PAX6 inhibits angiotensin II-induced cardiac fibroblast transdifferentiation

AIM To investigate the effects of paired box 6 （PAX6） on angiotensin II （Ang II）-induced transdifferentiation of cardiac fibroblasts （CFs） and its underlying mechanisms. METHODS Primary CFs were isolated from the hearts of adult mice, and Ang II was used to induce the transdifferentiation of CFs. The adenovirus vector carrying PAX6 was constructed and transfected into the CFs. The cells were divided into Ad-GFP+Ctrl group （transfected with control adenovirus vector）, Ad-GFP+Ang II group （transfected with control adenovirus vector and treated with Ang II）, Ad-PAX6+Ctrl group （transfected with adenovirus vector carrying PAX6） and Ad-PAX6+Ang II group （transfected with adenovirus vector carrying PAX6 and treated with Ang II）. The fluorescence expressed by transfected CFs was observed under an inverted fluorescence microscope. The protein levels of PAX6, α-smooth muscle actin （α-SMA）, collagen type I （Col I）, fibronectin （FN） and transforming growth factor β1 （TGFβ1） were detected by Western blot. The expression and distribution of α-SMA, Col I and FN were measured by immunofluorescence staining. The mRNA levels of PAX6 and TGFβ1 were determined by qPCR. RESULTS The fluorescence observed by inverted fluorescence microscopy confirmed the successful transfection of adenovirus vector into the CFs. qPCR and Western blot showed successful PAX6 overexpression in the CFs （P<0.01）. Ang II increased the myofibroblast marker α-SMA in the CFs, while overexpression of PAX6 significantly inhibited the expression of α-SMA induced by Ang II （P<0.01）. In addition, PAX6 overexpression significantly inhibited Ang II-induced synthesis of extracellular matrix （ECM） proteins Col I and FN （P<0.05）. Furthermore, Ang II treatment upregulated TGFβ1 mRNA and protein levels, while overexpression of PAX6 significantly inhibited TGFβ1 mRNA and protein expression induced by Ang II （P<0.05）. CONCLUSION PAX6 inhibits Ang II-induced CF transdifferentiation and ECM protein synthesis via inhibiting TGFβ1 expression.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

AIM：To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.     

Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Gefitinib inhibits epithelial-mesenchymal transition by up-regulating Foxo3a expression in mice with bleomycin-induced lung fibrosis

AIM：To identify the effect of gefitinib on the expression of forkhead box protein O3a (Foxo3a), α-smooth muscle actin (α-SMA) and related signal pathway molecules in the mice with bleomycin-induced lung fibrosis and to investigate the inhibition mechanism of gefitinib on lung epithelial-mesenchymal transition. METHODS：Thirty Kunming female mice were randomly divided into 3 groups:control group (received normal saline intratracheally), bleomycin group (received bleomycin intratracheally, 3 mg/kg), and bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally, 20 mg/kg). All the mice were sacrificed 14 d after the treatments. Pulmonary histological changes were evaluated by hematoxylin-eosin staining and Masson trichrome staining. The mRNA levels of Foxo3a and α-SMA in the lung tissues were detected by RT-PCR. Nuclear Foxo3a, α-SMA, and phosphorylation of EGFR, Akt and Foxo3a in the lung tissues were determined by Western blotting. RESULTS：Gefitinib inhibited bleomycin-induced lung fibrosis and significantly decreased the scores of lung inflammation and fibrosis. Foxo3a mRNA expression and total Foxo3a protein expression were increased, while the phosphorylated Foxo3a was decreased. Nuclear Foxo3a was increased significantly. Meanwhile, phosphorylated EGFR and Akt were decreased. The level of α-SMA was observably increased. CONCLUSION:Gefitinib restores Foxo3a activity and reduces α-SMA expression by modulating EGFR/Akt activity, thus inhibiting bleomycin-induced lung fibrosis.     

Regulation of rat pulmonary fibroblasts differentiation into myofibroblasts though RhoA/ROCK pathway mediated by TGF-β1

AIM：To investigate the regulatory effect of RhoA/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway mediated by transforming growth factor β1 (TGF-β1) on the differentiation of pulmonary fibroblasts into myofibroblasts. METHODS：Primarily cultured fibroblasts were obtained by trypsin digestion from the lung of neonatal rats. The fibroblasts were stimulated with TGF-β1 for different durations and were divided into control group, TGF-β1 induction group and Y-27632 treatment group. The distribution and expression of p-RhoA, ROCK, phosphorylated myosin binding subunit of myosin light chain phosphatase (p-MBS), serum response factor (SRF), α-smooth muscle actin (α-SMA),type I collagen and type Ⅲ collagen in the cells were detected by the methods of immunocytochemistry and Western blotting. RESULTS：A lot of parallel and cross arranged filaments labeled by α-SMA antibody appeared in the cells after TGF-β1 stimulation. The cultured cells stimulated with TGF-β1 were all myofibroblasts at 24 h determined by immunocytochemistry. The expression levels of p-RhoA, ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were increased gradually with the extension of TGF-β1 stimulation time. The expression of RhoA/ROCK signaling protein in the cells stimulated with TGF-β1 (peaking at 6 h of exposure) was 2.96 folds higher as compared with the non-stimulated cells. The expression of SRF protein (peaking at 12 h of TGF-β1 exposure) was 4.55 folds higher as compared with the non-sti-mulated cells. The expression levels of α-SMA and type I and type III collagens (peaking at 24 h of TGF-β1 exposure) were 4.06 folds, 2.19 folds and 3.04 folds higher as compared with the non-stimulated cells, respectively. Compared with TGF-β1 induction group, the protein expression levels of ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were significantly decreased at the corresponding time points in Y-27632 treatment group. CONCLUSION：TGF-β1 induces the differentiation of pulmonary fibroblasts into myofibroblasts, and then promotes the synthesis of collagen through the activation of ROCK pathway, which possibly plays an important role in the formation of pulmonary fibrosis.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

IL-1β induces human mesangial cell injury via exacerbating lipid-induced endoplasmic reticulum stress

AIM：To investigate the molecular mechanism that interleukin-1 beta (IL-1β) exacerbates lipid-induced endoplasmic reticulum stress (ERS) and the injury of human mesangial cells (HMCs). METHODS：HMCs were cultured and divided into control group, low-density lipoprotein (LDL) group, IL-1β+LDL group and 4-phenyl butyric acid (4-PBA)+IL-1β+LDL group. Oil red O staining was used to evaluate the accumulation of lipid droplet in the cells. The mRNA levels of glucose-regulated protein 78(GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK) and α-smooth muscle actin (α-SMA) were examined by real-time PCR. Immunocytochemistry was used to observe GRP78 expression. The protein level of NF-κB p65 was measured by Western blotting. The releases of IL-6 and TGF-β1 in the culture supernatants of HMCs were detected by ELISA. RESULTS：Compared with LDL group, the intracellular lipid accumulation, the mRNA levels of GRP78 and PERK, the protein expression of GRP78 and NF-κB p65, and the release of IL-6 were significantly increased in IL-1β+LDL group. Dramatically reduced intracellular lipid accumulation, down-regulated GRP78 and PERK mRNA expression, decreased protein levels of GRP78 and NF-κB p65, and suppressed IL-6 release were observed in 4-PBA+IL-1β+LDL group as compared with IL-1β+LDL group. The mRNA level of α-SMA was higher in IL-1β+LDL group than that in LDL group, and that in 4-PBA+IL-1β+LDL group was significantly depressed. CONCLUSION：IL-1β exacerbates lipid-induced ERS, thus promoting the injury of HMCs.     

Protective effects of Zhenwu decoction on kidney injury of diabetic rats induced by streptozotocin

AIM：To investigate the effects of Zhenwu decoction (ZWD) on the expression of α-smooth muscle actin (α-SMA) and NF-κB in diabetic nephropathy (DN) rats. METHODS：Diabetic rat model was induced by intrape-ritoneal injection of streptozotocin (STZ), and the animals were randomly divided into STZ group (n=22) and STZ+ZWD group (n=23). The normal rats served as control (n=16). All rats were sacrificed on 8 weeks after modeling. Biochemical assay and pathological observation (HE staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on the renal function and pathological morphology. The body weight, renal index, blood glucose, total urinary protein in 24 h, and superoxide dismutase (SOD), malondialdehyde (MDA),inducible nitric oxide synthase (iNOS) were determined as well. Western blotting was used to observe the effects of Zhenwu decoction on the expression of α-SMA and NF-κB in diabetic nephropathy (DN) rats. RESULTS：Compared with normal group, the renal index, blood glucose concentration, total urinary protein in 24 h, blood urea nitrogen (BUN), serum creatinine (SCr) and MDA were significantly higher and body weight was lower in DN rats (P<0. 05). Pathological examination of the kidneys in DN group showed glomerular hypertrophy, glomerular basement membrane thickening, tubular epithelial cell degeneration, mesangial matrix proliferation, protein cast formation in some renal tubules. The protein expression levels of α-SMA and NF-κB were markedly increased (P<0.05). After ZWD treatment, the level of renal index, total urinary protein in 24 h, BUN, SCr and the expression of α-SMA and NF-κB at the protein level were significantly decreased (P<0.05). The renal histological injury in ZWD group was significantly ameliorated. CONCLUSION：Zhenwu decoction might protect kidney against STZ-induced injury via decreasing the expression of α-SMA and NF-κB.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Imatinib attenuates DOCA/salt-induced rat myocardial fibrosis via PDGFs/PDGFRs signaling pathway

AIM：To investigate the role of imatinib (IMA) in reducing myocardial fibrosis and its relationship with platelet-derived growth factors (PDGFs)/PDGF receptors (PDGFRs) signaling pathway in deoxycorticosterone acetate (DOCA)/salt-induced hypertension in rats. METHODS:Sixty male uninephrectomized SD rats were treated with 1% NaCl and 0.2% KCl in the drinking water for 4 weeks and randomly divided into vehicle control (CON) group, DOCA treatment (DOCA) group and DOCA plus IMA treatment (DOCA+IMA) group. Systolic blood pressure (SBP) was mea-sured weekly using the tail-cuff method. The extent of myocardial interstitial fibrosis and the ratio of perivascular collagen area/vascular area (PVCA/VA) were analyzed by picro-Sirius red staining. The mRNA expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), procollagenⅠ, procollagenⅢ, PDGFRα and PDGFRβ in the myocardium was measured by real-time PCR. The protein levels of PDGFRα, PDGFRβ, p-PDGFRβ, vimentin and α-SMA were analyzed by immunohistochemical staining. The cell types of PDGFRα and PDGFRβ localizations were examined by immunofluorescence method. RESULTS：(1) SBP in DOCA and DOCA+IMA groups was higher than that in CON group on day 14 and day 28, and no difference of SBP between DOCA group and DOCA+IMA group was found. The extent of myocardial interstitial fibrosis and the ratio of PVCA/VA in DCOA group were higher than those in CON group, and those in DOCA+IMA group were significantly lower than those in DOCA group on day 28. (2) Compared with CON group, the expression of PDGFRα and PDGFRβ in DOCA group was increased on day 14. Compared with DOCA group, the expression of PDGFRα and PDGFRβ in DOCA+IMA group was attenuated. Compared with CON group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA group was increased significantly on day 28, but the expression of PDGFRα was not increased significantly, and the expression of PDGFRβ was higher than PDGFRα. Compared with DOCA group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA+IMA group was attenuated on day 28. (3) The fibroblasts in the myocardial interstitium of CON group were mainly vimentin positive, but not α-SMA positive on day 28,while the number of α-SMA-positive spindle-shaped cells (myofibroblasts) increased significantly in DOCA group. Compared with DOCA group, the number of myofibroblasts in DOCA+IMA group was attenuated on day 28. (4) PDGFRα was located in fibroblasts and myofibroblasts but not in vascular smooth muscle cells (VSMCs). PDGFRβ was not only located in myofibroblasts but also in VSMCs. CONCLUSION:PDGFRα takes effect in the early phase of myocardial fibrosis in the DOCA/salt hypertensive rats, while PDGFRβ takes effect in the entire process. PDGFRα and PDGFRβ are expressed in cardiac fibroblasts and myofibroblasts. Imatinib reduces the proliferation and transformation of fibroblasts, thereby attenuating myocardial fibrosis by inhibiting PDGFs/PDGFRs signaling pathway.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.