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).     

Involvement of extracellular signal regulated kinase in the regulation of amyloid precursor protein processing in PC12 cells by TPPB

AIM: To explore the signal transduction pathways involved in the regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) activator TPPB.METHODS: PC12 cells were treated with TPPB (PKC activator) for 3 h and various signal transduction inhibitors were added to the conditioned medium to investigate their effects on α-secretase form of soluble amyloid precursor protein (sAPPα) secretion after TPPB treatment via Western blotting. Extracellular signal regulated kinase (ERK, p42/44MAPK) and phospho-p42/44MAPK were also measured after TPPB treatment.RESULTS: TPPB (1 μmol/L) significantly increased sAPPα secretion as compared with control group. The increase in sAPPα secretion by TPPB was partially blocked by ERK inhibitor U0126, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and protein tyrosine kinase (PTK) inhibitor genistein, but not by p38MAPK inhibitor SB203580. TPPB (1 μmol/L) increased the expression of phospho-p42/44MAPK without altering total p42/44MAPK levels.CONCLUSION: ERK, JNK and PTK may be involved in the regulation of APP processing by TPPB.     

LPS induced B7-H1 expression in pancreatic carcinoma Panc-1 cells

AIM: To explore the mechanism of lipopolysaccharide (LPS)-induced B7-H1 expression in pancreatic carcinoma cell line Panc-1. METHODS: The levels of phosphorylated p38 mitogen-activated protein kinase (p-p38), phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) after stimulated with LPS or treated with mitogen-activated protein kinases (MAPKs) inhibitors were detected by Western blotting. The expression of B7-H1 in Panc-1 cells after LPS stimulation or MAPKs inhibitor treatment was measured by real-time PCR and Western blotting. RESULTS: The levels of B7-H1, p-p38, p-ERK and p-JNK were up-regulated with LPS stimulation. The promoted p-p38, p-ERK and p-JNK levels induced by LPS were inhibited by the corresponding MAPKs inhibitors. Furthermore, the inhibitors of p38 and ERK attenuated LPS-induced B7-H1 expression. However, JNK inhibitor had very little effect on LPS-induced B7-H1 expression. CONCLUSION: LPS induces B7-H1 expression in pancreatic carcinoma cell line Panc-1. ERK and p38 are involved in this regulation as the inhibitors of ERK and p38 attenuate LPS-induced B7-H1 expression.     

Combination of LPS and z-VAD-FMK mediates necroptosis in IFN-γ-pretreated macrophages

AIM:To explore the mechanism of necroptosis in M1 macrophages mediated by lipopolysaccharide (LPS) combined with z-VAD-FMK. METHODS:THP-1 cells were induced to differentiate into M0 and M1 macrophages with phorbol 12-myristate 13-acetate (PMA) and interferon-γ (IFN-γ). The release of lactate dehydrogenase (LDH) and TUNEL-positive cells at different time points after LPS (100 μg/L) treatment were detected, and the effects of different inhibitors were observed. The protein levels of receptor-interacting protein (RIP) 1, RIP3, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), P38 and NOD-like receptor protein 3 (NLRP3) were determined by Wes-tern blot. RESULTS:LPS mediated cell death, and its combination with z-VAD-FMK specifically mediated necroptosis in M1 macrophages rather than M0 ones. The expression of RIP3 and NLRP3 was upregulated by IFN-γ, and LPS-mediated phosphorylation of JNK was also enhanced by IFN-γ. The inhibitors against RIP3 and JNK partly blocked LDH release mediated by LPS combined with z-VAD-FMK. CONCLUSION:Combination of LPS and z-VAD-FMK mediates necroptosis in IFN-γ-pretreated macrophages possibly by upregulation of RIP3 and enhancement of LPS-mediated JNK phosphorylation.     

p38 MAPK involves in cepharanthine-induced apoptosis in cardiomyocytes

AIM: To investigate the apoptotic effect of cepharanthine (CEP) on neonatal rat cardiomyocytes(NRCMs) and the underlying mechanisms. METHODS: MTT assay was used to detect the viability of the cells. CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3. The phosphorylation levels of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK,were examined by Western blotting. The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes. RESULTS: CEP inhibited the viability of NRCMs in a dose-and time-dependent manners. Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs. The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs, but the change of JNK was not obvious. SB203580, an inhibitor of p38 MAPK, significantly alleviated the apoptotic effect induced by CEP. However, PD98059, an inhibitor of ERK1/2, did not significantly reduce the apoptotic effect.CONCLUSION: p38 MAPK is involved in CEP-induced apoptosis in NRCMs.     

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.     

Regulatory role of CCN1 in lipopolysaccharide-induced mouse acute lung injury

AIM: To observe the cellular location and expression change of cysteine-rich protein 61 (CYR61/CCN1) in lung tissues of the mice with lipopolysaccharide (LPS) intratracheal instillation, and to clarify the regulatory role of CCN1 expression in mediating inflammatory response. METHODS: The expression change of CCN1 in the lung tissues in vivo was observed by the method of immunohistochemistry, and immunofluorescence was employed to certify the cellular location of CCN1 in bronchial epithelial cells. Bronchial epithelial 16HBE cells were cultured in vitro, and the expression of CCN1 under the condition of LPS stimulation was quantified by RT-qPCR and Western blot with or without specific inhi-bitors of ERK1/2, JNK, P38 and PI3K signaling pathways. The mRNA expression levels of interleukin-6 (IL-6), IL-8, transformrg growth factor-β (TGF-β) and vascular endothlial growth factor (VEGF) were measured by RT-qPCR under the condition of recombinant CCN1 exposure or transfection with CCN1-siRNA. RESULTS: The results of immunohistochemistry indicated that CCN1 was primarily located in bronchial epithelium. The results of immunofluorescence revealed that CCN1 was localized in the cytoplasm. The specific inhibitors of ERK1/2, JNK, P38 and PI3K signaling pathways reversed the up-regulation of CCN1 upon LPS stimulation. Exposure to recombinant CCN1 resulted in the up-regulation of IL-6, IL-8, TGF-β and VEGF, while LPS-related up-regulation of IL-6, IL-8, TGF-β and VEGF was blocked by silencing of CCN1. CONCLUSION: Airway epithelium-derived CCN1 is up-regulated under the condition of lung injury and the regulatory mechanism involves ERK1/2, JNK, P38 and PI3K signal transduction pathways. CCN1 acts as an inflammatory mediator in amplification of inflammatory response, laying theoretical basis for the potential molecular therapeutic target of acute lung injury.     

Etanercept attenuates bleomycin-induced lung fibrosis in mice

AIM: To evaluate the effect of tumor necrosis factor α (TNF-α) antagonist etanercept on bleomycin-induced lung fibrosis in mice.METHODS: Forty-five Kunming female mice were randomly divided into 3 groups: the mice in control group were intraperitoneally injected with vehicle and intratracheally administered with saline aerosol, the mice in bleomycin group were intraperitoneally injected with vehicle and intratracheally administered with bleomycin (3 mg/kg) aerosol, and the mice in bleomycin+etanercept group were intraperitoneally injected with etanercept (4 mg/kg) every 3 d and intratracheally administered with bleomycin aerosol. All animals were sacrificed 28 d after treatments. The left lung was fixed in 10% neutral formalin and then stained with hematoxylin-eosin or Masson’s trichrome for the pathological examination. The tissues of right lung were sampled for measuring the content of hydroxyproline (HYP) by the method of alkaline hydrolysis. The serum concentrations of TNF-α and TGF-β were detected by ELISA. Total tissue protein was extracted for examination of ERK1/2, JNK and p38 by Western blotting.RESULTS: Etanercept prevented the collagen accumulation under the airway epithelium and decreased the scores of lung inflammation and fibrosis induced by bleomycin with significantly reduced the levels of tissue HYP, serum TNF-α and serum TGF-β. The protein phosphorylations of ERK/JNK/p38 in the lung tissues were remarkably decreased compared with BLM group.CONCLUSION: Etanercept decreases the phosphorylations of ERK1/2/JNK/p38 via inhibiting the expression of TNF-α and TGF-β. Etanercept might be useful in the treatment of pulmonary fibrosis.     

Effects of endothelin-1 on the induction of the transdifferentiation of human airway sub-epithelial fibroblasts

AIM: To explore the role of endothelin-1 (ET-1) in initiating transdifferentiation of sub-epithelial fibroblasts (SEFs) into myofibroblasts and its ionic and signal transduction mechanism.METHODS: Human SEFs or SEFs plated in collagen gels were co-cultured with human bronchial epithelial cells (16HBE) treated with lipopolysaccharide (LPS) plus mechanical scratch. ET receptor A inhibitor (BQ123) or the inhibitors specific for p38 MAPK, ERK1/2 were added, repectively. The expression of α-smooth muscle actin (α-SMA) in the SEFs and contractility of the collagen gels containing with SEFs as well as the effects of p38 MAPK or ERK1/2 on α-SMA expression were evaluated. Using Ca2+ sensitive Fluo-3/AM, dynamic changes of intracellular calcium concentration (［Ca²⁺］_i) were observed in the SEFs by laser confocal microscopy.RESULTS: Injured 16HBE induced the transdifferentiation of myofibroblasts, which expressd α-SMA and increased contractility. BQ123 blocked the induction to a certain extent. Injured 16HBE activated p38 MAPK and ERK1/2 pathways in SEFs, both inhibitors of p38 MAPK and ERK1/2 attenuated the induction of α-SMA by injured 16HBE. The addition of exogenous ET-1 enhanced α-SMA expression and activated p38 MAPK, ERK1/2 pathways in the SEFs. Additionally, ET-1 significantly facilitated Ca²⁺ inflow into the fibroblasts.CONCLUSION: Injured 16HBE induces the transdifferentiation of SEFs into myofibroblasts, which is involved in the activation of p38 MAPK and ERK1/2 pathways. The ET-induced influx of Ca²⁺ may be an early signal for initiating the myofibroblasts transdifferentiation.     

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.     

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 TGF-β1-activated p38 MAPK in up-regulation of PAI-1 expression by TGF-β1 in human ovarian cancer cells

AIM: To investigate the relationship between up-regulation of plasminogen activator inhibitor-1(PAI-1) expression and activation of p38 mitogen-activated protein kinase(p38 MAPK) and extracellular signal-regulated kinase(ERK) pathways by TGF-β1 in human ovarian cancer cells. METHODS: PAI-1 expression in human ovarian cancer cells treated with TGF-β1(10 μg/L)was assayed by real-time PCR and Western blotting. The activation of p38 MAPK and ERK was determined by Western blotting using phosphorylated p38 MAPK and phosphorylated ERK antibodies. Specific p38 MAPK inhibitor(SB203580) or ERK inhibitor(PD98059) was used to inhibit their activation. RESULTS: TGF-β1 up-regulated the expression of PAI-1, and activated p38 MAPK and ERK pathways in the ovarian cancer cells. Inhibition of p38 MAPK activation by SB203580 resulted in significant inhibition of the mRNA expression of PAI-1 induced by TGF-β1. However, inhibition of ERK activation did not significantly alter TGF-β1-induced increase in PAI-1 mRNA level. CONCLUSION: TGF-β1-activated p38 MAPK pathway contributes to the up-regulation of PAI-1 expression by TGF-β1 in ovarian cancer cells.     

Low concentration MNNG inhibition of JNK/SAPK is independent of a nuclear signal while MNNG activation of p38MAPK may depend on a nuclear signal

AIM:To identify the effect of alkylating agent N-methyl-N'- nitro-N-nitrosoguanidine (MNNG) with low concentration on JNK/SAPK and p38MAPK and the origins of JNK/SAPK and p38MAPK cascade.METHODS:p38 and JNK kinase activity were detected by immunoprecipitation and Western immunoblotting in intact and enucleated Vero cells.RESULTS:With the same experimental conditions, low concentration of MNNG inhibited JNK kinase in both intact cell and enucleated Vero cell. MNNG activated p38 kinase in intact cell while no effect on p38 kinase in enucleated cell was observed.CONCLUSION:Inhibition of JNK/SAPK by low concentration of MNNG was independ of a nuclear signal while MNNG activation of p38MAPK may depend on a nuclear signal.     

SHP-2 regulates PC12 cell survival and apoptosis in response to NGF via activation of ERK and JNK

AIM: To investigate the central role of the protein tyrosine phosphatase SHP-2 in the survival of PC12 cells upon NGF treatment and apoptosis after NGF withdrawal. METHODS: PC12 cells were treated with SHP-2 inhibitor (NSC87877). Cell survival rate was detected by MTT method and apoptosis was determined by flow cytometry. Moreover, pIRES-GFP (vector alone), pIRES-GFP-SHP-2 (wild type) and pIRES-GFP-SHP-2C459S (dominant negative mutant form) were transfected into PC12 cells. ERK, p-ERK, JNK and p-JNK were immunoblotted at 1 h in the presence of NGF and 5 h after NGF withdrawal. RESULTS: SHP-2 potentially enhanced survival and attenuated apoptosis of PC12 cells. The activation of ERK was significantly enhanced with NGF treatment either in the group without SHP-2 inhibitor or the SHP-2 wild type group. On the other hand, phosphorylation level of JNK was significantly increased after NGF withdrawal when PC12 cells were treated with SHP-2 inhibitor or transfected with SHP-2 mutant. CONCLUSION: SHP-2 may play a central role in mediating the survival and apoptosis of PC12 cells upon NGF exposure and withdrawal. The underlying mechanisms may be through the activation of ERK and JNK.     

Effects of oxidized high density lipoprotein on tissue factor expression in ECV304 cell line

AIM: To investigate the expression of tissue factor (TF) induced by oxidized high density lipoprotein (oxHDL) in human umbilical vein cell line, ECV304, and the related mechanisms. METHODS: Four main groups were designed: the negative, the positive (ECV304 with histamine), the HDL group and the oxHDL group. Quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting were used to detect the expression level of TF. The specific inhibitors of MAPKs, SP600125 (c-jun terminal NH2 kinase, JNK), SB203580 (p38 MAP kinase, p38 MAPK), PD98059 (extracellular signal-regulated kinase, ERK1/2) were used to investigate the underlying mechanisms. RESULTS: The TF expression in normal ECV304 cell line was not detected. Histamine administration resulted in a significant expression of TF in ECV304 cell line, with strongest effect after 1 h co-incubation at concentration of 1×10-5 mol/L histamine (about 4.8-fold higher expression of TF compared with that of 1×10-9 mol/L histamine). Expression level of TF was detected after stimulated with oxHDL in dose- and time- dependent manners. The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co-incubation, with 1.8-fold and 5.3-fold increase in TF expression, respectively, compared with that at 10 mg/L oxHDL and 2 h co-incubation. 20 mg/L oxHDL also caused an apparent augmentation of TF protein expression, about 1.5-fold higher compared with that stimulated by 40 mg/L oxHDL. HDL co-incubation did not cause a detectable expression of TF protein. The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%, 81.0%, 87.0%, respectively (all P<0.05) after application of inhibitors against p38MAPK, JNK and ERK1/2. CONCLUSION: The results suggest that oxHDL stimulates TF expression in ECV304 cell line in both dose- and time- dependent manners, in which MAPKs signal transduction may play an important role.     

Akebia saponin D promotes differentiation of bone marrow mesenchymal stem cells into osteoblasts through MAPK signaling pathways

AIM:To explore the role of Akebia saponin D(ASD) in the differentiation of rat bone marrow-derived mesenchymal stem cells(BMSCs) into osteoblasts. METHODS:The rat BMSCs were cultured using routine methods. The effects of ASD on the differentiation of MSCs into osteoblasts were observed. The p38 mitogen-activated protein kinase(p38 MAPK) inhibitor SB203580 and extracellular signal-regulated kinase(ERK) inhibitor PD098059 were used to evaluate the mechanisms. The activity of alkaline phosphate(ALP) and content of osteocalcin(OC) were assayed during differentiation. The mRNA expression of osteoprotegerin(OPG) and receptor activator of nuclear factor-κB ligand(RANKL) was determined by real-time fluorescence quantitative PCR. The activity of p38 MAPK and ERK was measured by Western blotting. RESULTS:Six days after treatment with ASD, the mRNA expression of OPG significantly increased, while the mRNA level of RANKL significantly decreased in induced cells. ASD increased the activity of ALP and the content of OC. Moreover, ASD enhanced the activity of both p38 MAPK and ERK, which was inhibited by SB203580 and PD098059. SB203580 and PD098059 also inhibited the positive role of ASD in the differentiation of MSCs into osteoblasts. CONCLUSION:Akebia saponin D significantly enhances differentiation of rat BMSCs into osteoblasts in vitro, which may be mediated by the p38 MAPK and ERK signaling pathways.     

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.     

LY367385 attenuates neuronal apoptosis after cerebral vasospasm in mice by inhibiting extracellular signal-regulated kinase pathway

AIM: To investigate the effects of LY367385,a selective antagonist of metabotropic glutamate receptor 1 (mGluR1), on the apoptosis of neuron, and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway after cerebral vasospasm (CVS) in mice. METHODS: The model of subarachnoid hemorrhage (SAH) in mice was established by endovascular perforation without opening cranium. Male ICR mice were randomly divided into 3 groups: sham operation group, NS+SAH group and LY367385+SAH group. Ten minutes after SAH, 5 μL of LY367385 or NS was microinjected into lateral cerebral ventricle, and then the neurological scores of the animals were examined. At different time points (6 h, 24 h, 48 h) after operation, the pathological changes in the brain tissues were observed under light and electron microscopes. The mRNA expression of mGluR1 was detected using RT-PCR, and Western blotting was used to examine the protein levels of mGluR1 and p-ERK1/2. Apoptotic cell number was determined by TdT-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In SAH+NS group, the scores of neurological function were significantly lower, while the mGluR1 mRNA, mGluR1 and p-ERK1/2 proteins and the number of apoptotic cells were significantly higher than those in sham operation group.Some neurons displayed histopathological changes of necrosis, myelin sheath internalization and disconnection. LY367385 decreased the mRNA expression of mGluR1 and protein levels of mGluR1 and ERK1/2.The neuronal apoptosis, neurological score and the ultrastructural changes were also improved. CONCLUSION: The increased expression of mGluR1 in hippocampus may induce apoptosis by activation of ERK1/2 signaling after CVS. LY367385 exerts good therapeutic effect on severe CVS.