Effects of Zuogui pill-medicated serum on proliferation and differentiation of MC3T3-E1 cells via ERK/TGF-β/Smads signaling cascade

AIM:To study the effects of Zuogui pill(ZG)-medicated serum on the proliferation and differentiation of MC3T3-E1 cells via ERK/TGF-β/Smads signaling pathway. METHODS:Using Premarin(conjugated estrogens tablets) as a positive control, the SD female rats were fed with high-, medium- or low-dose of ZG suspension. ZG-medicated serum was separated from abdominal aortic blood 7 d after feeding of ZG. MTT assay was applied to test the effect of ZG-medicated serum on the viability of MC3T3-E1 cells. The production of alkaline phosphatase(ALP) was detected by a modified calcium and cobalt dyeing method. The calcified nodules were observed by the method of alizarin red staining. The levels of core binding factor α1(Cbfα1) and collagen type I(Col I) protein were analyzed by Western blotting. The mRNA expression of TGF-β₁, Smad4 and Smad2 was measured by real-time RT-PCR. RESULTS:ZG-medicated serum promoted the proliferation of MC3T3-E1 cells in a dose-and time-dependent manner. Compared with other groups, treatment with 15% ZG(low dose) for 48 h increased the proliferation of MC3T3-E1 cells significantly. The protein levels of ALP, Cbfα1 and Col I,the calcified nodules, and the mRNA expression of TGF-β₁, Smad4 and Smad2 in MC3T3-E1 cells were all significantly increased after treatment with ZG-medicated serum. After the addition of PD98059(a specific blocker of ERK1/2 signaling pathway), all those were down-regulated except for mRNA expression of TGF-β₁. CONCLUSION:ZG regulates MC3T3-E1 cell proliferation and differentiation via the intervention of ERK/TGF-β/Smads signaling cascade, which may be one of the mechanisms that ZG effectively prevents and treats osteoporosis.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

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.     

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.     

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.     

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.     

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.     

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.     

Expression of Sonic hedgehog pathway-related genes in kidney tissues of rats with unilateral ureteral obstruction

AIM: To investigate the role of Sonic hedgehog (Shh) signaling pathway in renal interstitial fibrosis in the rats with unilateral ureteral obstruction (UUO). METHODS: Forty-eight male Sprague-Dawley rats were divided randomly into sham operation group and UUO model group with 24 rats each. The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fiber in the kidneys was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the expression of Shh signaling pathway-related proteins, including Shh, Smo,Ptch1 and Gli1. The contents of TGF-β₁ and Shh in the kidney tissues were determined by ELISA. Real-time RT-PCR was used to detect the mRNA expression of TGF-β₁, Col I, Col III and Shh signaling-related genes.RESULTS: Fibrosis observed with HE and Masson staining was obviously increased in UUO kidneys, and aggravated as time prolonged. The contents of TGF-β₁, Col I and Col III were also increased. In addition, the expression of Shh, Smo and Gli1 was markedly increased in obstructive kidneys, and the expression of Ptch1 was decreased (P<0.01), suggesting that Shh signaling was activated. The level of Shh in UUO rats was associated with the content of TGF-β₁. CONCLUSION: Shh signaling is activated in the progress of renal interstitial fibrosis in UUO rats, and the possible mechanism triggering the fibrogenic response is that Shh signaling promotes the expression of TGF-β₁.     

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.     

Effect of c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM: To investigate the effect of cellular Sloan-Kettering Institute (c-SKI) on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with transforming growth factor-β1 (TGF-β1) at varying concentrations for different time points. Western blot was used to test the expression of c-SKI and mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. Meanwhile, the endothelial marker E-cadherin was also detected. HCAECs were transfected with c-ski gene mediated by lentivirus (LV), the efficiency of LV-SKI transfection was detected by RT-qPCR. The HCAECs were divided into 4 groups:control group, TGF-β1 (5 μg/L) group, LV-SKI+ TGF-β1 group, LV-NC+ TGF-β1 group. The cell viability and colony formation were measured by MTT assay and colony formation assay. The protein levels of vimentin, α-SMA, E-cadherin, Smad2, Smad3, p-Smad2 and p-Smad3 were determined by Western blot. RESULTS: The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1 (P<0.01). Over-expression of c-SKI inhibited the proliferation of HCAECs (P<0.01). Compared with LV-NC group, over-expression of c-SKI down-regulated the expression of α-SMA and vimentin (P<0.01), up-regulated the expression of E-cadherin (P<0.01), and inhibited the protein phosphorylation of Smad2 and Smad3 (P<0.01), reversed the endothelial-mesenchymal transition induced by TGF-β1. CONCLUSION: The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition. Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs, the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.     

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.     

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.     

Transforming growth factor beta 1 reduces spontaneous recurrent seizures and inhibits activation of glial cells in rats

AIM: To investigate the mechanism that intranasal transforming growth factor beta 1 (TGF-β₁) reduces the occurrence of spontaneous seizures after status epilepticus (SE) induced by pilocarpine. METHODS: The rats received recombinant human TGF-β1 or the same volume of PBS, and were treated with pilocarpine to induce SE. All the rats were put into a special cage for video monitoring 7 days later. The determinations of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) positive cells by the method of immunohistochemistry were performed to evaluate the activation levels of the gliocytes in hippocampus. The neuron loss was measured by Nissl staining. RESULTS: TGF-β₁ reduced the average frequency, severity and duration of spontaneous seizures. The activated glia cells in the hippocampus were significantly reduced in TGF-β₁ group compared with pilocarpine group at 14 days after SE (P<0.05). TGF-β₁ significantly attenuated the loss of pyramidal neurons in hippocampal CA3 area at 14 days after SE (P<0.01). CONCLUSION: Intranasal TGF-β₁ reduces spontaneous recurrent seizures by inhibiting the activation of glia cells and attenuating the loss of pyramidal neurons.     

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.     

Effects of piceatannol on rat kidney with diabetic nephropathy in early stage

AIM: To observe the effect of piceatannol on the kidney of diabetic nephropathy rats in early stage, and to explore the possible mechanisms.METHODS: The rats were randomly divided into 5 groups:control group, model group, low dose of piceatannol treatment group, medium dose of piceatannol treatment group and high dose of piceatannol treatment group. The rat model of diabetic nephropathy was induced accordingly, and the rats received 20 mg/kg, 40 mg/kg or 60 mg/kg of piceatannol by gavage once a day for 4 weeks. Blood glucose was detected by glucometer. The urea nitrogen and creatinine levels in the serum were measured by urease-glutamate dehydrogenase enzymatic and inosine acid oxidase methods, respectively, and 24 h urinary microalbumin was analyzed by immune transmission turbidimetry test. Moreover, the pathological changes of the kidney tissues were observed under microscope with HE staining. The protein expression of TGF-β1 and Smad 7 and the phosphorylation levels of Smad2 and Smad3 were determined by Western blot. RESULTS: Compared with model group, piceatannol treatment significantly decreased the levels of blood glucose, blood urea nitrogen and urinary microalbumin, but had no effects on serum creatinine. Furthermore, HE staining showed that the increased mesangial cells, matrix hyperplasia and degenerated epithelial cells in model group were markedly inhibited after piceatannol treatment. Additionally, piceatannol treatment also reduced the protein expression of TGF-β1 and Smad 7, and the phosphorylation levels of Smad2 and Smad3. CONCLUSION: Piceatannol attenuates pathological progression in the kidney of diabetic nephropathy rats in early stage, which may be through inhibiting TGF-β/Smad signaling pathway.     

Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

Activation of renal tubular epithelial cells by monocytes/macrophages and the relative mechanisms

AIM: To observe the direct effects of peripheral blood monocytes/macrophages (MO/MAC) on renal tubular epithelial cells (RTEC),and further probe into the possible mechanisms. METHODS: Conditioned medium(M-CM) of human peripheral blood MO/MAC was collected and added to HK-2,a human renal proximal tubular cell line.After incubation with M-CM for 24 hours,HK-2 cells were detected for DNA synthesis by [³H]-TdR incorporation,osteopontin (OPN) and α-smooth muscle actin (α-SMA) expression by Western blot,and fibronectin(FN) secretion by ELISA.Furthermore,anti-TGFβ₁ neutralizing antibody and interlukin-10(IL-10) were used separately to antagonize the effects of M-CM on HK-2 cells. RESULTS: ①DNA synthesis,α-SMA expression and FN secretion were all increased in HK-2 cells when incubated with M-CM.②When adding with anti-TGFβ₁ neutralizing antibody (5 mg/L) in the M-CM,the degree of upregulation of α-SMA and FN in HK-2 cells was much lower than that stimulated by M-CM alone.③M-CM added with IL-10 (20 μg/L) had a weaker ability to induce the increasing in α-SMA expression and FN excretion in HK-2 cells, compared with M-CM itself alone.M-CM from MO/MAC preincubated with IL-10 caused a lower upregulation of α-SMA expression in HK-2 cells than M-CM from non-preincubated MO/MAC. CONCLUSION: MO/MAC can directly induce proliferation,transdifferentiation and extracellular matrix secretion in RTEC.TGFβ₁ and proinflammatory cytokines secreted by MO/MAC might be involved in the aboveeffects.