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.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Pyroptosis mediates high glucose-induced inflammation and injury in MC3T3-E1 osteoblasts

AIM To investigate whether pyroptosis contributes to the inflammation and injury in mouse embryonic osteoblastic cell line MC3T3-E1 induced by high glucose (HG; 45 mmol/L glucose). METHODS The cell viability was measured by CCK-8 assay. The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 (CASP1) were determined by Western blot. The secretion levels of interleukin-18 (IL-18) and IL-1β were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was detected by 2',7'-dichlorodihydrofluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. The alkaline phosphatase (ALP) activity was determined using the ALP kit, and the number of mineralized nodules was detected by alizarin red S staining. RESULTS After the MC3T3-E1 osteoblasts were treated with HG for 24 h, the protein expression levels of NLRP3 and CASP1, and the secretion levels of IL-18 and IL-1β were significantly increased. The decrease in cell viability, and the increases in ROS generation and MMP loss were also observed. Moreover, the differentiation and mineralization of MC3T3-E1 osteoblasts were inhibited, evidenced by decreases in both ALP activity and mineralized nodule number. Knockdown of CASP1 by siRNA attenuated the HG-induced osteoblast inflammation and injury mentioned above. CONCLUSION Pyroptosis mediates HG-induced inflammation and injury in MC3T3-E1 osteoblasts.     

Calcium overload and reactive oxygen species mediate high glucose-induced apoptosis of mouse osteoblast MC3T3-E1 cells

AIM: To investigate the role of reactive oxygen species (ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose. METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis. The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h. The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose (35 mmol/L) for 24 h. RESULTS: After high glucose treatment, the cell proliferation was inhibited. The early apoptosis and total cell death increased to (24.16?3.53)% and (63.74?4.32)%,respectively. High glucose treatment significantly increased intracellular levels of ROS and Ca²⁺. The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM. Inhibition of store-operated Ca²⁺ channels by La³⁺ also decreased the intracellular level of Ca²⁺ and cell apoptosis induced by high glucose. CONCLUSION: High glucose increases intracellular ROS level and the release of Ca²⁺ through the store-operated Ca²⁺ channels, thus resulting in intracellular Ca²⁺ overload and leading to apoptosis of osteoblasts.     

Changes of BMP signaling pathway in mammary glands of mice with mammary hyperplasia

AIM: To determine the expression patterns of bone morphogenetic protein (BMP) signaling components in the mammary glands with hyperplasia (MGH) and the activation of this signaling pathway. METHODS: The female C57BL/C mice (8-weeks-old) were randomly divided into control group and hormone group with 15 mice in each group. The mice in control group were treated with PBS, while the mice in hormone group were intragastric administration with estradiol valerate (2.5 mg/kg) for 25 d, followed by progesterone (4 mg/kg) peritoneal injection for 5 d. At the end of treatments, the mammary glands were collected to determine the morphological changes, the mRNA expression of BMP signaling components and the phosphorylation level of Smad1/5/9. RESULTS: In MGH, a part of BMP signaling pathway molecules were differentially expressed (P<0.05), including BMP ligands BMP2, 4, 5, 6, 7, 9, 13 and 14, BMP receptor BMPR1A, and antagonists Chrdl1 and Twsg, whereas the expression levels of some molecules, such as BMP3, BMP12, BMPR1B, BMPR2, Chrd, Chrdl2 and Noggin, were not altered. The phosphorylation levels of Smad1/5/9 was up-regulated in MGH (P<0.05). CONCLUSION: BMP signaling pathway is activated in MGH through the abnormal expression of its components. BMP signaling pathway may be a new target for MGH therapy.     

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.     

Role of BMP-7/Smads pathway in regulation of EndoMT in rats with HHPH

AIM: To investigate the role of bone morphogenetic protein 7 (BMP-7)/Smads pathway in the re-gulation of endothelial-mesenchymal transition (EndoMT) in rats with hypoxia-hypercapnia pulmonary hypertension (HHPH). METHODS: The rat pulmonary artery endothelial cells (RPAECs) were divided into normoxic control (Con) group, hypoxia-hypercapnia (HH) group, BMP receptor agonist rhBMP-7 group, BMP receptor inhibitor DMH-1 group and solvent DMSO group. After given the corresponding drugs in each group, the cells in Con group were cultured in a normal-oxygen incubator, and the cells in the remaining 4 groups were cultured in a low-oxygen and high-carbon-dioxide incubator. The expression levels of CD31 and α-smooth muscle actin (α-SMA) were observed by immunofluorescence staining. The mRNA and protein expression levels of α-SMA, CD31 and Smad1/5/8 were detected by RT-PCR and Western blot, respectively. The cell viability was measured by CCK-8 assay. The cell migration ability was detected by Transwell chamber assay. RESULTS: Compared with Con group, the expression of α-SMA at mRNA and protein levels in HH group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). Compared with HH group, the expression of α-SMA at mRNA and protein levels in rhBMP-7 group was decreased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were increased, the cell viability was increased and the number of migratory cells was reduced (P<0.05). Compared with rhBMP-7 group, the expression of α-SMA at mRNA and protein in DMH-1 group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). CONCLUSION: Hypoxia-hypercapnia conditions promote EndoMT in RPAECs, which promotes the development of hypoxia-hypercapnia pulmonary hypertension, and the underling mechanism may be related to the inhibition of BMP-7/Smads pathway.     

Effects of Rho kinase on p-Smad1 nuclear translocation in rat pulmonary artery smooth muscle cells

AIM: To explore the mechanism of bone morphogenetic protein (BMP) and Rho kinase signal pathways on the proliferation of pulmonary artery smooth muscle cells. METHODS: Pulmonary smooth muscle cells were isolated from the rat distal pulmonary artery and cultured. BMP and Rho kinase pathways were activated by BMP-2 and platelet-derived growth factor BB(PDGF-BB),respectively. Rho kinase inhibitor Y-27632 and MEK inhibitor U0126 were also used. Immunofluorescent staining was applied to observe p-Smad1 distribution across the nucleus, and the cells with positive p-Smad1 nuclear accumulation were counted and the nuclear translocation rate was calculated. The total p-Smad1 and its distribution across the nucleus were quantitatively determined by Western blotting. The cell proliferation was analyzed by CCK-8 assay. RESULTS: Exposure to BMP-2 significantly increased both the total amount of p-Smad1 and its nuclear accumulation in pulmonary smooth muscle cells. Pretreatment with PDGF-BB significantly decreased the nuclear accumulation of p-Smad1 induced by BMP-2 without decrease of total p-Smad1. However, pretreatment with Y-27632 or U0126 reversed the inhibitory effect of PDGF-BB on p-Smad1 nuclear accumulation. BMP-2 significantly inhibited the cell proliferation, but PDGF-BB blocked the effect of BMP-2 and significantly increased the cell proliferation. After pretreated with Y-27632 or U0126, the PDGF-BB-activated cell proliferation was suppressed.CONCLUSION: PDGF-BB-activated Rho kinase inhibits BMP-2-induced p-Smad1 nuclear translocation via MEK/ERK1/2, and increases pulmonary artery smooth muscle cell proliferation.     

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.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Strontium ranelate promotes osteogenic differentiation of rat bone mesenchymal stem cells through TGF-β1/Smad signaling pathway

[ABSTRACT] AIM: To study the roles of transforming growth factor β1 (TGF-β1)/Smad signaling pathway in strontium ranelate (Sr)-induced osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS: In the process of osteogenic differentiation of rat BMSCs, the expression of phosphorylated Smad2 (p-Smad2) and Runx2 was detected by Western blotting after the cells were treated with Sr. BMSCs were pretreated with SB431542, a selective inhibitor of TGF-β1, or Smad2 small interfering RNA (Smad2-siRNA), followed by Sr treatment, and then the expression of p-Smad2 and Runx2 was observed. At the same time, the activity of alkaline phosphatase (ALP) and the level of calcium nodules were detected to determine the osteogenic differentiation of BMSCs. RESULTS: The expression levels of p-Smad2 and Runx2 were enhanced under the action of Sr in the process of osteogenic differentiation of rat BMSCs. The expression of p-Smad2 reached to maximum when BMSCs were treated with Sr at concentration of 1 mmol/L for 1 h. The expression of Runx2 reached to maximum when BMSCs were treated with Sr at concentration of 1 mmol/L for 5 d. The pretreatment with SB431542 or Smad2-siRNA inhibited not only the expression of p-Smad2 and Runx2, but also the activity of ALP and the level of calcium nodules. CONCLUSION: Sr promotes the osteogenic differentiation of rat BMSCs through the TGF-β1/Smad signaling pathway.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Fructose promotes differentiation of 3T3-L1 preadipocytes by activating Akt signaling pathway

AIM: To study the effect of fructose on the differentiation of 3T3-L1 preadipocytes and the specific mechanism. METHODS: 3T3-L1 preadipocytes were cultured in vitro, induced to differentiate by cocktail method and treated with fructose at 1 g/L. The intracellular lipid content was identified and quantified by oil red O staining. The mRNA expression of perilipin-2 (Plin2), CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ was detected by RT-qPCR. The protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte protein 2 (aP2) was determined by Western blot. RESULTS: The volume of differentiated adipocytes and the accumulation of cytoplasmic lipid droplets in the 3T3-L1 cells with fructose intervention were increased compared with control group (P<0.05). Compared with control group, the expression levels of the marker proteins PPARγ and aP2 were up-regulated (P<0.01). The mRNA expression levels of Plin2, C/EBPα and C/EBPβ were up-regulated (P<0.05). In addition, the phosphorylation level of the key molecule Akt in the Akt signaling pathway was significantly increased (P<0.01) after the addition of fructose. After the addition of Akt blocker, the expression levels of PPARγ and aP2 were decreased. CONCLUSION: Fructose promotes the adipose differentiation of 3T3-L1 cells possibly by activating the Akt signaling pathway.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.     

Effects of Qiancaofang fumigation on expression of YAP/Smad3 in rats with knee osteoarthritis

AIM: To observe the effects of Qiancaofang fumigation on the expression of Yes-associated protein (YAP) and Smad3 in the cartilage of the rats with knee osteoarthritis (KOA), and to investigate the protective effects and mechanisms of Qiancaofang fumigation on the cartilage of the rats with KOA. METHODS: SD rats (n=32) were randomly divided into 4 groups:control group, model group, Chinese medicine group and hyaluronic acid (HA) group. The improved Hulth method was used to construct the rat model of KOA in model group, Chinese medicine group and HA group. The rats in Chinese medicine group were treated with Qiancaofang fumigation. The rats in HA group were treated with intra-articular injection of HA. After treatments, the cartilage tissues and serum of the rats in each group were taken. Hematoxylin-eosin (HE) staining and Mankin's scores were measured. The expression levels of cartilage oligomefic matrix protein (COMP) and C-telopeptide of type Ⅱ collagen (CTX-Ⅱ) in the serum were detected. The method of TdT-mediated dUTP nick-end labeling (TUNEL) was used to observe the apoptosis of the cartilage cells. Immunohistochemistry was performed to detect the expression of YAP and high mobility group protein B2 (HMGB2). Western blot was used to detect the expression of YAP, Smad3, bone morphogenetic protein 2 (BMP2), Bax and Bcl-2. RESULTS: The results of HE staining showed that the cartilage cells in model group were disordered and the structure was not clear. Compared with model group, the cartilage surface in Chinese medicine group was more smooth, the structure was clearer, and the staining of the cartilage matrix was more uniform. Compared with model group, the Mankin's scores in Chinese medicine group and HA group were significantly decreased (P<0.05), the serum concentration of CTX-Ⅱ was significantly increased, the serum concentration of COMP was significantly decreased, and the apoptosis rate of the cells was decreased (P<0.05). The results of immunohistochemistry and Western blot showed that the expression levels of YAP, HMGB2, Smad3 and Bcl-2 were increased in Chinese medicine group and HA group, and the expression levels of BMP2 and Bax were decreased as compared with model group (P<0.05). CONCLUSION: Qiancaofang fumigation relieves KOA partially through the YAP/Smad3 signaling pathway.     

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TGFBR1 signaling pathway activation in vascular smooth muscle cells

AIM:To investigate the effects of kaempferol-3-O-rutinoside (KR) on the proliferation, migration of vascular smooth muscle cells (VSMC) and the activation of transforming growth factor β receptor 1 (TGFBR1) signaling pathway in the cells. METHODS:The viability of VSMC was detected by MTT assay. The proliferation of VSMC was measured by EdU staining. The migration ability of VSMC was examined by Transwell assay. The protein levels of the migration-associated proteins matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) were detected by Western blot. Molecular docking study was conducted to explore the interaction between KR and TGFBR1. The protein le-vels of the phosphorylated TGFBR1, Smad2 and Smad3 were determined by Western blot. RESULTS:KR inhibited the viability of VSMC in a dose-and time-dependent manner. KR reduced the ratio of EdU-positive cells in a dose-dependent manner. KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP2 and MMP9 (P<0.05). KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280, ARG-215, ASP-290 and LYS-335 of TGBFR1. KR dose-dependently suppressed the activation of TGFBR1 and its downstream proteins Smad2 and Smad3 (P<0.05). CONCLUSION:KR inhibits the proliferation and migration of VSMC, possibly via blocking the TGFBR1 signaling pathway.     

Effect of HOCs implantation on protein expression of TGF-β/Smad signaling pathway in liver tissue of hepatic fibrosis rats

AIM: To investigate the effect of hepatic oval cells (HOCs) on the protein expression of TGF-β/Smad signaling pathway in the liver tissue of hepatic fibrosis rats.METHODS: The SD rat models of liver fibrosis were made by treating with carbon tetrachloride and combined factors. The HOCs was isolated from the model rats. HOCs suspension (0.5 mL at a density of 1×1012cells/L) were transplanted via portal vein into the hepatic fibrosis rats at 8th week and observed continuously for 30 days. Meanwhile, WuLing capsules were used for positive control. The blood samples were collected through trail vein at 8th day, 15th day, 23th day and 30th day after transplantation of HOCs. The levels of aspartate aminotransferase (AST) and alamine aminotransferase (ALT) in serum were determined by enzyme method. The morphological changes of hepatic tissues were observed under microscope with HE and Musson staining. The protein levels of collagen type I (Col-Ⅰ), extracellular-signal regulated protein kinase (ERK), phosphory-lation extracellular regulatedprotein kinases (p-ERK), TGF-β receptor type Ⅰ (TβRⅠ), TGF-β receptor type Ⅱ (TβRⅡ), mothers against decapentaplegic homolog 2/3 (Smad 2/3) and mothers against decapentaplegic homolog (Smad 7) were assessed in liver tissues by Western blotting. RESULTS: In HOCs and WuLing capsules treated groups, the levels of ALT and AST decreased significantly at 15th day, 23th day and 30th day after the transplantation of HOC. The damage degree of hepatic fiber hyperplasia of the liver histological structure reduced notably. The expression levels of Col-Ⅰ, ERK, p-ERK, TβRⅠ and TβRⅡ in liver tissues of hepatic fibrosis rats were down-regulated obviously while the expression of Smad 7 increased significantly.CONCLUSION: The implantation of HOCs prevents the progress of liver fibrosis in rats. The mechanism of action is to inhibit the protein expression of p-ERK, TβRⅠ, TβR Ⅱ for TGF-β/Smad signaling pathway of liver tissue.     

Activation of smad signaling and collagenⅠsynthesis in NRK52E cells induced by advanced glycation end products

AIM:To investigate the effects of advanced glycation end products on activation of Smad signaling pathway and collagenⅠ synthesis in proximal tubular epithelial cells. METHODS:Advanced glycation end products (AGE-BSA) were prepared by incubation of bovine serum albumin (BSA) with D-glucose. Normal rat proximal tubular epithelial (NRK52E) cells were cultured in RPMI-1640 medium with AGE-BSA. Phosphorylation and nuclear translocation of Smad2/3 were examined by immunocytochemistry. Levels of TGF-β1 in supernatant of cell culture were measured by enzyme-linked immunosorbent assay (ELISA). Expression of TGF-β1, Smad2, Smad3 and Smad7 mRNA were detected by RT-PCR. Expression of α-SMA , E-cadherin and collagenⅠproteins were detected by Western blotting.RESULTS:AGE-BSA induced Smad2/3 phosphorylation and nuclear translocation, two peaks occured at 30 min (68% vs 16%, P<0.05) and 24 h (76% vs 16%, P<0.05) compared to 0 min. The level of TGF-β1 markedly increased in supernatant of cell culture by induced AGE-BSA at 24 h and 48 h. The expression of TGF-β1 mRNA markedly increased at 24 h, and associated with high expression of Smad2, Smad3 and Smad7 mRNA at 48 h. AGE-BSA up-regulated significantly the expression of α-SMA and collagenⅠproteins, down-regulated the expression of E-cadherin protein. CONCLUSION:AGEs induces activation of Smad signaling, as well as transdifferentiation and collagenⅠ synthesis in proximal tubular epithelial cells.     

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.