Blockage of core fucosylation contributes to inhibition of epithelial mesenchymal transition of renal tubular epithelial cells

AIM: To investigate the role of inhibiting core fucosylation in the process of epithelial mesenchymal transition (EMT) in HK-2 cells.METHODS: An EMT cell model with transforming growth factor-β₁(TGF-β₁) was established and RNAi technique was used to silence the expression of α-1,6-fucosyltransferase ( FUT8) gene which is responsible to catalysation of core fucose. The morphological changes of HK-2 cells were observed under light microscope. The epithelial cell marker E-cadherin and fibrotic cell markers N-cadherin, fibroblast-specific protein-1(FSP-1) and α-smooth muscle actin(α-SMA) were detected by Western blotting and immunocytochemistry. The apoptosis induced by TGF-β₁ was determined by flow cytometry. RESULTS: After incubated with TGF-β₁ at the concentration of 5 μg/L for 48 h, HK-2 cells lost epithelial morphology and showed fibrotic morphology. The expression of α-SMA, FSP-1 and N-cadherin was markedly increased, while E-cadherin was decreased. Meanwhile, the expression of FUT8 was up-regulated, and the apoptosis of the cells increased. However, pre-incubation of the cells with FUT8 siRNA inhibited these changes above.CONCLUSION: The core fucosylation involves in the process of EMT in HK-2 cells. Blockage of core fucosylation results in the inhibition of EMT in HK-2 cells.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

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.     

Effect of all-trans-retinoic acid on the proliferation and expression of α-SMA in human embryonic lung fibroblasts

AIM: To investigate the effects of all-trans-retinoic acid (ATRA) on the proliferation and differentiation of transforming growth factor β₁(TGF-β₁)-stimulated human embryonic lung fibroblasts (HFL-I).METHODS: The HFL-I cells were cultured in vitro and were pretreated with ATRA for 3 days at the concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L. The proliferation of HFL-1 cells was detected by MTT method. The mRNA expression of α-smooth muscle actin(α-SMA) in HFL-I cells stimulated with TGF-β₁ for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h was detected by RT-PCR and the protein expression of α-SMA at the time points of 1,3 and 5 days was detected by Western blotting. The mRNA expression of α-SMA in HFL-I cells pretreated with different concentrations of ATRA for 24 h was detected the by RT-PCR and the protein expression at time point of 3rd day was detected by Western blotting. RESULTS: Different concentration of ATRA inhibited the proliferation of HFL-I in a dose-dependent manner (P<0.05). Both mRNA and protein expression of α-SMA in HFL-I cells pretreated with TGF-β₁ was up-regulated (P<0.05). ATRA down-regulated the mRNA and protein expression of α-SMA induced by TGF-β₁ in a dose-dependent manner (P<0.05). CONCLUSION: ATRA inhibits the proliferation and TGF-β₁-stimulated differentiation in HFL-I cells by down-regulating the mRNA and protein expression of α-SMA.     

TAK1 regulates fibrosis of renal tubular epithelial cells by regulating p38 MAPK signaling pathway

AIM:To investigate the effect of transforming growth factor-β (TGF-β) activated kinase 1(TAK1) on renal tubular epithelial fibrosis. METHODS:The renal tubular epithelial cell line HK-2 was used as the research object. After induced by TGF-β1, real-time PCR and Western blot were used to detect the expression of TAK1 in the HK-2 cells. TAK1 shRNA lentivirus was used to infect HK-2 cells, real-time PCR and Western blot were used to determine the interference effect on TAK1 expression in the HK-2 cells with TGF-β1 stimulation. Under the condition of treating with p38 MAPK activator anisomycin, the levels of type I collagen and type Ⅲ collagen in the supernatant, and the protein levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and p-p38MAPK^{Thr 180/Tyr 182} in the HK-2 cells with TAK1 knock-down were determined by ELISA and Western blot, respectively. RESULTS:TGF-β1 significantly increased the expression of TAK1 in the HK-2 cells(P<0.05). TAK1 shRNA significantly decreased the expression of TAK1 in the HK-2 cells with TGF-β1 stimulation. Type I collagen and type Ⅲ collagen secreted by the HK-2 cells after treatment with TGF-β1 were increased, the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} were also increased(P<0.05). Knock-down of TAK1 expression significantly inhibited the secretion of type I and type Ⅲ collagen, reduced the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} in the TGF-β1-induced HK-2 cells(P<0.05). Treatment with p38 MAPK activator reversed the inhibitory effect of TAK1 knock-down on the secretion of type I and type Ⅲ collagens, and the protein levels of α-SMA, CTGF and p-p38 MAPK^{Thr 180/Tyr 182} in the HK-2 cells(P<0.05). CONCLUSION:Knock-down of TAK1 expression attenuates the TGF-β1 induced fibrosis of renal tubular epithelial cells by inhibiting p38 MAPK signaling pathway.     

RTN1A induces renal tubular epithelial cells to secrete VEGF and IL-8 and promotes diabetic nephropathy renal fibrosis via ERK signaling pathway

AIM:To investigate the effects of reticulon 1A (RTN1A) on the secretion of vascular endothelial growth facter (VEGF) and interleukin-8 (IL-8) in renal tubular epithelial cells, and on the diabetic nephropathy (DN) renal fibrosis, and to explore the underlying mechanism. METHODS:The mouse model of DN was established, and the blood glucose, kidney index, urine microalbumin (UMA) and creatinine clearance (CCr) were measured. The protein levels of RTN1A, p-ERK, ERK, VEGF, IL-8 and renal fibrosis markers α-smooth muscle actin (α-SMA) and fibronectin (FN) were determined by Western blot. Human renal tubular epithelial cell line HK-2 was treated with high glucose, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were detected by Western blot and ELISA. The cells were treated with high glucose combined with RTN1A silencing or ERK inhibitor PD98059 for 24 h, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were also detected by Western blot and ELISA. RESULTS:The blood glucose, kidney index, UMA and CCr in the DN mice were significantly higher than those in control group (P<0.05), suggesting that DN model was successfully constructed. The protein levels of RTN1A and its downstream protein p-ERK, the cytokines VEGF and IL-8, and the fibrosis markers α-SMA and FN were significantly increased in the DN model mice (P<0.05). The protein levels of RTN1A, p-ERK, VEGF, IL-8, α-SMA and FN were also significantly increased in the HK-2 cells after treated with high glucose for 24 h, while these proteins were significantly decreased after silencing of RTN1A expression. CONCLUSION:RTN1A may be associated with the occurrence and development of DN. Silencing of RTN1A expression inhibits DN renal inflammation and fibrosis through ERK signaling. RTN1A may be an effective therapeutic target.     

Study on relationship between expression of PKCα in glomeruli and development of nephropathy in diabetic rats

AIM:To observe the dynamic changes of expression of PKCα, TGF-β₁ and α-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN).METHODS:Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKCα, TGF-β₁ and α-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy.RESULTS:The expression of PKCα and TGF-β₁ in renal tissue of diabetic groups were increased comparing with those of nomal control group(P<0.05). The mesangial cells expressed α-SMA in two months group. Chronologically the expression of PKCα, TGF-β₁ and α-SMA were positively correlative with each other and the impairment of kidney was also observed.CONCLUSIONS: During the DN process the expression of PKCα increased. PKCα raised GFR and the permeability of glomerular filtration membrane which enhanced urinary albumin excretion. PKCα also increased expression of TGF-β and therefore to induce the expression of α-SMA. The appearance of α-SMA was a marker of the phenotypic transform of renal cells.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Role of α1 adrenoceptor in proliferation of hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the role of α₁ and β₂ adrenoceptors(α₁AR and β₂AR) in the proliferation of hypoxic pulmonary artery smooth muscle cells (PASMCs).METHODS: PASMCs were isolated by an explant method from neonatal bovine pulmonary arteries. The cultured PASMCs were exposed to 6.6% O₂ for 6 h, 12 h and 24 h. The method of -TdR incorporation was used to measure the proliferation of PASMCs. _i was assayed with Fura-2/AM. The mRNA expression of α₁AR, β₂AR, c-fos and c-myc was determined by Northern blotting. The effects of activation of α₁AR and β₂AR, and inhibition of α₁AR on the above indexes were observed by treating PASMCs with different AR agonists and antagonists under hypoxic condition.RESULTS: Significant increase in TdR incorporation in hypoxic PASMCs with α₁AR activation was observed, and marked decrease in that was induced by α₁AR inhibition. However, no significant change was found after β₂AR activation. _i , the mRNA expression of c-fos, c-myc, α₁AR and β₂AR in PASMCs were increased after hypoxia.CONCLUSION: Hypoxia induces the increase in _i and mRNA expression of c-fos and c-myc, leading to the proliferation of PASMCs. The hypoxic proliferation of PASMCs is intervened by α₁AR, but not β₂AR. The remodeling of pulmonary arteriole and pulmonary hypertension may be involved in the processes of pulmonary arteriole constriction and proliferation induced by hypoxia through up-regulation of α₁AR.     

Effects of uremic serum on the proliferation and trans-differentiation of human renal tubular epithelial cells

AIM: To explore the effects of uremic serum on proliferation and trans-differentiation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line (HK-2) was cultured in RPMI-1640 medium. The proliferation effects of uremic serum at different concentrations were evaluated by methylene blue assay (MTT method) and flow cytometry. The positive cells percentage of α-smooth muscle actin(α-SMA)in different concentration uremic serum medium was also measured by flow cytometry in vitro. RESULTS: Absorbance 490 (A₄₉₀) was increased in 5%-20% uremic serum groups compared with that in normal controls with the use of MTT. Cells in G₁ phase were decreased, but proliferation index (PI) was increased in 10%-20% uremic serum groups compared with that in normal controls with the use of flow cytometry. No significant difference of cell proliferation index was found among uremic serum groups. The positive percentage of α-SMA cells was increased significantly in uremic serum groups compared with that in normal controls, and increased in parallel with the increasing of uremic serum concentration. CONCLUSION:These data suggest that AGEs could induce a high expression of MIP-1αm RNA and protein in cultured HUVECs in a dose-dependent and time-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.     

Effect of drug-containing serum of Liuwei Dihuang pills on TGF-β/Smad signaling pathway in HK-2 cells

AIM: To investigate the effect of the drug-containing serum of Liuwei Dihuang pills on the TGF-β₁/Smad signaling pathway in HK-2 cells.METHODS: The proliferation of HK-2 cell was detected by MTT method. Western blotting analysis was used to investigate the effect of the drug-containing serum of Liuwei Dihuang pills on the protein expression of the molecules of Smad signal transduction pathway.RESULTS: The drug-containing serum of Liuwei Dihuang pills promoted the proliferation of HK-2 cells. The level of Smad2 phosphorylation in HK-2 cells treated with 10% drug-containing serum of Liuwei Dihuang pills was significantly lower than that in the cells treated with TGF-β₁. Furthermore, SnoN, a negative factor in Smad signaling pathway, was up-regulated in HK-2 cells treated with 10% drug-containing serum.CONCLUSION: Drug-containing serum of Liuwei Dihuang pills inhibits TGF-β₁/Smad signaling pathway, including reducing Smad2 phosphorylation and promoting SnoN protein expression.     

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.     

Inhibitory effect of metformin on alveolar epithelial-mesenchymal transition in lung tissue of rats with pulmonary fibrosis

AIM: To study the inhibitory effect of metformin on alveolar epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis and the possible mechanism. METHODS: SD rats (n=48) were used, 12 of which were set up as normal control group, and 36 of which were induced by bleomycin (5 mg/kg) by tracheal instillation to establish pulmonary fibrosis. The pulmonary fibrosis rats were randomly divided into bleomycin group, low dose (100 mg/kg) of metformin group, and high dose (300 mg/kg) of metformin group. The rats in metformin groups were given the corresponding dose of metformin daily for 4 weeks. HE staining and Masson staining were used to observe the changes of lung histopathology and collagen deposition. Real-time PCR, Western blot and innunohistochemical staining were used to detect the mRNA and protein expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, zonula occludens-1 (ZO-1), collagen I, collagen III and transforming growth factor-β₁ (TGF-β₁), and the protein phosphorylation levels of Smad2/3 and extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined. RESULTS: Metformin up-regulated the expression of E-cadherin and ZO-1, down-regulated the expression of α-SMA, vimentin, collagen I and collagen III, and the protein phosphorylation levels of Smad2/3 and ERK1/2 were also decreased (P<0.05). CONCLUSION: Metformin inhibits alveolar EMT in the rats with pulmonary fibrosis, and its mechanism may be related to the inhibition of TGF-β₁ signal transduction pathway.     

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.     

Effects of thymosin α1 on the development and maturation of thymocytes

AIM: To investigate the effect of Thymosin α₁ on the development and matutation of thymocytes. METHODS: The proportion of CD4⁺CD8⁺ thymocytes and the expression of smoothened (Smo) of the hedgehog (Hh)-signaling in CD4^-CD8^-thymocytes were examined to observe the effect of thymosin α₁ on thymocyte development and matutation. RESULTS: Flowcytometric analysis showed that thymosin α₁ showed activity at a low dose of 30 μg/kg, and 30 μg/kg thymosin α₁ accelerated the replenishment and maturation of thymocytes according to the expression of Smo of the Hh-signaling in CD4^-CD8^-thymocytes, the potent negative regulator of proliferative responses. CONCLUSION: Thymosin α₁ can accelerates thymocyte development from CD4^-CD8^- to CD4⁺CD8⁺.     

Effects of epithelial cells treated with poly (I:C) on activation of fibroblasts and roles of EMMPRIN

AIM: To investigate the effects of epithelial cells treated with polyinosinic-polycytidylic acid [poly(I:C)] on the proliferation, transdifferentiation and signaling mechanisms of airway fibroblasts.METHODS:Human alveolar epithelial cells were treated with poly(I:C). The cell culture supernatants were used to stimulate the airway fibroblasts or the fibroblasts growing in collagen gels. The proliferation of the fibroblasts, the expression of a-smooth muscle actin (α-SMA) in the fibroblasts and the contractility of the collagen gels containing fibroblasts, as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) were observed. The proliferation of the fibroblasts and the expression levels of α-SMA, MMP-2 and MMP-9 in the fibroblasts stimulated with EMMPRIN were detected. The inhibitors specific for p38 MAPK or ERK1/2 were used to explore the effects on α-SMA expression and EMMPRIN secretion. RESULTS:The culture supernatants of the epithelial cells treated with poly(I:C) induced the proliferation, α-SMA expression and gel contraction as well as EMMPRIN secretion in the fibroblasts. EMMPRIN dose-dependently enhanced fibroblast proliferation, α-SMA expression and activity of MMP-2 and MMP-9. The supernatants of epithelial cells treated with poly(I:C) activated p38 MAPK and ERK1/2 signaling in the fibroblasts, as the specific inhibitors of MAPK or ERK1/2 signaling attenuated the expression of α-SMA and EMMPRIN in the fibroblasts. CONCLUSION: Poly(I:C) induces fibroblast proliferation, α-SMA expression and gel contraction by affecting the process mediated by p38 MAPK and ERK1/2 signaling pathways in epithelial cells. EMMPRIN may be the important media involved in this event.     

Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells

AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G. METHODS: The activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H₂O₂) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot. RESULTS: Mollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H₂O₂. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05). CONCLUSION: Mollugin may inhibit H₂O₂-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 expression.     

Phasic change of water soluble major crystallins with aging in rats

AIM: To study the correlation between phasic change of the relative quantity of major crystallins with aging in rats. METHODS: Six groups of SD rats (age 1 d， 8 d，2 weeks，8 weeks，8 months and 1.5 years) were raised routinely. Water soluble crystallins were extracted and separated by two－dimensional polyacrylamide gelelectrophoresis. After comassize blue staining，the crystallins patterns were scanned and analyzed. RESULTS: (1) Out of the eighteen water soluble major rat crystallins tested in each group, seven showed gradual phasic changes in relative quantity of crystallins, but there were no significant changes in total quantity of water soluble crystallins. (2) Phasic changes in these crystallins presented four different patterns: increasing (βB₄、αB₂、αA₂、βA₁), decreasing (β₇、β₈、γ_2,3、γ_5,6),relatively stable(βA₃、βB₅), and irregular. (3) The ratio of βB₄ /αA₂ increased gradually with the rat aging process. CONCLUSION: The gradual phasic changes in relative quantity of crystallins reflect the aging status of rat crystalline.