Study on the binding capacity of IgA1 from patients with IgA nephropathy to human mesangial cells

AIM: To examine and compare the ability of serum IgA 1, from both the patients with IgA nephropathy(IgAN) and the healthy control, to bind to human mesangial cells(HMC). METHODS: Serum IgA was isolated with jacalin column, heated to aggregated form(IgA₁) and labeled with [¹²⁵I]. Binding capacity of IgA₁ to primary HMC was evaluated by radioligant binding assay, specificity of binding was determined by competitive inhibition, and relative affinities was compared by cross competitive inhibition. RESULTS: Both IgA₁ from normal control and patients with IgAN bound to MC in a dose-dependent, saturatable manner, but the binding of IgA₁ from patients was saturated at approximately 200 pmol while that from healthy was at 400 pmol. The Scatchard analysis revealed a Kd of(8.9±2.1)×10^-8 mol/L for patient' s IgA₁ versus(4.3±1.2)×10^-7mol/L for normal IgA₁(P<0.05). Competition inhibition showed that both serum albumin and IgG could not block the binding of IgA₁ to HMC while mIgA 1 could partially block that. Cross competition inhibition demonstrated that patient' s IgA₁ blocked normal IgA₁ binding significantly(P<0.01), however, normal IgA₁ was unable to inhibit the binding of patient' s IgA₁. CONCLUSIONS: ①There are IgA binding proteins or receptors on mesangial cells. ②IgA₁ from patients with IgAN has a higher binding capacity than that from healthy.     

Effect of tea-polyphenols on the activation of NF-κB and the expression of TGF-β1 mRNA in THP-1 cells incubated with VLDL,LDL or ox-LDL

AIM:To investigate the effect of tea-polyphenols (TP) on the activation of NF-κB and the expression of TGF-β₁ mRNA in THP-1 cells (a human acute monocytic leukemia cell line). METHODS:THP-1 cells were incubated with the different concentrations of TP, VLDL, LDL or ox-LDL. In the THP-1 cellls, the nuclear malposition rate of NF-κB was detected with immunohistochemistry technique, the positive index of the TGF-β₁ mRNA expression was detected by hybridization in situ, and accumulation of total cholesterol (TC) in cells incubated with 0.4-40 μg/L TP was determined with oxidase assay. RESULTS:The nuclear malposition rate of NF-κB, the positive index of the TGF-β₁ mRNA expression and TC in THP-1 cells incubated with 0.4-40 μg/L of TP were lower than those with 0 μg/L of TP in TP-V group, TP-L group and TP-O (P＜0.05). The differences of these markers in THP-1 cells incubated with more than 40 μg/L TP in TP-V group, TP-L group and TP-O were not statistically significant, compared with TP-C group (P＞0.05). CONCLUSION:TP inhibited the activation of NF-κB, the expression of TGF-β₁ mRNA and the foam cell formation in the mono-macrophage.     

Recombinant human defensin α1 induces proliferation of rat glomerular mesangial cells in vitro

AIM: To study the effects and mechanism of recombinant human defensin α₁ on cell proliferation in cultured rat glomerular mesangial cells.METHODS: The influences of defensin α₁ at various concentrations on rat 1097 mesangial cell line cultured in vitro were evaluated with MTT assay.The different concentrations of U0126,signal-regulated protein kinase (MEK) inhibitor,were added into the culture mediums of mesangial cells to do blocking test.Incubated with a final concentration of 3 mg/L defensin α₁,the phosphorylation of extracellular signal regulated kinase (ERK)1/2 and type IV collagen of mesangial cells in different times were evaluated by Western blotting.RESULTS: Defensin α₁ at 3-20 mg/L enhanced proliferation of rat glomerular mesangial cells.The incubation times for the maximum effect on proliferation was 12 h (P<0.01),whereas defensin α₁ concentration >20 mg/L decreased cell proliferation.The cell proliferation induced by defensin α₁ was inhibited by U0126.Stimulation of the cells with defensin α₁ at concentration of 3 mg/L for 5 minutes induced a maximum effect on a ratio of phosphorylation of ERK1/2 to total ERK.After 12 h incubation with defensin α₁,an increase in type IV collagen was observed by Western blotting and continued to increase at 24 h and 48 h (P<0.01).CONCLUSION: Defensin α₁ enhances rat glomerular mesangial cell proliferation and induces type IV collagen production by MAPK signaling pathway.     

TGF-β1 upregulates Smad2 expression in peritoneal mesothelial cells

AIM: To investigate the expression of Smad2 signal protein in peritoneal mesothelial cells and how transforming growth factor β1 (TGF-β1) affects its expression. METHODS: Rat peritoneal mesothelial cells were cultured in different levels of TGF-β1 (0,1.25,2.5,10 μg/L) for different time (0,5,15,30,60,120 min). Endogenous Smad2 expression was evaluated by RT-PCR and immunohistochemical assay. The alteration of subcellular location of Smad2 was determined by immunohistochemical assay. RESULTS: TGF-β1 induced Smad2 mRNA expression, which increased at 5 min, peaked at 30 min, and declined to baseline levels by 120 min, in a time-dependent manner. Smad2 mRNA expression induced by TGF-β1 was also in a dose -dependent manner. TGF-β1 induced Smad2 phosphorlylation and nuclear localization in both time-dependent and dose-dependent manner, which was concordant with mRNA expression. Smad2 translocated from cytoplasm to nuclear accumulation in response to TGF-β1, and peaked at 30 min. CONCLUSIONS: Smad2 is present in peritoneal mesothelial cells. TGF-β1 may activate Smad2 expression and translocation to nuclear in a time-dependent and dose-dependent manner.     

Roles of gap junction in TGF-β1-induced proliferation of vascular smooth muscle cells from spontaneous hypertensive rats

AIM: To investigate whether gap junction participates in transforming growth factor β₁(TGF-β₁)-induced proliferation of spontaneous hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). METHODS: The thoracic aorta of the rats were sampled. The primary SHR VSMCs were isolated and cultured in vitro. The cells were divided into 4 groups: control group, TGF-β₁ group,18α-glycyrrhetinic acid(18α-GA) group and TGF-β₁+18α-GA group. The proliferation of SHR VSMCs was observed by the methods of MTT and flow cytometry. The protein expression and co-localization of connexin(Cx)43 and Cx40 in SHR VSMCs were detected by immunofluorescence staining. The protein levels of Cx43 and Cx40 in the cells were also measured by Western blotting. The method of molecular dye transfer (scrape dye transfer method) was applied to detect the function of gap junction in SHR VSMCs. RESULTS: The protein expression of Cx43 and Cx40 in SHR VSMCs was positive and co-localized in the cytoplasm. Compared with control group, the percentage of S-phase detected by cell cycle and A value detected by MTT in TGF-β₁ group were obviously increased (P<0.05), indicating that the proliferation of the cells was enhanced. However, the proliferation of the cells decreased in 18α-GA group (P<0.05). Compared with TGF-β₁ group, the percentage of S-phase and A value in TGF-β₁+18α-GA group were both significantly decreased (P<0.05), indicating that the proliferation of the cells decreased. Compared with control group, the protein expression of Cx43 in TGF-β₁ group was increased (P<0.05), whereas the protein expression of Cx40 was not changed (P>0.05), and the protein expression of Cx43 and Cx40 in 18α-GA group were decreased (P<0.05). Compared with TGF-β₁ group, the expression of Cx43 in TGF-β₁+18α-GA group was significantly decreased (P<0.05),but no difference of the Cx40 protein levels between the two groups was observed. Compared with control group, the function of gap junction detected by scrape dye transfer method in TGF-β₁ group was enhanced (P<0.05), and weakened in 18α-GA group (P<0.05). Compared with the TGF-β₁ group, the function of gap junction in TGF-β₁+18α-GA group was significantly attenuated (P<0.05). CONCLUSION: TGF-β₁ enhances the function of gap junction to stimulate the proliferation of SHR VSMCs through the expression of Cx43 protein. The expression of Cx40 protein may not play a major role in this process.     

Effects of TGF-β1 and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells(HSPC)

AIM:To study the effect of TGF-β₁ and TNF-α antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cells (HSPC). METHODS:CD₃₄⁺cells were purified from fresh umbilical cord blood by immunomagnetic beads, and mononuclear cells were purified from bone marrow by Ficoll-hypaque. The effects of TGF-β₁ and /or TNF-α antisense PS-ODNS on ex vivo expansion of CD₃₄⁺ cells、CFU-GEMM、CFU-GM、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems.RESULTS:TGF-β₁ antisense PS-ODNS cooperated with cytokines increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E, which was as 4, 2.6, 2.7, 1.8, 2.1 times as that of the control (the cytokines combination), respectively. TNF-α antisense PS-ODNS cooperated with cytokines respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 4, 2.9, 2.6, 1.7, 1.8 times as that of the control. The above two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD₃₄⁺ cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5.3, 2.1, 2.7, 1.9, 1.8 times as that of the control.CONCLUSION:Inhibition of endogenous TGF-β₁ and TNF-α by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.     

Roles of TGF-β1 and singal protein SMADs in rat myocardial hypertrophy

AIM: To investigate the role of SMADs singal pathway in rat myocardial hypertrophy. METHODS: The rat model of myocardial hypertrophy was produced by constriction of the abdominal aorta. The wet left vertricular/body weight ratio (LVMI) was measured. The expression of TGF-beta l and Smad 2, 3, 7 mRNA were assessed by RT-PCR. RESULTS: The LVMI and the expression of TGF-beta l and Smad 2, 3, 7 mRNA in cardiomyothy were increased in 3 day after the operation and continued at last 4 weeks. The peak expression of TGF-beta l and Smad 2, 3, 7 mRNA was in 2 weeks after operation. The expression of Smad 7 was increased in 3 days after operation, but the peak was in 1 week after operation, then decreased. CONCLUSION: The signal protein Smad 2, 3, 7 are involved in the progress of rat myocardial hypertrophy produced by constriction of abdominal aorta.     

Dynamical observation of the expression of TGF-β1 and MAPK1/3 in the renal tubules of rats with diabetes

AIM: To observe the expression of transforming growth factor β₁ (TGF-β₁), MAPK_1/3 and fibronectin (FN) in the development of renal tubulointerstitial disease. METHODS: Wistar male rats were randomly divided into normal control group, diabetic group of 1week, 2 weeks, 4 weeks and 8 weeks. Diabetic model was induced by peritoneal injection of streptozotocin. Immunohistochemistry was employed to detect the expression of TGF-β₁, MAPK_1/3 and FN in the kidney. TGF-β₁ protein in the renal cortex was checked by Western blot. BG, Scr and UP were analysed by biochemical methods, and the morphological changes in renal tubulointerstitium were also examined under microscopy on sections stained with HE and PAS. RESULTS: The expression of MAPK_1/3 and FN was observed, but not the expression of TGF-β₁ in normal renal tissue. Positive staining of TGF-β₁ was observed in the renal tubulo-interstitium in 1-week diabetic group and thereafter it increased in the course of diabetes. A continuous increase in the expression of MAPK_1/3 and FN was also observed in two - week diabetic rats. Chronologically the expression of TGF-β₁,MAPK_1/3 and FN and the ratio of KW/BW were positively correlative with each other in diabetic animals except one -week diabetic rats. There was also a positive correlation between MAPK_1/3 and FN in l -week diabetic rats. CONCLUSION: Our data suggest that TGF-β₁ appears in the renal tubulointerstitium in early period of diabetes and then its signal is mediated by MAPK_1/3 cascades to accelerate production of FN ,and in turn leads to renal hypertrophy and tubulointerstitial fibrosis.     

Localization and expression of TGF-β1,2 in fetal and adult skins

AIM:To explore the localization and expression of transforming growth factor-β_1,2 (TGF-β_1,2) and alpha-smooth muscle actin (α-ASMA) in fetal and adult skins. METHODS:Skins of 15 cases of fetuses with different gestational ages and 5 cases of adults were taken, embedded with paraffin wax, and sectioned. Immunohistochemistry method and pathological method were used to detect the expression intensity and distribution of TGF-β_1,2 and α-ASMA. RESULTS: Positive immunohistochemical signals of TGF-β_{1, 2} and α-ASMA were found in fetal and adult skins. In skins derived from young fetus, the positive signals of these three proteins were very weak. Along with the increment in gestational age, the positive cellular rates of TGF-β_1,2M and α-ASMA were elevated progressively. In elder fetal and adult skins, TGF-β_1,2 were mostly distributed in epidermal cells, endothelial cells and some fibroblasts, while α-ASMA was mainly located in myofibroblasts and sweat gland epithelial cells. CONCLUSION:The endogenous TGF-β_1,2might be involved in the cutaneous development at embryonic stage, in the cutaneous structure maintenance at adult stage, and in the wound healing after injury.     

Brain damage of cerebral ischemia/reperfusion and expression of TGF-β1 mRNA in diabetic rats

AIM:To observe the difference of cerebral inj ury following ischemia/reperfusion and mR-NA expression of TGF-β₁ between diabetic and non-diabetic rats.METHODS:At first,Wistar rats weredivided i nto two groups,non-diabetes and diabetes,and then two groups followed by sham,middle cerebralartery occlusion(MCAO) 2h and reperfusion 24 h after MCAO 2 h respectively.TGF-β₁ mRNA expressionwas measured by semi-quantitative reverse transcri ption polymerase chain reaction(RT-PCR);Cerebraldamage was eval uated by histopathology.RESULTS:In the same condition of ischemia or ischemia/reperfu-sion,inj uried area enlarged in DMgroups;The expressionlevel of TGF-β₁ mRNA increased at the ti me of 2h after MCAOi n non-diabetic group and diabetic group,especialy significantly in non-diabetic group withMCAO 2 h,and decreased at the time of reperfusion 24 h after MCAO 2 h,but still higher than that in theshamgroup.CONCLUSION:Diabetes mellitus exacerbated brain lesion following ischemia/repefusion,in-creased TGF-β₁ mRNA expresion after MCAO may be an anti-injury reaction,and anti-injury abilityis de-creased under diabetic condition.     

Effects of Bene Jones protein and TGF-β1 on the proliferation of rat renal proximal tubular cells in vitro

AIM:To study the effect of Bene Jones protein (BJP) from multiple myeloma(MM) patient and TGF-β₁ on cultured renal proximal tubular cell(PTC) proliferation.METHODS:[H³]TdR incorporation was used to study the effect of λBJP and TGF-β₁ on cultured rat NRK.52E PTC proliferation, the expression of TGF-β₁ in the supernatant of PTC cultured with BJP was assessed with ELISA.RESULTS:① [H³]TdR incorporation of PTC was inhibited by BJP in a dose-dependent manner, when co-cultured with 100-800 μmol/L BJP and 2.0 μg/L TGF-β₁, the [H³]TdR incorporation was lower than that of BJP alone, especially when BJP≥400 μmol/L;②The expression of TGF-β₁ in the supernatant of PTC cultured with BJP was increased, especially when BJP≥400 μmol/L(P＜0.05);③ The [H³]TdR incorporation of PTC was also inhibited by exogenous TGF-β₁ in a dose-dependent manner.CONCLUSION:λBJP has antiproliferative effect on rat PTC in vitro, The effect is related with stimulating the PTC to produce excessive TGF-β₁, which also has antiproliferative effect on PTC in some degree.     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.     

Reverse effect of FOXC2 silencing on epithelial-mesenchymal transition induced by TGF-β1 in MCF-7 cells

AIM: To study the reverse effect of FOXC2 silencing on epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1(TGF-β1) in MCF-7 cells. METHODS: Cultured MCF-7 cells were treated with TGF-β1 at concentration of 5 μg/L for 6 d. The cell morphological changes were observed under phase-contrast microscope. The changes of EMT-related marker proteins were assessed by immunofluorescence staining assay. TGF-β1-induced MCF-7 cells were transfected with FOXC2-siRNA mediated by recombinant lentivirus. In addition, the expression levels of FOXC2 and EMT-related marker proteins E-cadherin, claudin-1 and fibronectin-1 were also measured by RT-PCR and Western blotting. The invasion of MCF-7 cells was detected by Transwell assay. RESULTS: TGF-β1 induced the morphological alteration in MCF-7 cells from epithelial phenotype to mesenchymal phenotype,up-regulated the expression of mesenchymal marker fibronectin-1, and down-regulated the expression of epithelial markers E-cadherin and claudin-1. FOXC2 silencing reversed and restored the mesenchymal MCF-7 cells to epithelial phenotype and reduced the tumor invasion. CONCLUSION: EMT model induced by TGF-β1 in breast cancer MCF-7 cells is successfully established, which increases the invasion of MCF-7 cells. The effect of TGF-β1 is reversed by FOXC2-siRNA and the invasion of the cells is reduced.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

TGF β1 inhibits the maturation of dendritic cells and down-regulates TLR4 expression

AIM: To investigate the effects of transforming growth factor β1 (TGF-β1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to develop TGF β-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD80, CD86, I-Ab and CD40 in TGF β-DC were lower. The TGF β-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF β-DC was also found. CONCLUSION: TGF-β1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.     

Role of TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

Effect of eleutheroside B/E on proliferation and expression of PPARγ and TGF-β1 in HBZY-1 cells cultured with high glucose

AIM: To explore the effects and mechanism of eleutheroside (ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose. METHODS: The HBZY-1 cells were cultured under high glucose condition. The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth regulation curve of the cells. The cells were divided into 6 groups: low glucose (LG) group, high glucose (HG) group, high glucose plus ETS-B/E (low dose, medium dose and high dose) groups, and high glucose plus losartan (LTG) group. After all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγ was detected by immunocytochemistry and Western blotting. RESULTS: The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation. At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells (P<0.05). The expression of TGF-β1 was significantly inhibited, and the expression of PPARγ was significantly promoted by ETS-B/E (P<0.05). ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration- and time-dependent manner. CONCLUSION: ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγ expression.