| Acute lung injury (ALI) is mainly characterized by diffusive injuries to lung epithelium and increased permeability of alveolar-capillary membranes caused by various factors, which lead to pulmonary edema and pulmonary closure. It expresses as distress of respiratory and refractory hypoximia clinically. There are many etiological factors that can evoke ALI/acute respiratoty distress syndrome (ARDS) directly or indirectly. Lipopolysaccharide (LPS), the main component of the cell wall of gram-negative bacteria, is one of the most important factors causing pulmonary infection and systemic infection. LPS plays an important role in initiating inflammatory response through binding to its receptors and causing systemic inflammatory response syndrome (SIRS) which can induce ALI. However, it remains incompletely illuminated the definite mechanism of LPS initiating inflammatory over-reaction and inducing ALI.Cholecyetokinin (CCK), a typical braingut peptide, is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to play a pivotal role in many physiological and pathophysiological processes. Sulfated cholecystokinin-octapeptide (CCK-8) is the minimum sequence for biological activity. Particular emphasis had been laid on its regulatory actions in nervous system, digestive system, and endocrine system in the previous studies of CCK. CCK is also demonstrated to be located in the lung. Our laboratory has been studying the effect of CCK-8 against endotoxin shock (ES) and inflammation. It was well known that the actions of CCK are mainly mediated by two distinct receptors, CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR). However the mechanism of alleviating the inflammatory response by CCK-8 is not clear.Endogenous gaseous transmitters, a unique class of biomaterials in regulating homeostasis, are found to play important roles in a variety of physiological and pathological events. Up to now, three gaseous transmitters have been recognized, namely nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S). Many evidence confirmed the important role of NO and CO in ALI. H2S is the third to be included in the family of endogenous gaseous transmitters, following NO and CO. Two pyridoxal-5'-phosphate-dependent enzymes-cystathionine-synthase (CBS ) and cystathionineγ-lyase (CSE) are responsible for the majority of the endogenous production of H2S in mammalian tissues that use L-cysteine as the main substrate. Researches about physiological functions and pathological effects of H2S became frequent only recently. It was proved that H2S participated in the regulation of neural function and vasomotion, as well as the pathogenesis of hypertension, pulmonary artery hypertension and ES, etc. However, there were few reports about the role of H2S in LPS-induced ALI, which was the major objective of this study.Recent years, a series of studies showed that the activation and damage of lung microvascular endothelial cells (LMVEC) has the effect in ALI induced by LPS. The signal transduction mechanisms through which LPS activate endothelial cells are anfractuous. However it is undoubtful that the LPS—CD14—TLR4—IRAK—IKK—IκB—NF-κB—proinflammatory cytokines pathway plays a pivotal role in the endothelial cells activation induced by LPS. NF-κB is a center of various signal pathways. LPS binds to its receptor CD14 on the membrane of endothelial cells and activates TLR4 to form LPS-receptor complex. Upon activation, TLR4 most likely form homodimers itself, resulting in the recruitment of an adapter named MyD88. The death domain of MyD88 then recruits downstream IL-1 receptor–associated kinase (IRAK) to the receptor complex. IRAK is then autophosphorylated and dissociated from the receptor complex, and recruits TNF receptor-associated factor 6 (TRAF6) that in turn activates downstream kinases. Several such kinases have been found to be involved in TLR/NF-κB signaling pathways including NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) . Activated MEKK1 or NIK are individually capable of activating the IKK complex. Subsequently, IκB is phosphorylated and degraded, leading NF-κB to translocate to the nucleus, and initiating the related gene transcription.However, it hadn't been reported that whether CCK-8 could influence CSE expression induced by LPS in the lung of rats and exert its action by regulating CSE/H2S, and whether TLR4/NF-κB signal pathway was involved in the regulatory effect of CCK-8 on CSE expression induced by LPS. The present study was designed to observe the ameliorative effect of CCK-8 on ALI and injury of LMVEC induced by LPS, the location and expression of CCK-R in LMVEC, the regulatory effect of CCK-8 on the expression of CSE/H2S and the role of TLR4/NF-κB signal pathway at different levels including in vivo and in vitro, in order to explore the role of CSE/H2S system in the ameliorative effect of CCK-8 ALI induced by LPS and provide new method for clinical doctor to prevent and treatment ALI.1 The roles of endogenous and exogenous H2S in the ameliorative effect of CCK-8 on ALI induced by LPSThe objective of this study was to investigate the role of endogenous and exogenous H2S in the ameliorative effect of CCK-8 on ALI induced by LPS. Eighty-four SD rats were divided into seven groups randomly (n=12 in each group):①control group: pyrogen-free normal saline (NS, 200μl per rat) was instilled intratracheally;②LPS group: LPS (200μg·200μl-1 each) was instilled intratracheally;③NaHS (sodium hydrosulfide, donor of H2S)+LPS group: 0.5 mL of NaHS (28μmol·kg-1) was injected intraperitoneally 10 min before LPS instillation;④PPG (L-propargylglycine, CSE inhibitor)+LPS group: 0.5 ml of PPG (45μmol·kg-1) was injected intraperitoneally 10 min before LPS instillation;⑤CCK-8+LPS group: a bolus dose (40μg·kg-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;⑥PPG+CCK-8+LPS group: PPG was injected 20 min before LPS administration, and CCK-8 was injected 10 min after PPG administration;⑦CCK-8 group: a bolus dose (40μg·kg-1) of CCK-8 was injected through lingual vein 10 min before NS instillation. Each parameter was observed respectively 4 h, 8 h after LPS administration. Blood sample was collected to test the H2S concentration in plasma. Bronchoalveolar lavage (BAL) was done on half of the rats in each group to detect polymorphonuclear neutrophils (PMN) number and protein content in bronchoalveolar lavage fluid (BALF). As for the other rats in each group, which did not receive BAL, the ratio of lung weight to body weight (LW/BW) was calculated, and lung MDA content, MPO activity and P-selectin (P-SLT) level were determined. The morphological changes and the index of quantitative assessment (IQA) of lung injuries were also observed through light microscopy.The results as follows:①diffusive inflammatory cellular infiltration was observed 4 h and 8 h after LPS instillation, accompanied with widened alveolar septum, damaged alveolar structure and local emphysema, and the process was worsened as time lengthened; compared to control group, the IQA, LW/BW, PMN and protein content in BALF and MDA content, MPO activity and P-SLT level in lung tissue were all increased in LPS group (P<0.05 or P<0.01).②Compared with LPS group at the same time points, the severity of lung injuries and the IQA were decreased in NaHS+LPS group and CCK-8+LPS group. NaHS and CCK-8 also lead to a decrease in LW/BW, PMN number and protein content in BALF, as well as lung MDA content, MPO activity and P-SLT level compared with those of LPS group (P<0.05 or P<0.01). PPG could enhance the rise of PMN numbers, MDA content, MPO activity and P-SLT level in the lung (P<0.05 or P<0.01), worsen the injury induced by LPS and inhibit the protective action of CCK-8. CCK-8 alone had no effect on the lung of rats.③Compared with control group, LPS instillation resulted in a decrease in plasma H2S concentration. Pretreatment with CCK-8 and NaHS reversed the changes caused by LPS administration. It could elevate the H2S content in plasma (P<0.01). However, PPG could enhance the effect of downregulating H2S content in plasma (P<0.05). CCK-8 alone had no effect on H2S level.The results above indicated that, LPS instillation could cause obvious lung injuries and downregulate H2S concentration in plasma. NaHS, the H2S donor and CCK-8 could upregulate the H2S content and aggravated the injuries. There maybe exist internal relationship between the protective effect of CCK-8 and H2S. CCK-8 could attenuate LPS-induced ALI by means of anti-oxidation and inhibition of PMN aggregation, which were both mediated by H2S.2 The mechanisms of CSE/H2S system in CCK-8 attenuating ALI induced by LPS2.1 Changes of CSE mRNA expression in rats with CCK-8 attenuating ALI induced by LPSIn the present study, we investigated CSE activity and the expression of CSE mRNA by RT-PCR in the lung during the process of CCK-8 attenuating lung injury induced by LPS, to explore the molecular mechanism and biology significance of CCK-8 regulating LPS-induced H2S production and to verify the non-respiratory function of the lung at molecular level. Sixty SD rats were divided into five groups randomly (n=12 in each group):①control group: pyrogen-free normal saline (NS, 200μl per rat) was instilled intratracheally;②LPS group: LPS (200μg·200μl-1each) was instilled intratracheally;③CCK-8+LPS group: a bolus dose (40μg·kg-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;④proglumide(CCK-R-non-specific inhibitor) +CCK-8 +LPS group: proglumide(1mg·kg-1) was injected 20 min before LPS administration, and CCK-8 was injected 10 min after proglumide administration;⑤CCK-8 group: a bolus dose (40μg·kg-1) of CCK-8 was injected through lingual vein 10 min before NS instillation. 4 h or 8 h after administration, each rat was executed. Part of mid-upper of left lung lobe was used to prepare homogenate of lung tissue for detecting CSE activity, part to extract total RNA and to test CSE mRNA expression with RT-PCR method.Results were as follows: compared with control group, CSE mRNA expression, CSE protein activity in the lung of LPS group were reduced, especially significantly in groups of 8 h (p<0.05); compared to LPS group, CSE mRNA expression, CSE protein activity of CCK-8+LPS were all increased (all p<0.01); compared with CCK-8+LPS group, CSE mRNA expression, CSE protein activity of proglumide+CCK-8+LPS group were reduced (p<0.05); compared with control group, there were no significant changes of CCK-8 group about CSE activity and CSE mRNA expression (all p>0.05).It suggested that CSE/H2S system participated in the pathophysiological process of ALI caused by LPS. CCK-8 could enhance the increased expression of CSE mRNA and CSE activity in rats with ALI induced by LPS. CCK-8 enhanced the H2S content through up-regulating CSE expression2.2 The role of NF-κB in CCK-8 enhancing CSE mRNA expression in the lung of rats with ALI induced by LPS and its influence on TLR4It was not elucidated about the signal pathway underlying CCK-8 enhancing CSE expression with ALI induced by LPS. NF-κB regulates expression of numerous components of the immune system. These include proinflammatory cytokines, chemokines, adhesion molecules and inducible enzymes. NF-κB acts at the crossroads of many signalling pathways. The continuing efforts to increase our molecular appreciation of the regulation of NF-κB will be of great value in learning to fully exploit this transcription factor as a therapeutic target. mCD14 is a surface glycoprotein anchored to the plasma membrane by a phosphotidy inositol without transmembrane domain. Several independent lines of evidence from biochemical and genetic studies support the hypothesis that LPS functions by interacting with a second CD14-associated signaling receptor. The homologue of Drosophila Toll (dToll) in human was first identified in 1997 and was named as Toll like receptor (TLR). Then several lines of evidence suggest that TLR4 functions as a specific co-receptor of CD14 in recognizing and mediating the effect of LPS in mammals. These data support an LPS signaling complex that minimally includes CD14 and TLR4 to respond to LPS to elicit the full spectrum of gene expression. It was worthwhile to study whether TLR4/NF-κB was involved in the up-regulation of CSE expression by CCK-8 in ALI rats induced by LPS. In the present study, we therefore have studied the signal pathway involved in CCK-8 up-regulating CSE expression using NF-κB inhibitor PDTC. Eighty-four SD rats were divided into seven groups randomly (n=12 in each group):①control group: pyrogen-free normal saline (NS, 200μl per rat) was instilled intratracheally;②LPS group: LPS (200μg·200μl-1 each) was;③PDTC + LPS group: a bolus dose (15 mg·kg-1, ip) of PDTC was injected 2 h before LPS administration;④CCK-8+LPS group: a bolus dose (40μg·kg-1) of CCK-8 was injected through lingual vein 10 min before LPS instillation;⑤PDTC+CCK-8+LPS group: PDTC was injected 2 h and 10 min before LPS administration, and CCK-8 was injected 2 h after PDTC administration;⑥CCK-8 group: a bolus dose (40μg·kg-1)) of CCK-8 was injected through lingual vein 10 min before NS instillation;⑦PDTC group: a bolus dose (15 mg·kg-1, ip) of PDTC was injected 2h before NS administration. Animals were sacrificed 8 h after agent instillation.The results showed that PDTC upgraduated the CSE mRNA expression and CSE activity induced by CCK-8, which suggested that NF-κB signal pathway played an important role in CCK-8 up-regulating CSE expression in the lung of rats with ALI induced by LPS; PDTC and CCK-8 inhibited the TLR4 expression induced by LPS, so maybe there is modulate mechanism between NF-κB and TLR4 in LPS-induced ALI.2.3 The effect of H2S on CCK R mRNA expression with ALI induced by LPSOur laboratory previous reported that lung tissue expressed CCK receptor gene, and the expression could be upregulated by LPS, indicating that CCK-8 might bind to CCK receptors and interfere with inflammatory response induced by LPS. The aim of this study was to investigate the effect of H2S on CCK R mRNA expression with ALI induced by LPS, using RT-PCR, in order to verify that the roles of H2S in the ameliorative effect of CCK-8 on ALI induced by LPS from another point of view. Eighteen SD rats were divided into six groups randomly (n=3 in each group):①control group: pyrogen-free normal saline (NS, 200μl per rat) was instilled intratracheally;②LPS group: LPS (200μg·200μl-1 each) was instilled intratracheally;③NaHS + LPS group: 0.5 mL of NaHS (28μmol·kg-1) was injected intraperitoneally 10 min before LPS instillation;④PPG+LPS group: 0.5 mL of PPG (45μmol·kg-1) was injected intraperitoneally 10 min before LPS instillation;⑤NaHS group: 0.5 mL of NaHS (28μmol·kg-1) was injected intraperitoneally 10 min before NS instillation;⑥PPG group: 0.5 mL of PPG (45μmol·kg-1) was injected intraperitoneally 10 min before NS instillation. Animals were sacrificed 4 h after agent instillation.The results showed that LPS upregulated CCK-A/BR mRNA expression, and H2S could enhance the increased expression of CCK-A/BR mRNA induced by LPS. It suggested that there is positive feedback protective mechanism between H2S and CCK-8. 3 The roles of CSE/H2S system in CCK-8 attenuating LMVEC injury induced by LPS and its mechanisms3.1 CCK-8 attenuating LPS-induced LMVEC injury and modulating CSE/H2S systemLMVEC played an important role in anti-inflammation during ALI. However, it was not reported whether LMVEC was involved in the protective effect of CCK-8. The effect of CCK-8 was mediated by its receptor. There are two main isoforms of CCK receptor (CCK-R), CCK-AR and CCK-BR. But whether the two kinds of CCK-R and CSE exist in LMVEC and the change of its expression induced by LPS were not elucidated. The purpose of the present study was to observe the roles of H2S in CCK-8 attenuating LMVEC injury induced by LPS and its mechanism, as well as location and expression of CCK-AR and CCK-BR in LMVEC. The cultured LMVEC were divided randomly into five groups treated with different agents: Control group, LPS group, CCK-8+LPS group, CCK-8 group and LPS+proglumid (CCK-R inhibitor) group. For group receiving LPS or CCK-8, LPS (10 mg·L-1) or CCK-8 (10-6 mol·L-1) was added into the culture medium (DMEM) respectively. For group receiving CCK-8 plus LPS or LPS plus proglumide, CCK-8 or proglumide (10 mg·L-1) was added into the culture medium (DMEM) respectively 10 min before LPS administration. Negative control group received saline. Then cells of each group were incubated for eight hours. MDA content and release rate of lactate dehydrogenase (LDH) of LMVEC were investigated using test kit. H2S content and soluble E-selectin (sE-SLT) in the superment and the rate of trypan blue uptake and CSE activity of LMVEC were also detected. Expression of CSE mRNA and CCK-R of LMVEC was examined by RT-PCR.The results were as follows:①compared with control group, the cell vitality was reduced, while the LDH release rate and MDA content of LMVEC were increased in LPS group. Meanwhile, LPS lead to a decrease in CSE mRNA expression, CSE activity and H2S concentration. Compared with LPS group, the cell vitality was reduced, while the LDH release rate and MDA content of LMVEC were increased in proglumide +LPS group. The changes of the cell vitality, LDH release rate and MDA content caused by LPS were attenuated and the CSE/H2S system was upregulated in CCK-8+LPS group. CCK-8 alone had no effect.②There was no CCK-AR mRNA (1.3 Kb) expression in each group. CCK-BR mRNA (480bp) expression products were observed in control group and increased significantly in CCK-8+ LPS group as much as that of control group. LPS upregulated the expression of CCK-BR mRNA also.These data confirmed that CCK-8 functioned the cytoprotective actions for mitigating lipoperoxide damage of LMVEC induced by LPS, which was mediated by CCK-R. It suggested that LMVEC played an important role in CCK-8 attenuating ALI referring to the results of the former part. It was firstly found that there existed CCK-BR in LMVEC and the cytoprotection of CCK-8 may be mediated by up-regulating CCK-BR mRNA expression. It was also firstly found that there existed CSE mRNA in LMVEC. The underlying mechanism of CCK-8 for cytoprotection was also associated with enhancing the generation of CSE/H2S induced by LPS.3.2 Effect of H2S on NF-κB binding activity in the rat LMVEC stimulated by LPSTo elucidate the anti-inflammatory mechanism of H2S in the present study, NF-κB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) and the IκBαprotein level in the cytoplasma was detected by Western blot. The cultured LMVEC were divided randomly into six groups:①control group: incubated with regular culture medium;②LPS group: LPS (10 mg·L-1) was added to the culture medium;③NaHS+LPS group: NaHS (0.5 mM) was added to the culture medium 10 min before LPS (10 mg·L-1) addition;④PPG+LPS group: PPG (10 mM) was added to the culture medium 10 min before LPS (10 mg·L-1) addition;⑤NaHS group: NaHS (0.5 mM) was added to the culture medium;⑥PPG group: PPG (10 mM) was added to the culture medium. Then cells of each group were incubated for one hour.Results:①The NF-κB binding activity was significantly higher in LMVEC stimulated with LPS in comparison with unstimulated cells, and additional treatment with NaHS markedly reduced the binding activity. The effect of NaHS was abrogated by PPG. NaHS or PPG alone had no effect on the NF-κB binding activity. The binding specificity was confirmed by using homologous (NF-κB) and nonhomologous (AP-2) oligonucleotides as competitors.②The IκBαprotein level in LMVEC was markedly decreased 1 h after incubation with LPS , and NaHS obviously increased IκBαprotein level in LMVEC stimulated by LPS. The effect of NaHS was attenuated by PPG. NaHS or PPG alone had no effect on the IκBαprotein level.These results showed that NaHS inhibited NF-κB activity and IκBαdegradation in rat LMVEC.3.3 The role of NF-κB in CCK-8 enhancing CSE mRNA expression in the rat LMVEC stimulated by LPS and its effect on TLR4The purpose of the study was to verify that NF-κB signal pathway was involved in CCK-8 enhancing CSE expression in LMVEC induced by LPS and its effect on TLR4. The cultured LMVEC were divided randomly into seven groups treated with different agents: control group, LPS group, LPS+PDTC group, CCK-8+LPS group, CCK-8+LPS+PDTC group, CCK-8 group and CCK-8+PDTC group. For group receiving LPS or CCK-8, LPS (10 mg·L-1) or CCK-8 (10-6 mol·L-1) was added into the culture medium (DMEM) respectively. For group receiving CCK-8 plus LPS, CCK-8 was added into the culture medium (DMEM) 10 min before LPS administration. PDTC (50μmol·L-1) was added 2 h before LPS or CCK-8 administration. Negative control group received saline. Then cells of each group were incubated for eight hours. CSE mRNA, TLR4 mRNA and protein expression was detected using RT-PCR, Western blot methods. The results showed that PDTC upregulated CSE expression induced by CCK-8, which demonstrated that NF-κB signal pathway played an important role in CCK-8 up-regulating CSE expression in LMVEC; meanwhile, CCK-8 and PDTC inhibited the expression of TLR4 mRNA and TLR4 protein, so maybe there is a modulate mechanism between NF-κB and TLR4 in LPS-induced LMVEC.CONCLUSIONThe roles of CSE/H2S system in CCK-8 attenuating ALI and injury of LMVEC induced by LPS was examined systemically in vivo and in vitro, its signal pathway and receptor mechanisms were explored as well. The results provided novel and reliable experimental evidence for the clinical application.1 Intratracheal instillation of LPS could cause severe lung injuries and downregulate the H2S content in plasma. Inhibition of endogenous H2S by PPG exacerbated the lung injuries induced by LPS, while exogenous H2S ameliorated the lung injuries. It was suggested that abnormally low levels of H2S might contribute to the ALI induced by LPS, exogenous H2S could protect the lung against the injuries, the possible mechanism of which may be associated with the role of H2S in inhibiting PMN recruitment and the following inflammation and oxidation. CCK-8 could alleviate the lung injury while upregulated the H2S content in plasma.There maybe exist internal relationship between the protective actions of CCK-8 and H2S.2 CCK could enhance the expression of CSE mRNA in the lung of ALI induced by LPS. CCK-8 enhanced LPS-induced CSE expression in the lung of rat and in LMVEC through NF-κB signal pathway. Maybe there is a modulate mechanism between NF-κB and TLR4 in this process. H2S could upregualte the expression of CCKA/BR mRNA in the rat lung of ALI. 3 CCK-8 functioned the cytoprotective actions for mitigating lipoperoxide damage of LMVEC induced by LPS, which was mediated by CCK-R and upregualte the expression of CSE mRNA. It suggested that LMVEC played an important role in CCK-8 attenuating ALI.4 There existed CCK-BR in LMVEC and LPS could directly increase CCK-BR mRNA expression, which is one of the important receptor mechanism underlying endogenous and exogenous CCK-8 affording cytoprotection for mitigating lipoperoxide damage of LMVEC induced by LPS and regulating CSE/ H2S system.5 There existed CSE mRNA in LMVEC, thus providing a basical work for developing the molecular mechanisms of anti-inflammation in H2S.
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