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.     

Baicalein inhibits monocrotaline-induced vascular wall thickening in rats with pulmonary hypertension

AIM:To investigate the effects of baicalein on pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) in rats, and its molecular mechanism was further explored. METHODS:Male SD rats (n=28) were randomly divided into 4 groups:control group, MCT group, MCT+baicalein 50 mg/kg group and MCT+baicalein 100 mg/kg group. The PAH model was established by subcutaneous injection of MCT. After 2 weeks of modeling, the rats in baicalein treatment groups were gavaged baicalein 50 and 100 mg·kg^-1·d^-1 for 14 d, the rats in control group were administered with saline. After 4 weeks of modeling, right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI) and right ventricular mass index (RVMI) were detected. Masson staining was used to detect the degree of lung fibrosis. The pathomorphological changes of the pulmonary vessels were observed by HE staining. Western blot was used to detect the expression of α-smooth muscle actin (α-SMA) in the lung tissue and the phosphorylation p38, ERK and JNK in the artery. RESULTS:Compared with the control group, RVSP, RVHI and RVMI increased significantly in the MCT group (P<0.01). Pulmonary fibrosis and the thickening of pulmonary artery wall were observed. α-SMA was up-regulated and p38, ERK and JNK was activated significantly (P<0.01). Compared with the MCT group, baicalein (50 and 100 mg/kg) significantly decreased the RVSP, RVHI and RVMI (P<0.01). Lung fibrosis was reduced and the vascular wall thickening was decreased in baicalein-treated groups. Baicalein (50 and 100 mg/kg) inhibited the phosphorylation of p38, ERK and JNK compared with the MCT group (P<0.01). CONCLUSION:Baicalein ameliorates MCT-induced PAH by the inhibition of pulmonary artery wall thickening at least partially via MAPK signaling pathway.     

JNK pathway promotes apoptosis of rat hippocampal neurons after cerebral ischemia and reperfusion

AIM:To investigate the effect of c-Jun N-terminal kinase(JNK) pathway on the apoptosis of hippocampal neurons after cerebral ischemia-reperfusion(IR) in SD rats. METHODS:Ninety rats were randomly divided into 5 groups:sham group, cerebral IR group,cerebral IR+JNK inhibitor(SP600125) group,cerebral IR+JNK agonist(anisomycin) group and cerebral IR+vehicle group. The brain samples were collected 24 h after reperfusion. The protein level of caspase-3 in hippocampal neurons was measured by immunohistochemical and Western blotting techniques. The mRNA expression of caspase-3 in the hippocampus was determined by real-time fluorescence quantitative PCR. The apoptosis of hippocampal neurons was detected by TUNEL staining. RESULTS:Compared with sham group, the expression of caspase-3 at mRNA and protein levels in cerebral IR group increased obviously(P<0.05). Compared with cerebral IR group, the expression of caspase-3 at mRNA and protein levels in cerebral IR+JNK inhibitor group decreased obviously(P<0.05), and those in cerebral group increased obviously(P<0.05). However, the expression of caspase-3 at mRNA and protein levels in cerebral IR+vehicle group had no obvious change(P>0.05).The apoptosis of hippocampal neurons in each group was consistent with the changes of caspase-3 at mRNA and protein levels. CONCLUSION:Activation of JNK pathway enhances caspase-3 expression in rat hippocampal neurons after cerebral IR,thus promoting the apoptosis of the neurons.     

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.     

Expression of genes in MAPKs pathway in development of neural tube defects induced by hyperthermia

AIM: To study the expression and the role of ERK1/2 and JNK1/2 of MAPKs pathways in the development of neural tube defects induced by hyperthermia. METHODS: The animal models of golden hamster were produced by hyperthermia. The expression of ERK1/2 and JNK1/2, and levels of their phosphorylation were measured by Western blotting in control group and hyperthermia group. RESULTS: p-ERK1/2 steadily expressed in each control group, and the expression of p-ERK1/2 significantly decreased, which was different from that in the corresponding control group (P<0.05). The activity of p-JNK1/2 increased in hyperthermia group and the amount of p-JNK1/2 increased as compared to control group. The peak appeared at 16 h after exposed to hyperthermia (P<0.05). CONCLUSION: Hyperthermia, which induces a decrease in p-ERK1/2 expression and increases the expression of p-JNK1/2 of MAPKs pathway, results in the unbalance of cell proliferation and apoptosis, and induces neural tube defects.     

Effect of Kupffer cell blockade on activation of mitogen-activated protein kinases in response to lipopolysaccharide

AIM: Using the mouse model of lipopolysaccharide(LPS) attack,we study the effect of Kupffer cell (KC) blockade on the activation of mitogen-activated protein kinases(MAPKs) signal transduction pathway induced by LPS.METHODS: GdCl₃ (10 mg/kg) or the same volume of NS was continually injected intravenously at 48 h and 24 h before LPS (5 mg/kg) was injected into the male mice of Kunming species.The liver was then took out and KCs were isolated 30 minute after LPS was injected.The KCs isolated from the mice were cultured,and pretreated with GdCl₃ (100 μmol/L) for 1 h.The culture medium containing LPS (100 μg/L) was added and continuously incubated for 30 minute.The protein expression and phosphorylation level of ERK1/2 and p38MAPK in liver or KCs were assayed in vivo and in vitro,and effect of GdCl₃ on the phagocytosis function was observed,respectively.RESULTS: LPS induced the protein phosphorylation of ERK1/2 and p38MAPK in KCs or liver,no effect on the protein expression was observed.GdCl₃ treatment inhibited LPS-induced KCs activation and secretion of TNF-α,however,it had no effect on ERK1/2 and p38MAPK in KCs or liver,neither at the protein expression nor the phosphorylation.KCs secreted a few TNF-α with short time treatment with GdCl₃ alone in vitro.CONCLUSION: KC blockade with GdCl₃ alleviates LPS-induced KCs activation and the release of TNF-α not through modulating intracellular ERK1/2 or p38MAPK signal transduction pathways.We presume that GdCl₃ might reduce liver injury through cross talk of other intracellular signal transduction pathways (JNK,NF-кB,GPCR,etc).     

Icaritin induces differentiation of MC3T3-E1 subclone 14 cells via estrogen receptor and BMP/Smad signaling pathway in vitro

AIM: To observe the effects of icaritin (ICT) on the proliferation and differentiation of MC3T3-E1 subclone 14 cells (a pre-osteoblast cell line) and to observe the role of estrogen receptor (ER) and bone morphogenetic protein(BMP)/Smads signaling pathways in the differentiation of the cells. METHODS: The methods of WST-8 and BrdU were used to observe the viability and proliferation of MC3T3-E1 subclone 14 cells after treatment with different concentrations of ICT. The effects of ICT and noggin on the levels of alkaline phosphatase(ALP), type I collagen (Col I) and bone Gla protein (BGP) in MC3T3-E1 subclone 14 cells were observed after ER was blocked by ICI182780. The relative mRNA levels of BMPs (2, 4, 7) were detected by real-time PCR. The protein phosphorylation of Smad1/5/8 was determined by Western blotting after ER signaling pathway was blocked by ICI182780. RESULTS: ICT at concentrations of 0.1 μmol/L and 1 μmol/L increased the levels of ALP, Col I and BGP, and the numbers of mineralized nodules in MC3T3-E1 subclone 14 cells, indicating that ICT-promoted the differentiation, but did not affect the cell viability and proliferation. After the ER receptor signaling was blocked, ICT-promoted differentiation was significantly decreased. ICT improved the mRNA expression of BMP-2, 4, but did not affect the mRNA expression of BMP-7. After the ER receptor signaling was blocked, ICT-promoted phosphorylation of Smad1/5/8 was significantly decreased. Blockage of BMP/Smad signaling inhibited the effect of ICT on the differentiation. CONCLUSION: Icaritin induces the differentiation of MC3T3-E1 subclone 14 cells by activating BMP/Smad signaling pathway through ER.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Role of ghrelin in cell protection by up-regulating HSP70 through ERK1/2 signaling pathway in PC12 cells

AIM:To study the role of ghrelin in cell protection by up-regulating heat shock protein 70 (HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathway in the PC12 cells. METHODS:Sodium nitoprusside (SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group (incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group (ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group (quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group (ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group (added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS:Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased (P<0.05). Compared with SNP-injured group, ghrelin (100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells (P<0.05), and significantly up-regulated the protein expression of HSP70 (P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin (P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression (P<0.05). CONCLUSION:Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.     

Role of Oct3/4 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: Lentivirus (LV) vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non-transfection group, transfection group (transfected with Oct3/4 -LV) and negative control group (transfected with FU-PCG-NC-LV). β-mercaptoethanol (β-ME) was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in transfected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin-associated protein 2(MAP-2) at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of Oct3/4 and the neural cell specific markers neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP) were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTT assay. RESULTS: The results of PCR confirmed that the Oct3/4 -LV was successfully constructed and the virus titer was 2×10¹¹ TU/L. The best transfection efficiency and survival rate appeared when multiply of infection(MOI) was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4%±2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups (P<0.05). MSCs were induced by β-ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP-2 were higher than those in other groups (P<0.05). Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased (P<0.01). Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction (P<0.05). CONCLUSION: Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

Age-associated changes of p38 MAPK and JNK signal pathways in cardiac fibroblasts treated with transforming growth factor beta 1

AIM:To investigate the effect of aging on p38 mitogen-activated protein kinase(MAPK) and c-Jun N-terminal kinase(JNK) signal pathways in rat cardiac fibroblasts(CFs). METHODS:Cardiac fibroblasts obtained from neonatal and aged rats were cultured and randomly divided into 4 groups:neonatal PBS control group(N1 group), neonatal TGF-β₁ treatment group(N2 group), aged PBS control group(A1 group) and aged TGF-β₁ treatment group(A2 group). Proliferation of CFs was detected by MTT coloricmetric assay. The expression levels of total p38 MAPK, JNK, phospho-p38 and phospho-JNK were measured by Western blotting. RESULTS:The proliferative capacity of aged CFs was significantly decreased as compared with neonatal CFs after stimulated with TGF-β₁. In response to TGF-β₁, the expression levels of phospho-p38 and phospho-JNK were significantly increased in N2 group and A2 group as compared with N1 group and A1 group, respectively. The levels of total p38 and nonphosphorylated JNK in N2 group were similar to those in A2 group. Compared with N2 group, the levels of phospho-p38 and phospho-JNK markedly decreased in A2 group. CONCLUSION:These data indicate that p38 MAPK and JNK signal pathways are impaired in aged CFs.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

Effect of normal mesenteric lymph on multiple organ injury in mice with endotoxic shock

AIM: To observe the effects of normal mesenteric lymph (NML) on the lung, heart and liver injuries and the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) in the mice with endotoxic shock (ES). METHODS: The NML was drained form health male BALB/c mice for the intervention of ES after the removal of cellular constituent. Lipopolysaccharide (LPS, 35 mg/kg) was intraperitoneally injected into the mice for the establishment of ES model. After 60 min of LPS injection, the administration of NML (1/15 of whole blood volume) was performed through the femoral artery in NML+ES group. Meanwhile, the mean arterial pressure (MAP) was monitored during the experiment. At 6 h after intraperitoneal injection of LPS or the corresponding time point, blood samples were harvested from the heart through apical centesis for determination of the biochemical indexes to reflect myocardial and hepatocyte injuries. Simultaneously, the lung, heart and liver tissue specimens from a fixed location were harvested for the observation of histomorphology and the measurement of phosphorylation levels of p38 MAPK, ERK1/2 and JNK. RESULTS: Compared with sham shock (SS) group, MAP in ES group and NML+ES group remarkably decreased at multiple time points after intraperitoneal injection of LPS. However, MAP in NML+ES group at 80 min, 90 min, 190 min, 210 min, 240 min, 250 min, 340 min, 350 min, and 360 min were significantly increased compared with ES group. There were normal structures in the lung, liver and myocardium of the mice in SS group, while the morphological damages of these tissues appeared in ES group. Meanwhile, the damages were attenuated in the mice of NML+ES group. The activities of AST, ALT and CK-MB in the plasma in ES group were remarkably higher than those in SS group. The CK-MB activity in NML+ES group was also increased compared with SS group, and the activities of AST and LDH-1 were lower than those in ES group. At 6 h after LPS injection, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in the lung tissues were remarkably increased. Meanwhile, no statistical difference of these indexes between the myocardial and hepatic tissues was observed. NML intervention decreased the phosphorylation levels of p38 MAPK in the lung tissues, and p38 MAPK, ERK1/2 and JNK in the myocardial tissues. CONCLUSION: The NML administration alleviates multi-organ injuries and reduces the phosphorylation level of p38 MAPK in the lung tissues in the mice subjected to ES.     

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

AIM: To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 (MCP-1) induced by oxidized low density lipoprotein (ox-LDL) in the vascular smooth muscle cells (VSMCs). METHODS: mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RT-PCR and ELISA, respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies (a specific TLR4 inhibitor), PD98059 (ERK1/2 specific inhibitor), SB23015 (p38MAPK specific inhibitor) and SP600125 (JNK specific inhibitor) were used to investigate the underlying mechanisms. RESULTS: The mRNA and protein expressions of MCP-1 in VSMCs were up-regulated by ox-LDL (P<0.05), while those were inhibited by TLR4 neutralizing antibodies, PD98059 or SB23015 (P<0.05), but not by SP600125 (P>0.05). TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION: ox-LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4/ERK1/2 and TLR4/p38MAPKs pathways.     

Akt-eNOS-NO signal pathway mediates regulatory effect of Ang-1 and Ang-2 on vascular hyperreactivity in early hemorrhagic shock rats

AIM:To observe the role of endothelial nitric oxide synthase(eNOS) in the regulatory effect of angiopoietin-1(Ang-1) and angiopoietin-2(Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS:The protein expression of eNOS was measured in the superior mesenteric artery(SMA) after hemorrhagic shock by Western blotting. The effect of eNOS inhibitor on the vascular reactivity of SMA treated with Ang-1 and Ang-2 in the early(hyperreactivity) and late(hyporeactivity) periods of hypoxia were observed via an isolated organ perfusion system. The protein levels of eNOS in the hypoxic mixture of vascular endothelial cells(VECs) and vascular smooth muscle cells(VSMCs), and the concentration of nitric oxide(NO) in the medium supernatant of the mixture cells treated with Ang-1, Ang-2 and the inhibitors of Tie-2, Akt, p38 MAPK and ERK were measured. RESULTS:The protein expression of eNOS in SMA was low in normal control group, and increased significantly after hemorrhagic shock, which was 1.84, 3.55, 4.75, 5.96 and 6.33 folds of the normal control level in shock 10 min, 30 min, 1 h, 2 h and 4 h groups, respectively(P<0.01). Inhibitor of eNOS decreased the vascular hyperreactivity in hypoxia 10 min group, in which the E_max of norepinephrine(NE) was decreased from 13.479 mN to 9.043 mN(P<0.05). It also repressed the maintenance effect of Ang-1 on vascular reactivity in hypoxia 10 min group, in wihich the E_max of NE was decreased from 15.283 mN to 11.219 mN(P<0.01). The effect of Ang-2 on the vascular hyperreactivity in hypoxia 10 min group, the vascular hyporeactivity in hypoxia 4 h group, or the effect of Ang-1 or Ang-2 on the vascular reactivity in hypoxia 4 h group did not change. The protein expression of eNOS was increased 10 min after hypoxia as compared with the normal control, which was decreased by Ang-2 and the inhibitors of Tie-2 and Akt(P<0.01), but was not decreased by p38 MAPK and ERK inhibitors. The concentration of NO in the medium supernatant was increased 10 min after hypoxia, and was significantly decreased by Ang-2 and the inhibitors of Tie-2, Akt and eNOS, while the inhibitors of p38 MAPK and ERK had no influence on it. CONCLUSION:Ang-1 and Ang-2 regulate the vascular hyperreactivity in the early hemorrhagic shock rats through Akt-eNOS-NO pathway.     

Signaling mechanism of dendritic cell maturation stimulated by uric acid

AIM: To investigate the effect of uric acid on the signal molecule expression involved in MAPKs and NF-κB pathways during the maturation of dendritic cells (DCs). METHODS: DCs were obtained from murine bone-marrow and cultured in vitro. After the immature DCs were stimulated with uric acid (200 mg/L) and NF-κB inhibitor PDTC, or MAPKs inhibitors SB203580, PD98059 or SP600125 for 15 min, 30 min or 45 min, the cytoplasmic and nuclear extracts of the cells were collected and were subject to immunoblot analysis with the antibodies specific for NF-κB p65 or phosphorylated forms of p38, ERK1/2 and JNK. The cell lysates from DCs treated with LPS or DMSO served as controls. After treated with uric acid and PDTC, SB203580, PD98059 or SP600125 for 48 h, DCs were collected. The cell surface markers were analyzed by flow cytometry. The production of IL-12 p70 in the culture supernatants was detected by ELISA. RESULTS: Within 15 min of uric acid conditioning in the immature DCs, increased expression of NF-κB p65 and the phosphorylation of p38, ERK1/2 and JNK in the nuclear or cytoplasmic extracts of DCs were observed. The expression of these proteins reached their peak at 30 min after stimulation. Pretreatment of DCs with PDTC, SB203580, SP600125 or PD98059 blocked the expression of NF-κB p65 and phosphorylation of p38, ERK1/2 and JNK in response to uric acid stimulation. Treatment of DCs with SB203580, SP600125 or PDTC reduced the uric acid-induced up-regulation of CD83, CD86 and IA/IE, and inhibited the effect of uric acid on the secretion of IL-12 p70 (P<0.05 or P<0.01). SB203580 and PDTC possessed a significant inhibitory effect on uric acid. Nevertheless, PD98059 increased the up-regulation of CD83, CD86, IA/IE and IL-12 p70 induced by uric acid (P<0.05). CONCLUSION: Uric acid controls the balance of signal molecule phosphorylation of p38 MAPK, ERK1/2 and JNK, and NF-κB pathways. A possible mechanism of the DCs maturation stimulated by uric acid may be the modulation of the threshold and duration of MAPKs and NF-κB signaling.     

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.     

Calreticulin is involved in the protection of hypoxic preconditioning against cardiomyoblast H9c2 cell oxidative injury

AIM: To investigate whether hypoxic preconditioning (HPC) protects cardiomyoblast H9c2 cells against oxidative injury, and to discuss whether calreticulin (CRT) contribute to this protection through p38 MAPK signaling pathway. METHODS: Cardiomyoblast H9c2 cells were randomly divided into eight groups as follows: hydrogen peroxide stress (H₂O₂); brief hypoxic exposure of 20 min to simulate hypoxic preconditioning (HPC); 20 min of hypoxic exposure followed by 24 h of normoxic reoxygenation before hydrogen peroxide stress (HPC+H₂O₂), SB203580 (the specific inhibitors of p38 MAPK)+HPC+H₂O₂, antisense oligonucleotides transfection of calreticulin (AS), AS+H₂O₂, AS+HPC+H₂O₂ and control. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess the cell apoptosis and necrosis. RT-PCR and Western blotting analysis was used to detect calreticulin expression and phosphorylation of p38 MAPK. RESULTS: The results obtained are as follows: (1) HPC relieved cell injury caused by H₂O₂. Compared with those in H₂O₂ group, apoptosis rate and LDH leakage in culture medium in HPC + H₂O₂ group decreased 13.4% and 44.0%, respectively (P<0.05), and cell survive rate increased 12.7% (P<0.05). SB203580, a selective p38 MAPK inhibitor presented before HPC, eliminated the cytoprotection of HPC. Compared with HPC+H₂O₂ group, apoptosis rate and LDH leakage increased 5.4% and 45.0%, respectively (P<0.05), and cell survive rate decreased 5.0%(P<0.05). (2) Brief hypoxia intimating HPC resulted in mild CRT up-regulation (1.4-fold increased vs control group, P<0.05), but this up-regulation was lower than that of 3.6-fold increase induced by oxidative stress. HPC relieved the over-expression of CRT induced by H₂O₂ (26% decreased vs H₂O₂ group, P<0.05). (3) Transfection of antisense oligonucleotides of CRT before HPC reduced cytoprotection against oxidative stress. Correlative analysis indicated that mild up-regulation of CRT induced by HPC was positively correlated with survive rate (r=0.8573, P<0.05). (4) SB203580 suppressed CRT up-regulation (the expression of CRT decreased 38% or 23%, vs HPC+H₂O₂ group or HPC group, respectively). CONCLUSION: These results suggest that hypoxic preconditioning up-regulates calreticulin expression through p38 MAPK signaling pathway and protects cardiomyoblast H9c2 cells against oxidative injury.