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.     

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.     

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.     

Effect of p38 protein kinase on the activation of rat alveolar macrophages by lipopolysaccharide

AIM:To investigate the role of p38 protein kinase in the activation of rat alveolar macrophages(AMs) induced by lipopolysaccharide(LPS).METHODS:Nuclear protein was extracted, p38 protein kinase in nuclear extracts was analyzed by Western blot. The concentrations of TNF-α and IL-8 in supernatant were measured by radioimmunoassay.RESULTS:The concentrations of TNF-α, IL-8 in supernatant and p38 protein kinase in nuclear extracts were increased significantly induced by LPS and blocked by SB203580, a selective inhibitor of p38 protein kinase.CONCLUSION:The inductoin of TNF-α and IL-8 in alveolar macrophages by LPS may be mediated through the activation of p38 protein kinase.     

Role of p38MAPK in production of pro-inflammatory cytokines in Kupffer cells from severe acute pancreatitis rats

AIM:To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in the Kupffer cells (KCs) production of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β), in severe acute pancreatitis (SAP) rats.METHODS:Sprague-Dwaley rats were randomized into three groups:①sham operation rats, ②SAP rats, ③SAP rats given the p38 MAPK inhibitor CNI-1493(10 mg/kg, iv). The SAP model was induced by the bili-pancreatic duct infusion with 5% sterile soduim taurocholate solution. Rats from each group were killed at 12 h after sham operation or SAP and Kupffer cells (KCs) were isolated. The mRNA expressions of TNF-α and IL-1β (by quantitative real-time RT-PCR) and p38 MAPK activity (by Western blot analysis) in KCs were examined. The levels of TNF-α and IL-1β in plasma were determined by ELISA.RESULTS:There was a significant acvitation of p38 MAPK in KCs harvested from SAP rats than those from sham operation rats. SAP also promoted the mRNA expressions of TNF-α and IL-1β in KCs and the plasma levels of TNF-α and IL-1β. These events were significantly inhibited by treatment with CNI-1493.CONCLUSIONS:p38 MAPK activation is one important aspect of the signaling events that may mediate the KCs production of pro-inflammatory cytokines, TNF-α and IL-1β, in SAP rats. The inhibition of the p38 MAPK may be a potential target in the prevention and treatment of SAP.     

Role of ERK on proliferation of airway smooth muscle cells in chronic asthmatic rats

AIM: To investigate the roles of extracellular signal-regulated kinase(ERK) signaling pathway on regulating proliferation of airway smooth muscle by observing the expression of ERK in airway smooth muscle(ASM) in chronic asthmatic rats.METHODS: Airway remodeling was detected in chronic asthmatic rats by using image analysis system. The expressions of ERK and proliferating cell nuclear antigen(PCNA) in lung tissue from chronic asthmatic rats were observed by immuocytochemistry staining. The expressions of ERK1/2, p ERK1/2 and PCNA were detected in airway smooth muscle (ASM) by immunofluorescence double staining with confocal microscopy, and the expressions of protein or mRNA of ERK and PCNA in ASM were also detected by immunoblotting and hybridization in situ,respectively.RESULTS: The thickening of smooth muscle and structural remodeling in airway were observed in chronic asthmatic rats by image analysis. The enhanced expressions of ERK and PCNA appeared obviously increased in same lung tissue and the expressions of protein or mRNA of ERK and PCNA were significantly increased in ASM.CONCLUSION: ERK signal pathway might be an important pathway on regulating cell proliferation of ASM resulting in asthmatic airway remodeling.     

Effect of epigallocatechin-3-gallate on LPS-induced p38 MAPK pathway in mouse macrophages

AIM: To investigate the effect of epigallocatechin-3-gallate (EGCG) on lipopolysaccharide (LPS)-induced p38 MAPK activation and tumor necrosis factor-α (TNF-α) secretion in macrophages. METHODS: Western blotting was used to detect the phosphorylation of p38 MAPK in mouse macrophages cultured in vitro. Enzyme linked immunosorbent assay was used to determine the secretion of TNF-α in macrophages. Electron microscopy was used to study the effect of EGCG on the structure of LPS. RESULTS: LPS caused activation of p38 MAPK and more production of TNF-α, EGCG inhibited LPS-induced phosphorylation of p38 MAPK and TNF-α production and had no effect on the structure of LPS. CONCLUSIONS: EGCG has no direct effect on LPS, but blocks cellular signal pathway. The inhibition of EGCG on LPS-induced TNF-α production is mediated, at least in part, through blocking of p38 MAPK pathway.     

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.     

Regulation of ERK MAPK in evodiamine-induced A375-S2 cell death

AIM: To compare the cytotoxic effect of evodiamine with chemotherapy drugs on A375-S2 cells, and to examine the relationship between the effects of PKC and ERK on evodiamine-induced cell death. METHODS: MTT assay and Western blot analysis were applied. RESULTS: Compared to actinomycin D, cisplatin and 5-FU, evodiamine showed less cytotoxic effects on A375-S2 cells, but it induced more significant inhibition of proliferation in A375-S2 cells incubated with evodiamine for 24 h, followed by continuous culture in drug-free medium. The activation of PKC induced by 10 μg·L^-1 PMA partially blocked evodiamine-induced cell death, which was reversed by PKC and ERK inhibitors. Moreover, evodiamine down-regulated the expressions of ERK and phosphorylated ERK. CONCLUSION: Evodiamine has a strong inhibitory influence on proliferation of A375-S2 cells, even after removal of evodiamine. Evodiamine blocks the protective role of ERK to A375-S2 cells through the downregulation of ERK and phosphorylated ERK expression.     

TGF-β-activated MAPK cascade and its regulation on cells

Transforming growth factor-β(TGF-β)is a multifunctional growth factor.It plays a very important role in the growth, differentiation, migration, apoptosis of cells and production of extracellular matrix throughout many signaling pathways.MAPK cascade is one of those signaling pathways.TGF-βcan activate MAPKs and fulfill its multiple regulation on a variety of cells.     

Role of p38 mitogen-activated protein kinase in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is debilitating, often fatal, expensive to treat and common. The intracellular signals following diabetes that lead to diminished contractility, apoptosis, fibrosis and ultimately heart failure are not fully understood but probably involve p38 mitogen-activated protein kinase (p38), one of serine/threonine kinases which, when activated, cause cardiomyocyte contractile dysfunction and death. Pharmacological inhibitors of p38 suppress inflammation and are undergoing clinical trials of rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis and acute coronary syndrome. In this review, we discuss the mechanisms, circumstances and consequences of p38 activation in DCM. The purpose is to evaluate p38 inhibition as a potential therapy for DCM.     

Effect of prostate-specific membrane antigen on phospho-ERK,growth and migration of prostate cancer LNCaP cells

AIM: To study the expression of prostate-specific membrane antigen (PSMA) on the level of phospho-ERK, cell growth and migration of prostate cancer LNCaP cells.METHODS: The method of silencing PSMA was established by lentivirus-mediated RNAi in our early experiment. The cells were divided into 3 groups.In experimental group, the expression of PSMA in LNCaP cells was stably blocked by lentivirus-mediated RNAi. In negative control group, the cells were transfected with lentivirus-mediated control RNAi (without any interference to PSMA).The normal LNCaP cells served as blank control. The cells in these 3 groups were cultured in both 2 environments: normal medium and medium with PD98059 (an inhibitor of ERK phosphorylation). The phospho-ERK was detected by Western blotting and immunocytochemistry. Furthermore, the growth and migration of the cells were evaluated by MTT and transwell assays,respectively.RESULTS: In normal medium, the expression of phospho-ERK was attenuated in experimental group (P<0.05) and the quantity of "positive" cells was less than those in other 2 groups (P<0.05). Furthermore, the growth curves of the cells showed that the growth ability in experimental group was significantly decreased (P<0.05, after 48 h) and the migration ability in experimental group was reduced (P<0.05). In the inhibitory medium, the cells in all 3 groups expressed phospho-ERK at a lower level. Moreover, the abilities of growth and migration in these 3 groups were poorly displayed. These inhibitory effects on phosphorylation of ERK were similar to the cells in experimental group cultured in normal medium.CONCLUSION: PSMA may play a role in up-regulation of phospho-ERK and it may take an advantage in growth and migration of prostate cancer LNCaP cells.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.     

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.     

Recent progresses on the study of endotoxin-induced p38 MAPK signal transduction

The diseases caused by endotoxin have seriously affected human health. Previous studies have shown that p38 MAPK pathway is involved in the intracellular signal transduction induced by lipopolysaccharide (LPS), which plays an important role in the activation of inflammation-related cells to release inflammation mediator. Recently there have been some progresses in the isoforms distribution, substrate, molecular mechanism of regulating the release of inflammatory mediators, cellular specific activation and levels of p38 MAPK.     

Mitogen-activated protein kinase is involved in the changes of intracellular free calcium concentration induced by wound exudate in cultured rat epidermal stem cells

AIM: To observe the effects of harvested wound exudate on intracellular free Ca²⁺ in epidermal stem cells (ESCs) in vitro, and to investigate the relationship between mitogen-activated protein kinases (MAPKs) signal pathways and Ca²⁺ mobilization in this condition.METHODS: Wound exudate was harvested from the 80 full-thickness wounds produced on both sides of the back in 40 adult Wistar rats. ESCs were isolated, purified from neonatal Wistar rats by referring to the formerly records and binding our ideas. When the cultured cells showed up clone growing, they were divided into five groups as follows: group A: control group (no-treatment); group B: only treatment with wound exudate; group C: treatment with wound exudate and PD98059; group D: treatment with wound exudate and SB203580; group E: treatment with wound exudate, PD 98059 and SB203580. Then, the cells were incubated with fluorescence Ca²⁺ dye fluo-3/AM at 37 ℃ for 30 min, and measured by using laser scanning confocal microscope.RESULTS: The results showed that the fluorescent intensity of group B was higher than that in group A. A phenomenon of calcium oscillation was found in group C and group D. Furthermore, a rapid decrease of fluorescent intensity was observed in the cells that were preincubated with PD98059 and SB203580 at the same time. CONCLUSION: Based on above results, we propose that wound exudate can directly induce an increase in intracellular free Ca²⁺concentrations of ESCs. MAPKs signaling pathway has an important function of feedback regulation for free Ca²⁺ mobilization of ESCs in this condition, and also is capable of affecting the biological behaviour of epidermal stem cells.     

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).     

Effects of isoflurane and sevoflurane on apoptosis of cortical neuron in neonatal rats and the role of MAPKs pathway

AIM: To investigate the effects of isoflurane and sevoflurane at the same dose on apoptosis of cortical neuron in neonatal rats and the role of mitogen-activated protein kinases (MAPKs) pathway.METHODS: Eleven neonatal rats were selected at postnatal day 7 from 1 litter (altogether 5 litters) and assigned randomly into control group (C group), isoflurane group (Ⅰ group) and sevoflurane group (S group). The rats in Ⅰ group, S group or C group were exposed to 1.1% isoflurane, 1.8% sevoflurane and room air for 4 h, respectively. The brain of neonatal rats were perfused and embedded by paraffin. Caspase-3 positive expression in the retrosplenial cortex (RS) of the brain was observed by immunohistochemical staining. Meanwhile, the fresh cortex was separated at 0 h in C group and at 2 h and 4 h in Ⅰ group and S group. The levels of phospho-SAPK/JNK and SAPK/JNK, phospho-p38 and p38 in fresh cortex were detected by Western blotting.RESULTS: Caspase-3 positive cells in the the cortex were increased by 441% in Ⅰ group (P<0.01) and 151% in S group (P<0.01) as compared to C group, and increased by 115% in Ⅰ group (P<0.05) as compared to S group. The protein levels of phospho-SAPK/JNK in the cortex were increased by 219% at 2 h (P<0.05) and 181% at 4 h (P<0.05) in Ⅰ group, while no significant difference between S group and C group was observed. The phospho-p38 protein in the cortex was increased by 38.9% at 2 h (P<0.05) and 36.9% at 4 h (P<0.05) in Ⅰ group, and increased by 32.6% (P<0.05) at 2 h and 128.0% at 4 h (P<0.01) in S group as compared to C group.CONCLUSION: Isoflurane induces more apoptotic neurons in the cortex of the brain in neonatal rats at postnatal day 7 than sevoflurane. Isoflurane induces apoptosis mainly by activating SAPK/JNK phosphorylation, while sevoflurane induces aopotosis by activating p38 phosphorylation.