Nontypeable haemophilus influenzae induces IL-8 expression in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner

AIM:To investigate the key molecular mechanism of inflammatory response in alveolar epithelial cells induced by nontypeable haemophilus influenzae (NTHi). METHODS: A549 cells were co-cultured with NTHi (multiplicity of infection, MOI: 10) and harvested 15 min and 30 min after stimulation. The phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) in A549 cells was detected by Western blotting. The intracellular expression of nuclear factor-κB (NF-κB) p65 was examined by flow cytometry 4 h after stimulation. A549 cells were preincubated with p38 inhibitor (SB203580) or NF-κB inhibitor (PDTC) for 1 h and then stimulated with NTHi for 24 h. The level of interleukin 8 (IL-8) in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The phosphorylation of p38 MAPK was rapidly induced by NTHi stimulation. The expression of NF-κB p65 in A549 cells after NTHi stimulation was significantly up-regulated compared with control group (P<0.05). The level of IL-8 in the supernatants was increased 24 h after bacterial stimulation compared with control group (P<0.05). Blockage of p38 MAPK or NF-κB remarkably decreased IL-8 secretion in A549 cells (P<0.05). CONCLUSION:NTHi induces inflammatory response in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner.     

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.     

Minimally modified low-density lipoprotein up-regulates endothelin receptors in mouse mesenteric artery through p38 MAPK pathway

AIMTo investigate whether minimally modified low-density lipoprotein (mmLDL) affects the quantity and activity of endothelin （ET） type A (ET_A) and type B (ET_B) receptors in mouse mesenteric artery by activating p38 mitogen-activated protein kinase (MAPK) inflammatory pathway. METHODSThe KM mice were divided into normal saline (NS) group (injection of NS via caudal vein), mmLDL group (injection of mmLDL via caudal vein), LDL group (injection of LDL via caudal vein), mmLDL+SB 203580 group (injection of mmLDL via caudal vein and intraperitoneal injection of p38 MAPK pathway specific inhibitor SB 203580) and mmLDL+DMSO group (injection of mmLDL via caudal vein and intraperitoneal injection of DMSO). Mesenteric artery ring segment vasoconstriction dose-response curves affected by sarafotoxin 6c (S6c) and ET-1 were recorded by the myography system. The mRNA levels of ET_B receptor, ET_A receptor and interleukin-6 (IL-6) were detected by RT-qPCR. The protein levels of ET_B receptor, ET_A receptor, IL-6, p38 MAPK, p-p38 MAPK, NF-κB and p-NF-κB were determined by Western blot. The serum concentration of IL-6 was measured by ELISA. RESULTSThe contractile responses of the blood vessel segments to S6c and ET-1 were significantly increased by mmLDL (P<0.01). The mRNA and protein expression levels of ET_A receptor, ET_B receptor, and IL-6 significantly increased (P<0.01). The protein levels of p-p38 MAPK and p-NF-κB were significantly increased (P<0.01). The serum level of IL-6 was significantly increased (P<0.01). These effects of mmLDL were inhibited by p38 MAPK inhibitor SB 203580. CONCLUSION mmLDL increses the serum concentration of IL-6, up-regulates the expression of IL-6, ET_A receptor and ET_B receptor in mouse mesenteric artery, and enhances the vasoconstriction function medi?ated by ET_A and ET_B receptors, which is related to the activation of p38 MAPK inflammatory pathway and downstream NF-κB 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.     

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.     

Effects of simvastation on homocysteine-induced endothelial dysfunction and inflammatory response and its molecular mechanisms

AIM: To investigate the effects of simvastation on homocysteine-induced endothelial dysfunction and inflammatory response and the underlying mechanisms of such effects. METHODS: MTT assay was used to detect cell viability, and DCFH-DA assay was used to examine the levels of reactive oxygen species (ROS). Furthermore, Western blotting was performed to detect protein expression and electrophoretic mobility shift assay (EMSA) was used to detect NF-κB DNA binding activity. RESULTS: Homocysteine (0.1-1 mmol/L) decreased the human umbilical vein endothelial cell (HUVEC) viability and increased the levels of ROS. Western blotting and ELISA showed that homocysteine significantly increased the expression of TNF-α, IL-6, MCP-1 and ICAM-1. However, pretreatment with simvastation (1-20 μmol/L) reversed the decreased cell viability and markedly suppressed an increase in the ROS level and the expression of TNF-α, IL-6, MCP-1 and ICAM-1 induced by homocysteine. Homocysteine induced p38 phosphorylation and such phosphorylation was also inhibited by simvastation and antioxidant NAC. EMSA and Western blotting showed that homocysteine induced NF-κB activation due to the increased phosphorylation of the inhibitory protein (IκBα) as well as the degradation of IκBα, while simvastation pretreatment almost completely blocked the NF-κB activation as well as the phosphorylation and degradation of IκBα. CONCLUSION: Simvastation inhibits homocysteine-induced endothelial dysfunction and inflammatory response through interfering with ROS-p38-NF-κB pathway.     

Effects of zanthoxylum seed oilA2 on NF-кB signaling pathway and inflammatory factor in lung tissue of asthmatic mice

AIM: To investigate the dynamic influence of zanthoxylum seed oilA2 (ZSOA2) on NF-кB signaling pathway and inflammatory factor in pulmonary tissue of asthmatic mice. METHODS: The suspensoid (0.2 mL containing 20% albumin hydroxide and 10% ovalbumin) was administered by intraperitoneal injection to sensitize the BALB/c mice on day 1, then 0.4% ovalbumin solution (50 μL in phosphate buffer fluid) was dripped into the respiratory tract through nasal cavity to establish the asthmatic mouse model. After dripped ovalbumin for 24 h, 48 h, 3 d, 7 d and 14 d, the mice were killed at specified time points. The contents of interleukin-4 (IL-4), interleukin-5 (IL-5) and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were determined by ELISA. The pathological changes of the lung tissues were observed with HE staining. The inflammatory cell counts were conducted by Eosin staining. The protein levels of adhesion molecule and the molecules of NF-κB signaling pathway in lung tissues were determined by Western blotting. RESULTS: In ZSOA2 treated mice, the pathological injury of the lung was significantly attenuated as compared to the model mice, the counts of eosinophils and lymphocytes were reduced obviously in lung bronchial area of asthmatic mice at all observed time points (P<0.05). The levels of IL-5 and IL-4 decreased and IFN-γ increased in BALF. The results of Western blotting showed that ZSOA2 down-regulated the protein levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, I kappa B kinase alpha-α and phosphorylation inhibitory-κB. ZSOA2 also up-regulated the protein levels of tumor necrosis factor receptor 1 and phosphorylation nuclear factor-kappaB in lung tissue at all observed time points. CONCLUSION: ZSOA2 has therapeutic effect on asthma by down-regulating the protein expression of IκB-β and p-IκB, inhibiting the releases of cytokines and chemotactic factors, and attenuating the infiltration of inflammatory cells in the lungs of ovalbumin challenged asthma mice.     

Nontypeable Hemophilus influenzae induces inflammatory response of bronchial epithelial cells via NF-κB activation and histone deacetylase activity reduction

AIM: To investigate the effect of nontypeable Hemophilus influenzae (NTHi) on the inflammatory response of bronchial epithelial cells, and to preliminarily explore its mechanism. METHODS: The human normal bronchial epithelial BEAS-2B cells were cultured, and the expression levels of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant were measured by ELISA and RT-qPCR after infection with NTHi at MOI=10. The levels of IκBα and the phosphoacetylation of histones were determined by Western blot, and then the expression of histone deacetylase (HDAC) and the enzyme activity of HDAC were detected. The binding activity of NF-κB and IL-8 was measured by electrophoretic mobility shift assay and chromatin immunoprecipitation. Finally, the expression levels of GM-CSF and IL-8 were measured after pretreatment with NF-κB and HDAC inhibitors. RESULTS: The expression levels of IL-8 and GM-CSF in the culture supernatant of BEAS-2B cells were significantly increased (P<0.05) after infection with NTHi at MOI=10. In addition, infection with NTHi significantly down-regulated the expression of cytoplasmic IκBα and enhanced the DNA-binding activity of NF-κB. The phosphoacetylation of histones H3 and H4 and the binding of IL-8 and RNA polymerase II were also significantly increased after infection with NTHi. The expression level and enzyme activity of intracellular HDAC were also significantly reduced (P<0.05) after infection with NTHi. The expression levels of GM-CSF and IL-8 were significantly reduced after pretreatment with NF-κB inhibitor (P<0.05), while the secretion of IL-8 was significantly increased after pretreatment with HDAC inhibitor (P<0.05). CONCLUSION: NTHi inhibits the expression and activity of HDAC by activating the NF-κB signaling pathway, and promotes the secretion of IL-8 and GM-CSF in bronchial epithelial cells, thereby aggravating the inflammatory response.     

Inhibitory effect of NF-κB decoy ODN on expressions of TLR4 and IL-8 in LPS- induced intestinal epithelial cells

AIM: To study the effect of NF-κB decoy oligodeoxynucleotides(ODNs) on TLR4 and IL-8 expression in LPS-induced SW480 cells. METHODS: SW480 cells were cultured in vitro and stimulated for 3 h with LPS (10 μg/L). NF-κB decoy oligodeoxynucleotides mediated by lipofectin 2000 were added into the cell culture for 6 h. The supernatants were collected and messured for IL-8 by ELISA. TLR4 mRNA and IL-8 mRNA were examined by RT-PCR, respectively. The results were compared with control group, Scrambled ODNs group and lipofectin 2000 group. RESULTS: After SW480 cells were stimulated by LPS, TLR4 mRNA, IL-8 mRNA and IL-8 expressions were significantly increased, and the difference compared with control group was obvious. After treated with NF-κB decoy oligodeoxynucleotides, TLR4 mRNA, IL-8 mRNA and IL-8 expressions were significantly inhibited. The Scrambled ODNs group and lipofectin 2000 group had no effect on them. CONCLUSION: NF-κB decoy ODNs will become a new gene drug for treating inflammatory bowel disease(IBD).     

Cucurbitin E relieves airway inflammation by inhibiting MAPKs/NF-κB signaling pathways in asthmatic mice

AIMTo investigate the effects of cucurbitacin E on airway inflammation and the signaling pathways of MAPKs and NF-κB in asthmatic mice. METHODSHealthy mice (n=40) were randomly divided into control group, model group, low-dose cucurbitacin E group, high-dose cucurbitacin E group and dexamethasone group. Ovalbumin sensitization was used to induce asthma in the mice. The protein levels of p-JNK, p-ERK1/2, p-p38 MAPK and p-p65 in the lung tissues were determined by Western blot. RESULTSCompared with control group, the numbers of inflammatory cells, such as eosinophils, lymphocytes and neutrophils, were significantly increased in model group, and the activity of MAPKs and NF-κB signaling pathway-related proteins was significantly enhanced. Cucurbitin E at high dose attenuated airway inflammation in asthmatic mice, and significantly inhibited the activity of MAPKs and NF-κB signaling pathway-related proteins. Histopathological results showed proliferation of goblet cells and bronchial mucosal epithelial cells, infiltration of inflammatory cells in the alveoli, and narrow alveolar cavity in model group, while the pathological changes were significantly alleviated in cucurbitin E treatment groups. CONCLUSION Cucurbitin E improves airway inflammation in asthmatic mice, and its mechanism may be related to the inhibition of MAPKs and NF-κB signaling pathways.     

Inhibitory effects of CCK-8 on NF-κB activities stimulated by LPS in rat PIMs

AIM: To investigate the inhibitory effects of cholecystokinin octapeptide (CCK-8) on nuclear factor-κB (NF-κB) activities stimulated by lipopolysaccharide (LPS) by using forskolin, the activator of adenylate cyclase, and PKA inhibitor H-89 in rat pulmonary interstitial macrophages (PIMs).METHODS: PIMs were isolated and purified.EMDA was applied to detect NF-κB activities and Western blotting was used to analyze the IκB-α protein level in rat PIMs.RESULTS: The NF-κB activity was not detected in normal control rat PIMs.The NF-κB activity in LPS-treated rat PIMs was obviously higher than that in control group (P<0.01).The IκB-α protein level in endochylema decreased obviously compared to control group (P<0.01).No obvious change of NF-κB activity and IκB-α protein level in CCK or Fsk treated rat PIMs was observed (P>0.05).The NF-κB activity in CCK＋LPS group and LPS＋Fsk group were obviously lower than that in LPS group (P<0.05).The IκB-α protein level was obviously higher (P<0.01).In LPS＋CCK＋H-89 group, the NF-κB activity in rat PIMs was obviously higher than that in CCK＋LPS group (P<0.01), while the IκB-α protein level decreased (P<0.01).CONCLUSIONS: The activation of cAMP-PKA signaling pathway inhibits the increase in NF-κB activity and the decrease in IκB-α protein level stimulated by LPS in rat PIMs.The anti-inflammatory effects of CCK-8 were taken effect by activating cAMP-PKA signaling pathway and further inhibiting the NF-κB activity.     

Klebsiella pneumoniae capsular polysaccharide induces secretion of cytokines in bronchial epithelial cells via EGFR signaling pathway

AIM: To investigate the role of epidermal growth factor receptor (EGFR) in the secretion of inflammatory cytokines in human bronchial epithelial BEAS-2B cells induced by Klebsiella pneumoniae (KP) capsular polysaccharide (CPS). METHODS: KP was cultured in vitro, and the CPS was extracted. The BEAS-2B cells were stimulated with CPS at different concentrations, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in the supernatant were measured by ELISA. The phosphorylation level of EGFR was detected by Western blot at different time points after stimulation. After pretreatment of the BEAS-2B cells with EGFR inhibitor AG1478, the phosphorylation level of ERK was detected by Western blot, the nuclear translocation of P65 was detected by indirect immunofluorescence, and the levels of TNF-α and IL-8 in the supernatant of the cells were measured. Finally, the levels of TNF-α and IL-8 in the culture supernatant of CPS-stimulated cells were detected by ELISA after pretreated with ERK inhibitor PD98059 and NF-κB inhibitor PDTC. RESULTS: Exposure to CPS at 10 mg/L for 12 h significantly induced the BEAS-2B cells to secret TNF-α and IL-8. The phosphorylation levels of EGFR and ERK and the nuclear translocation of p65 in the BEAS-2B cells were significantly increased after CPS stimulation (P<0.05). The phosphorylation level of ERK and the nuclear translocation of p65 were significantly reduced in the cells pretreated with EGFR inhibitor AG1478. Furthermore, the levels of TNF-α and IL-8 in the supernatant were significantly decreased after pretreated with the inhibitors of EGFR, ERK and NF-κB. CONCLUSION: Klebsiella pneumonia capsular polysaccharide activates the ERK and NF-κB signaling pathways via EGFR, and then induced the secretion of inflammatory cytokines TNF-α and IL-8 in the bronchial epithelial cells, indicating that EGFR may be a key factor in the inflammatory response induced by KP infection.     

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).     

NF-κB activation participates in the activated Akt signaling induced cardiac hypertrophy in vivo

AIM: To explore whether NF-κB activation participates in the activated Akt signaling-induced cardiac hypertrophy in vivo. METHODS: We used two in vivo models of cardiac hypertrophy, namely, aortic banding in Sprague-Dawley rats for 3 weeks and transgenic mice with cardiac specific expression of constitutively active Akt (caAkt). Electrophoretic mobility shift assay (EMSA) was used to determine NF-κB binding activity with nuclear proteins extracted from heart tissues. Western blots were performed to examine the phosphorylation of Akt and phosphor-IκBα with appropriate specific anti-phospho antibodies. RESULTS: ① The heart weight/body weight (HW/BW) ratio was significantly increased by 34.5% (P<0.01) in aortic banded 3 weeks rats compared to sham operated control. The level of phosphor-Akt in hypertrophic heart was significantly increased compared to sham operated control (P<0.01). ② The ratio of HW/BW in caAkt transgenic mice was significantly increased by 123.4% (P<0.01), compared to wild type control. NF-κB binding activity and the level of phospho-IκBα were also significantly increased in caAkt mice compared to wild type control (P<0.01). CONCLUSION: NF-κB activation participates in the activated Akt signaling-induced cardiac hypertrophy in vivo.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

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.