PHC inhibits the defects of alveolar type II epithelial cells and activation of ERK in lung tissue of ALI rat induced by LPS

AIM: To investigate the effects of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS) induced the changes of ultrastructure of alveolar type II epithelial cells (ATII) and activation of extracellular signal-regulated protein kinase (ERK) in lung tissue in rats. METHODS: Acute lung injury (ALI) was induced successfully by intravenous administration of LPS (5 mg/kg) in rats. PHC (3.0, 1.0, and 0.3 mg/kg) was administered to rats 0.5 h prior and then again concomitant with LPS exposure. The changes of ultrastructure of ATII, lung permeability index (LPI), wet to dry weight (W/D) ratio in lung were measured at 6 h after LPS application. Western blotting analysis was performed to determine the phosphorylations of ERK in lung tissue at 6 h after LPS application. To examine whether the effects of PHC on activation of ERK was in a time-dependent manner, lung tissues at 0 h, 2 h, 4 h, 6 h, and 12 h were collected for measuring the level of phosphorylated ERK. RESULTS: Challenge with LPS alone resulted in a significant increase in W/D ratio in lung and LPI. The defects of ATII with no lamellar bodies in cytoplasm, the lack of microvilli along its margin, severely swollen endoplasmic reticulum, nuclear cisterna and loss of integrity of the basement membrane induced by LPS were observed under transmission electron microscope. LPS also triggered activation of ERK at 2 h. Pre-treatment with PHC significantly abolished increase in W/D ratio in lung, LPI and attenuated pathological changes of ATII in a dose-dependent manner. Moreover, pre-treatment with PHC efficiently blunted the activation of ERK induced by LPS at 6 h. CONCLUSION: These results suggest that pre-treatment with PHC significantly attenuates the lung permeability and defects of ATII in LPS-induced ALI in rats, and these effects are partly responsible for the inhibition of ERK activation by LPS.     

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.     

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 p38 MAPK-HSP27 signaling pathway in rat acute lung injury induced by lipopolysaccharide

AIM: To observe the role of p38 mitogen-activated protein kinase (p38 MAPK)-heat-shock protein 27 (HSP27) signaling pathway in lipopolysaccharide-induced acute lung injury (ALI) in rats. METHODS: Wistar rats were randomly divided into control group, ALI group and ALI+SB203580 group. After the experimental model was established, the rats were sacrificed. The pathological changes of the lung and the changes of F-actin and G-actin in the endothelial cells were observed. The ratio of wet weight to dry weight (W/D) of the lung tissues was measured. The protein levels in bronchoalveolar lavage fluid (BALF) were detected. The levels of IL-6 and TNF-α in serum and BALF were tested. The concentrations of p-p38 and p-HSP27 in the lung were determined. RESULTS: In ALI group, the protein levels in BALF and W/D ratio of the lung increased significantly at 2 h. The levels of TNF-α and IL-6 in serum and BALF began to increase at 2 h, which had significant difference as compared with control group. Aleolar epithelial swelling, alveolar walls widening, alveolar interstitial and cavity edema, and the exudation of alveolar inflammation cells, red blood cells and protein were observed in ALI group. The protein levels in BALF and W/D ratio of the lung in ALI+SB203580 group were much less than those in ALI group. The exudation of alveolar inflammation cells, red blood cells and protein, and the interstitial and alveolar edema in ALI+SB203580 group alleviated as compared with ALI group. The expression of p-p38 MAPK and p-HSP27 in the lung at 2 h in ALI group was higher than that in control group. F-actin expression in ALI group obviously increased than that in control group at time points of 0 h and 8 h. Compared with ALI group, the expression of p-HSP27 and F-actin in ALI+SB203580 group was reduced. CONCLUSION: Lipopolysaccharide activates p38 MAPK-HSP27 signaling pathway and induces lung injury. Blockage of p38 MAPK-HSP27 signaling pathway may reduce lung injury.     

Glucocorticoid attenuates LPS-induced acute lung injury by inhibiting p38MAP kinase in rats

AIM: To examine the role of glucocorticoid receptor (GR) in regulation of lipopolysaccharide (LPS)-induced lung injury. METHODS:Male Sprague-Dawley rats were divided into six groups randomly: control group (n=6), LPS group (n=6 each), Dex+LPS group (n=6 each), RU486 group (n=6), RU486+LPS group (n=6 each) and RU486+Dex+LPS group (n=6 each). All groups were subjected into 1 h, 3 h, 6 h and 12 h time point subgroups after LPS administration, except of control group and RU486 group. The concentrations of TNF-α and IL-6 in bronchoalveolar lavage fluids (BALF) were detected by ELISA. The histopathologic changes of lung tissues, the activation of p38MAPK and the expression of MKP-1 in lung tissue were also observed. Further, to confirm the role of GR in this model, the mortality of rats in LPS group vs RU486+LPS group and in Dex+LPS group vs RU486+Des+LPS group was compared. RESULTS: LPS induced lung injury and the secretions of TNF-α and IL-6 in BALF, which were significantly enhanced by pretreatment of RU486 (P<0.05). RU486 pretreatment also significantly increased the LPS-induced lethality (P<0.05). Dexamethasone attenuated LPS-induced lung damage, cytokine release and mortality rates, and the protective effects might be mediated by GR. Western blotting analysis showed dexamethasone inhibited the phosphorylation of p38MAPK in lung tissues by induction of MKP-1, and these actions were also GR dependent. CONCLUSION: GR plays an essential role in regulation of LPS-induced acute lung injury. Anti-inflammatory effects of hormone-activated GR may be mediated by inhibition of p38MAPK phosphorylation/activation, which is associated with the induction of MKP-1.     

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).     

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.     

Relationship of p38MAPK,NF-κB and HO-1 in LPS-induced acute lung injury in rats

AIM: To investigate the relationship of p38 mitogen-activated protein kinases (p38MAPK), nuclear factor-kappa B (NF-κB) and heme oxygenase-1 (HO-1) in acute lung injury (ALI) in rats.METHODS: Forty male Wistar rats were divided into 5 groups (n=8) at random: control group or normal saline group (NS group), lipopolysaccharide group (LPS group), Hemin (inducer of HO-1)+LPS group, ZnPPIX (inhibitor of HO-1)+LPS group and SB203580 (inhibitor of p38MAPK)+LPS group (SB+LPS group). Six hours after endotracheal instillation of LPS or NS, the ratio of neutrophils and the protein contents in bronchoalveolar lavage fluid (BALF) of right lung, the ratio of wet/dry weight (W/D) of the superior lobe of right lung, and arterial blood gas analysis (ABG) were examined. The protein levels of p38MAPK and NF-κB in the lower lobe of right lung were detected by Western blotting. The protein expression of HO-1 in the middle lobe of right lung was measured by the method of immunohistochemisty. The structure of the lung was evaluated under light microscope. RESULTS: Compared with NS group, the ratio of neutrophils and protein contents in BALF, the ratio of W/D, the protein levels of HO-1, p38MAPK and NF-κB were obviously higher, and arterial oxygen pressure (PaO₂), partial pressure of carbon dioxide in artery (PaCO₂) and bicarbonate content (HCO^-₃) were significantly lower in LPS group, Hemin+LPS group, ZnPPIX+LPS group and SB+LPS group (P<0.05 or P<0.01). The ratio of neutrophils and proteins in BALF, the ratio of W/D, the protein levels of p38MAPK and NF-κB were significantly lower, the protein level of HO-1 was obviously higher in Hemin+LPS group and SB+LPS group than those in LPS group (P<0.05), while the changes of the parameters in ZnPPIX+LPS group were in a contrary manner (P<0.05). No significant difference of the parameters between Hemin+LPS group and SB+LPS group (P>0.05) was found. The structures of the lung tissues in LPS group were severely damaged and even severer damages were observed in ZnPPIX+LPS group. The structural changes of the lung tissues in Hemin+LPS group and SB+LPS group were slighter. CONCLUSION: p38MAPK/NF-κB and HO-1 are inhibited by each other and the effects of them are independent on the acute lung injury.     

Change of ICAM-1 expression in intestine tissue of mice induced by LPS and role of p38 MAPK in its expression

AIM: To study the change of intercellular adhesion molecule-1(ICAM-1) expression in intestine tissues of mice induced by LPS and regulatory effect of p38 mitogen-activated protein kinase(p38 MAPK) on ICAM-1 expression. METHODS: Protein and mRNA of ICAM-1 were measured using Western blotting and RT-PCR respectively in intestine tissue of BALB/c mice treated by lipopolysaccharide(LPS) or LPS plus SB203580, a specific inhibitor of p38 MAPK. RESULTS: Compared with control group, the expression of ICAM-1 protein and mRNA was increased significantly by LPS stimulation in dose- and time-dependent manner. ICAM-1 expression reached peak value at 12-36 h after LPS stimulation. 20.0 mg/kg of LPS could induce the maximum of ICAM-1 expression. Pretreatment of mice with SB203580 for 30 min could inhibit significantly LPS-induced expression of ICAM-1 protein and mRNA expression in mouse intestine tissues. CONCLUSIONS: These data highlight that LPS could up-regulate ICAM-1 protein and mRNA expression in intestine tissue of mice in dose- and time-dependent manner, and p38 MAPK signal pathway plays an important role in ICAM-1 expression induced by LPS. It suggests that inhibition of p38 MAPK might be a useful principle for the prevention and treatment of intestine damage of endotoxic shock.     

Effect of LPS on intracellular localization of p38 protein kinase in Raw264.7 cells

AIM: To investigate the activation dynamics and the intracellular localization of p38 protein kinase in Raw264.7 cells after lipopolysaccharide (LPS) stimulation.METHODS: Protein kinase assay and immunogold electron microscope technique were used to check the activation dynamics and distribution of p38 MAPK in Raw264.7 cells before and after LPS stimulation. RESULTS: The kinase assay results showed that a marked increase in p38 activity was detected 15 min after LPS treatment, and reached maximal activity 30 min post stimulation, then dropped down and got closed to the pre-stimulated level 2 h later. The optimal LPS concentration for treatment was 100 μg/L. The immunogold electron microscope data showed that p38 spread evenly in every part of the cytosol of the non-stimulated and EGF stimulated Raw264.7 cells, such as endoplasmic reticulum, mitochondria, lysosome, while the golden granules intensity in the cytosol area decreased and in the nuclear area increased significantly after LPS stimulation.CONCLUSION: p38 MAPK moves to the nuclei of Raw264.7 cells on account of stimulation by LPS.     

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.     

Change of endogenous hydrogen sulfide/cystathionine-γ-lyase system in acute lung injury induced by LPS in rats

AIM: To observe the changes of endogenous hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) system while acute lung injury induced by LPS in rats. METHODS: Eighty rats were randomly divided into six groups (n=8): Ⅰ, control group;Ⅱ, LPS 1 h group; Ⅲ, LPS 3 h group; Ⅳ, LPS 6 h group; Ⅴ, LPS 9 h group; Ⅵ, LPS 12 h group. The ALI model of rats was prepared with LPS. The rats were respectively killed at 1, 3, 6, 9 or 12 h after administration of LPS. The morphological changes of lung tissues were observed by light and electron microscope. The lung coefficient and the wet-to-dry weight ratio were measured. The contents of IL-1β and IL-10 in serum, the H2S level in plasma and the CSE activity in lung tissue were respectively detected. RESULTS: ⑴ In LPS 1 h group, the morphology, the lung coefficient, the wet-to-dry weight ratio, the H2S level and the CSE activity showed no changes compared with the control group. The contents of IL-1β and IL-10 were increased compared with the control group (IL-1β, P<0.05;IL-10, P<0.01). ⑵ In LPS 3 h, 6 h, 9 h and 12 h groups, compared with the control group, the lung tissues were significantly damaged, the lung coefficient and the wet-to-dry weight ratio were significantly increased respectively (LPS 3 h, P<0.05; LPS 6 h, 9 h, 12 h, P<0.01). The contents of IL-1β and IL-10 in serum were markedly increased (P<0.01). The H2S level in plasma and the CSE activity in lung tissue were significantly decreased (P<0.01).CONCLUSION: The changes of inflammatory cytokines may be the pathological foundation of the ALI induced by LPS and the endogenous hydrogen sulfide/cystathionine-γ-lyase system is possibly involved in the formation of the ALI.     

Effect of propolis on the expression of CD54 and activation of NF-κB p65 of lung tissue in acute lung injury rats

AIM: To study the effect of propolis on the expression of CD54 and activation of NF-κB p65 in lung tissue of acute lung injury (ALI) rats. METHODS: 40 male Wistar rats were divided into 5 groups: normal control, model control, dectancyl group, water soluble derivative of propolis (WSP) group and ethanol extracted propolis (EEP) group. ALI animal model was performed by oleic acid and LPS twice attack. The pathologic slice was observed with light microscope and the NF-κB p65 activity and CD54 expression were tested by immunohistochemistry (SABC and SP). RESULTS: Both EEP and WSP antagonized the lung edema, decreased the inflammation and inhibited the expression of CD54 and activation of NF-κB p65. CONCLUSION: The increase in the expression of CD54 and the activation of NF-κB p65 in the lung tissues of ALI were involved in the formation of ALI. Propolis ameliorated the lung damage, which maybe related to the inhibition of CD54 expression and NF-κB p65 activation.     

Effects of neutrophil extracellular traps on acute lung injury in neonatal rats

AIMTo investigate the role of neutrophil extracellular traps (NETs) in neonatal rats with acute lung injury (ALI). METHODSThirty 7-day-old SD rats were randomly divided into normal saline control group, ALI group and ALI+deoxyribonuclease (Dnase) group (each n=10). The rats in ALI group were intraperitoneally injected with lipopolysaccharides (LPS) at 20 mg/kg, and the rats in ALI+Dnase group were intraperitoneally injected with Dnase at 5 mg/kg after LPS injection. After 6 h, the rats were anesthetized with chloral hydrate, bronchial alveolar lavage fluid (BALF) was collected, and the content of cell-free DNA (cf-DNA) in BALF was detected by fluorescence microarray. The right lung tissues were fixed in 4% paraformaldehyde, and the morphological structure of the lung tissues were observed by HE staining. The content of interleukin-6 (IL-6) and tumor necrosis factor-α （TNF-α） in the left lung homogenate was measured by ELISA. Immunofluorescence and Western blot were used to detect the production of citrullinated histone H3 (CitH3) and myeloperoxidase (MPO) in the rat lung tissues. RESULTSCompared with control group, the levels of cf-DNA, CitH3, MPO, IL-6 and TNF-α in lung tissues of neonatal rats in ALI group and ALI+Dnase group were all increased (P<0.05), and severe inflammatory infiltration in the lung tissues was observed. Compared with ALI group, the levels of cf-DNA, CitH3, MPO, IL-6 and TNF-α in ALI+Dnase group were decreased (P<0.05), and the inflammatory infiltration was attenuated. CONCLUSION In neonatal rats with ALI, the level of NETs is an important indicator of lung tissue injury, and NETs may be a new target for the treatment of neonatal ALI.     

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

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

Establishment of human lung infection model in vitro and mechanisms of NTHi-induced inflammatory responses

AIM: To investigate the key signal pathways of inflammatory responses in lung tissues induced by the infection of nontypeable Haemophilus influenzae(NTHi). METHODS: Human lung tissues were co-incubated with NTHi (10¹⁰ CFU/L) for 4 h and 24 h, respectively. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected by Western blotting. The nuclear translocation of nuclear factor (NF)-κB was examined by electrophoretic mobility shift assay (EMSA). The expression of Toll-like receptor (TLR) 2 was measured by real-time quantitative RT-PCR, and the level of interleukin (IL)-8 was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, lung tissues were incubated with anti-TLR2 monoclonal antibody (5 mg/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or NF-κB inhibitor PDTC (25 μmol/L) for 2 h, then stimulated with NTHi (10¹⁰ CFU/L) for another 24 h. The supernatants were collected for IL-8 detection. RESULTS: The TLR2-p38 MAPK-NF-κB signaling pathway in lung tissues was rapidly activated 4 h after NTHi stimulation. IL-8 secreted from lung tissues infected with NTHi was significantly increased compared with uninfected lungs (P<0.05). The pre-incubation with anti-TLR2 antibody, p38 MAPK inhibitor or NF-κB inhibitor markedly decreased IL-8 production induced by NTHi. CONCLUSION: NTHi induces inflammatory responses in lung tissues by activation of TLR2-p38 MAPK-NF-κB signaling pathway. Human lung infection model provides a new research tool for the study of interaction between pathogens and hosts.     

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.     

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.