Strontium ranelate promotes osteogenic differentiation of rat bone mesenchymal stem cells through Hedgehog/Gli1 signaling pathway

AIM: To explore whether strontium ranelate(Sr) promotes osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through the Hedgehog/Gli1 signaling pathway. METHODS: BMSCs were isolated from 4-week-old rats by adherent culture and induced to differentiate into osteoblasts. According to the experimental purposes, the cells were exposed to different concentrations of Sr, cyclopamine(Cy, an inhibitor of Hedgehog receptor) or Gli1-siRNA. The expression of Gli1 and Runx2 in the cells was detected by Western blotting. The activity of alkaline phosphatase(ALP) was measured by the method of colorimetry, and the mineralized nodules were observed under microscope with alizarin red staining. RESULTS: Exposure to Sr at concentrations of 0.1 to 5 mmol/L for 7 d markedly increased the expression of Gli1 in the BMSCs, and the increase in Gli1 expression was the most obvious following Sr exposure at concentration of 3 mmol/L. Cy at concentration of 10 μmol/L inhibited Sr-induced up-regulation of Gli1 expression. Transfection of the BMSCs with Gli1-siRNA not only obviously inhibited Sr-induced up-regulation of Gli1 and Runx2(a downstream protein of Gli1) expression, but also antagonized Sr-induced enhancement of ALP activity and the formation of mineralized nodules. CONCLUSION: The Hedgehog/Gli1 pathway is involved in Sr-induced osteogenic differentiation of rat BMSCs.     

Strontium renelate promotes osteogenesis via regulation of Sonic hedgehog signaling molecules in rat bone marrow mesenchymal stem cells

AIM：To study the role of Sonic Hedgehog (Shh) in strontium ranelate (Sr)-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：BMSCs was isolated from 4-week-old rats by adherent culture. The cells in the 3rd～5th generations were induced to differentiate into obteoblasts, and then were treated with different concentrations of Sr and cyclopamine (Cy). The activity of alkaline phosphatase (ALP) was mea-sured by colorimetry. Mineralized nodules were observed by alizarin red staining. The cellular Shh and Runx2 expression was detected by Western blotting. RESULTS：Sr at concentration of 3 mmol/L increased the activity of ALP and induced the formation of mineralized nodules. Sr at concentrations ranging from 0.1 to 5 mmol/L increased the expression of Shh and Runx2 in the BMSCs at 7 d. Furthermore, the peak expression of Shh occurred following the exposure of Sr (1 mmol/L) or Runx2 (3 mmol/L). On the other hand, Sr at concentration of 1 mmol/L showed a time-dependent increase in the expression of Shh and Runx2 from 1 d to 7 d. Cy at concentration of 10 μmmol/L not only obviously inhibited Sr-induced expression of Shh and Runx2, but also antagonized the increase in the ALP activity and mineralization induced by Sr in the BMSCs. CONCLUSION：Sr promotes osteogenic differentiation of BMSCs by increasing the expression of Shh and Runx2.     

Strontium ranelate promotes differentiation of bone marrow mesenchymal stem cells to osteoblasts by increasing expression of bone morphogenetic protein 2

AIM: To explore whether strontium ranelate (Sr) promotes differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by increasing the expression of bone morphogenetic protein 2 (BMP-2). METHODS: Rat BMSCs were isolated, purified and cultured, then were induced to differentiate into osteoblasts. The cells were treated with different concentrations of Sr or noggin (an inhibitor of BMP-2) according to the experimental purposes. The activity of alkaline phosphatase (ALP) was measured by colorimetry. Mineralized nodules were measured by alizarin red staining. The expression of BMP-2 was detected by Western blotting. RESULTS: Treatment with Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d obviously increased the activity of ALP,and Sr at concentration of 3 mmol/L produced the maximum effect. Exposure of the cells to Sr at concentration of 3 mmol/L for 21 d significantly increased mineralized nodules. Exposure of the cells to Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d markedly increased the expression of BMP-2. Preconditioning with noggin at concentration of 100 μg/L for 2 h not only inhibited Sr-induced expression of BMP-2, but also antagonized the increase in the activity of ALP and mineralization induced by Sr in BMSCs. CONCLUSION: Up-regulation of the expression of BMP-2 may be one of the mechanisms by which Sr promotes differentiation of rat BMSCs to osteoblasts.     

Effect of rhizoma drynariae total flavonoids on osteogenesis in cultured bone masenchymal stem cells

AIM: To explore the effects of total flavonoids of rhizoma drynariae on osteogenic differentiation of bone mesenchymal stem cells (BMSCs) by intervening rat BMSCs with osteogenesis differentiated induction in certain conditions in vitro. METHODS: BMSCs were isolated and purified by the whole marrow culture method, and identified by flow cytometry. BMSCs were treated with different concentrations of total flavonoids of rhizoma drynariae. The expression of alkaline phosphatase (ALP) was quantitatively detected at 7th and 14th day after intervention by ALP and mineralized nodules straining methods. RESULTS: Uniform and stable BMSCs were separated successfully by the whole marrow culture method. After intervention for 7 days and 14 days, the different concentrations of total flavonoids of rhizoma drynariae manifested different expression levels of ALP. BMSCs induced by rhizoma drynariae total flavonoids at dose of 10-5 g/L were all positive with ALP and mineralized nodules straining methods. CONCLUSION: Total flavonoids of rhizoma drynariae at lower concentration promote the osteogenic differentiation of BMSCs. The expression of ALP exhibits a gradually increased tendency accompanied with a decrease in the concentration of total flavonoids of rhizoma drynariae.     

Inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblasts

AIM：To investigate the inhibitory effect of Ophiopogon japonicus on rat cardiac fibroblast (CFs) and the underlying mechanism. METHODS：Cultured CFs from Sprague-Dawley (SD) rats were randomized into 4 groups: control group (normal rat cardiac fibroblasts), Ophiopogon japonicus of 10 μg/L group, Ophiopogon japonicus of 20 μg/L group and Ophiopogon japonicus of 30 μg/L group. Cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 in CFs were determined. RESULTS：Compared with control, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 10 μg/L group. Compared with Ophiopogon japonicus of 10 μg/L group, the cell vitality, ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 20 μg/L group. Compared with Ophiopogon japonicus of 20 μg/L group, the cell vitality,［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3 were significantly decreased in Ophiopogon japonicus of 30 μg/L group. CONCLUSION：Ophiopogon japonicus may inhibit CFs. These actions are related to the changes of ［3H］-proline incorporation, and the protein expression of TGF-β1, p-Smad2/3 and total Smad2/3.     

Runx2 promotes osteogenic differentiating C2C12 cells through inhibiting macroautophagy

AIM：To explore the relationship between macroautophagy and Runx2-induced osteogenic differentiation of C2C12 cells.METHODS：The cells of C2C12/Runx2Dox subline were used in the study, which expresses Runx2 in response to doxycycline (Dox). The cells were treated with Dox (10 mg/L) for 0 d, 1 d, 3 d, and 6 d, and the expression levels of LC3b, Beclin-1, p62, and LAMP-2 were detected at each time point using real-time qPCR. The LC3-I/LC3-II ratio was measured by Western blotting. The cells were treated with different concentrations of 3-methyladenine (3-MA) or rapamycin (Rap) for 14 days in the presence of Dox, and the alkaline phosphatase (ALP) activity was measured. The cells were treated with 3-MA (5 mmol/L) or Rap (10 μmol/L) for 1 d, 3 d, and 6 d in the presence of Dox, and the expression levels of ALP and osteocalcin (OC) were detected.RESULTS：The expression levels of LC3b and Beclin-1 were dramatically down-regulated during Runx2-induced osteogenic differentiation of C2C12 cells, and the levels of p62 and LAMP-2 were not changed during this process. The conversion of LC3-I into LC3-II was inhibited. The ALP activity was increased in 3-MA (5 mmol/L)-treated cells, but decreased in Rap (10 μmol/L)-treated cells. The expression levels of ALP and OC were up-regulated by 3-MA treatment and were down-regulated by Rap treatment.CONCLUSION：Runx2 induces osteogenic differentiation of C2C12 cells by inhibiting the formation of autophagosome through down-regulating LC3 and Beclin-1 as well as inhibiting the conversion of LC3-I to LC3-II.     

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.     

Mechanisms of catalpol-induced osteogenic differentiation in SD rat bone marrow mesenchymal stem cells

AIM: To investigate the mechanisms for catalpol-induced osteogenic differentiation of SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: The cells were divided into control group, osteoinduction group and catalpol group. The activity of alkaline phosphatase (ALP) was measured at 7 d, 14 d and 21 d after catapol treatment, meanwhile ALP positive cell numbers and calcium nodes were counted at 14 d and 21 d after catapol treatment,respectively. The mRNA expression of Runx2, osteocalcin, Wnt3a, β-catenin, Wnt5a and Wnt11 was detected at 7 d, 14 d and 21 d after catapol treatment by real-time PCR. RESULTS: Catalpol at 2.0 mg/L increased ALP activity and ALP positive cell numbers significantly(P<0.05), meanwhile, it also increased calcium nodes numbers in cultured BMSCs (P<0.05). Compared with control group, catalpol increased the mRNA expression of Runx2 significantly at 14 d, but not at the 7 d and 21 d. Catapol also promoted the mRNA expression of osteocalcin significantly from 7 d to 21 d. Compared with control group, the mRNA expression of Wnt3a and β-catenin in catalpol group was increased at 14 d and 21 d. In addition, the mRNA expression of Wnt5a and Wnt11 in catalpol group was higher than that in control group at 14 d, but that was decreased at 21 d. CONCLUSION: Catalpol induces differentiation of BMSCs into osteoblast by increasing the mRNA expression of Runx2, and promotes the differentiation and mature of these osteoblasts by increasing ALP secretion, osteocalcin mRNA expression and calcium deposition. The activation of Wnt signaling pathway may be involved in this pro-osteogenic differentiation process.     

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 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.     

Lentiviral vector CV072-mediated Mfn2 over-expression inhibits hepatic stellate cell activation

AIM:To construct a lentiviral vector carrying mitofusin 2 (Mfn2), and to investigate the inhibitory effect of Mfn2 on the activation of rat hepatic stellate cells and its mechanism of reducing the formation of hepatic fibrosis-related factors. METHODS:The lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP containing Mfn2 was constructed and transfected into the hepatic stellate cells. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was evaluated. The protein levels of Bax, Bcl-2, cleaved caspase-3, α-SMA, TGF-β1, Smad2 and Smad3 were detected by Western blot. The levels of type I collagen, type Ⅲ collagen and type IV collagen in the cell culture supernatants were determined by ELISA. RESULTS:Compared with control group, the apoptosis of the hepatic stellate cells transfected with lentivirus over-expression vector CV072-pCMV-Mfn2-EGFP was increased, and the protein levels of proapoptotic molecules Bax and cleaved caspase-3 were increased (P<0.01). TGF-β1/Smad pathway-related proteins TGF-β1, p-Smad2 and p-Smad3 were decreased, and the levels of fibrosis-related proteins α-SMA, type I collagen, type Ⅲ collagen and type IV collagen were decreased (P<0.01). CONCLUSION:Transfection of lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP effectively inhibits hepatic stellate cell activation in vitro and may reduce the production of hepatic fibrosis-related factors by inhibiting TGF-β1/Smad pathway.     

Comparison of 2 methods for inducing iPSC to differentiate into neural stem cells

AIM: To select an efficient way of promoting induced pluripotent stem cells (iPSC) to differentiate into neural stem cells (NSC) by comparing 2 methods. METHODS: The culture system in method A contained SB431542 (5 mmol/L) and drosomophorin (5 mmol/L) with 100% initial cell density, while that in method B contained SB431542 (5 mmol/L) and drosomophorin (1 mmol/L) with 30%~50% initial cell density. For comparison and identification of the 2 methods, the growth state was observed under microscope, and the expression of Pax6, nestin, Sox1 and Sox2 was quantitatively detected by real-time PCR and flow cytometry. The related protein expression and the ability of spontaneous differentiation were determined by immunofluorescence analysis. RESULTS: The cells derived from method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density achieved the higher expression of Pax6, nestin, Sox1 and Sox2. The growth state was better and the cells differentiated into neurons and astrocytes normally. CONCLUSION: The method A was superior to method B, and we recommend the method A with 5 mmol/L of SB431542 and drosomophorin and 100% initial cell density as the method for differentiating NSC.     

Effects of rhTGF-β1 on proliferation and osteogenic differentiation of MSCs in SD rats

AIM：To investigate the effects of recombinant human transforming growth factor β1 (rhTGF-β1) on the ability of proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), as well as its effects on the expression of bone morphogenetic protein 2 (BMP-2), Smad4 and core binding factor α1 (Cbfa1). METHODS：SD rat MSCs were isolated and purified by the differential time adherent method. MTT assay was used to confirm the optimal concentration of rhTGF-β1 for the proliferation of MSCs. The optimal concentration for differentiation of MSCs into osteoblast was also determined by observing the activity and positive staining of alkaline phosphatase. According to the different induction conditions, MSCs were divided into 4 groups:control group, classic group, rhTGF-β1 group, and rhTGF-β1+classic group. Alkaline phosphatase, type I collagen, bone Gla protein and calcium nodes were detected to evaluate the osteogenic differentiation. BMP-2 was detected by ELISA and the mRNA expression of Smad4 and Cbfa1 was analyzed by real-time quantitative PCR. RESULTS：The optimal concentrations of rhTGF-β1 for the proliferation of MSCs and for the osteogenic differentiation of MSCs were 10 and 5 μg/L, respectively. The MSCs in classical group and rhTGF-β1 group were promoted to osteogenic differentiation, and the mRNA expression of BMP-2, Smad4 and Cbfa1 was increased. rhTGF-β1 induced osteogenic differentiation of MSCs in the early and middle terms. However, in rhTGF-β1+classic group, the osteogenic differentiation of MSCs was more obvious in the late term. CONCLUSION:The induction conditions of classical group, rhTGF-β1 group and rhTGF-β1+ classical group promote the differentiation of MSCs by increasing BMP-2 secretion and starting the TGF-β superfamily/Smads signaling pathway to regulate the differentiation of MSCs.     

Retinoid X receptor agonist inhibits TGF-β1-induced collagen synthesis in cardiac fibroblasts by repressing Smad2 activation

AIM: To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms. METHODS: CFs were cultured using myocardial tissue with dry method. Hypoxic environment was established for CFs by continuous nitrogen supplement. Type I and type III collagens in supernatants were detected by ELISA. Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents. The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining. RESULTS: Under hypoxic condition, TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-dependent manner in the CFs. At the concentration of 5 μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01). RXR agonist 9-cis-retinoic acid (9-cis-RA; 10^-9~10^-6mol/L) decreased TGF-β1 (5 μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic condition. The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01). Smad2 inhibitor (20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF-β1 under hypoxic condition. Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly increased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05). CONCLUSION: Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

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.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.