Wnt signaling pathway is involved in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells

AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.     

Expression of Akt and ERK1/2 in human osteoarthritic chondrocytes

AIM: To investigate the expression of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) in normal and osteoarthritic chondrocytes. METHODS: The samples of knee cartilage were obtained from the normal donors (n=5) and the patients (n=18) undergoing total knee arthroplasty with the diagnosis of osteoarthritis (OA). The expression of p-Akt and p- ERK1/2 in the normal and osteoarthritic cartilage tissues was detected by the method of immunohistochemistry. The chondrocytes were isolated and identified by toluidine blue staining and immunohistochemical method. The expression levels of Akt, p-Akt, ERK1/2,p-ERK1/2,phosphorylated 70-kD ribosomal protein S6 kinase(p-p70S6K) and proliferating cell nuclear antigen(PCNA) were tested in normal and osteoarthritic chondrocytes by Western blotting. Real-time fluorescence quantitative PCR was used to measured the expression levels of aggrecan and type II collagen gene in normal and osteoarthritic chondrocytes. RESULTS: The expression of p-Akt in normal cartilage was higher than that in OA cartilage. The expression of p- ERK1/2 in OA cartilage was higher than that in normal cartilage. Compared with the normal chondrocytes, the expression of p-Akt and p-p70S6K, and the mRNA levels of aggrecan and type II collagen were increased (P<0.05), and the expression of p-ERK1/2 and PCNA was decreased in OA chondrocytes (P<0.05). CONCLUSION: Akt might regulate aggrecan and type II collagen synthesis via p-p70S6K, and ERK1/2 might regulate OA chondrocyte proliferation through PCNA. Both Akt and ERK1/2 play important roles in the pathogenesis of OA.     

Effect of TNF-α on the cartilage endplate chondrocytes of New Zealand white rabbits

AIM:To evaluate the biological roles of TNF-α on the cartilage endplate cells (chondrocytes). METHODS:The chondrocytes were isolated and harvested from the cartilage endplate of New Zealand rabbits and then the biological characteristics of cells were identified by methods such as toluidine blue staining for type Ⅱ collagen. After different concentrations of TNF-α were added to culture medium respectively, the rate of the proliferation of chondrocytes in different time was measured with MTT. The protein expressions of Bax, Bcl-2, Fas and caspase-3 were measured by immunocytochemistry. The changes of the mRNA of aggrecan and type Ⅱ collagen were measured by RT-PCR. RESULTS:The TNF-α at concentration of 50 μg/L and 100 μg/L decreased the rate of the proliferation on chondrocytes. Though TNF-α at concentrations of 10 μg/L and 50 μg/L increased the level of Bax, Fas and caspase-3, only 50 μg/L TNF-α decreased the level of Bcl-2. TNF-α at concentrations of 10 μg/L and 50 μg/L decreased the level of collagen IIa mRNA and only 50 μg/L TNF-α decreased the level of aggrecan. CONCLUSION:TNF-α not only inhibits the proliferation and the matrix synthesis in chondrocytes, but also increases the expression of pro-apoptotic factors in chondrocytes.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Induction of stem cells from adult Beagle canine bone marrow into chondrocytes in vitro

AIM：To explore the methods of the differentiation from adult Beagle canine bone marrow stem cells (BMSCs) into chondrocytes in vitro and determine the factors involved in the differentiation process.METHODS：About 10 mL BMSCs were aspirated from canine femoral bone,primarily cultured and subcultured in vitro.TGF-β1 was added into the culture medium.BMSCs were cultured and expanded in the medium until they reached the required quantity.BMSCs were induced to differentiate into chondrocytes at high cell density.Matrix of cartilage cells was detected by toludine blue stain,and cartilage specific collagen Ⅱ was detected by immunohistochemistry.RESULTS：The structure of cellular cartilage form BMSCs was uniformly positive of toludine blue staining.Immunohistochemical staining was positive for the collagenⅡ.CONCLUSION：Application of TGF-β1 may induce canine bone marrow stem cells into chondrocytes in vitro,which can be used as seeding cells in cartilage tissue engineering.     

Effects of bone mesenchymal stem cell transplantation on silicosis fibrosis in rats

AIM: To study the effects of exogenous bone mesenchymal stem cell (BMSC) transplantation on silicosis fibrosis in rats, and to explore the dose-effect relationship. METHODS: BMSCs were isolated and cultured from male 5-week-old SD rats in vitro. Fifty healthy female SD rats were randomly divided into 5 groups: control group, silicosis model group, BMSCs treatment A group (1×10⁹ cells/L), BMSCs treatment B group (3×10⁹ cells/L) and BMSCs treatment C group (5×10⁹ cells/L). The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal intubation, and different doses of BMSCs were given for intervention therapy. All the rats were sacrificed on the 21st day after the model was established. The morphological changes of the lung tissues were observed by HE staining and Masson staining. The localization and distribution of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were determined by the method of immunohistochemistry. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III were detected by Western blotting. The sex-determining region (SRY) protein was searched by an immunofluorescence method to confirm the homing of BMSCs. RESULTS: Compared with control group, the silicosis model group had significant alveolitis changes, silicon nodule formation, collagen deposition and other pathological characteristics. Compared with silicosis model group, the pathological changes in BMSCs treatment A group were improved. The conditions of BMSCs treatment B group were also improved significantly. However,the pathological changes in BMSCs treatment C group were increased obviously. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III in the lung tissues ranked as follows: BMSCs treatment C group > silicosis model group > BMSCs treatment A group > BMSCs treatment B group > control group. The difference between BMSCs treatment C group and silicosis model group was not statistically significant, and the differences between the other groups were statistically significant. The SRY-positive cells were observed in BMSCs treatment B group, but no significant expression in the heart, liver, spleen and kidney tissues was observed. CONCLUSION: The exogenous BMSC transplantation antagonizes the development of silicosis fibrosis in rats, which has dose-effect relationship.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

AIM: To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells (HNPCs). METHODS: Cultured HNPCs were subjected to oxygen-glucose deprivation (OGD) to mimic the micro-environment of degenerative HNPCs. The morphological changes of the cells in control group and OGD group were observed under optical microscope. The cells were treated with ginsenoside Rg1 at concentrations of 25, 50 and 100 μmol/L. The expression of collagen Ⅱ and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis. The cell viability was measured by CCK-8 assay. The mRNA level of Ki67 was detected by real-time PCR. The apoptosis was analyzed by flow cytometry. The activity of caspase-3 was measured by a caspase-3 kit. The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot. Furthermore, the expression of Wnt/β-catenin pathway-related proteins, the cell viability and apoptosis, and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1. RESULTS: Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm. However, the cell adherence was slower, and the cells were long fusiform with decreased cytoplasm after OGD treatment, indicating that the model of degenerative HNPCs was successfully established. Compared with normal HNPCs, the expression of collagen Ⅱ and aggrecan at mRNA and protein levels was decreased in OGD group (P<0.05), which was then increased after the cells were treated with ginsenoside Rg1 at 25, 50 and 100 μmol/L (P<0.05). Compared with normal HNPCs, the cell viability and Ki67 expression were decreased in OGD group (P<0.05), which were increased after treatment with ginsenoside Rg1 (P<0.05). Meanwhile, the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells (P<0.05), which were decreased after treatment with ginsenoside Rg1 (P<0.05). In addition, the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100 μmol/L (P<0.05). LiCl, a Wnt/β-catenin pathway agonist, obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells (P<0.05), indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs. CONCLUSION: Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway. This study will provide a new idea for prevention and treatment of degenerative HNPCs.     

Protective effect of β-asarone on primary rat hippcampal neurons against hypoxia/hypoglycemia and reperfusion injury

AIM：To investigate the protective effect of β-asarone against hypoxia/hypoglycemia and reperfusion injury in primary rat hippocampal neurons. METHODS：Cell viability, the activity of caspase-3, the protein expression of p-JNK and Bcl-2, and the mRNA expression of Bcl-2 and caspase-3 were determined by MTT assay, spectrophoto-metry, Western blotting and real-time PCR. RESULTS：Compared with normal control group, the cell viability decreased and the activity of caspase-3 increased obviously, the expression of p-JNK protein and caspase-3 mRNA increased obviously, and the expression of Bcl-2 protein decreased obviously in model group (P<0.05). Compared with model group, different doses of β-asarone inhibited the changes of the above indexes (P<0.05). CONCLUSION：β-asarone inhibits JNK-mediated chondrosome signaling pathway, thereby attenuating the process of hippocampal neuron apoptosis after hypoxia/hypoglycemia and reperfusion.     

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor (MIF) knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids.
METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α (cPLA2α) and phospho-cPLA2α were evaluated by Western blotting.
RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone (Dex) on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α.
CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.     

MEF2A/2D regulates depolarization induced differentiation via Rho signaling pathway in VSMCs

AIM：To investigate the role of myocyte enhance factor 2 (MEF2) in the regulation of depolarization-induced differentiation via Rho signaling pathway in vascular smooth muscle cells (VSMCs). METHODS： In primarily cultured mouse portal vein VSMCs, the techniques of immunofluorescence, Western blotting, real-time RT-PCR and siRNA transfection were applied to determine the membrane translocation of RhoA, the phosphorylation of LIM kinase (LIMK) and cofilin-2, and the mRNA expression of 4 MEF2 isoforms, myocardin and SM22α under high KCl-induced depolarization. RESULTS：RhoA membrane translocation occurred 30 s after depolarization, while phosphorylation of LIMK and cofilin-2 peaked at 10 min and 30 min, with increment of 42.20% and 32.75%， respectively. The mRNA expression of MEF2A and 2D was increased by 47.63% and 48.15%, respectively. In MEF2A/2D knockdown VSMCs, the mRNA expression of myocardin was not sensitive to depolarization, while SM22α expression was not affected. CONCLUSION：MEF2A/2D, acting on myocardin, is involved in the regulation of depolarization-induced differentiation via Rho signaling pathway in VSMCs     

Changes of canonical Wnt signaling pathway in brains of rats with autism induced by prenatal exposure to valproic acid

AIM：To investigate the roles of the canonical Wnt pathway in autism. METHODS：Using an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of the canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus formation (HF) of autistic rats. The expression levels of glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β, β-catenin and phosphorylated β-catenin were observed by Western blotting. The mRNA expression of GSK-3β, β-catenin, c-Myc and cyclin D1 was assessed by semi-quantitative RT-PCR. RESULTS：The results of Western blotting showed that inactivated GSK-3β (Ser9) phosphorylation was significantly increased, and inhibitory β-catenin (Ser33/37/Thr41) phosphorylation was obviously decreased compared with control group. The results of RT-PCR showed that the mRNA levels of β-catenin, c-Myc and cyclin D1 increased, and GSK-3β was significantly enhanced in VPA-exposed rats compared with the controls. CONCLUSION：Increased activity of canonical Wnt pathway in the PFC and HF of autistic rats may contribute to the susceptibility to autism.     

Effect of GRK5 on activation of rat astrocytes

AIM: To study the effect of G-protein-coupled receptor kinase 5 (GRK5) on the activation of astrocytes in the brain cortex of newborn Wistar rats. METHODS: GRK5 gene was silenced in the model of rat brain cortex astrocytes in vitro for 24 h. N-acetylcysteine (NAC), which is a known inhibitor of NF-κB, was added into the culture medium according to gene silencing for 24 h. The expression levels of GFAP and caspase-3 were detected by the method of immunofluorescence, and the mRNA levels of NF-κB, TNF-α, IL-1β and iNOS were determined by real-time PCR. Moreover, the activity of SOD and concentrations of TNF-α and NO were measured. RESULTS: GRK5 gene silencing increased the expression of NF-κB at mRNA and protein levels obviously (P<0.01), and the mRNA levels of IL-1β and iNOS increased synchronously (P<0.01). Furthermore, caspase-3-positive cells in GRK5 siRNA group were increased compared with control siRNA group (P<0.01). Treatment with NAC obviously reduced the activity of NF-κB and weakened the effects induced by GRK5 siRNA (P<0.05). CONCLUSION: GRK5 siRNA increases NF-κB activity and induces the activation of astrocytes.     

Role of caveolin-1 on down-regulation of LPS-induced monocyte chemotactic protein 1 by 17β-estradiol in vascular smooth muscle cells

AIM: To elucidate the effect of caveolin-1 on the down-regulation of LPS-induced monocyte chemotactic protein 1 (MCP-1) by 17β-estradiol (E₂) in vascular smooth muscle cells (VSMCs).METHODS: The primary-cultured VSMCs were exposed to E₂ at concentrations of 10^-9-10^-6 mol/L. LPS-induced MCP-1 production was assayed by ELISA. The protein expression of caveolin-1 was determined by Western blotting and was silenced by β-methyl cyclodextrin(β-MCD) or caveolin-1 specific siRNA. RESULTS: LPS significantly enhanced MCP-1 production. E₂ at concentrations of 10^-9-10^-6 mol/L inhibited LPS-induced MCP-1 production. The use of caveolin-1 inhibitor β-MCD or silencing the protein expression of caveolin-1 by specific siRNA largely impaired LPS-enhanced MCP-1 production, while E₂ markedly inhibited caveolin-1 expression. CONCLUSION: Inhibition of LPS-induced MCP-1 production by E₂ is related to the suppression of caveolin-1.     

Minocycline inhibits BV-2 cell activation by regulating P2X7 receptor

AIM：To explore the role of P2X7 receptor in inhibition of lipopolysaccharide (LPS)-stimulated BV-2 cell activation by minocycline. METHODS：BV-2 cells were divided into 5 groups: control group, LPS group, LPS+0.1 μmol/L Mino group, LPS+1 μmol/L Mino group and LPS+10 μmol/L Mino group. The expression of P2X7 receptor was determined by real-time PCR and Western blotting. The levels of TNF-α and IL-1β in the microglia culture supernatants were measured by ELISA. The morphological changes of the cells were also observed. RESULTS：After exposed to LPS, the expression of P2X7 receptor increased in BV-2 cells at mRNA and protein levels. The concentrations of TNF-α and IL-1β in the microglia culture supernatants also increased. Meanwhile, 0.1~10 μmol/L minocycline inhibited those changes in a dose-dependent manner. CONCLUSION：Minocycline inhibits the activation of microglia. The mechanism may be related to the P2X7 receptor.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.