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).     

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.     

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 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.     

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.     

Alteration of p38 MAPK activity in lung tissue in diabetic rats

AIM: To observe the pathologic changes in lung and the role of p38 MAPKinase signal pathways in pulmonary alteration in diabetic rats. METHODS: Diabetic rats were induced by intraperitoneally injected streptozotozin (STZ). After 4 weeks, we observed the pathologic changes in lungs, tested protein kinase C (PKC) activities by isotope in lungs of model rats, tested transforming growth factor (TGF-β₁) by Western blotting and immunohistochemical analysis, and determined the expression of p38 MAPKinase mRNA using in situ hybridization.RESULTS: After STZ administration for 4 weeks, we observed thickened pulmonary capillary basal lamina and increased number of fibre in Diabetes mellitus (DM) rats. TGF-β₁ levels, PKC and p38 MAPK activities were also found increased. CONCLUSION: The increased activities of TGF-β₁ and p38 MAPK suggeste that TGF-β₁ may play an important role in diabetic lung, and hyperglycemia-PKC-p38 MAPK signal pathways may be involved in the pathogenesis of diabetes.     

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.     

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.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Rapid effects of dexamethasone on activation of ERK and p38 in HO-8910cells

AIM: To investigate the effects of synthetical glucocorticoid dexamethasone(Dex) on the activation of two members of mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated protein kinase1/2(ERK1/2) and p38 MAPK (p38) in human ovarian cancer cell line HO-8910. METHODS: The activation of ERK1/2 and p38 was determined by Western blot. RESULTS: Inhibition of activation of ERK1and ERK2 by10^-7 mol/L Dex occurred at 5 min, with maximum up to 41% and 54% respectively at 30min (P<0.01), and sustained until 4 h. On the contrary, p38 activity was rapidly stimulated by 10^-7 mol/L Dex, with maximum to 84% at15 min (P<0.01), and sustained till1h. Furthermore, these effects increased with the concentration of Dex(10^-10-10^-6 mol/L). RU486, an antagonist of glucocorticoid receptor (GR), did not affect these effects. CONCLUSION: Dex can rapidly inhibit ERK1/2 and stimulate p38 activation in a GR-independent manner in HO-8910cells, which might play a role in Dex-mediated growth inhibition in these cells.     

Regulation of dopamine receptors on cocaine-induced MAPK signal ing pathway and c-fos gene expression

AIM:To investigate the role of D1 and D3 d opamine receptor on MAPK signal transduction and c-fos gene expression after acute cocaine treatment.METHODS:Activations of extracellular signal-regulated kinase (E RK),the c-Jun N-terminal kinase (JNK),p38 activation and expression of c-fos in wild type and D1 and D3 receptor mutant mice after acute cocaine treatment were checked by Western blotting.RESULTS:ERK activation and c-fos induction was enhanced in D3 mutant mice and abolished in D1 mutant mice by acute cocaine treatment,while p38 and JNK activation was not obviously modulated by the D1 and D3 receptors b y acute cocaine treatment.Meanwhile,c-fos induction was inhibited when SL3 27,a specific MEK inhibitor,was injected before cocaine treatment.CONCLUSION:D1 and D3 receptors play opposite roles in the regul ation of ERK activation and c-fos gene expression after acute cocaine treatm ent.The expression of c-fos gene depends on ERK signal pathway after acute cocaine treatment.     

Inhibitory effect of sodium ferulate on Aβ25-35-induced p38 MAPK activation

AIM: To investigate effect of sodium ferulate on Aβ_25-35-mediated signaling pathway. METHODS:The isolated peritoneal macrophages from mice were cultured. p38 MAPK protein kinase in nuclear extracts was analyzed by Western blotting. The concentration of TNF-α and NO in supernatant were measured by ELISA and Griess reaction technique. The expression of iNOS protein was detected by immunochemical technique. RESULTS:Aβ_25-35 significantly increased the concentrations of TNF-α and NO in supernatant, expression of iNOS in macrophages and p38 MAPK protein kinase in nuclear extracts, which were blocked by sodium ferulate. CONCLUSION:Sodium ferulate inhibits p38 MAPK activation triggered by Aβ_25-35.     

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.     

Effects of cyclic tensile strain on expression of p38 MAPK and phospho-p38 MAPK in osteoarthritis chondrocytes

AIM: To observe the effect of cyclic tensile strain (CTS) on the expression of p38 MAPK and phospho-p38 MAPK in rabbit osteoarthritis (OA) chondrocytes in vitro. METHODS: The animal model of OA was induced by anterior cruciate ligament transection in New Zealand white rabbits. The animals in all groups were evaluated 10 weeks later. The rabbits in OA group were randomly divided into 3 groups, low CTS (0.5 Hz, sin10%, 6 h/d) group, high CTS (1.0 Hz, sin10%, 6 h/d) group and control group. Both CTS groups were stimulated by a Flexercell-4000 tension system. The expression of p38 MAPK and phospho-p38 MAPK of the chondrocytes was analyzed by RT-PCR and Western blotting at the time points of 24 h, 1 week and 2 weeks. RESULTS: The knee joints of the rabbits in OA group had obvious degeneration of articular cartilage. The expression of p38 MAPK in normal group was significantly lower than that in control group (P<0.01), and the difference between low CTS group and high CTS group 1 week after stimulation (P<0.05) was observed. Meanwhile, significant difference was found between low CTS group and control group 2 weeks after CTS treatment (P<0.01). The expression of phospho-p38 MAPK was decreased at different time points in low CTS group. CONCLUSION: Different cyclic tensile strains lead to different effects on the expression of p38 MAPK and phospho-p38 MAPK in the chondrocytes. p38 MAPK signaling pathway plays an important role in the development of osteoarthritis in chondrocytes.     

Effect of FSD-C10 on modulation of inflammatory microenvironment in an Alzheimer disease double transgenic mouse model

AIM: To explore the therapeutic effect of a novel Rho kinase inhibitor FSD-C10 on β-amyloid protein precursor (APP)/presenilin-1 (PS1) double transgenic mice. METHODS: The transgenic mice overexpressing human APP with the Swedish mutation (695) and human PS1 with ΔE9 mutation at the age of 8 months were used in this study. The mice were randomly divided into model group and FSD-C10 intervention group, and wild-type mice at the same age served as normal controls. The mice in FSD-C10 intervention group were treated with FSD-C10 (25 mg·kg^-1·d^-1) for 2 months by intraperitoneal injection. The mice in model group and the wild-type mice were injected with saline in the similar manner. Morris water maze (MWM) test was applied to examine the capacity of learning and memory. The Aβ_1-42 deposition, Tau protein phosphorylation, and the expression of β-site APP-cleaving enzyme (BACE) as well as inflammatory molecules, such as TLR-4 and NF-κB, and M1/M2 microglial markers, such as iNOS and Arg-1, were determined by the methods of immunohistochemistry and Western blot. RESULTS: Compared with model group, FSD-C10 significantly improved the learning and memory abilities of APP/PS1 double transgenic mice, accompanied by reduced Aβ_1-42 deposition, Tau protein phosphorylation and BACE expression in the hippocampus. The intervention of FSD-C10 decreased the protein levels of TLR-4 and p-NF-κB, reduced the expression of iNOS and increased the expression of Arg-1 in the brain tissues. CONCLUSION: The novel Rho kinase inhibitor FSD-C10 improves the capacity of spatial learning and memory in APP/PS1 double transgenic mice, which may be related to the inhibition of TLRs/NF-κB signaling pathway, the reduction of the secretion of inflammatory molecules and the polarization of anti-inflammatory M2 microglia, thus improving the inflammatory microenvironment of the brain in APP/PS1 double transgenic mice.     

Role of ZIPK in the regulatory effects of PKCα and PKCε on calcium sensitivity during hemorrhagic shock in rats

AIM: To observe the role of zipper-interacting protein kinase (ZIPK) in the regulatory effects of protein kinase Cα (PKCα) and protein kinase Cε (PKCε) on calcium sensitivity during hemorrhagic shock(HS) in rats. METHODS: The skinned first class arborization of superior mesenteric artery (SMA) from HS rats were adopted to observe the influence of inhibitor of ZIPK on the effects of PKCα and PKCε agonists on calcium sensitivity after shock via measuring the contraction initiated by Ca2+ with isolated organ perfusion system, hypoxic vascualr smooth muscle cells (VSMCs) were adopted to measure the protein expression and activity of ZIPK after applying PKCα and PKCε agonists following hypoxia via Western blotting. RESULTS: (1) The calcium sensitivity of SMA was decreased after 2 h shock, and increased by agonists of PKCα and PKCε. Emax of Ca2+ was increased from 47.2％to 66.5％ (P<0.01) and 66.3％ (P<0.01) of normal control respectively as compared with 2 h shock group. The increasing effects of PKCα and PKCε agonists on calcium sensitivity of SMA after 2 h shock were weakened by the inhibitor of ZIPK. The cumulative dose-response curve of Ca2+ was shifted to the right, the Emax of Ca2+ was decreased to 42.6％ and 47.5％ of normal control (P<0.01), respectively. (2) The protein expression and activity of ZIPK in VSMCs were decreased after 2 h hypoxia, and were increased by the agonists of PKCα and PKCε following 2 h hypoxia. CONCLUSION: PKCα and PKCε regulate the calcium sensitization probably through changing the protein expression and activity of ZIPK following HS in rats.     

Mechanisms of PGF2α on the glucose-induced insulin secretion in NIT-1β cells

AIM:To investigate the cellular mechanisms by which PGF₂α promotes glucose-stimulated insulin secretion in NIT-1 beta cells. METHODS:Using the radioimmunoassay (RIA), the amount of the PGF₂α augmentation of glucose stimulated insulin secession was determined in different conditions, and the confocal laser scanning methods by Fluo-3AM as a fluorescent probe were used to analyze the changes of intracellular calcium in NIT-1β cells. RESULTS:At the lower glucose (0, 5.5 mmol/L), PGF₂α (5 μmol/L) failed to potentiate insulin secretion (P>0.05). However, in the presence of 16.5 mmol/L glucose, PGF₂α increased significantly in insulin secretion (P<0.05). Neither the AC inhibitor ddA nor the GC inhibitor Ly-83583 altered PGF₂α-potentiated insulin secretion in the presence of 16.5 mmol/L (P<0.05 or P<0.01). Otherwise, the PLC inhibitor U-73122 and the PKC blocker calphostin C both counteracted the insulinotropic of PGF₂α (P<0.01 or P<0.05). Moreover, exposure of the NIT-1β cells to 5 μmol/L PGF₂α induced a rapid increase of intracellular calcium (P<0.01). The inhibitor, ddA or Ly-83583 had no impact on PGF₂α-induced elevation of the intracellular calcium (P<0.01). Pretreatment of the cells with U-73122 completely prevented the calcium response induced by PGF₂α (P<0.01). CONCLUSION:Efects of PGF₂α was independent of cAMP or cGMP, potentiated glucose (16.5 mmol/L)-induced insulin secretion in NIT-1β cells through stimulation of phospholipase C, which subsequently mediated the elevation of intracellular calcium and activation of protein kinase C.     

Neural differentiation of bone marrow mesenchymal stem cells from an Alzheimer disease mouse model

AIM：To estimate the neural differentiation efficiency of bone marrow mesenchymal stem cells (MSCs) derived from amyloid precursor protein (APP) transgenic mice and to investigate the correlation with Notch1 signaling and the autophagy activity during the differentiation. METHODS：The MSCs were divided into APP group (MSCs from APP transgenic mice) and WT group (MSCs from wild-type mice). MSCs were treated with β-mercaptoethanol as an inducer for differentiating into neurons. The levels of Aβ40 and Aβ42 were measured using enzyme-linked immunosorbent assay kits. The expression of neural cell-specific markers, neuron-specific enolase (NSE) and microtubule-associated protein 2 (MAP-2), was measured by immunocytochemistry and Western blotting. The expression levels of Notch1, Notch intracellular domain (NICD), Hes5, LC3 and p62 (a selective substrate of autophagy) were also detected by Western blotting. RESULTS：The neural differentiation capacity and the Aβ expression level of the MSCs in APP group were higher than those in WT group, and stronger inhibition of Notch1 signaling pathway in the MSCs from APP group was observed. However, the process of autophagy, which is essential for the survival and function of the neural cells, was impaired in the neural differentiated counterpart of the MSCs in APP group. CONCLUSION:Over-expression of APP might contribute to the high neural differentiation capacity of MSCs by inhibiting Notch1 signaling pathway in vitro. However, autophagy is impaired in the differentiated MSCs from APP transgenic mice.     

Effect of neuregulins on mtp53 and HIF-1α in MDA-MB-231 cells

AIM: To explore the effect and significance of neuregulins /ErbB2 receptor signal transduction pathway on mtp53 and hypoxia-iducible factor-1α (HIF-1α) in none-overexpression ErbB2 breast cancer cell MDA-MB-231. METHODS: The expression of neuregulin was detected by immunocytochemistry and Western blotting. MDA-MB-231 cells were treated with ErbB2 kinase inhibitor AG825. Proliferation was measured by MTT assay. The cell cycle and apoptosis were determined by flow cytometry. The expressions of mtp53 and HIF-1α were detected by Western blotting. The mRNA expression of HIF-1α was detected by RT-PCR. RESULTS: MDA-MB-231 cells expressed a relative higher level of neuregulin. In the results of Western blotting, the positive reaction band was found in 44 kD which coincides with the molecular weight of neuregulin. When MDA-MB-231 cells were treated with AG825, the proliferation was inhibited in time and dose dependent manners (P<0.01). The cell cycle was arrested in G0/G1 phase (P<0.05). The apoptosis rate was increased (P<0.05). The protein expression levels of mtp53 and HIF-1α were decreased (P<0.05), and the mRNA level of HIF-1α was also decreased (P<0.05).CONCLUSION: Our study indicates that neuregulins are synthesized in MDA-MB-231 cells as transmembrane proteins. Neuregulins activate ErbB2 receptor signal transduction pathway by ligand autocrine or paracrine actions, and play an important role in proliferation of none-overexpression ErbB2 breast cancer cell MDA-MB-231. Proliferation and survivorship, and inhibition apoptosis can be induced with up-regulation of mtp53 and HIF-1α level.