Influence of IFN-γ combined with M-pred on human embryonic lung fibroblasts

AIM: To look for a better method to deal with interstitial lung disease, interferon-gamma (IFN-γ) combined with methylprednisolone (M-pred) to influence human embryonic lung fibroblast on proliferation, collagen synthesis and the expression of transforming growth factor-β1 (TGF-β1) protein and mRNA were investigated. METHODS: Exponentially growing cells were preincubated for 48 h before harvested. The microculture tetrazolium (MTT) assay was used to measure the inhibition ratios of M-pred combined with different concentrations of IFN-γ. The expression of proliferation cell nuclear antigen (PCNA) was detected by immunocytochemical analysis. Hydroxyproline kit was adopted to detect collagen synthesis. The expressions of TGF-β1 mRNA and protein were detected respectively by RT-PCR and Western blotting. RESULTS: Methylprednisolone, IFN-γ as well as the combination of methylprednisolone and IFN-γ inhibited the proliferation of HELF and the expression of PCNA in comparison with control group (P<0.05). That also decreased the expression of hydroxyproline, inhibited the expression of TGF-β1 mRNA and protein (P<0.05). The effectiveness of IFN-γ became stronger when the concentration increased. The synergistic effect between IFN-γ and methylprednisolone was observed (P<0.05). CONCLUSION: The combination effect of IFN-γ and methylprednisolone is augmented compared with using IFN-γ or methylprednisolone alone, suggesting that the combination use of both drugs has better anti-fibrous degeneration effect than use either alone.     

Effect of pioglitazone on glucose metabolism and PPAR-γ expression in lipid-infused rats

AIM: To investigate the effect of pioglitazone (Pio) on glucose metabolism and peroxisome proliferators-activated receptor (PPAR)-γ expression in free fatty acid (FFA) -induced insulin resistance in rats. METHODS: A hyperinsulinaemic-euglycaemic clamp and ［3-3H］-glucose tracing technique were used in awake rats. Glucose metabolism in vivo and PPAR-γ in adipose tissue expression were assessed with elevation FFA by lipid infusion over 4 h in rats pretreated with or without Pio.RESULTS: During steady-state of clamp, there was a significant increase in plasma FFA in two lipid-infused groups, compared to control rats (P<0.01). The glucose infusion rates (GIR) in Pio-treated rats (P/L group), compared with controls, were significantly reduced ［(20.6±0.4) mg·kg^-1·min^-1 vs (33.6±0.6)mg·kg^-1· min^-1, P<0.01］, whereas the GIR was lower in the lipid group (L group) than that in the P/L group［(12.6±0.8) mg·kg^-1·min^-1 vs (20.6±0.4) mg·kg^-1·min^-1, P<0.01］. The hepatic glucose production (HGP) was significantly suppressed (85%) ［(18.3±2.1)mg· kg^-1·min^-1 (basal) vs (2.7±2.4)mg· kg^-1·min^-1, and (17.5±2.6) mg· kg^-1·min^-1 vs (2.6±1.0)mg· kg^-1·min^-1］, all P<0.01 during clamp in control and P/L groups. The suppressive effect of insulin on HGP was significantly blunted in L group［(17.3±2.1)mg· kg^-1·min^-1 vs (15.8±1.5)mg· kg^-1·min^-1］. The rate of glucose disappearance (GRd) was significantly reduced in two lipid-infused rats compared with controls［(26.6±1.6)mg· kg^-1·min^-1 and (23.2±0.9)mg· kg^-1·min^-1 vs (37.7±2.6)mg·kg^-1·min^-1,P<0.01］. The PPAR-γ expression of adipose tissue in P/L group was significantly upregulated. CONCLUSION: Lipid-infusion induces an acute insulin-resistance in vivo. Pio treatment upregulates the PPAR-γ of adipose tissue and suppresses HGP. Pio can protect partly against lipid-induced insulin resistance.     

TCRVβ7.1 transfection activates signal protein ERK and insreases IFN-γ expression in PBMCs

AIM: To investigate the effect of tumor-specific T cell receptor (TCR) gene transfection on production of cytokine and signaling activation in T cells.METHODS: TCRVβ7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vβ7.1 was detected by flow cytometry before and after transfection. The total quantities of protein and phosphorylation of ERK1/2 were detected by Western blotting. The expressions of IL-4 and IFN-γ were detected by ELISA.RESULTS: The results of flow cytometry showed that TCRVβ7.1 protein was efficiently expressed after transfection. The phosphorylation level of ERK increased significantly in TCRVβ7.1-modified PBMCs, and was related with the activation of T cells. The expression of IFN-γ was significantly higher in TCR-transfected cells than that in non-transfected cells. The expression of IL-4, however, has no distinct difference between groups.CONCLUSION: The transfection of TCRVβ7.1 induces phosphorylation of ERK1/2 and production of IFN-γ, and activates T lymphocytes.     

Influence of siRNA-mediated PPARγ gene knockdown on invasion ability of chorioepithelioma JEG-3 cells

AIM: To investigate the effect of peroxisome proliferator-activated receptor γ(PPARγ) on the invasion ability of trophoblasts. METHODS: A segment of PPARγ siRNA was synthesized. Three groups were designed: experiment group, negative control group and blank control group. The PPARγ siRNA was transfected into JEG-3 cells. Real-time quantitive PCR was used to detect the mRNA levels of PPARγ and mucin-1(MUC1). The Transwell culture inserts were used to detect the invasion ability of JEG-3 cells 24 h after treated with PPARγ siRNA. RESULTS: After transfected with PPARγ siRNA, the mRNA expression levels of PPARγ and MUC1 were significantly depressed by (75.0±0.8)% and (65.0±1.3)% (P<0.05),respectively, and the invasion ability of JEG-3 cells was significantly strengthened (P<0.05). CONCLUSION: The depression of PPARγ gene down-regulates the expression of MUC1, and affects the invasion ability of trophoblasts, indicating that PPARγ may regulate the invasion of trophoblasts by MUC1 and involve in the development of placental defects such as preeclampsia.     

Protective role of 14-3-3γ in burn and LPS-induced rat myocardial injury

AIM: To study the protective role of 14-3-3γ in burn or LPS-induced myocardial injury. METHODS: The rat model of burn or LPS-induced injury was established. The heart functions and 14-3-3γ protein expression were detected 3 h, 6 h, 12 h and 24 h after treatment. Primary neonatal rat cardiomyocytes were used in vitro. pFLAG-14-3-3γ plasmid was constructed and transfected into the cardiomyocytes 24 h before LPS-induced injury. The injury in the cardiomyocytes was evaluated by measuring the cell viability and the level of lactate dehydrogenase(LDH). Apoptosis of cardiomyocytes was detected by flow cytometry. Opening of mitochondrial permeability transition pore (mPTP) was also determined by Ca²⁺-induced swelling of isolated myocardial mitochondria. RESULTS: The expression of 14-3-3γ was elevated following the burn or LPS-induced myocardial injury in vivo. In vitro, transfection with pFLAG-14-3-3γ plasmid in to the cardiomyocytes significantly protected against LPS-induced injury. Compared with the cardiomyocytes without transfection with pFLAG-14-3-3γ plasmid, higher cell viability rate and lower LDH release, cell apoptosis and mPTP opening were observed in the cardiomyocytes transfected with pFLAG-14-3-3γ plasmid. CONCLUSION: The 14-3-3γ protein protects the heart against burn or LPS-induced injury by inhibiting the mPTP opening.     

Effects of rosiglitazone,a PPARγ ligand,on chemoprevention of MNNG-induced gastric cancer in rats

AIM: To examine the chemo-preventive effects of peroxisome proliferator-activated receptor γ(PPARγ) ligand rosiglitazone (RSG) on a rat model of gastric carcinogenesis induced by chemical carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). We also attempted to identify novel anti-cancer mechanisms of rosiglitazone.METHODS: Ninety male Wistar rats were randomly allocated into six groups: group A (control group); group B (MNNG group); group C, D and E (RSG group, given different concentrations of rosiglitazone). The treatment procedures were terminated at 40th week. Stomach was harvested and gastric carcinoma was verified by histology. The gastric cancer incidence in different groups was calculated. To elucidate the mechanisms underlying the chemo-preventive effects of PPARγ ligand, we examine the gene expression profiles of MNNG induced gastric cancer and the rosiglitazone treated gastric cancer with Uniset Rat I Bioarray microarray.RESULTS: Incidence of gastric cancer in group A-E was 0% (0/10), 70% (14/20), 15% (3/20), 30% (6/20) and 30% (6/20), respectively. Gastric cancer incidence in group C, D and E was significantly lower than that in group B (P<0.01). A gene that showed prominent responses in rosiglitazone treated group was identified. The hypertension-related, calcium-regulated gene (HCaRG) was significantly upregulated in rat gastric carcinoma in rosiglitazone treated group when compared to MNNG group. The expression of HCaRG was down-regulated in human gastric cancerous tissue. CONCLUSION: PPARγ ligand rosiglitazone has a potent chemo-preventive effect against gastric cancer development in rats. Upregulation of HCaRG may be one of the mechanisms underlying the chemo-preventive effect of rosiglitazone in gastric cancer.     

Protective role and mechanism of peroxysome proliferator activated receptor-γ in injury of cultured rat cortical neurons induced by hypoxia/ reoxygenation

AIM: To observe the role of peroxysome proliferator activated receptor-γ (PPAR-γ) and the relationship of cyclooxygenase-2 (COX-2) and PPAR-γ in injury of cultured rat cortical neurons induced by hypoxia/reoxygenation. METHODS: Primary rat cortical neurons were cultured. Experiments include control group, hypoxia/ reoxygenation group and hypoxia/ reoxygenation with PPAR-γ agonist group. Cell viability was surveyed by MTT assay. COX-2 protein expression was measured by Western blotting.RESULTS: Neuron viability raised dramatically in hypoxia/reoxygenation with PPAR-γ agonist group, compared with hypoxia/reoxygenation group (P<0.05). The COX-2 protein expression in hypoxia/ reoxygenation with PPAR-γ agonist group decreased significantly compared with hypoxia/ reoxygenation group (P<0.05). CONCLUSION: PPAR-γ agonist inhibits the expression of COX-2 and reduces obviously cortical neuron injury induced by hypoxia/ reoxygenation. It may protect cortical neurons by down-regulating the expression of COX-2.     

PPARγ affects epithelial-mesenchymal transition in renal tubular epithelial cells under high glucose by regulating AKT/FAK signaling pathway through PTEN

AIM To investigate the role of peroxisome proliferator-activited receptor γ （PPARγ） in the regulation of PTEN/AKT/FAK signaling pathway and epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells grown in high-glucose environment. METHODS Renal tubular epithelial cells （NRK52E cells） cultured in high glucose were used as an in vitro model system. PPARγ was over-expressed or knocked down in these cells, and its effect on PTEN expression was determined by RT-qPCR, immunofluorescence and Western blot. The changes of EMT-related proteins were also measured. The PPARγ inhibitor GW9662 and the PPARγ agonist rosiglitazone were used along with PTEN over-expression or knockdown to determine whether the effects of PPARγ were mediated through PTEN. RESULTS PPARγ over-expression resulted in the increased expression of PTEN at mRNA and protein levels, the up-regulation of E-cadherin, and the down-regulation of vimentin and α-SMA. Knockdown of PPARγ expression reduced the mRNA and protein levels of PTEN, down-regulated E-cadherin, and up-regulated vimentin and α-SMA （P<0.05）. Treatment of the NRK-52E cells with GW9662 decreased PTEN expression and increased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN over-expression. Treatment of the NRK-52E cells with rosiglitazone increased PTEN expression and decreased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN knockdown. These changes were all statistically significant （P<0.05）. CONCLUSION PPARγ regulates the mRNA and protein expression of PTEN in renal tubular epithelial NRK52E cells, and affects EMT in renal tubular epithelial cells. The regulation of AKT/FAK signaling pathway by PPARγ is primarily mediated by PTEN.     

Effect of S-nitrosylation induced by IL-1β and IFN-γ on DNA binding activity of cAMP response element binding protein in fibroblast-like synoviocytes

AIM: To investigate the effect of S-nitrosylation induced by recombinant interleukin-1β (rIL-1β) and recombinant interferon-γ (rIFN- γ) on DNA binding activity of cAMP response element binding protein (CREB) in fibroblast-like synoviocytes (FLSs). METHODS: (1)FLSs were incubated with rIL-1β and rIFN-γ in the presence or absence of inducible nitric oxide synthase inhibitor aminoguanidine (AG) for 12 h. The supernatant of the cell culture was collected to determine the contents of nitric oxide (NO). The total proteins prepared from each group ［only the total proteins prepared from rIL-1β+rIFN-γ group was reacted with dithiothreitol (DTT) for 15 min in vitro］ were subjected to the biotin switch assay, and the level of S-nitrosylation was determined by Western blotting. (2)FLSs were incubated with rIL-1β, rIL-1β+ rIFN-γ and AG respectively for 12 h. The nuclear extracts from each group were prepared. The nuclear extracts from each group were subjected to electrophoresis mobility shift assay to analyze the DNA binding activity of CREB (only the nuclear extracts from rIL-1β +rIFN-γ group was reacted with DTT for 15 min in vitro before assaying). RESULTS: rIL-1β plus rIFN-γ increased the production of NO and the level of S-nitrosylation, which was inhibited by AG. Administration of DTT in the total proteins reversed the induction of S-nitrosylation by rIL-1β and rIFN-γ. Co-incubation with rIL-1β and rIFN-γ inhibited the CREB activity induced by rIL-1β alone, which was reversed by AG. Administration of DTT in the nuclear extracts reversed the effect of co-incubation of the cytokines. CONCLUSION: Co-incubation with rIL-1β and rIFN-γ may increase the level of S-nitrosylation through inducing the production of endogenous NO, leading to reversible thiols modification of CREB and inhibit the DNA binding activity of CREB in FLSs.     

Association of IFN-γ and IL-4 gene polymorphisms with childhood bronchial asthmatic susceptibility and the levels of plasma IFN-γ,IL-4 and IgE

AIM: To investigate the gene polymorphisms of interferon-γ(IFN-γ) and interleukin-4(IL-4) and the association with asthmatic susceptibility and the levels of plasma IFN-γ, IL-4 and IgE of asthmatic children. METHODS: 100 asthmatic children and 122 control children were enrolled the study. The genotypes of IFN-γ gene-179G/T polymorphism, IL-4 gene-33C/T and-589C/T polymorphisms were tested by PCR-RFLP.The genotype of IFN-γ gene +874A/T polymorphism was tested by AS-PCR.The CA repeat polymorphism of IFN-γ gene was detected by capillary electrophoresis technique.The levels of serum IFN-γ, IL-4 and IgE were measured by ELISA. RESULTS: 100 asthmatic children and 122 control children were all GG homozygotes at -179 locus of IFN-γ gene.-179 locus of IFN-γ gene has no mutation. The genotypes and allele frequency of IFN-γ gene +874A/T and CA repeat polymorphisms showed no significant difference between asthmatic children and the control(P>0.05). An association was revealed between IFN-γ gene +874A/T polymorphism and the level of plasma IFN-γ.The level of IFN-γ was lower in AA genotype than in AT genotype(P<0.05). The genotypes and allele frequency of IL-4 gene -33C/T and -589C/T polymorphisms showed significant difference between asthmatic children and the control(P<0.05).The levels of plasma IL-4 and IgE were higher in TT genotype at -33 locus and -589 locus than those in CT genotype, but only -33C/T polymorphism was associated with the level of plasma IL-4(P<0.05). CONCLUSION: The IFN-γ gene +874A/T and CA repeat polymorphisms were not correlated with asthmatic susceptibility, but there is significant correlation between the level of IFN-γ and +874A/T polymorphism. TT genotype of IL-4 gene -33 locus and -589 locus maybe the susceptible genotype of asthma in children, and the -33 locus polymorphism is associated with the level of IL-4.