| Bacterial sepsis and its more severe complications are clinically commonand potentially lethal diseases. The common denominator for all of thesecomplications is injury or dysfunction of pulmonary microvascular endothelialcells (PMVECs). PMVECs are not only the primary targets of injury, but alsothe active and effective inflammatory cells, which play an important role inthe development of diseases. So, it is crucial in the study of pulmonarycirculation diseases to further study the mechanisms of PMVEC injury.Infection, especially gram negative sepsis, is a major cause of pulmonarydiseases such as ALI/ARDS. Bacterial lipopolysaccharide (LPS), a kind ofglycoprotein on the outer membrane of gram negative bacilli, can cause astrong inflammatory response in the body, damage the PMVECs directly orindirectly, and play a key role in the pathological process of ALI/ARDS.Complex signaling pathways are involved in the injury of PMVECs inducedby inflammation. Studies suggested that RhoA/ROCK pathway was involvedin the occurrence and development of ALI/ARDS, pulmonary arterialhypertension (PAH) and lung cancer. RhoA/ROCK signaling pathway caninduce the rearrangement of the actin cytoskeleton, regulate the genetranscription and cell cycle progression, and act as the signal converter ormolecular switch in the cell signal transduction. However, further studies arerequired for full understanding of the mechanisms of RhoA/ROCK pathway inthe damage of rat PMVECs induced by LPS, as well as the cross-talk ofRhoA/ROCK pathway with other signaling pathways. So, in the present study,LPS were used to induce the model of PMVEC damage and to investigate thechanges of RhoA/ROCK pathway in the process. Moreover,immunocytochemistry, flow cytometry, confocal laser and molecular cell biology technology were also used to investigate the influence of ROCKinhibitor fasudil on LPS-induced PMVECs damage, and to further discuss themechanisms of downstream signal transduction.Part1RhoA/ROCK pathway is involved in LPS-induced damage of ratPMVECsObjective: To study the changes of RhoA/ROCK pathway inLPS-induced damage of rat PMVECs and the mechanism of action.Methods: The tissue-explant technique was used to culture the primaryrat PMVECs. The identification of rat PMVECs was carried out byimmunocytochemical staining of factor Ⅷrelated antigen and transmissionelectron microscope. The viability of PMVECs was determined by MTT andlactate dehydrogenase (LDH) method. The mRNA expression of RhoA andROCK1was detected by RT-PCR. The phosphorylation of MYPT1wasanalyzed by Western blot.Results:1The culture and identification of rat PMVECsWe successfully cultured rat PMVECs by tissue-explant technique withsome modification. After removal of explants, the PMVECs were cultured for3~5days and displayed the shape of paving stone at this moment. The resultsof immunocytochemical staining of factor Ⅷ related antigenshowed thatabout95%of the cells were positive. The results of electron microscopeshowed that the plasma membrane protrusions, also namely microvilli, couldbe observed in most cells, W-P bodies were also observed in the cytoplasm. Inaddition, a large number of plasma membrane vesicles (also called pinocytoticvesicles) were found in the cytoplasm of cells. All the above structures wereconsistent with the typical endothelial cell ultrastructure. Considering that thetissue explants were obtained from the lung edge, it could be confirmed thatthe cultured cells were indeed PMVECs.2Effect of LPS on the viability of rat PMVECs and the intervention effect offasudilMTT results suggested that the OD value in control group was0.95± 0.12,0.01,0.1and1μg/ml of LPS had no obvious effect on the viability ofPMVECs, while the OD value in LPS10and100μg/ml groups decreased to0.75±0.19(P <0.05) and0.50±0.12(P <0.01) respectively. LPS10μg/mlcould cause the injury of PMVECs, but sufficient cells were still existed forsubsequent test. So, LPS (10μg/ml) was used to induce the damage ofPMVECs. Compared with the OD value (0.93±0.15) in LPS10μg/ml (0h)group, the OD value did not change obviously at6h and12h after LPStreatment, but decreased significantly at24h (0.73±0.19), and stillmaintained at the low level at48h and72h. Fasudil (25,50μM) improved theLPS-induced injury of PMVECs in different degree (compared with the LPSgroup，P <0.05).3Effect of LPS on the activity of LDH and the intervention effect of fasudilThe activity of LDH in control group was127.6±17.3U/L, which wasincreased to155.1±23.4U/L (P <0.05),162.3±34.3U/L (P <0.05),189.1±25.1U/L (P <0.01) and194.7±22.7U/L (P <0.01) respectively after thecells had been treated with LPS0.1,1,10and100μg/ml for24h. The LDHrelease began to increase at6h after the cells had been treated with LPS10μg/ml and the activity of LDH increased to139.7±19.2U/L, which wassignificantly higher (P <0.05) than that (114.1±21.1U/L) before LPStreatment. With the time prolongation, the activity of LDH increasedcontinuously, suggesting that PMVECs were damaged increasingly. Fasudil(10,25and50μM) pretreatment significantly decreased the LDH activity inthe supernatant (P <0.05or P <0.01), which suggested that fasudil inhibitedthe release of LDH, reduced the injury of PMVECs induced by LPS.4The changes of mRNA expression of RhoA and ROCK1in LPS-inducedinjury of rat PMVECsCompared with the control group (LPS0min), the mRNA expression ofRhoA began to increase at15min after LPS (10μg/ml) treatment (P <0.05),and reached the highest level at60min. While the mRNA expression ofROCK1began to increase after LPS (10μg/ml) treatment for5min (P <0.05),and appeared two peaks at15min and60min respectively (P <0.01). With the prolongation of time, the RhoA and ROCK1mRNA expression slightlydecreased, but still maintained at a higher level than that in the control group(LPS0min) at240min after LPS treatment, suggesting that RhoA/ROCKsignal pathway might be involved in the injury of PMVECs induced by LPS.5Effect of LPS on the phosphorylation of MYPT1and the intervention effectof fasudilActivated ROCK can further phosphorylate the downstream substrateMYPT1. Therefore, the phosphorylation level of MYPT1reflects the degreeof activation of ROCK. Western blot analysis showed that the phosphorylationof MYPT1began to increase after LPS (10μg/ml) treatment for5min (P <0.05), and reached the highest level at15min (P <0.01). With theprolongation of time, the expression of p-MYPT1slightly decreased, but stillhigher than that of the control group (LPS0min) at120min after LPStreatment (P <0.01). Fasudil (10,25and50μM) pretreatment significantlydecreased the expression of p-MYPT1induced by LPS treatment for15min(P <0.05or P <0.01).Conclusion: The primary rat PMVECs were successfully cultured bytissue-explant technique in the present experiment. LPS markedly induced thedamage of rat PMVECs, and RhoA/ROCK signaling pathway was involved inthe process.Part2Protective effect of fasudil against LPS-induced apoptosis andcytoskeleton rearrangement of rat PMVECs and the underlyingmechanismsObjective: To study the effects of ROCK inhibitor fasudil onLPS-induced apoptosis and cytoskeleton rearrangement and the underlyingmechanisms.Methods: The apoptosis of PMVECs was evaluated by AnnexinV/PI andHoechst33258fluorescent staining assay. The change of cytoskeleton wasdetected by F-actin staining. The phosphorylation of ERK1/2, JNK1/2/3, p38and the expression of apoptosis related proteins Bax and Bcl-2were detectedby Western blot. Results:1Effect of fasudil on LPS-induced apoptosis of rat PMVECs1.1Effect of different duration of LPS exposure on apoptosis of rat PMVECsThe results of AnnexinV-FITC staining showed that compared with thecontrol group (LPS0h，the early apoptosis rate was3.1±0.7%and lateapoptosis rate was1.0±0.3%),10μg/ml LPS treatment for6h did not affectthe apoptosis of PMVECs. While the early apoptosis rate of PMVECsincreased to15.1±3.8%(P <0.01),24.3±3.6%(P <0.01),34.8±4.5%(P<0.01) and43.2±8.2%(P <0.01), and the late apoptosis rate increased to2.1±0.9%(P <0.05),9.6±2.1%(P <0.01),12.2±4.7%(P <0.01),29.1±7.4%(P <0.01) respectively at12,24,48and72h after LPS treatment,which suggested that LPS obviously induced the apoptosis of rat PMVECs.1.2Fasudil decreased LPS-induced apoptosis of PMVECs significantlyCompared with the control group (the early apoptosis rate and lateapoptosis rate were2.1±0.7%and3.0±0.7%respectively), the earlyapoptosis rate increased to37.8±8.1%(P <0.01) and the late apoptosis rateincreased to10.7±2.6%(P <0.01) after LPS treatment for24h, which weredecreased in different degree by fasudil pretreatment. The early apoptosis ratedecreased to27.7±4.6%(P <0.05)，21.8±5.9%(P <0.01) and18.6±3.9%(P<0.01), and the late apoptosis rate decreased to7.5±1.3%(P <0.05)，6.4±1.3%(P <0.05) and5.3±1.4%(P <0.01) respectively after10,25and50μM of fasudil pretreatment, suggesting that fasudil has some interventioneffect on LPS-induced apoptosis injury of rat PMVECs.1.3Fasudil improved LPS-induced apoptotic morphological changes of ratPMVECsFrom the results of Hoechst33258staining we observed that the nucleusof PMVECs in control group was round or oval with neat edge withhomogeneous and low-intensity blue fluorescence. No obvious apoptotic cellswere found in control group. After treatment with LPS for24h, the nucleus ofPMVECs was condensed and hyperchromatic with high-brightnessfluorescence. Part of nuclear chromatin condensed highly and marginalized, and the number of PMVECs with crescent-shaped nuclei increased, whichsuggested that LPS induced apoptosis of rat PMVECs significantly. Fasudil(10,25and50μM) pretreatment significantly weakened the apoptoticmorphological changes of PMVECs induced by LPS, further illustrating thatfasudil can prevent the apoptosis of rat PMVECs induced by LPS.1.4Effect of fasudil on the expression of Bax and Bcl-2proteinsCompared with the control group, treatment of PMVECs with10μg/mlLPS increased the protein expression of Bax (P <0.01) and decreased theprotein expression of Bcl-2(P <0.01). Fasudil (10,25and50μM)pretreatment significantly reversed the imbalance of protein expression of Baxand Bcl-2caused by LPS, with the decreased Bax expression and increasedBcl-2expression (P <0.05or P <0.01), which indicated that fasudil mightinhibit LPS-induced apoptosis of rat PMVECs by regulating the expression ofanti-apoptotic and pro-apoptotic proteins.2Effect of fasudil on the distribution of skeleton protein F-actin in ratPMVECsThere are a few of actin filaments in the control group, which mainlydistributed in the periphery of cells. After treatment with LPS, the cytoplasmicF-actin fibers increased significantly, and most of them arranged along thelongitudinal of PMVECs. The F-actin fibers around the cells graduallydisappeared, and intensive fasciculate stress fibers appeared in the cytoplasm.With the prolongation of time, the arrangement of F-actin became more andmore disorderly and dispersive, and the normal connections between cellswere almost lost. Meanwhile, the formation of stress fiber and themorphological changes of cytoskeleton induced by LPS were inhibited by10,25and50μM of fasudil pretreatment in different degree, and fasudil alsoreduced the fluorescence intensity of F-actin.3Effect of fasudil on the activation of MAPKs in LPS-treated rat PMVECsLPS (10μg/ml) caused obvious phosphorylation of ERK1/2at15minafter treatment and with a peak arrived at60min (P <0.01). Whereas,pretreatment with10,25and50μM of fasudil did not inhibit the ERK1/2 phosphorylation, suggesting that ERK1/2was not as the downstream signal ofRhoA/ROCK in LPS-induced apoptotic injury. In addition, thephosphorylation of JNK1/2/3and p38began to increase after LPS treatmentfor5min and with the highest level both arrived at30min, which was allinhibited by fasudil (10,25and50μM) pretreatment (P <0.05or P <0.01),suggesting that JNK1/2/3and p38might act as the downstream signals ofRhoA/ROCK in LPS-induced apoptotic injury.4The activation of JNK and p38MAPKs participated in the apoptotic injuryinduced by LPS in rat PMVECsIn order to further clarify that JNK and p38were involved inLPS-induced apoptosis of rat PMVECs, we observed the effects of SB203580(p38inhibitor) and SP600125(JNK inhibitor) on apoptosis and the expressionof apoptosis-related proteins induced by LPS. SB203580(10μM) andSP600125(20μM) pretreatment significantly reduced the phosphorylation ofp38and JNK MAPKs respectively, suggesting that SB203580and SP600125can inhibite the activation of p38and JNK MAPKs respectively. MeanwhileSB203580and SP600125pretreatment significantly inhibited the apoptosisinduced by LPS in rat PMVECs, and the early apoptosis rate and lateapoptosis rate were all decreased (P <0.05or P <0.01). SB203580andSP600125pretreatment also reversed the imbalance of the expression of Baxand Bcl-2proteins induced by LPS, with a decreased expression of Bax and anincreased expression of Bcl-2(P <0.01), further suggesting that activation ofJNK and p38participated in the LPS-induced apoptosis of rat PMVECs.Conclusion: Activation of JNK and p38MAPKs, the downstreamsignaling molecules of RhoA/ROCK signaling pathway, played an importantrole in LPS-induced apoptosis of rat PMVECs. Fasudil, a potent and selectiveinhibitor of ROCK, exerted an anti-apoptotic effect and inhibited thecytoskeletal rearrangement in rat PMVECs, which were mediated by theinhibition of RhoA/ROCK and its downstream JNK and p38MAPKs. Part3Effects of fasudil on the expression of inflammatory factorsinduced by LPS in rat PMVECs and the underlying mechanismsObjective: To study the effects of fasudil on the expression ofinflammatory factors induced by LPS in rat PMVECs and the antioxidativemechanisms.Methods: The mRNA expression of IL-6, TNF-αand MCP-1wasevaluated by RT-PCR; The content of IL-6and MCP-1in supernatant wasdetermined by ELISA assay kit; ROS was measured by confocal microscopywith DCFH-DA staining. The activity of SOD and GSH-PX and content ofMDA were measured using kits supplied by Nanjing JianchengBiotechnological Company. Western blot analysis was used to determine theprotein expression of NF-κB p65in the cytoplasm and nucleus.Results:1Effects of fasudil on the mRNA expression of IL-6, TNF-αand MCP-1induced by LPS in rat PMVECsCompared with the control group (LPS0h), the mRNA expression ofIL-6and MCP-1began to increase after LPS treatment for3h (P <0.01), andmaintained at a higher level until the end of the experiment. The highest levelof mRNA expression of MCP-1arrived at6h, and that of IL-6arrived at12h,which were both reduced by10,25and50μM of fasudil pretreatment (P <0.05or P <0.01). LPS exerted no obvious effect on the expression of TNF-α.2Effect of fasudil on the secretion of IL-6and MCP-1induced by LPS in ratPMVECsCompared with the control group (LPS0h, the concentration of IL-6andMCP-1was475±76pg/ml and1.082±0.389μg/ml respectively), thesecretion of IL-6began to increase after LPS treatment for3h (P <0.05), andmaintained at a high level until48h (P <0.01), with the highest level arrivedat12h (1098±81pg/ml, P <0.01). The secretion of MCP-1also increased to1.988±0.017μg/ml (P <0.05),2.026±0.066μg/ml (P <0.05) and2.038±0.072μg/ml (P <0.05) at3,6and12h respectively. Meanwhile, the contentof both IL-6and MCP-1increased at different time points after LPS treatmentcompared with that in the corresponding control group (P <0.05or P <0.01).Fasudil (10,25and50μM) pretreatment decreased the secretion of IL-6and MCP-1induced by LPS at different time points (P <0.05or P <0.01).3Effect of LPS on the production of ROS in rat PMVECs and the interventioneffect of fasudilCompared with the control group (LPS0h), the fluorescence intensity ofROS was significantly increased from3h to48h after LPS treatment, andwith the highest level arrived at6h. After that, the fluorescence intensity hadsome decrease at24h and48h after LPS treatment, but still stronger than thatin control group (P <0.01), suggesting that the production of ROS increasedobviously after the cells were treated with LPS. Compared with the LPS (6h)group, pretreatment with fasudil (10,25and50μM) obviously reduced thefluorescence intensity of ROS induced by LPS treatment for6h (P <0.01),which suggested that fasudil significantly inhibited the production of ROSinduced by LPS in rat PMVECs.4Effect of fasudil on the activity of SOD and GSH-PX and the content ofMDACompared with the control group (LPS0h), the activity of SODdecreased significantly from3h to48h after LPS treatment (P <0.05or P <0.01), and the lowest level arrived at12h. The activity of GSH-PX began todecrease at6h (P <0.05), and maintained the low level at12,24and48hafter LPS treatment (P <0.01). On the contrary, the content of MDA began toincrease after LPS treatment for6h (P <0.01), and reached the highest levelat12h (P <0.01), which maintained until48h after LPS treatment. Whilefasudil (10,25and50μM) pretreatment increased the activity of SOD andGSH-PX reduced by LPS treatment for12h (P <0.05or P <0.01), anddecreased the content of MDA in different degree (P <0.05or P <0.01).5Effect of fasudil on the nuclear translocation of NF-κB p65induced by LPSCompared with the control group (LPS0h), the expression of NF-κB p65in the whole cell extracts of rat PMVECs showed no obvious changes afterLPS treatment for different time, while that in the nuclear proteins wasobviously increased after LPS treatment for3h (P <0.01). The expression ofNF-κB p65in the nuclear proteins arrived the highest level at12h, and maintained at a high level until48h after LPS treatment (P <0.01).Meanwhile, fasudil (10,25and50μM) pretreatment significantly decreasedthe expression of NF-κB p65in the nuclear proteins of rat PMVECs, whichsuggested it might be through inhibiting the nuclear translocation of NF-κBp65that fasudil reduced the secretion of inflammatory factors.6Effects of N-acetylcysteine on the mRNA expression of IL-6and MCP-1and the nuclear translocation of NF-κB p65induced by LPSN-acetylcysteine (5,10mM) pretreatment significantly decreased theexpression of NF-κB p65in the nuclear proteins of rat PMVECs induced byLPS treatment for12h (P <0.05or P <0.01). Meanwhile, the mRNAexpression of IL-6and MCP-1induced by LPS was also reduced byN-acetylcysteine (5,10mM) pretreatment (P <0.05or P <0.01).Conclusion:(1) N-acetylcysteine, the inhibitor of ROS, obviouslyinhibited the production of ROS and the expression of IL-6and MCP-1induced by LPS in rat PMVECs, which suggested that ROS was involved inLPS-induced inflammatory injury of rat PMVECs.(2) Fasudil inhibited theproduction of ROS. The increased level of MDA and the decreased activity ofSOD and GSH-PX induced by LPS were reversed by fasudil. Meanwhile,fasudil decreased the expression of IL-6and MCP-1by inhibiting the nucleartranslocation of NF-κB p65, suggesting that fasudil ameliorated theinflammatory injury induced by LPS through its antioxidant effect. |
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