| Study background and objectiveThe acute lung injury and the respiratory distress syndrome (ALI/ARDS) is a criticalclinical syndrome with progressive dyspnea, refractory hypoxemia and higy mortality, which pathological features mainly include inflammatory reaction and alveolar-capillary membrane injury resulted from severve infection, trauma, shock, etc. The difference of the both lies in the pathogenetic condition, the latter is more severe than the former. The essence of ALI/ARDS is a vascular leak syndrome induced by excessive inflammatory response in lung tissue. Infection especially gram-negative bacterial sepsis is a key etiological factor for ALI/ARDS. Bacterial lipopolysaccharide (LPS) or endotoxin, being of a component of the outer envelope of all gram-negative bacteria is a highly proinflammatory molecule. LPS may induce excessive inflammatory response of the body tissue and cells, which results in a series of pathophysiological changes including functional disorder, disorganization, apoptosis and even necrosis.Among the LPS signal transduction pathway, Toll-like receptor-4 (TLR-4) is known as 'the door protein', which controls the transduction of LPS inflammatory signal into the cell and subsequently initiates inflammatory response and induces a series of pathophysiological changes. TLR-4 is integrally involved in LPS signaling and has a requisite role in the activation of caspases (eg. caspase-3) and nuclear transcription factor-κB (NF-κB), both of which are the key signaling events that mediates an array of cell responses. Caspase-3 is known as 'the apotosis executioner' and NF-κB promotes the expression of proinflammatory gene products.Among the enormous lung parenchymal cells, pulmonary microvascular endothelial cells (PMVECs) and alveolar epithelial cells (AECs) are two important cell groups. They are not only the target cells of inflammatory cells and mediators, but are active inflammatory cells and effector cells as well. They play important roles in the occurance and progress of ALI/ARDS. Regrettably however, the roles of the two cell groups in the pathogenesis of ALI/ARDS have been paid little attention to in the past.Though unceasing researches have been being done by scholars all over the world, the therapeutic methods for treating ALI/ARDS have not been got a breakthrough. And respiratory support is still one of the major methods for treating the disease. Being as a kind of liquid breathing medium, perfluorocarbon (PFC) is a promising method in treating ALI/ARDS. In addition to its high ability at carrying and dissolving oxygen and carbon dioxide, PFC has shown a wide non-specific biological effects both in vivo and in vitro. But the mechanism of the biological effects of PFC is little known. Besides, the probable protective roles of PFC in PMVECs and AECs and the mechanism underlined are needed to research.In view of the above-mentioned, the study objective is:①To investigate the apoptosis injury and inflammatory resopnses of PMVECs and AECs induced by LPS. And to detect the activation of LPS/TLR-4 signal pathway so as to elucidate the role of the signal pathway in mediating the apoptosis injury and inflammatory responses of the both cells.②To study and research the protective effects of PFC on the injury of PMVECs and AECs induced by LPS and the effects of PFC on LPS/TLR-4 signal pathway, namely to search for the mechanism of the protective effects of PFC.Mehtods1. Methods investigating the protective effects of PFC on the injury of PMVECs induced by LPS and the mechanism of PFC's protective effects1.1 Method of isolation, culture, purification and identification of PMVECs from rat lung: Primary cells of PMVECs were obtained after isolated peripheral lung tissues of SD rat, digested with type IV collagenase and dispase and aggregated by serum stimulation. The purified was done by serial subcultivation. According to typical cobble morphology of endothelial cells, VIII factor (also known as von willebrand factor, vWF) in endothelial cells detected by immunocytochemical staining, and ultrastructure characteristics of PMVECs observed under transmission electronic microscopy, PMVECs were identified.1.2 Methods investiagting the protective effects of PFC on the apoptosis injury induced by LPS: cells apoptosis rate was detected by Flow cytometry (FCM) and the ultrastructure changes of apoptosis were observed under TEMThe third and/or fourth generation PMVECs were used in our research. The cells were divided into four groups:①control group: cells did not receive any intervention,②PFC group: PFC was added to the cell culture medium to a final volume concentration [vol/vol, PFC: culture media] of 30%. After vortexing, the mixed liquor containing PFC and culture media was transfered into the culture flask. As PFC is not miscible with the medium, cells exposed to PFC were constantly shaken (60 times/min).③LPS group: cells were incubated with LPS at a final concentration of 10 ng/ml, 100ng/ml, 1μg/ml, 10μg/ml, and④LPS+PFC group (coculture group): cells were incubated with both LPS and PFC according to the above-mentioned percentage. The samples were collected at 6,12h after intervention for FCM and 6h for TEM.1.3 The expression of TLR-4 and caspase-3 mRNA were detected by real time PCR and the protein level of the two were detected by western blotting. The groups and the intervention methods were identical with those in FCM detection. The samples were collected at 2, 6,12h after intervention for real time PCR and 0.5, 2, 6, 12h for western blotting. LPS concentration was 10μg/ml in the experiment.1.4 Methods researching for the effects of PFC on the inflammatory response of PMVECs induced by LPS1.4.1 The release of proinflammatory mediators of ICAM-1, TNF-αand IL-8 were tested by ELISA or radioimmunity method;1.4.2 The expression of TLR-4 mRNA and TLR-4 protein were detected by real time PCR and western blotting respectively;1.4.3 the activities of NF-κB were determined by western blotting and by immuno-cytochemical double staining.The groups and the intervention methods were identical with those in FCM detection. LPS concentration was 10μg/ml in the experiment.2. Methods investigating the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC's protective effectsBeing as a parallel study, we investigated the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC's protective effects. The cell groups, intervention methods and the observing parameters were all identical to that in PMVECs experiment. The only difference was that A549 cell line was used in this test.Results1. results of the protective effects of PFC on the injury of PMVECs induced by LPS and the mechanism of PFC's protective roles1.1 Isolation, culture, purification and identification of PMVECs from rats lung: Methods of isolation and identification of PMVECs from rat lung were established. And the purified PMVECs from rats presented a typical cobblestone morphology with monolayer growth and contact inhibition. vWF were expressed in cytoplasm of the purified cells, which was shown by immunocytochemical staining. Purified cells presented some typical ultrastructure characteristics of endothelial cell, including Weibel-Palade (W-P) bodies and abundant heterophagic vacuoles in cytoplasm, abundant mirovilli on PMVECs plasma membrane, etc..1.2 apoptosis results detected by FCM and TEM①results of FCM: The early apoptosis rates of PMVECs induced by LPS 10 ng/ml, 100ng/ml, lμg/ml, 10μg/ml, 12h after stimulated, were 6.23%, 6.07%, 9.51% and 23.48% respectively. The late apoptosis and necrosis rates were 3.93%, 6.03%, 5.84% and 10.89% respectively; and the viable cell rates were 88.91%, 87.33%, 84.19% and 64.86% respectively. This indicated that apoptosis damage of PMVECs induced by LPS was elevated at dose dependent, whereas the viable cell rate was decreased at dose dependent.Compared with Control, PFC and LPS+PFC groups, the early apoptosis rate and the late and necrosis rate in LPS groups were significantly increased (only there was no difference of late apoptosis rate at 6h between LPS and PFC group, p=0.066), the viable rate was markedly decreased. And the injury degree at 12h was more severe than that at 6h group. At 6 and 12h, there was no statistical difference of apoptosis and viable cell rate between the control and PFC groups and PFC groups each other. Compared with control and PFC groups, the early apoptosis rate of coculture group (LPS + PFC) at 6, 12h was raised, and the viable rate was decreased.②resuls of TEM: There were no apoptotic ultrastructure signs were observed under TEM in both cotrol and PFC groups. In LPS group, heterochromatin condensation, nuclear membrane unclear, mitochondrion swollen, etc., which representing the signs of early and intermediate stage of apoptosis were observed under TEM. And in coculture group, the nuclear membrane was clear, chromatin was even. Although heterochromatin was much more but no condensation, which indicated that there was no apoptotic ultrastructure signs were observed in the coculture group.1.3 results of the expression of TLR-4 and caspase-3 mRNA detected by real time PCR and the protein level of the two detected by western blotting:①results of real time PCR: the expression of TLR-4 and caspase-3 mRNA in LPS groups was markedly increased, the former increased at 2,6,12h, and the latter increased at 6,12h. There was no significant effect of PFC alone on the expression of the two genes. Whereas the expression of the two genes in the coculture groups was significantly decreased.②results of western blotting: In LPS groups, TLR-4 protein was markedly elevated at 0.5, 2, 6h, and caspase-3 precursor was significantly degraded and activated caspase-3 (17 kDa and 11 kDa subunits) was significantly increased at 0.5, 2, 6, 12h; There was no obvious effect of PFC alone on the two proteins. But in the coculture groups, the proteins of TLR-4 and activated caspase-3 were markedly decreased.1.4 Results of the protective effects of PFC on the inflammatory responses of PMVECs induced by LPS1.4.1 results of cytokines tested: The proinflammatory mediators were significantly increased in LPS groups. ICAM-1 and TNF-αwere increased at 2, 6, 12h, the secrete peaks were at 2h, IL-8 increased at 6h, and the secrete peak at 12h. There was no obvious impact of PFC alone on the above mediators. In the coculture groups, however, all the above mediators were markedly decreased.1.4.2 results of the expression of TLR-4 mRNA and TLR-4 protein: read the result of 1.3 please.1.4.3 results of activeties of NF-κB①results of activeties of NF-κB detected by western boltting: Being as the inhibitor of NF-κB, IkB-αwas significantly degraded at 0.5h, NF-κB P65 was released and translocated into the nucleus. Except for 2h, IκB-α. was degraded markedly and the level of P65 in nucleus was increased. The changes of the two proteins were approximately in-synchronism. There was no obviously effect of PFC alone on the activeties of NF-κB. But in coculture groups, the activeties of NF-κB were obviously decreased, which indicated by the degradation of IicB-αmarkedly decreased and the reduction of NF-κB P65 in the nucleus.(2) results of immunocytochemical double staining of NF-κB: In control group, IκB-αwas observed in cytoplasm and there was nearly no P65 protein in the nuclus, similar results were observed in PFC groups. In LPS groups, from 30min to 6h after stimulated, the coloration of IκB-αwas obviously thinningz and even no, whereas the heavy nuclear staining of P65 was observed, which indicated P65 was activated and translocated into the nucleus. In coculture groups, cytoplasmic immunocytochemical staining of IκB-αwas mainly observed and there was nearly no coloration of NF-κB P65 was observed in nucleus at 0.5 and 2h. At 6h, in addition to the coloration of IKB-αin cytoplasm, NF-κB P65 protein in nucleus was also observed in a few cells.2. results of the protective effects of PFC on the injury of AECs induced by LPS and the mechanism of PFC's protective roles2.1 apoptosis results detected by FCM and TEM①results of FCM: The early apoptosis rates of A549 cells induced by LPS 10 ng/ml, 100ng/ml, lμg/ml, 10μg/ml, 12h after stimulated, were 4.12%, 6.42%, 7.53% and 16.12% respectively. The late apoptosis and necrosis rates were 3.49%, 5.22%, 4.31% and 5.67% respectively, and the viable cell rates were 91.34%, 87.87%, 87.78%, and 77.74% respectively. This indicated that the apoptosis injury of A549 cells induced by LPS was elevated at dose dependent, whereas the viable cell rate was decreased at dose dependent.Compared with control group, PFC and LPS+PFC groups, the early apoptosis rate and the late and necrosis rate in LPS groups were significantly increased at 6 and 12h, the viable cell rates were markedly reduced. And the injury degree at 12h was more severe than that at 6h group. There was no statistical difference of apoptosis and viable cell rate between the control and PFC groups at both 6 and 12h and PFC groups each other. Compared with control and PFC group, the early apoptosis rate of coculture group at 12h was obviously increased and the viable rate was decreased.②results of TEM: There were no apoptotic ultrastructure signs were observed under TEM in both cotrol and PFC groups. In LPS group, heterochromatin condensation, especially a typical horseshoe shape of chromatin condensation, nuclear membrane unclear, etc., which representing the signs of early and intermediate stage of apoptosis were observed under TEM. And there was no obviously apoptotic ultrastructure signs were observed in the coculture group.2.2 results of the expression of TLR-4 and caspase-3 mRNA detected by real time PCR and the protein level of the two detected by western blotting.①results of real time PCR: After stimulated by LPS, the expression of TLR-4 and caspase-3 mRNA was markedly increased, the former increased at 2, 6,12h, and the latter increased at 6,12h. There was no effect of PFC alone on the expression of the two genes. Whereas the expression of the two genes in the coculture group was significantly decreased.②results of western blotting: After stimulated by LPS, TLR-4 protein was markedly elevated at 0.5, 2, 6,12h, caspase-3 precursor was significantly degraded and activated caspase-3 (17 kDa and 11 kDa subunits) was synchronicly increased at 0.5, 2, 6 and 12h. There was no effect of PFC alone on the two proteins. In the coculture group, however, the proteins of TLR-4 and activated caspase-3 were markedly decreased.2.3 Results of the effects of PFC on the inflammatory responses of A549 cells induced by LPS2.3.1 results of cytokines tested: The proinflammatory mediators were significantly increased in LPS groups. ICAM-1, TNF-αand IL-8 were increased at 2, 6,12h; and the secrete peaks of ICAM-1, TNF-αwere all at 2h, and IL-8 was at 12h; There was no effect of PFC alone on the above mediators. In the coculture groups, however, all the above mediators were markedly decreased.2.3.2 results of the expression of TLR-4 mRNA and TLR-4 protein: read the result of 2.2 please.2.3.3 results of activeties of NF-κB①detected by western boltting: IκB-αwas significantly degraded at 0.5,2, 6 and 12h. Being as the in-synchronism response, NF-κB P65 was released and translocated into the nucleus. But there was statistical significance only at 2 and 6h. There was no effect of PFC alone on the activeties of NF-κB. But in coculture groups, the activeties of NF-κB were obviously decreased, indicated by the degradation of IκB-αmarkedly decreased and the reduction of NF-κB P65 in the nucleus.(2) results of the immunocytochemical double staining of NF-κB: IκB-αwas observed in cytoplasm and there was nearly no P65 protein in nuclus in control and PFC groups. In LPS group, the heavy nuclear staining of P65 was observed at 6h. In coculture group, double coloration cells , IκB-αpositive staining cells and P65 cells were all observed. It should be pint out that staining result of NF-kB in A549 cells was not as good as that in PMVECs, and we failed to succeed at staining A549 cells at 0.5 and 2h. The result was maybe caused by some reasons we still unable to definite.Conclusions1. LPS can induce apoptosis injury in both PMVECs and ECs in vitro, and the injury degree is at dose- and time-dependant. The expression of TLR-4 and caspase-3 mRNA and protein level were markedly increased after stimlated by LPS. As TLR-4 is the initiator of the LPS/ TLR-4 signal pathway, and caspase-3 is known as the apotosis executioner, which suggests that LPS induces apoptosis in PMVECs and AECs maybe via mediating the pathway of LPS/ TLR-4→caspases activation in proper order→ apoptosis.2. PFC alone does not induce apoptosis injury in both PMVECs and AECs in vitro and has no obviously effects on the expression of TLR-4 and caspase-3 mRNA and the protein level of the both two signal molecules. In the coculture groups, however, the apoptosis rates of the both cell populations were significantly decreased, the viable cells were markedly increased. Meanwhile, the up-regulation of the expression of TLR-4 and caspase-3 mRNA and the protein level induced by LPS were significantly reduced. This means that PFC is able to protect PMVECs and AECs from LPS-induced apoptosis injury via blocking the initiation of LPS signal pathway .3. LPS activates both PMVECs and A549 cells in vitro, make the two cell populations release proinflammatory mediators. Being as the priming signal molecule, the expression of TLR-4 mRNA and protein was markedly increased after stimlated by LPS. Meanwhile, the activities of NF-κB, the key downstream effector molecule of the signal pathway were increased rapidly stimulated by LPS as well. This suggests that LPS induces inflammory responses in PMVECs and AECs via the signal transduction pathway of LPS/ TLR-4→NF-κB activation→proinflammatory mediators production.4. PFC alone does not induce the release of cytokines in PMVECs and AECs in vitro and has no effects on the expression of TLR-4 gene and protein. Meanwhile PFC alone does not affect the activities of NF-κB as well. When the two population cells were incubated with LPS and PFC, however, the release of proinflammatory cytokines was obviously reduced. And the expression of TLR-4 gene and protein as well as the activities of NF-κB were significantly down-regulated. Which suggests that PFC is able to prevent PMVECs and AECs from LPS-induced inflammatory responses via blocking the initiation of LPS signal pathway. |
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