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.     

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.     

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.     

Effect of Xueshuantong injection on LPS-induced DIC in rabbits

AIM：To evaluate the effect of Xueshuantong injection on lipopolysaccharide (LPS)-induced experimental disseminated intravascular coagulation (DIC) in rabbits. METHODS：To establish the LPS-induced DIC model, LPS was continuously injected into the rabbit autricular vein for 6 h. The survival rate of the rabbits was recorded after 24 h. The plasma levels of alanine aminotransferase (ALT) and blood urine nitrogen (BUN) were detected. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were measured. The activity of protein C, antithrombin Ⅲ (ATⅢ) and the concentration of tumor necrosis factor α (TNF-α) were also determined. RESULTS：The survival rate of DIC rabbits was only 26.7%. BUN and ALT increased obviously. APTT and PT became much longer, platelet count, fibrinogen, protein C and ATⅢ decreased obviously, and plasma TNF-α increased remarkably. The intravenous administration of Xueshuantong injection increased the survival rate of DIC rabbits up to 66.7% in a dose-dependent manner. Xueshuantong injection also decreased the plasma levels of BUN, ALT, APTT, PT and TNF-α significantly, while increased the levels of fibrinogen, platelet, protein C and ATⅢ in plasma. CONCLUSION： Xueshuantong injection has therapeutic effect on LPS-induced DIC in rabbits.     

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.     

Effect of Qingkailing injection on LPS-induced disseminated intravascular coagulation in rabbits

AIM: To study the effect of Qingkailing injection on lipopolysaccharides(LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. METHODS: The LPS-induced DIC model in rabbits was performed by continuous infusion of LPS at a dose of 100 μg·kg^-1·h^-1 for 6 h. The survival rate of the rabbits was observed for 24 h. The treatment with Qingkailing injection or heparin was started simultaneously with LPS infusion through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were measured. The plasma levels of alanine aminotransferase (ALT) and blood urine nitrogen (BUN) were detected. The activity of protein C, antithrombi III (ATIII) and the concentration of tumor necrosis factor α(TNF-α) were also determined. RESULTS: Continuous infusion of LPS induced gradual impairment of hemostatic parameters. The APTT, PT, BUN, ALT and plasma TNF-α increased obviously,while platelet count, fibrinogen, protein C and ATIII decreased obviously. The intravenous administration of Qingkailing injection attenuated the increase in levels of APTT, PT, BUN, ALT and plasma TNF-α, and decreased the plasma levels of fibrinogen, platelet, protein C and ATIII induced by LPS infusion. CONCLUSION: Qingkailing injection has the effect against DIC induced by LPS in rabbits.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

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.     

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).     

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.     

Mechanisms underlying protection of mouse myocardium against LPS-induced injury by Siduqing

AIM: To investigate the protective effect of Siduqing, a Chinese medicine, on LPS-induced myocardium injury in mice and its mechanisms. METHODS: Mice were divided into 4 group: control, LPS, Siduqing treatment and Siduqing group, and administered intragastrically with Siduqing decoction or distilled water (0.2 mL/10 g) twice a day for 3 days, two hours after Chinese herbal medicine treatment on day 3, LPS (30 mg/kg) or normal saline was injected intraperitoneally. The serum creatine kinase (CK) and myocardial superoxide dismutase (SOD) activities were determined, and myocardial tumor necrosis factor α (TNFα) and malondialdehyde (MDA) contents were also detected. In addition, the histological changes and ultrastructure of heart were examined. RESULTS: Histological examination showed edema in myocardium and architectural disarray at 12, 24 h after LPS injection, mitochondrial swelling, condensation and margination of chromatin, irregular nuclear envelope and loss of contractile filaments at 24 h post LPS administration, while Siduqing treatment attenuated the above pathological changes of myocardium. CK activity in serum and myocardial TNFα content were higher in LPS group than control and Siduqing treatment group. Myocardial SOD activity in siduqing treatment group was higher than that in LPS group, but there was no difference in myocardial MDA content between control, LPS and Siduqing treatment group. CONCLUSION: These data suggest that Siduqing protects myocardium against LPS- induced injury via inhibiting myocardial TNFα production.     

EGFR-p38 MAPK signaling pathway is involved in expression of HMGB1 in pulmonary tissues of rats with ventilator-induced lung injury

AIM: To investigate the role of epidermal growth factor receptor (EGFR)-p38 mitogen-activated protein kinase (MAPK) pathway in the expression of high mobility group box 1 protein (HMGB1) in the lung tissues of rats with ventilator-induced lung injury (VILI).METHODS: Thirty-two healthy Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=8 each): group A, spontaneous breathing; group B, small tidal volume ventilation (VT=8 mL/kg); group C, high tidal volume ventilation (VT=40 mL/kg); group D, high tidal volume ventilation plus EGFR antagonist AG-1478. The rats in group B, group C and group D were mechanically ventilated for 4 h and then all animals were sacrificed.Total protein content and white blood cell (WBC) count in bronchoalveolar lavage fluid (BALF), the lung wet/dry weight ratio (W/D) and myeloperoxidase (MPO) activity were determined. The histological changes of lung tissues were observed by HE staining. The EGFR protein and mRNA expression, p38 MAPK activity and HMGB1 protein expression in the lung tissues were also detected.RESULTS: The inflammatory responses as evidenced by lung HE staining, total protein and WBC in BALF, the lung W/D and MPO activity were significantly higher in group C than those in group A (P<0.05). The mRNA expression of EGFR, EGFR activity, p38 activity and HMGB1 protein level also significantly increased in group C (P<0.05) as compared with group A. Significant decreases in the above indexes in group D were observed as compared with group C.CONCLUSION: High tidal volume ventilation induces acute lung injury, which may be related to up-regulation of HMGB1 expression through EGFR-p38 MAPK signal pathway.     

Effects of lipopolysaccharide binding protein on activation of p38 signaling pathway induced by LPS in macrophages

AIM:To investigate the regulatory effects of lipopolysaccharide binding protein(LBP)on activation of p38 signaling pathway induced by lipopolysaccharide(LPS)in alveolar macrophages.METHODS:The LBP from actue phase rat serum was purified by ammonium sulphate precipitation, Bio-Rex70 resin and the MonoQ column. Rat alveolar macrophages were exposed to LPS (0.01 mg/L or 1 mg/L) the various concentrations of LBP(0 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L and 10 mg/L).Western blotting were used to detect phospho-p38 in alveolar macrophages. RESULTS:SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and the molecu-lar weight was 60 kD.The binding of lipopolysaccharide to mononuclear cells were enhanced by purified rat LBP.Stimu-lation of rat alveolar macrophages with LPS at concentration of 0.01 mg/L was LBP dependent.LBP at concentrations up to 1 mg/L was able to increase the activation of p38.However, when LBP concentrations were further increased to 10mg/L, the phosphorylation levers of p38 were lower as compared with that in the presence of 1 mg/L.Stimulation of ratalveolar macrophages with LPS at concentrations of 1 mg/L was LBP-independent.CONCLUSION:The activation of p38 induced by LPS at lower concentration(0.01 mg/L) was LBP-dependent, meanwhile, LPS at higher concentration(1 mg/L) was LBP-independent.     

Propofol inhibits activation of p38 MAPK in rat brain tissues with LPS-induced brain injury

AIM: To investigate the effects of propofol on lipopolysaccharide (LPS)-induced activation of p38 mitogen-activated protein kinase (p38 MAPK) and inducible nitric oxide synthase (iNOS) in brain tissues of rats. METHODS: Sprague-Dawley rats of both sexes were randomly divided into 3 groups (n=24 each): control group, LPS group and LPS+propofol group. The models of LPS-induced brain injury were established by injecting LPS (1 mg/kg) via left internal carotid artery in LPS group. Propofol (100 mg/kg) was given intraperitoneally immediately after the LPS was given in LPS+propofol group. The same volume of normal saline was given to the rats in control group. The rats were decapitated 6 h, 24 h, 48 h and 72 h after administration. The brains were immediately isolated to detect the water content, activation of p38 MAPK and the exepression of iNOS protein. Meanwhile, the pathological changes were observed under light microscope. RESULTS: The water content of the brain was higher in LPS group than that in control group. The protein levels of phosphorylated p38 MAPK(p-p38 MAPK) and iNOS in LPS group increased 6 h after LPS administration, reached the peak at 24 h, and still higher than those in control group at 48 h and 72 h (P<0.05). The levels of those indexes were all lower in LPS+propofol group at various time points than those in corresponding LPS group (P<0.05). The pathological changes were slighter than those in LPS group. The water content of the brain was positively correlated with the levels of p-p38 MAPK and iNOS (r=0.603, r=0.727,P<0.05). CONCLUSION: Propofol attenuates LPS-induced brain injury by inhibiting the activation of p38 MAPK and down-regulating iNOS 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.     

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.     

Effects of NF-κB "decoy" oligodeoxynucleotides on TNF-α and IL-6 expression in LPS-induced mouse macrophages

AIM: To study the effect of NF-κB "decoy" oligodeoxynucleotides on TNF-α and IL-6 expression in LPS-induced mouse macrophages. METHODS: Mouse macrophage cell line J774.1 cells were cultured with LPS and liposome-mediated oligodeoxynucleotides, and the levels of TNF-α and IL-6 measured in the different culture supernatant by enzyme linked immunosorbent assay. RNA was extracted from macrophages, and the mRNA expression of TNF-α and IL-6 in macrophages was observed by RT-PCR. RESULTS: NF-κB "decoy" oligodeoxynucleotides decreased the expression of TNF-α and IL-6 in LPS-induced macrophages and inhibited generation of TNF-α and IL-6. The level of TNF-α and IL-6 did not change in control group. CONCLUSIONS: NF-κB "decoy" oligodeoxynucleotides inhibit the expression of TNF-α and IL-6 in LPS-induced macrophages, which is probably due to the specific inhibition of activated NF-κB binding sites .     

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.