Role of connective tissue growth factor in high glucose-induced epithelial-mesenchymal transition in podocytes

AIM: To investigate the role of connective tissue growth factor (CTGF) in high glucose-induced epithelial-mesenchymal transition (EMT) in podocytes. METHODS: The differentiated podocytes cultured under different conditions for 24 h at 37 ℃ were divided into 4 groups: control (5 mmol/L glucose solution), 5 mmol/L glucose solution supplemented with 50 μg/L CTGF, high glucose (30 mmol/L glucose solution) and high glucose supplemented with CTGF (50 μg/L). The morphology of cultured podocytes was observed under phase-contrast microscope. To study the relevant markers of EMT, the mRNA and protein expression was analyzed by real-time RT-PCR and Western blotting, respectively. In addition, the effect of CTGF inhibition with anti-CTGF antibody on high glucose-induced EMT in podocytes was investigated. RESULTS: High glucose induced phenotypic transition in the podocytes from an arborization morphology to a cobblestone morphology. After addition of CTGF to high glucose culture, the podocytes developed a feature of spindle. Under high glucose condition, podocytes underwent EMT, as the epithelial marker (nephrin) was decreased and the mesenchymal marker (desmin) was increased. Exogenous CTGF showed the effect of synergy on high glucose-induced EMT. Moreover, high glucose-induced EMT in podocytes was prevented by CTGF inhibition with anti-CTGF antibody. CONCLUSION: CTGF is involved in high glucose-induced EMT in podocytes. Inhibition of CTGF might prevent podocytes from injury in diabetes.     

Expression and modulation of connective tissue growth factor in renal interstitial fibrosis

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstream effector of TGF-β acting on interstitial cells to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

Impact of hydrogen sulfide donor on endothelin-1 and connective tissue growth factor expression in rats with pulmonary hypertension induced by high pulmonary blood flow

AIM: To explore the possible impact of hydrogen sulfide (H2S) donor-sodium hydrosulfide (NaHS) on endothelin-1 (ET-1) and connective tissue growth factor (CTGF) expressions in rats with pulmonary hypertension induced by high pulmonary blood flow. METHODS: Thirty-two male SD rats were randomly divided into 4 groups: shunt group, shunt+NaHS group, sham group and sham+NaHS group. Rats in shunt group and shunt+NaHS group were subjected to an abdominal aorta-inferior vena cava shunt to create an animal model of high pulmonary flow. After 11 weeks of experiment, rat systolic pulmonary artery pressure (SPAP), lung tissue H2S, plasma ET-1 concentration and lung tissue ET-1mRNA expression, as well as pulmonary artery CTGF protein expression were detected.RESULTS: After 11 weeks of experiment, SPAP, lung tissue ET-1mRNA, plasma ET-1 as well as pulmonary artery CTGF expressions were increased markedly, respectively, whereas H2S in lung tissue decreased significantly in rats of shunt group as compared with that in sham group (all P<0.05). After administration of NaHS for 11 weeks, H2S in lung tissue increased significantly, whereas SPAP, plasma ET-1 and lung tissue ET-1 mRNA expression as well as pulmonary artery CTGF protein expression decreased significantly, respectively, in rats of shunt+NaHS group as compared with that in shunt group (all P<0.05).CONCLUSION: NaHS might be involved in the development of pulmonary hypertension induced by high pulmonary blood flow by down-regulating vasoactive peptides ET-1 and CTGF expressions in lung tissues of rats.     

Effect of Ginkgo biloba extract on the expression of connective tissue growth factor in the initiating stage of fibrosis in lungs of rats

AIM: To study the effect of Ginkgo biloba extract (GbE) on the expression of connective tissue growth factor (CTGF) in the initiating stage of pulmonary fibrosis of rats after administration of bleomycin (BLM).METHODS: The expression of CTGF in lungs was detected by Western blotting. The content of hydroxyproline was assayed by the method of chloramines T. The content of malondialdehyde (MDA) in plasma was investigated by colorimetry.RESULTS: On day 14 after administration of BLM, the contents of CTGF in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (P<0.05; P<0.01). On day 30 after BLM, the contents of hydroxyproline in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (both P<0.01). Treatment with GbE ameliorated the above changes induced by BLM. CONCLUSION: GbE ameliorates the up-regulation of CTGF in the initial stage of fibrosis in lungs of rats after administration of BLM. GbE prevents the hyperoxidative injury in lungs of rats after BLM, which might be one of mechanisms underling the effect of GbE on CTGF.     

Role of transforming growth factor-β in the airway inflammation and remodeling in asthma

Transforming growth factor-β (TGF-β) was reported to be increased in asthma in some studies. Accumulation of TGF-β in airway promotes smooth muscle cell mitogenesis and hyperplasia, and induces fibroblast and myofibroblast and smooth muscle proliferation as well as increase in protein synthesis in connective tissue (such as collagen deposition on the reticular basement membrane). The autocrine induction of collagen expression by smooth muscle may contribute to the thickening of the reticular basement membrane, irreversible fibrosis and remodeling seen in the airways in some asthmatics. TGF-β is considered to be a major fibrogenic cytokine. It can increase smooth muscle mass and lead to severe bronchial obstruction in an asthma attack.     

Effects of uremic serum of different molecular weight groups ongene and protein expression of connective tissue growth factor gene and proteinexpression in human renal tubular epithelial cells

AIM:To investigate the effects of uremic serum of different molecular weight groups on gene and protein expression of connective tissue growth factor (CTGF) in human renal tubular epithelial cells.METHODS:The serum from 40 chronic renal failure patients and 20 healthy volunteers were collected and uremic serum was segregated to three groups: >10 000 D,5 000-10 000 D,<5 000 D by 10 000 D and 5 000 D molecular weight Centricon Plus 20 Centrifugal Filter Devices.The protein expression of CTGF was examined by Western blotting.The mRNA expression of CTGF was detected by RT-PCR.RESULTS:CTGF gene expression were increased in 2.5%-20% uremic serum groups compared with that in normal control group,and it was the highest in 10% uremic serum groups.CTGF gene expression was increased significantly in molecular weight >10 000 D and 5 000-10 000 D groups,and the highest was in >10 000 D group,but it was no significant difference in <5 000 D group compared with that in normal control group.CTGF protein was increased in different molecular weight uremic serum groups compared with that in normal control group,and gradually increased following the increasing of uremic serum concentration and it was the highest in molecular weight >10 000 D group.CONCLUSION:Human renal tubulointerstitial fibrosis was accelerated significantly by uremic toxin,especially molecular weight >10 000 D uremic toxin through promoting the gene and protein expression of CTGF in renal tubular epithelial cells in patients with chronic renal failure.     

Effect of TRPC6 on PDGF-induced proliferation of rat airway smooth muscle cells

AIM:To investigate the role of canonical transient receptor potential channel 6 (TRPC6) in the proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor (PDGF). METHODS:Rat ASMCs were cultured by enzyme digestion and tissue adhesion. The method of indirect immunofluorescence was applied to identify the ASMCs and to detect the expression of TRPC6 in ASMCs. The proliferation of ASMCs was determined by CCK-8 assay. The mRNA expression of TRPC6 was tested by real-time PCR. The protein expression of TRPC6 was analyzed by Western blotting. The influence of TRPC6 blocker at different concentrations on the proliferation of ASMCs was measured by CCK-8 assay. RESULTS:The results of immunofluorescence indicated that TRPC6 expression in ASMCs was positive. PDGF at concentration of 20 μg/L induced the proliferation of ASMCs compared with control group (P<0.05). When ASMCs were treated with both PDGF and different concentrations of TRPC6 blocker SKF96365, the proliferation of ASMCs was attenuated in dose-dependent and time-dependent manners as compared with the cells treated with PDGF alone (P<0.05). The mRNA expression of TRPC6 in PDGF group was significantly increased (P<0.05) after ASMCs were treated with PDGF for 12 h, 24 h and 48 h. The protein level of TRPC6 in PDGF group was significantly increased after ASMCs were treated with PDGF for 24 h and 48 h compared with control group (P<0.05). CONCLUSION: Up-regulation of TRPC6 at mRNA and protein levels is most possibly related to the proliferation of ASMCs induced by PDGF. Therefore, TRPC6 is involved in the proliferation of ASMCs.     

Effect of Kechuanning on airway remodeling and protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus

AIM:To investigate the effect of Kechuanning on airway remodeling and the protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus. METHODS:The asthmatic rat model induced by respiratory syncytial virus was established. The experimental rats were divided into normal group, asthma model group, low dose (0.33 mL/kg), middle dose (3.0 mL/kg) and high dose (10 mL/kg) of Kechuanning groups, and PD98059 (3 mg/kg) group. The airway responsiveness of the rats was measured by animal ventilator. The pathological changes of the lung tissues were observed by HE staining. PAS staining and Masson staining were used to observe goblet epithelial cells metaplasia and airway collagen deposition. The expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the lung tissues of the rats was detected by immunohistochemical staining. The protein levels of ERK1/2 and p-ERK1/2 were determined by Western blot. RESULTS:Compared with model group, the airway responsiveness of the rats in middle dose and high dose of Kechuanning groups was significantly decreased (P<0.01), the injury of lung tissues was significantly decreased, the goblet epithelial cells metaplasia and airway collagen deposition were significantly reduced (P<0.01), and the expression of MMP-9 and TIMP-1 in the lung tissues was also significantly decreased (P<0.01). In addition, the protein level of p-ERK1/2 in high dose of Kechuanning group was significantly decreased compared with model group (P<0.01). CONCLUSION:Kechuanning may treat asthma by regulating the expression of p-ERK1/2 in the lung tissues and improving the airway remodeling symptoms of asthmatic rats induced by virus.     

Role of hypoxia stimulated expression of connective tissue growth factor in renal interstitial fibrosis

AIM: To study the expression of connective tissue growth factor (CTGF) induced by hypoxia, and the role and mechanism of hypoxia on promoting renal interstitial fibrosis. METHODS: Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in rat animal model for 9 days as in vivo studies; marker of hypoxia-HIF-1α mRNA and protein, the expression of CTGF in the obstructed kidneys were assessed by RT-PCR, immunohistochemistry and Western blotting respectively. In vitro, normal rat kidney interstitial fibroblast cells (NRK-49F) were exposed to hypoxia (1%O2) for up to 6 hours, hypoxia was confirmed by detecting the expression of HIF-1α protein in cells, cellular level of CTGF mRNA and protein were assessed by RT-PCR and Western blotting respectively. RESULTS: Neither HIF-1α mRNA nor HIF-1α protein was expressed in the kidney from sham-operated group of rats. High level of HIF-1α mRNA were occurred, and strongly HIF-1α positive immunostaining were seen in the tubular and interstitial cells in kidney from UUO rats. Expression and location of CTGF protein were paralleled and relevant with the expression of HIF-1α protein in kidney of UUO rats. In cultured NRK-49F cell line, subjected to hypoxia even for 6 hours stimulated the expression of CTGF mRNA and protein. CONCLUSION: Our results indicated that hypoxia could stimulate the expression of CTGF mRNA and protein in kidney from UUO rats, which may in turn contribute to renal interstitial fibrosis.     

Expression of connective tissue growth factor in the renal tissue of rats with unilateral ureteral obstruction

AIM: To detect the expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat unilateral ureteral obstructive (UUO) nephropathy animal model, and to observe the kinetic changes at different stages of firosis. METHODS: Male SD rats were subjected to either left ureteral ligation or sham operation, then killed at 3, 7, 14, 21 or 28 days after UUO or sham operation (n=6 at each time point). HE, Masson or PAS staining were applied to the renal tissue sections. The extent of tubulointerstitial injury was determined by Banff classification. RESULTS: The extent of tubulinterstitial fibrosis became serious with the time of obstruction. Tubules were mostly atropic and replaced by proliferative fibrous tissue at day 28. The expression of CTGF and α-SMA were consistent with the damage of tubulointerstitial. The positive correlation among CTGF or α-SMA and the tubulointerstitial injury scores were significant. The expression of TGF-β1 came to peak at day 7 to 14, and gradually decreased at day 21 and 28. CONCLUSION: These results indicate that the expression of CTGF may be upregulated by TGF-β in UUO rats, and CTGF may be involved in tubulointerstitial fibrosis through the development of myofibroblasts.     

Dynamic observation of asthma model induced by allergen in mice from acute inflammation to chronic remodeling

AIM: To dynamically observe and compare the relative changes of the indexes from the process of acute inflammation to chronic remodeling in asthmatic mice induced by ovalbumin (OVA).METHODS: Female BALB/c mice (n=60) were randomly divided into normal control group and asthma group. The mice in asthma group were sensitized and challenged by OVA, while the mice in normal group received equal volume of normal saline (NS). The challenge was performed for 3 consecutive days from the 21th day to observe the response of acute inflammation, and then the mice in different groups were challenged once per week for 5 weeks. Detailed comparisons of the dynamic changes of cell infiltration, cytokine expression and airway remodeling were conducted.RESULTS: Compared with NS group, the mice in OVA group showed a predominantly eosinophilic infiltration into the airway lumen, increased production of Th2-type cytokines, secretion of epithelial mucus and deposition of subepithelial collagen. In OVA challenge groups, the levels of inflammatory cells and inflammatory factors were remarkably higher in 24 d group, whereas the most obvious changes of goblet cell hyperplasia and airway remodeling were observed in 52 d group.CONCLUSION: Acute asthma model is sufficiently induced by 3 consecutive days of OVA challenge protocol, which is accompanied with high levels of inflammatory cells and inflammatory factors. The OVA challenge protocol once per week for 5 weeks could induce a chronic asthma model with obvious airway remodeling.     

Epidermal growth factor receptor pathway participates in growth promotion of chondrocytes by growth hormone from sex hormone-inhibited adolescent rats

AIM: To investigate the possibility that epidermal growth factor receptor pathway participates in the growth promotion by growth hormone (GH) of growth plate chondrocytes cultured in vitro from adolescent rats treated with gonadotropin-releasing hormone analogue (GnRHa). METHODS: The chondrocytes from tibial growth plate of 5-8 female Sprague-Dawley (SD) rats treated with GnRHa were cultured in monolayer. Specific pharmacological inhibitor of Janus kinase (JAK2 tyrphostin AG490, 1, 10, 100 nmol/L), EGFR kinase inhibitor AG1478 (0.1, 1, 10 nmol/L) and neutralizing antibodies against EGF (0.1, 1, 10 mg/L) were added before GH stimulation. The proliferation of chondrocytes was investigated by the methods of MTT and immunohistochemical staining for PCNA. Phosphorylations of ERK1/2 and EGFR were detected by Western blotting. RESULTS: GH enhanced the proliferation of chondrocytes and the levels of ERK1/2 and EGFR phosphorylation in a dose dependent manner. The effect peaked at the concentration of 100 μg/L. Pretreatment with tyrphostin AG490 and AG1478 almost completely inhibited the proliferation of chondrocytes and phosphorylation of ERK1/2 and EGFR by GH. However, the neutralizing antibodies against EGF only partially inhibited the effects of GH.CONCLUSION: GH achieves its direct effect on the promotion of cell proliferation by the activation of JAK2 pathway and the downstream end of MAPK-ERK signaling molecules. The promotion of cell proliferation can be mediated by activation of EGFR pathway. The results suggest that there is signaling cross-talk between GH and EGF-EGFR pathway.     

Role of PARP and caspase-3 in the airway epithelial injury induced by peroxynitrite

AIM:To study the mechanism responsible for ONOO^--induced the airway epithelial injury. METHODS:Effects of 3-aminobenzamide(3-AB), a poly-(ADP-ribose) polymerase(PARP) inhibitor, and Ac-DEVD-CHO, a caspase-3 inhibitor, on LDH release and apoptosis of cultured rat tracheal epithelial (RTE) cells induced by ONOO^- were examined. The cleavage of PARP was analysed by Western blot. RESULTS:3-AB inhibited the release of LDH induced by ONOO^- partially, and had no effect on the apoptosis of RTE cells. Caspase-3 inhibitor Ac-DEVD-CHO obviously prevented the apoptosis of RTE cells induced by ONOO^- in a dose-dependent manner. The cleavage of PARP was observed in the process of apoptosis of RTE cells induced by ONOO^-. CONCLUSIONS:PARP activation represents one of the pathways of ONOO^--mediated epithelial injury, and the excessive activation of PARP contributes to the necrosis in RTE cells induced by ONOO^-. Cleavage of PARP by activated caspase-3 plays a crucial role in the apoptosis of RTE cells induced by ONOO^-.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

Nerve growth factor attenuates cochlear injury induced by brain ischemia and reperfusion in rats

AIM: To investigate the effect of nerve growth factor (NGF) on auditory and cochlear damage induced by brain ischemia and reperfusion. METHODS: The rats with brain ischemia and reperfusion were divided into two groups at random. In the experimental group, the rats were injected intramuscularly with NGF . In control group, the animals were injected with normal saline instead of NGF. Then the hearing loss and cochlear structural changes of rats in both groups were compared. RESULTS: It was found that the hearing loss of rats in NGF group were less significantly than that of control group ( P< 0.01) after 60 min and 24 h reperfusion following 30 min ischemia. Scanning and transmission microscopy showed that the damage of the outer hair cells and the spiral neurons of rats in NGF group was much slighter than that of control group. CONCLUSION: NGF prevents auditory and cochlear injury induced by brain ischemia and reperfusion.     

Effect of hydrogen sulfide on airway inflammation induced by ozone in mice

AIM: To investigate the effect of hydrogen sulfide (H₂S) on airway inflammation induced by ozone (O₃) exposure and its mechanisms.METHODS: C57BL/6 mice (n=32) were randomly divided into control group, O₃ group, NaHS+O₃ group and NaHS group. The mice in O₃ group and O₃+NaHS group were exposed to 2.14 mg/m³ O₃ for 3 h on days 1, 3 and 5, while the mice in control group and NaHS group were exposed to filtered air. NaHS (14 μmol/kg) was administered intraperitoneally to the mice in NaHS group and O₃+NaHS group 30 min before each exposure. After the last exposure for 24 h, the airway responsiveness was determined, and bronchoalveolar lavage fluid (BALF) was collected for counting inflammatory cells and measuring total protein concentration. The lung tissues were collected for observing the morphological changes with HE staining. The levels of interleukin-6 (IL-6), interleukin-8 (IL-8), malondialdehyde (MDA) and NF-κB p65 protein in the lungs were determined.RESULTS: Compared with control group, the airway responsiveness, inflammatory cells, protein concentration, inflammation score, levels of IL-6, IL-8, MDA and NF-κB p65 in O₃ group increased significantly, but these in NaHS+O₃ group decreased compared with O₃  group.CONCLUSION: The present findings suggest that H₂S attenuates O₃ induced airway inflammation by inhibiting NF-κB expression and preventing lipid peroxidation.