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 Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Transforming growth factor-β1 regulates hypoxia-induced bronchial epithelial-mesenchymal transition by activation of lysys oxidase

AIM: To investigate whether transforming growth factor-β₁ (TGF-β₁) participates in hypoxia-induced bronchial epithelial-mesenchymal transition (EMT) through lysyl oxidase (LOX). METHODS: Sprague-Dawley (SD) rats were exposed to hypoxia to establish the animal model and were treated with LOX inhibitor β-aminopropionitrile (β-APN). Furthermore, primary rat bronchial epithelial cells were cultured in vitro and exposed either to normoxia or to hypoxia. TGF-β₁, TGF-β₁ receptor inhibitor (SB431542) or β-APN was used in the cell experiments. The content of collagen was measured by colorimetric method. The expression of TGF-β₁, LOX, and 2 EMT-related proteins (namely, the epithelial marker E-cadherin and the mesenchymal marker vimentin) were determined by immunohistochemistry and We-stern blot, respectively. RESULTS: The expression of TGF-β₁, vimentin and LOX and cross-linking of collagen were enhanced in hypoxia-exposed rat and in hypoxia-exposed bronchial epithelial cells, but the enhancement was impaired by the treatment with β-APN. In contrast, the expression of E-cadherin was reduced in hypoxia-exposed rat, and was reversed by treatment with β-APN. In vitro experiments demonstrated that TGF-β₁ and hypoxia led to the morphological phenotype characteristic of EMT in rat bronchial epithelial cells, in which the morphology of rat bronchial epithelial cells was switched from cobble-stone shape in normoxia-exposed group to spindle fibroblast-like morphology in hypoxia-or TGF-β₁-exposed group (P<0.01). Additionally, both β-APN and SB431542 partially prevented TGF-β₁ and hypoxia induced EMT in rat bronchial epithelial cells. TGF-β₁was able to dose-dependently up-regulate LOX expression in rat bronchial epithelial cells, which was blocked by concurrent incubation with SB431542. The up-regulation of TGF-β₁, vimentin, LOX and cross-linking of collagen and down-regulation of E-cadherin in hypoxia-exposed rat bronchial epithelial cells was significantly reversed by incubation with SB431542. CONCLUSION: TGF-β₁ regulates hypoxia-induced EMT in bronchial epithelial cells via activation of the LOX.     

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.     

Effects of PI3K/PKB signaling pathway on expression of osteopontin in human hepatic stellate cells induced by transforming growth factor-β1

AIM: To investigate the regulatory effects of phosphatylinositol 3-kinase/protein kinase B (PI3K/PKB) signaling pathway on the expression of osteopontin (OPN) in transforming growth factor-β₁ (TGF-β₁)-induced human hepatic stellate cells. METHODS: Human hepatic stellate cell line LX-2 was cultured in DMEM and stimulated by TGF-β₁ at the final concentration of 2.5, 5, 10 and 20 μg/L for 24 h or at final concentration of 10 μg/L for 12 h, 24 h and 48 h. LX-2 cells were pretreated with wortmannin, a specific inhibitor of PI3K/PKB signaling pathway, at final concentration of 0.1 μmol/L for 1 h, followed by incubation with TGF-β₁ at final concentration of 10 μg/L for 24 h. The cells were collected. The expression of OPN was detected by real-time PCR and Western blotting. RESULTS: In LX-2 cells, the expression of OPN was apparently elevated when incubated with TGF-β₁. With the increase in TGF-β₁ concentration or the extension of incubation hours, the expression of OPN was increased gradually in a dose-and time-dependent manner with certain limits. LX-2 cells pretreated with wortmannin and incubated with TGF-β₁ had a significant decrease in the OPN expression as compared with control group (P<0.01). CONCLUSION: The expression of OPN in TGF-β₁-induced LX-2 cells is regulated by the PI3K/PKB signaling pathway.     

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.     

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.     

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.     

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.     

Antagonistic effect of thalidomide on TGF-β1-induced change of CTGF gene promoter-binding proteins in HELF cells

AIM:To investigate the antagonistic effect of thalidomide (THD) on the activation of connective tissue growth factor (CTGF) gene promoter induced by transforming growth factor-β₁ (TGF-β₁) in human embryonic lung fibroblasts (HELF). METHODS:Luciferase reporter vector driven by CTGF gene promoter was used to detect the effects of TGF-β₁ and THD on the activity of CTGF gene promoter, and DNA pull-down assay with CTGF gene promoter as a probe was used to analyze the changes of CTGF promoter-binding proteins under different conditions. RESULTS:TGF-β₁ increased the activity of reporter driven by CTGF gene promoter (P<0.01), while THD significantly inhibited TGF-β₁-induced increase in the reporter activity dose-dependently (P<0.01). At the same time, THD had inhibitory effect on the TGF-β₁-induced change of CTGF gene promoter-binding proteins (P<0.05). CONCLUSION:Regulation of CTGF gene promoter-binding proteins takes part in TGF-β₁-induced activation of CTGF gene promoter, while THD has antagonistic effect on 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.     

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.     

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.     

The role of adrenomedullin in diabetic nephropathy

AIM: To investigate the role of adrenomedullin (AM) in diabetic nephropathy. METHODS: We observed the changes in the expression and secretion of AM, TGF-β₁ in the cultured human mesangial cells under high glucose condition and the contents of the laminin and type IV collagen in the supernatants. The effect of intervention with AM was also observed. RESULTS: High glucose condition resulted in increase in the expression and secretion of AM、 TGF-β₁、 laminin and type IV collagen. AM reversed the influence of high glucose on the cultured human mesangial cells. CONCLUSION: These results showed that high glucose condition is one of stimulating factors of AM and the renal protective action of AM may be associated with suppression of TGF-β₁ and reducing excessive accumulat ion of laminin and type IV collagen.     

Effects of endogenous carbon monoxide on the expression of transforming growth factor-beta 3 and type Ⅰ collagen of pulmonary artery in rats under hypoxia

AIM: To explore the impact of endogenous carbon monoxide (CO) on the expression of transforming growth factor-beta 3 (TGF-β₃) and type Ⅰcollagen in pulmonary artery of rats under hypoxia. METHODS: In the model of rats under hypoxic pulmonary hypertension, the measurement of pulmonary artery mean pressure (PAMP) and carboxyhemoglobin (HbCO) formation within pulmonary tissue homogenates was performed. TGF-β₃ and collagen Ⅰexpressions were detected by immunohistochemical assay. The expressions of TGF-β₃, type Ⅰ procollagen mRNA, and tissue inhibitor of metalloproteinase -1 (TIMP-1) mRNA were detected by in situ hybridization. RESULTS: ZnPP significantly increased PAMP and markedly decreased HbCO formation within lung tissue homogenates in rats under hypoxia( P< 0.01). Meanwhile, ZnPP promoted the expression of TGF-β₃ and collagen Ⅰprotein in pulmonary arteries in rats under hypoxia ( P< 0.01). ZnPP obviously elevated the expressions of TGF-β₃ mRNA, type Ⅰ procollagen mRNA, and TIMP-1 mRNA in pulmonary arteries in rats under hypoxia ( P< 0.01). CONCLUSION: Endogenous CO plays an important role in decreasing collagen synthesis and promoting degradation in pulmonary artery of rats under hypoxia by inhibiting the expression of TGF-β₃.     

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.     

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.     

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.     

Effects of Aβ1-42-induced microglial cells on survival of neural stem cells in vitro

AIM: To explore the effects of β-amyloid protein 1-42 (Aβ_1-42)-induced microglia on the survival of cultured neural stem cells (NSCs) in vitro . METHODS: Using the Transwell chambers to build a coculture system of NSCs and microglia, we detected the proliferation, differentiation and apoptosis of the NSCs with the microglia before and after induction by Aβ_1-42. RESULTS: Compared with non-intervention group, the proliferation rate of NSCs in Aβ_1-42 intervention coculture group decreased, as well as the positive expression rates of microtubule-associated protein 2 (MAP-2) and choline acetyltransferase. CONCLUSION: The inflammation mediated by Aβ_1-42 inhib their the proliferation of NSCs and induces their apoptosis. Inflammation also significantly reduces the ratio of NSCs differentiating to neurons, especially to cholinergic neurons.