原含量显著升高（P〈0.01）,羟脯氨酸含量升高（P〈0.05）,TGF-β1表达显著增高并持续保持在较高水平（P〈0.01）,纤维化反应在第42天达到高峰,其后有好转趋势。结论 CAL法能成功建立可靠的心肌纤维化动物模型,其机制可能与上调TGF-β1表达有关。     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.     

白细胞介素1β抑制心肌成纤维细胞胶原合成并促进其分解

本文应用3H-胸腺嘧啶核苷(3H-thymidine, 3H-TdR)掺入法及3H-脯氨酸(3H-proline, 3H-Pro)掺入法观察白细胞介素1β(interleukin-1β, IL-1β)对Spague-Dawley乳鼠心肌成纤维细胞DNA及胶原合成的影响,并用明胶酶谱法和Western blot检测基质金属蛋白酶(matrix metalloproteinases, MMPs) MMP-2、 MMP-9活性及MMP-2和MMP-9蛋白表达,用RT-PCR检测MMP-2、 MMP-9的mRNA表达.结果显示:(1)0.1、1、10、100ng/mL的IL-1β作用于细胞24h后,各组3H-TdR掺入量明显较对照组低(P<0.05, P<0.01),同时3H-Pro掺入量明显降低(P<0.05, P<0.01);而0.01ng/mL的IL-1β作用于细胞后,对3H-TdR掺入量和3H-Pro掺入量无明显影响.(2)不同剂量(0.01～100ng/mL)的IL-1β均刺激MMP-2和MMP-9活性升高,并呈剂量依赖性.IL-1β增加MMP-2和MMP-9蛋白表达(P<0.05, P<0.01).(3)IL-1β(0.01～100ng/mL)刺激MMP-2和MMP-9 mRNA表达升高(P<0.05, P<0.01).以上结果表明,IL-1β通过减少心肌成纤维细胞的细胞分裂来降低胶原的合成,同时促进MMP-2和MMP-9的转录及转录后的表达来促进胶原的分解,提示其在心肌重塑过程中起一定作用.     

A novel role of vimentin filaments: binding and stabilization of collagen mRNAs

The stem-loop in the 5' untranslated region (UTR) of collagen α1(I) and α2(I) mRNAs (5'SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5'SL of α1(I) and α2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5'SL- and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5'SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen α1(I) and α2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen α1(I) and α2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies.     

The linker region of Smad2 mediates TGF-β-dependent ERK2-induced collagen synthesis

Transforming growth factor (TGF)-β1 can cause fibrosis diseases by enhancing production of collagen. However, the intracellular signaling mechanism for TGF-β1 stimulation of this process has not been fully elucidated. The present study focused on this mechanism and the cross-talk between the MAPK and Smad pathways. Extracellular signal-regulated kinase (ERK)2 ablation by a small interfering RNA led to marked inhibition of TGF-β1-induced collagen synthesis and enhanced phosphorylation of the Smad2 linker site in NIH/3T3 fibroblast cells. However, ERK1 ablation had minimal effects. Ablation of either ERK2 or ERK1 had no effect on the phosphorylation of the Smad2 C-terminal site. Furthermore, a Smad2 mutant with reduced phosphorylation of the Smad2 linker site inhibited TGF-β1-induced collagen synthesis. These results indicate that ERK2, rather than ERK1, plays a predominantly positive role in TGF-β1-induced collagen synthesis, and that ERK2 enhances collagen synthesis, at least partially, through activation of the Smad2 linker site.     

Resveratrol ameliorates early diabetic nephropathy associated with suppression of augmented TGF-β/smad and ERK1/2 signaling in streptozotocin-induced diabetic rats

Diabetic nephropathy (DN) is the major cause of end-stage renal disease. The early changes in DN are characterized by an increased in kidney size, glomerular volume, and kidney function, followed by the accumulation of glomerular extracellular matrix, increased urinary albumin excretion (UAE), glomerular sclerosis, and tubular fibrosis. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. In the present study, we examined the beneficial effects of RSV on DN and explored the possible mechanism of RSV action.Male Sprague–Dawley rats were injected with streptozotocin at 65 mg/kg body weight. The induction of diabetes mellitus (DM) was confirmed by a fasting plasma glucose level ≥300 mg/dL and symptoms of polyphagia and polydipsia. The DM rats were treated with or without RSV at 0.75 mg/kg body weight 3 times a day for 8 weeks. Animals were sacrificed and kidney histology was examined by microscopy. Urinary albumin excretion, glomerular hypertrophy and expressions of fibronectin, collagen IV, and TGF-β in the glomeruli were alleviated in RSV-treated DM rats, but not in untreated DM rats. In addition, RSV treatment reduced the thickness of the glomerular basement membrane (GBM) to the original thickness and increased nephrin expressions to normal levels in DM rats. Moreover, RSV inhibited phosphorylation of smad2, smad3 and ERK1/2 in diabetic rat kidneys. This is the first report showing that RSV alleviates early glomerulosclerosis in DN through TGF-β/smad and ERK1/2 inhibition. In addition, podocyte injuries of diabetic kidneys are lessened by RSV.     

High glucose promotes the production of collagen types I and III by cardiac fibroblasts through a pathway dependent on extracellular-signal-regulated kinase 1/2

Hyperglycemia promotes fibrosis by increasing collagen synthesis, a process involving mitogen activated protein kinases (MAPKs). Several studies of diabetic cardiomyopathy have demonstrated an accumulation of collagen, including collagen types I and III, in the myocardium, leading to interstitial fibrosis, which is related to left-ventricular diastolic dysfunction. However, the mechanisms of hyperglycemia-induced collagen production in cardiac fibroblasts are poorly defined. In the present study, neonatal rat cardiac fibroblasts treated with high glucose (25 mM) were assessed by real time PCR and enzyme linked immunosorbent assay (ELISA) showed an increase in both the mRNA and protein level of collagen types I and III. These effects were not due to changes in osmotic pressure. Extracellular signal regulated kinase 1/2 (ERK1/2) was activated by high glucose level (25 mM), and treatment with PD98059 to block ERK phosphorylation significantly inhibited the mRNA and protein expression of collagen types I and III. These results suggest that high glucose accelerates the synthesis of collagen types I and III, and an ERK1/2 cascade in cardiac fibroblasts play an essential role in the control of collagen deposition by high glucose.     

Mesenchymal stem cells ameliorate silica-induced pulmonary fibrosis by inhibition of inflammation and epithelial-mesenchymal transition

Silicosis is a devastating occupational disease caused by long-term inhalation of silica particles, inducing irreversible lung damage and affecting lung function, without effective treatment. Mesenchymal stem cells (MSCs) are a heterogeneous subset of adult stem cells that exhibit excellent self-renewal capacity, multi-lineage differentiation potential and immunomodulatory properties. The aim of this study was to explore the effect of bone marrow-derived mesenchymal stem cells (BMSCs) in a silica-induced rat model of pulmonary fibrosis. The rats were treated with BMSCs on days 14, 28 and 42 after perfusion with silica. Histological examination and hydroxyproline assays showed that BMSCs alleviated silica-induced pulmonary fibrosis in rats. Results from ELISA and qRT-PCR indicated that BMSCs inhibited the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in lung tissues and bronchoalveolar lavage fluid of rats exposed to silica particles. We also performed qRT-PCR, Western blot and immunohistochemistry to examine epithelial-mesenchymal transition (EMT)–related indicators and demonstrated that BMSCs up-regulate E-cadherin and down-regulate vimentin and extracellular matrix (ECM) components such as fibronectin and collagen Ⅰ. Additionally, BMSCs inhibited the silica-induced increase in TGF-β1, p-Smad2 and p-Smad3 and decrease in Smad7. These results suggested that BMSCs can inhibit inflammation and reverse EMT through the inhibition of the TGF-β/Smad signalling pathway to exhibit an anti-fibrotic effect in the rat silicosis model. Our study provides a new and meaningful perspective for silicosis treatment strategies.     

ERK1/2通路在低氧上调大鼠PASMCs钙激活性氯离子通道表达的作用

目的：探讨钙激活性氯离子通道（CLCA2）在大鼠低氧性肺动脉平滑肌细胞（PASMCs）中mRNA和蛋白表达的变化及其与ERK1/2信号通路的关系。方法：PASMCs随机分为：常氧组（N组）,低氧组（H组）,DMSO对照组（D组）,U0126干预组（U组）,Staurosporine aglycone干预组（SA组）,采用免疫印迹法检测CLCA2蛋白的表达;选用半定量逆转录-聚合酶链反应（RT-PCR）技术测定CLCA2 mRNA水平的表达。结果：PASMCs中CLCA2 mRNA和蛋白的表达量,H组较N组明显上调（P<0.01）;U组较D组明显上调（P<0.01）;SA组较D组mRNA的表达显著下调（P<0.01）,蛋白的表达轻微下调。结论：低氧可上调CLCA2中mRNA和蛋白在PASMCs的表达;ERK1/2通路激活剂-Staurosporine aglycone能下调CLCA2在PASMCs中mRNA和蛋白的表达量;ERK1/2通路抑制剂-U0126可上调CLCA2在PASMCs中mRNA和蛋白的表达量。     

Ca^2+介导肾小管ICAM-1高表达参与肾结石的形成

本研究旨在探讨细胞间黏附分子1 (intercellular cell adhesion molecule-1, ICAM-1)在高钙尿肾结石(genetic hypercalcium renal stones, GHS)大鼠中的表达以及Ca^2+对肾小管上皮细胞ICAM-1的影响。取GHS大鼠和SD大鼠,荧光定量PCR检测肾组织ICAM-1 mRNA表达水平,免疫组化检测ICAM-1蛋白表达。比色法检测大鼠肾组织SOD活力和MDA水平。通过ICAM-1 siRNA转染大鼠肾小管上皮细胞系NRK-52E构建ICAM-1低表达细胞模型,Ca^2+(5 mmol/L)处理NRK-52E细胞,检测细胞SOD活力和MDA水平,通过Western blotting检测细胞ICAM-1蛋白表达水平。荧光定量PCR结果显示,与SD对照组相比,GHS组大鼠肾组织ICAM-1 mRNA水平显著升高,差异具有统计学意义(p<0.01);免疫组化结果显示,ICAM-1蛋白在GHS大鼠肾组织中呈阳性表达;氧化应激检测结果显示,与SD对照组比较,GHS组大鼠肾组织SOD活性显著降低,MDA含量显著升高,差异具有统计学意义(p<0.01)。Western blotting结果显示,与对照组比较,Ca^2+组NRK-52E细胞ICAM-1表达蛋白显著升高,差异具有统计学意义(p<0.01);与Ca^2+处理NC-siRNA组比较,Ca^2+处理ICAM-1 siRNA组NRK-52E细胞ICAM-1表达蛋白显著降低;与ICAM-1 siRNA组NRK-52E细胞比较,Ca^2+处理ICAM-1 siRNA组NRK-52E细胞后ICAM-1表达蛋白水平无显著性变化(p>0.05)。细胞氧化应激检测结果显示,与对照组比较,Ca^2+组NRK-52E细胞SOD活性显著降低,MDA含量显著升高,差异具有统计学意义(p<0.01);与Ca^2+处理NC-siRNA组比较,Ca^2+处理ICAM-1 siRNA组SOD活性显著升高,MDA含量显著降低,差异均具有统计学意义(p<0.01);与ICAM-1 siRNA组NRK-52E细胞比较,Ca^2+处理ICAM-1 siRNA组NRK-52E细胞SOD活力和MDA含量无显著性变化(p>0.05)。ICAM-1在GHS肾小管上皮细胞中高表达,Ca^2+诱导肾小管上皮细胞ICAM-1高表达,促进细胞氧化应激水平。     

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis

Background and aims

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

Methods

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Results

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Conclusions

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.     

Neurexin‐2 is a potential regulator of inflammatory pain in the spinal dorsal horn of rats

Chronic pain is one of the serious conditions that affect human health and remains cure still remains a serious challenge as the molecular mechanism remains largely unclear. Here, we used label‐free proteomics to identify potential target proteins that regulate peripheral inflammatory pain and reveal its mechanism of action. Inflammation in peripheral tissue was induced by injecting complete Freund''s adjuvant (CFA) into rat hind paw. A proteomic method was adopted to compare the spinal dorsal horn (SDH) in peripheral inflammatory pain (PIP) model rats with controls. Differential proteins were identified in SDH proteome by label‐free quantification. The role of screened target proteins in the PIP was verified by small interfering RNA (siRNA). A total of 3072 and 3049 proteins were identified in CFA and normal saline (NS) groups, respectively, and 13 proteins were identified as differentially expressed in the CFA group. One of them, neurexin‐2, was validated for its role in the inflammatory pain. Neurexin‐2 was up‐regulated in the CFA group, which was confirmed by quantitative PCR. Besides, intrathecal siRNA‐mediated knock‐down of neurexin‐2 attenuated CFA‐induced mechanical and thermal hyperalgesia and reduced the expression of SDH membrane glutamate receptors (eg mGlu receptor 1, AMPA receptor) and postsynaptic density (eg PSD‐95, DLG2). These findings increased the understanding of the role of neurexin‐2 in the inflammatory pain, implicating that neurexin‐2 acts as a potential regulatory protein of inflammatory pain through affecting synaptic plasticity in the SDH of rats.     

Activation of PERK in ET-1- and thrombin-induced pulmonary fibroblast differentiation: Inhibitory effects of curcumin

In the present study, we investigated the role of PKR-like endoplasmic reticular kinase (PERK), an endoplasmic reticulum (ER) stress kinase, in endothelin 1 (ET-1)- and thrombin-induced pulmonary fibrosis (PF), and the preventive effects of curcumin (CUR). Using the human embryonic WI-38 lung fibroblast cell line, ET-1 and thrombin induced the expression of ER stress-related proteins (CCAAT-enhancer-binding protein homologous protein, PERK, and binding immunoglobulin protein), a profibrogenic factor (cellular communication network factor 2 [CCN2]), and differentiation markers including α-smooth muscle actin (α-SMA), collagen I (Col I), and Col IV. Knockdown of PERK expression via small interfering RNA (siRNA) significantly reduced the increases in CCN2, α-SMA, Col I, and Col IV proteins in WI-38 cells according to western blot analysis and immunohistochemistry (IHC). Activation of c-Jun N-terminal kinase (JNK) was observed in ET-1- and thrombin-treated WI-38 cells, and the addition of a JNK inhibitor (SP) suppressed the induction of the indicated proteins by ET-1 and thrombin. Thapsigargin (TG), an ER stress inducer, elevated expressions of PERK and ER stress-related proteins with increased differentiation of WI-38 cells. Knockdown of PERK by siRNA or the PERK inhibitor glycogen synthesis kinase reduced expressions of the differentiation markers, α-SMA and Col IV, in WI-38 cells. CUR concentration-dependently inhibited ET-1- or thrombin-induced CCN2, α-SMA, and vimentin proteins with decreased levels of phosphorylated mitogen-activated protein kinase and PERK in WI-38 cells. An in vivo bleomycin-induced PF study showed that an intraperitoneal injection of CUR (30 mg/kg) reduced expressions of α-SMA, CCN2, Col IV, and vimentin in lung tissues via IHC staining using specific antibodies. This study is the first to demonstrate that PERK activation contributes to pulmonary fibroblast differentiation elicited by ET-1 or thrombin, and the inhibitory activity of CUR against PF is demonstrated herein.     

Interplay between H6PDH and 11β-HSD1 implicated in the pathogenesis of type 2 diabetes mellitus

Extensive studies have been performed on the role of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in metabolic diseases. Our previous study reported glucose could directly regulate hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. Recently, we further investigated the interplay of H6PDH and 11β-HSD1 and their roles in hepatic gluconeogenesis and insulin resistance to elucidate the importance of H6PDH and 11β-HSD1 in pathogenesis of type 2 diabetes mellitus (T2DM). T2DM rats model and H6PDH or 11β-HSD1 siRNA transfected in CBRH-7919 cells were used to explore the effect of H6PDH and 11β-HSD1 in T2DM. The results showed that the expression and activity of H6PDH and 11β-HSD1 in livers of diabetic rats were increased, with the expressions of PEPCK and G6Pase or liver corticosterone increased apparently. It also showed that H6PDH siRNA and 11β-HSD1 siRNA could inhibit the protein expression and enzyme activity by each other. With H6PDH siRNA, the enhancement of gluconeogenesis was blocked and insulin resistance stimulated by corticosterone was reduced. H6PDH and 11β-HSD1 might be the effective and prospective targets for T2DM and metabolic syndromes, based on the interplay between these two enzymes.     

Urotensin-2 promotes collagen synthesis via ERK1/2-dependent and ERK1/2-independent TGF-β1 in neonatal cardiac fibroblasts

U2 (urotensin-2) is the most potent vasoconstrictor in mammals which is involved in cardiac remodelling, including cardiac hypertrophy and cardiac fibrosis. Although the cellular mechanisms of the U2-induced vasoconstriction have been extensively studied, the signalling pathways involved in U2-induced TGF-β1 (transforming growth factor-β1) expression and collagen synthesis remain unclear. In this study, we show that U2 promoted collagen synthesis and ERK1/2 (extracellular signal-regulated kinase 1/2) activation in neonatal cardiac fibroblasts. The U2-induced collagen synthesis and TGF-β1 production were significantly but not completely inhibited by blocking ERK1/2. Both ERK1/2 inhibitor and TGF-β1 antibody could separately inhibit U2-induced collagen synthesis, and the synergistic inhibition effect was observed by blocking ERK1/2 and TGF-β1 simultaneously. These data suggest that U2 promotes collagen synthesis via ERK1/2-dependent and independent TGF-β1 pathway in neonatal cardiac fibroblasts.     

Suppression of type I collagen production by microRNA-29b in cultured human stellate cells

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through imperfect base pairing with the 3′ untranslated region (3′UTR) of target mRNA. We studied the regulation of alpha 1 (I) collagen (Col1A1) expression by miRNAs in human stellate cells, which are involved in liver fibrogenesis. Among miR-29b, -143, and -218, whose expressions were altered in response to transforming growth factor-β1 or interferon-α stimulation, miR-29b was the most effective suppressor of type I collagen at the mRNA and protein level via its direct binding to Col1A1 3′UTR. miR-29b also had an effect on SP1 expression. These results suggested that miR-29b is involved in the regulation of type I collagen expression by interferon-α in hepatic stellate cells. It is anticipated that miR-29b will be used for the regulation of stellate cell activation and lead to antifibrotic therapy.