钙激活氯通道蛋白ANO1在心肌成纤维细胞分化过程中的表达及其钙激活氯通道电生理特性变化*

目的: 研究钙激活氯通道蛋白ANO1在心肌成纤维细胞(CFs)向成肌纤维细胞(MFs)分化过程中的表达规律及其电生理特性,探索ANO1在心肌纤维化进程中的作用。方法: 以刚分离贴壁的大鼠原代CFs及其分化的MFs为研究对象,利用全细胞膜片钳检测钙激活氯通道电流变化,采用免疫荧光标记技术和Western blot检测ANO1、α-SMA和vimentin在CFs和MFs细胞中的表达特征。结果: 刚贴壁培养的CFs钙激活氯通道电流密度远高于MFs;ANO1主要表达于CFs和MFs的细胞核周围及细胞核上,少量表达于细胞膜;ANO1在刚贴壁的CFs中表达较弱,而在有分裂增殖趋向的MFs细胞中表达较为明显。结论: ANO1的表达与MFs的分化过程密切相关,提示其可能参与调节心肌纤维化进程。     

Serelaxin inhibits differentiation and fibrotic behaviors of cardiac fibroblasts by suppressing ALK-5/Smad2/3 signaling pathway

Serelaxin, a recombinant form of human relaxin-2, is currently regarded as a novel drug for treatment of acute heart failure. However, whether therapeutic effects of serelaxin are achieved by inhibiting cardiac fibrosis remains unclear. In this study, we investigate effects of serelaxin on inhibiting cardiac fibrosis. Cardiac fibroblasts (CFs) were isolated from the hearts of adult rats. Effects of serelaxin on differentiation of CFs towards myofibroblasts (MFs) and their fibrotic behaviors after induction with TGF-β1 were examined. Synthesis and degradation of collagens, secretion of IL-10, and expression of ALK-5 and p-Smad2/3 of TGF-β1-induced cells were assessed after treatment with serelaxin. Serelaxin inhibited differentiation of TGF-β1-induced CFs towards MFs, and reduced proliferation and migration of the induced cells. Moreover, serelaxin down-regulated expression of collagen I/III and TIMP-2, and up-regulated expression of MMP-2 and MMP-9 in the cells. After treatment with serelaxin, activity of MMP-2 and MMP-9 and secretion of IL-10 increased, expression of ALK-5 and the level of Smad2/3 phosphorylation was reduced significantly. These results suggest that serelaxin can inhibit differentiation of TGF-β1-induced CFs towards MFs, reduce production of collagens by suppressing ALK-5/Smad2/3 signaling pathway, and enhance extracellular matrix degradation by increasing MMP-2/TIMP-2 ratio and IL-10 secretion. Serelaxin may be a potential therapeutic drug for inhibiting cardiac fibrosis.     

CCN1 protects cardiac myocytes from oxidative stress via beta1 integrin-Akt pathway

CCN1 (Cyr61) is a secreted matricellular protein, mediating angiogenesis and cell survival through interaction with integrins. Although CCN1 expression is induced in the heart during ischemia and pressure overload, its function in cardiac myocytes remains to be elucidated. We hypothesized that CCN1 may not only induce angiogenesis but may also have a direct effect on cardiac myocytes during ischemia. In this study, we investigated the effect of CCN1 on survival of cardiac myocytes under oxidative stress and examined a signal transduction pathway downstream of CCN1. A solid-phase binding assay demonstrated that CCN1 was bound to cardiac myocytes in a dose-dependent, saturable manner. Inactivation of beta1 integrin in cardiac myocytes inhibited binding with CCN1, indicating that CCN1 was bound to cardiac myocytes via beta1 integrin. Knockdown of endogenous CCN1 decreased the number of surviving cells under oxidative stress, while pretreatment of cardiac myocytes with recombinant CCN1 significantly increased the number of surviving cells. Moreover, TUNEL staining showed that CCN1 significantly decreased apoptotic cells. Furthermore, treatment of cardiac myocytes with CCN1 induced phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Inactivation of beta1 integrin inhibited CCN1-induced phosphorylation of these kinases and abolished the protective effect of CCN1. Moreover, pretreatment of cells with wortmannin completely blocked the protective effect of CCN1 on cardiac myocytes under oxidative stress, indicating that the protective effect of CCN1 was mainly mediated by activation of Akt. The antiapoptotic effect of CCN1 on cardiac myocytes together with its proangiogenic property could be beneficial in the treatment of ischemic heart disease.     

miR-34a attenuates myocardial fibrosis in diabetic cardiomyopathy mice via targeting Pin-1

Diabetic cardiomyopathy (DCM) is characterized by myocardial hypertrophy and fibrosis. This study aimed to investigate the effects of microRNA (miR)-34a on myocardial fibrosis in DCM and its potential mechanism of targeting Pin-1 signaling. Vimentin and Pin-1 proteins in mouse cardiac tissues were detected by immunohistochemical staining. Locked nucleic acid in situ hybridization was used to measure miR-34a expression in cardiac tissues. Primary mouse cardiac fibroblasts (CFs) were transfected with a mimics control/miR-34a mimics or Pin-1 plasmid and cultured in high-glucose (HG) Dulbecco's modified Eagle's medium. The miR-34a levels were measured by quantitative polymerase chain reaction. The apoptosis and viability of transfected cells were detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling and Cell Counting Kit-8 assays respectively. A cell migration experiment and dual-luciferase reporter assay were also performed. The body weight and fasting blood glucose of DCM mice were significantly higher than those in the control (CTL) group. In addition, DCM mice had decreased serum insulin levels and impaired cardiac function. The number of CFs in the DCM group was higher than in the CTL group and Pin-1 expression was upregulated. The expression level of miR-34a in the cardiac tissue of mice in the DCM group was obviously downregulated compared with the CTL group. The HG stimulation of CFs for 48 h significantly downregulated the expression level of miR-34a and was associated with increased Type I collagen expression, cell viability, and migration and decreased apoptosis. However, these effects could be reversed by overexpressing miR-34a in HG-induced CFs. Furthermore, we found that Pin-1 was a direct target of miR-34a. Our results suggest that miR-34a can attenuate myocardial fibrosis in DCM by reducing Type I collagen production, cell viability, and migration and increasing the apoptosis of CFs by targeting Pin-1 signaling.     

Fasting up‐regulates ferroportin 1 expression via a Ghrelin/GHSR/MAPK signaling pathway

The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft‐L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O‐acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft‐L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft‐L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre‐treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway.     

Overexpression of peptidase inhibitor 16 attenuates angiotensin II–induced cardiac fibrosis via regulating HDAC1 of cardiac fibroblasts

Cardiac hypertrophy and fibrosis are the major causes of heart failure due to non‐ischaemia heart disease. To date, no specific therapy exists for cardiac fibrosis due to the largely unknown mechanisms of disease and lack of applicable therapeutic targets. In this study, we aimed to explore the role and associated mechanism of peptidase inhibitor 16 (PI16) in cardiac fibrosis induced by angiotensin II. In cardiac fibroblasts (CFs), overexpressed PI16 significantly inhibited CF proliferation and the levels of fibrosis‐associated proteins. Further analysis of epigenetic changes in CF revealed that overexpressed PI16 decreases the nuclear level of histone deacetylase 1 (HDAC1) after angiotensin II treatment, resulting in increased histone 3 acetylation in K18 and K27 lysine. However, overexpression of HDAC1 by an adenovirus vector in CFs reversed these changes. Echocardiography showed that PI16 transgenic (Tg) mice have smaller left ventricle mass than wild‐type mice. Histological analysis data showed that PI16 Tg mice demonstrated smaller cardiomyocyte size and less collagen deposition than wild‐type mice. The effects of PI16 on HDAC1 and histone 3 were also confirmed in PI16 Tg mice using immunostaining. Generally, PI16 is a HDAC1 regulator specifically in CFs, and PI16 overexpression prevents cardiac hypertrophy and fibrosis by inhibiting stress‐induced CF activation.     

AICAR stimulates IL-6 production via p38 MAPK in cardiac fibroblasts in adult mice: a possible role for AMPK

Though known as a sensor of energy balance, AMP-activated protein kinase (AMPK) was recently shown to limit damage and apoptotic activity and contribute to the late preconditioning in heart. Interleukin-6 was also reported to involve in anti-apoptosis and cardio-protection in myocardium. Interestingly, both AMPK activity and IL-6 level were increased in response to ischemia, hypertrophy and oxidative stress. To determine whether AMPK activation will promote IL-6 production, cardiac fibroblasts (CFs) from mice were incubated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR). The results demonstrated that AICAR time and dose-dependently stimulated IL-6 production by ELISA and immunofluorescence. Pretreatment with p38 mitogen-activated protein kinase (MAPK) inhibitor blocked AICAR-induced IL-6 production; furthermore, AICAR-activated p38 MAPK phosphorylation by Western blot. To confirm that the increase in IL-6 production is ascribed to AMPK activation, we used another known AMPK activator, metformin. It also dose-dependently potentiated IL-6 production in CFs, and this potentiation could be reversed by p38 MAPK inhibitor. In conclusion, AMPK activation promoted IL-6 production in CFs via p38 MAPK-dependent pathway.     

Tongguan capsule‐derived herb reduces susceptibility to atrial fibrillation by inhibiting left atrial fibrosis via modulating cardiac fibroblasts

Tongguan capsule is a compound Chinese medicine used to treat ischaemic heart diseases. This study aimed to investigate whether Tongguan capsule‐derived herb (TGD) has a preventive effect on atrial fibrillation (AF) in post‐myocardial infarction (MI) rats and to determine the underlying mechanisms. MI was induced by ligation of the left anterior descending coronary artery. TGD was administered to the post‐MI rats over a 4‐week period. The TGD‐treated rats had lower rates of AF inducibility and shorter AF durations than the MI rats. TGD improved the left atrial (LA) conduction velocity and homogeneity. It reduced the fibrosis‐positive areas and the protein levels of collagen types I and III in the left atrium. In vitro, it inhibited the expression of collagen types I and III by inhibiting the proliferation, migration, differentiation and cytokine secretion of cardiac fibroblasts (CFs). In conclusion, the current study demonstrated that TGD reduces susceptibility to AF and improves LA conduction function in rats with post‐MI by inhibiting left atrial fibrosis and modulating CFs. Targeting the CF population may be a novel antiarrhythmic therapeutic approach.     

TRIM72 contributes to cardiac fibrosis via regulating STAT3/Notch-1 signaling

Cardiac fibrosis is a pathophysiological process characterized by excessive deposition of extracellular matrix. We developed a cardiac hypertrophy model using transverse aortic constriction (TAC) to uncover mechanisms relevant to excessive deposition of extracellular matrix in mouse myocardial cells. TAC caused upregulation of Tripartite motif protein 72 (TRIM72), a tripartite motif-containing protein that is critical for proliferation and migration. Importantly, in vivo silencing of TRIM72 reversed TAC-induced cardiac fibrosis, as indicated by markedly increased left ventricular systolic pressure and decreased left ventricular end-diastolic pressure. TRIM72 knockdown also attenuated deposition of fibrosis marker collagen type I and α-smooth muscle actin (α-SMA). In an in vitro study, TRIM72 was similarly upregulated in cardiac fibroblasts. Knockdown of TRIM72 markedly suppressed collagen type I and α-SMA expression and significantly decreased the proliferation and migration of cardiac fibroblasts. However, TRIM72 overexpression markedly increased collagen type I and α-SMA expression and increased the proliferation and migration of cardiac fibroblasts. Further study demonstrated that TRIM72 increased phosphorylated STAT3 in cardiac fibroblasts. TRIM72 knockdown in cardiac fibroblasts resulted in increased expression of Notch ligand Jagged-1 and its downstream gene and Notch-1 intracellular domain. Inhibition of Notch-1 abrogated sh-TRIM72-induced cardiac fibrosis. Together, our results support a novel role for TRIM72 in maintaining fibroblast-to-myofibroblast transition and suppressing fibroblast growth by regulating the STAT3/Notch-1 pathway.     

ANO1对口腔鳞癌SCC-25细胞株的影响

采用免疫组化检测160例口腔鳞癌组织及相应正常组织的ANO1表达,并进行多项体外买验,以明确ANO1对SCC-25细胞迁移的影响。结果显示,正常组织中的ANO1阳性表达明显低于口腔鳞癌组织;有淋巴结转移的口腔鳞癌组织的ANO1阳性表达显著高于无转移的口腔鳞癌组织;多项体外实验表明,ANO1过表达有利于SCC-25细胞的迁移;尼氟灭酸能减缓细胞的迁移速度。综上所述,ANO1促进了口腔鳞癌患者体内的肿瘤转移,并增加了SCC-25细胞的体外移动、侵袭、伸展、剥脱能力,有望成为治疗口腔鳞癌的新的靶点。     

Macrophage migration inhibitory factor regulates joint capsule fibrosis by promoting TGF-β1 production in fibroblasts

Joint capsule fibrosis caused by excessive inflammation results in post-traumatic joint contracture (PTJC). Transforming growth factor (TGF)-β1 plays a key role in PTJC by regulating fibroblast functions, however, cytokine-induced TGF-β1 expression in specific cell types remains poorly characterized. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in inflammation- and fibrosis-associated pathophysiology. In this study, we investigated whether MIF can facilitate TGF-β1 production from fibroblasts and regulate joint capsule fibrosis following PTJC. Our data demonstrated that MIF and TGF-β1 significantly increased in fibroblasts of injured rat posterior joint capsules. Treatment the lesion sites with MIF inhibitor 4-Iodo-6-phenylpyrimidine (4-IPP) reduced TGF-β1 production and relieved joint capsule inflammation and fibrosis. In vitro, MIF facilitated TGF-β1 expression in primary joint capsule fibroblasts by activating mitogen-activated protein kinase (MAPK) (P38, ERK) signaling through coupling with membrane surface receptor CD74, which in turn affected fibroblast functions and promoted MIF production. Our results reveal a novel function of trauma-induced MIF in the occurrence and development of joint capsule fibrosis. Further investigation of the underlying mechanism may provide potential therapeutic targets for PTJC.     

Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway

Cardiac vascular microenvironment is crucial for cardiac remodelling during the process of heart failure. Sphingosine 1‐phosphate (S1P) tightly regulates vascular homeostasis via its receptor, S1pr1. We therefore hypothesize that endothelial S1pr1 might be involved in pathological cardiac remodelling. In this study, heart failure was induced by transverse aortic constriction (TAC) operation. S1pr1 expression is significantly increased in microvascular endothelial cells (ECs) of post‐TAC hearts. Endothelial‐specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis in myocardium. In vitro experiments demonstrated that S1P/S1pr1 praxis activated AKT/eNOS signalling pathway, leading to more production of nitric oxide (NO), which is an essential cardiac protective factor. Inhibition of AKT/eNOS pathway reversed the inhibitory effect of EC‐S1pr1‐overexpression on angiotensin II (AngII)‐induced cardiomyocyte (CM) hypertrophy, as well as on TGF‐β‐mediated cardiac fibroblast proliferation and transformation towards myofibroblasts. Finally, pharmacological activation of S1pr1 ameliorated TAC‐induced cardiac hypertrophy and fibrosis, leading to an improvement in cardiac function. Together, our results suggest that EC‐S1pr1 might prevent the development of pressure overload‐induced heart failure via AKT/eNOS pathway, and thus pharmacological activation of S1pr1 or EC‐targeting S1pr1‐AKT‐eNOS pathway could provide a future novel therapy to improve cardiac function during heart failure development.     

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.     

Caveolin‐1 negatively regulates inflammation and fibrosis in silicosis

Inhalation of crystalline silica causes silicosis, the most common and serious occupational disease, which is characterized by progressive lung inflammation and fibrosis. Recent studies revealed the anti‐inflammatory and anti‐fibrosis role of Caveolin‐1 (Cav‐1) in lung, but this role in silicosis has not been investigated. Thus, this study evaluated Cav‐1 regulatory effects in silicosis. It was found that Cav‐1 levels were significantly reduced in the lung from silicosis patients and silicotic mice. The silicosis models were established in C57BL/6 (wild‐type) and Cav‐1 deficiency (Cav‐1 ^−/−) mice, and Cav‐1 ^−/− mice displayed wider alveolar septa, increased collagen deposition and more silicotic nodules. The mice peritoneal‐derived macrophages were used to explore the role of Cav‐1 in silica‐induced inflammation, which plays a central role in mechanism of silicosis. Cav‐1 inhibited silica‐induced infiltration of inflammatory cells and secretion of inflammatory factors in vitro and in vivo, partly by downregulating NF‐κB pathway. Additionally, silica uptake and expression of 4‐hydroxynonenal in silicotic mice were observed, and it was found that Cav‐1 absence triggered excessive silica deposition, causing a stronger oxidative stress response. These findings demonstrate the protective effects of Cav‐1 in silica‐induced lung injury, suggesting its potential therapeutic value in silicosis.     

PDK1 promotes apoptosis of chondrocytes via modulating MAPK pathway in osteoarthritis

3-Phosphoinositide dependent protein kinase-1 (PDK1), a serine threonine kinase, belongs to the AGC kinase family and is associated with apoptosis. The aim of this study was to investigate the expression of PDK1 (3-Phosphoinositide dependent protein kinase-1) in articular cartilage with osteoarthritis (OA) and to analyze the relationship between PDK1 and chondrocyte apoptosis. Immunohistochemistry and RT-PCR analysis showed that the expression of PDK1 in articular cartilage of OA patients and healthy controls. IL-1β-stimulated SW1353 cells were used to imitate the OA-like chondrocyte injury in vitro, and IL-1β-induced the expression of PDK1, apoptotic markers(PARP, caspase-3), and phosphorylated p38 were detected by Western blot. The co-localization of PDK1 and Cleaved-caspase3 was confirmed through immunofluorescence. Knocking down PDK1 expression through PDK1 siRNA. Western blot was performed to detect the knockdown efficiency of PDK1 and the impact of PDK1 knockout on IL-1β-induced expression of apoptotic markers and phosphorylated p38 in SW1353 cells. Flow Cytometry-Based Annexin V/PI Staining was used to exam chondrocyte apoptosis. Our experimental results suggested that PDK1 may promote chondrocyte apoptosis in OA via p38 MAPK signaling pathway.