Effects Of Endotoxin, Levofloxacin And Tea Polyphenol On The Lung Of Rats With Hepatopulmonary Syndrome

Part I The effects of endotoxin on hepatopulmonary syndrome in the rats IntroductionHepatopulmonary syndrome(HPS)is characterized by gas exchange abnormalities,intrapulmonary vascular dilatations and an increased alveoloarterial oxygen difference(AaPO₂).Microvascular dilatation within the pulmonary arterial circulation may result from decreased pre-capillary arteriolar tone alone or additional mechanisms such as angiogenesis,remodelling,and vasculogenesis,which have been recently suggested.In HPS,the vasodilatation is assumed to result from excessive vascular production of vasodilators,particularly nitric oxide(NO)and so on.These are increased in cirrhotic patients HPS and normalized after OLT.But microvascular dilatation within the pulmonary arterial circulation is not recovered.So maybe other mechanisms involved in this syndrome.In cirrhosis,intestinal bacterial overgrowth,impaired host defenses,and disruption of the gut mucosal barrier may promote bacterial translocation so there maybe more endotoxin or bacteria in serum.Normally,the lung vascular bed is not exposed to large amounts of bacterial product.We hypothesize that endotoxin and bacteria can injure the lung in HPS rats.So in this study we explored how endoxin affect the lung of HPS rats.ObjectiveTo establish the model of HPS in the rats and study the effects of endotoxin on the lung in this model.Materials and MethodsRats model of HPS were induced by ligating the bile duct to result in the biliary cirrhosis.Twenty male SD rats were randomly divided into two groups with 10 animals in each one:sham operation group rats were isolated the bile duct;HPS model group rats were isolated the bile duct and ligated.After 6 weeks the portal pressure,ratios of dry weight to wet weight of lung(D/W),weight of spleen,endotoxin, contents of plasma,MPO active and MDA contents in the lung,plasma and lavage fluid of lung were examined.In addition,the results of the blood-gas analysis,the bacterial culture of blood,bile and ascites and the evaluation of pathologic change of lung also were investigated.ResultsLarge quantities macrophages in the lung of HPS rats were found by Hematoxylin-Eosin staining. Compared with sham operation group,portal pressure,D/W,endotoxin contents of plasma,MPO active and MDA contents in the lung,BLAF and serum were significantly increased In HPS group.And the inflammation of lung in HPS group was more severe than that in sham operation group.DiscussionThe results of this study showed that the inflammation rates of HPS rats was higher than that in sham group rats in that the plasma endotoxin level in HPS rats was ten folds higher than that in the latter group. Pathological features,portal hypertension,splenomaglay,disturbance of acid-base balance and the D/W of the lung showed the model was successfully established.Intestinal endotoxemia occurred because intestinal bacterial overgrowth,impaired host defenses in cirrhosis rats more bacteria translocated into blood.More macrophages,which should not be existed in non-HPS effected lung,were found in the lung of HPS rats.The bacteria produced endotoxin and initiated inflammation.More macrophages accumulated in pulmonary could release inflammatory factors and oxygen radicals that injured the lung.We found endotoxin in the plasma of HPS rats was ten times higher than sham operation rats.More content of MPO and MDA released by macrophages,the severity inflammatory and fibrosis in HPS rats lung and the higher ratio of wet/dry of lung maybe be caused by endoxin and translocation of bacteria.Conclusion:Translocation of bacteria and endotoxin play important roles in HPS.Partâ…¡Effects of levofloxacin on the lung of rats with hepatopulmonary syndrome and its mechanism study IntroductionIn Partâ… of this study we found that bacteria tanslocation and endotoxin involved the pathogenesis of HPS.In HPS,the development of portosystemic shunts and the dramatic decrease in the phagocytic capacity of the liver allow circulating bacteria or bacterial endotoxins to enter the pulmonary circulation.In this situation,the lungs clear the blood of gut bacteria and endotoxins thereby compensating for the decrease in liver phagocytic function.The increased phagocytic activity in pulmonary is ascribable to extensive accumulation of pulmonary intravascular macrophages that adhere to the pulmonary endothelium.Pathogens that are translocated from the gut to the pulmonary circulation probably cause macrophage sequestration by inducing coordinated expression of macrophage and endothelium adhesion molecules,as well as local release of inflammatory factors,such as TNF-a,PDGF-BB,ET-1,TGF-β,ICAM-1 and so on.Furthermore,those maybe injure the lung and lead to fibrosis.Translocation of gut bacteria in HPS rats and endotoxin may be an important step in the pathogenesis of HPS.Norfloxacin, predominantly active against Gram-negative bacteria,has been shown to prevent bacterial translocation in rats.The aim of the present study was to determine whether giving prophylactic levofloxacin treatment to rats with HPS would decrease pulmonary pathological changes and how it did.ObjectiveEffects of levofloxacin on the lung of rats with hepatopulmonary syndrome and its mechanism study.Materials and MethodsRat model of HPS was induced by ligating the bile duct to result in the biliary cirrhosis.Thirty male SD rats were randomly divided into three groups with 10 animals in each one:sham operation group (rats were isolated the bile duct and intraperitoneally injected 1.2ml NS),HPS model group(rats were ligated the bile duct and intraperitoneally injected 1.2ml NS)and levofloxacin group(rats were intraperitoneally injected levofioxacin 20mg/kg).After 6 weeks the portal pressure,ratios of dry weight to wet weight of lung(D/W),weight of spleen,endotoxin,contents of plasma,MPO active,the content of MDA,TNF-a,TGF-β,IL-1β,PDGF-BB,NO,and ET-1 in the lung homogenates,plasma and lavage fluid of lung were examined.The mRNA levels of ICAM-1,TGF-βand IL-1βwere detected by ISH. ED-1,PDGF-BB and TGF-βwere immunohistochemically located.TNF-a,PDGF-BB,ET-1 and ICAM-1mRNA were semi-quantified using RT-PCR.The expression of ET-1,PDGF-BB and TGF-βwere detected by Western blot.In addition,the results of the blood-gas analysis,the bacterial culture of blood,bile and ascites and the evaluation of pathologic change of lung also were investigated.Effect of endotoxin on mortality was tested by intravenously injected of LPS(0.01,0.1,1 and 10 mg/kg in BDL and levofloxacin rats;0.01,0.1,1,10,and 40 mg/kg in sham operation rats). ResultsIn levofloxacin group,portal pressure,lung Wet-to-Dry Weight Ratio,endotoxin contents of plasma were significantly decreased compared with those in HPS group.MPO activity,the contents of MDA, TNF-a,TGF-β,IL-1β,PDGF-BB,NO,and ET-1 were also reduced significantly in the serum,BALF and lung homogenate.In comparison with HPS group,the mRNA level of PDGF-BB,NO,TNF-a,ET-1 and ICAM-1 in the lung was down-regulated in levofloxacin group.The protein level of TGF-β,PDGF -BB,ET-1,ED-1 were also lower in levofloxacin group than that of HPS group.But all parameters mentioned of above were higher in HPS rats than that in sham operation rats.Particularly,the plasma endotoxin level in HPS rats was ten folds higher than that in the latter group.For the endotoxin sensitivity,as expected,sham operation rats were highly resistant to endotoxin with 40%mortality at 10 mg/kg of LPS,i.v.,and no death at 1 mg/kg or lower doses of LPS.In contrast,LPS doses as low as 10 mg/kg caused significant mortality in BDL rats.Furthermore,the mortality in the levofloxacin group rats was lower than HPS rats.Masson staining showed that the collagen was less in levofloxacin group than that in HPS.TEM suggested that the lamellar bodies were more destroyed and vacuolizationed in the HPS group in comparison with levofloxacin group.DiscussionBacterial infection,especially with intestinal-type bacterial fora,is a common complication in HPS. Bacterial translocation,that is,extra-intestinal dissemination of gut bacteria,occurs in most rats with HPS could lead to endoxemia.In this study we found that the plasma endotoxin level in HPS rats was ten folds higher than that in the sham operation group.For the endotoxin sensitivity,sham operation rats were highly resistant to endotoxin with 40%mortality at 10 mg/kg of LPS,i.v.,and no death at 1 mg/kg or lower doses of LPS.In contrast,LPS doses as low as 10 mg/kg caused significant mortality in BDL rats. In other word,the resistant ability of HPS group to LPS was very low in that the death-causing LPS doses of HPS group was 1000 folds lower than that of sham operation group.Meanwhile,endoxemia could activate ED-1 positive macrophage which released oxygen free radical and many more inflammatory factors in the lung.By those ways the lipid,protein and nuclear acid would be destroyed. However,the rats improve the ability of resistance endotoxin and ameliorate the blood gas analysis by the way of administration levofloxacin to reduce endotoxin.All above proved endoxin plays a significant role in HPS and reduction endotoxin could palliate the injury of lung. During phagocytosis,activated macrophages release numerous secretory products into the extracellular environment,including cytokines and nitric oxide(NO),inducible NO-synthase(iNOs)expressed in the pulmonary intravascular macrophages of cirrhotic rats.Activated inducible(iNOs)was expressed in the pulmonary intravascular macrophages of cirrhotic rats may explain the increase in lung production of NO. Large quantity of activated iNOS and NO could lead to hyoxemia,hyperventilation and scaled-up P[(A-a)O₂]. It can released oxygen radical which damage the lung and aggravated inflammation by the reaction NO with O₂.ET-1 must be producted more in the local lung with NO increasing,which could contract the blood vessel and promoted fiber generation,likewise augmented PDGF-BB,TGF-β,TNF-a,IL-1β,NO could progressed the lung fibrosis.All of those were significantly decreased in levofloxacin group than HPS group.As expected,we found the fiber in the levofloxacin group lung was lessened,the inflammation palliated,pathological changes of lamellar bodies softened.Conclusion1 levofloxacin could reduce plasma endotoxin2 levofloxacin can attenuate the injury of lung in HPS rats.Partâ…¢Effects of tea polyphenol on the lung of rats with hepatopulmonary syndrome IntroductionSepsis is a common complication of HPS with a high mortality.The increased risk of infection is secondary to impairment of several of the host defense mechanisms including impaired neutrophil function.Furthermore,low grade endotoxemia may occur because of impaired Kupffer cell removal of gut-derived endotoxin,which in cirrhosis may be released into the peripheral circulation because of portal systemic shunting activated polymorphonuclear neutrophils into the liver.We have showed that reduction endotoxin can protect the HPS rat's lung by administered levofloxacin.However,the antibiotic could not be used for a long time in fact because it would bring up severe complications,such as dual infection which could lead rats to death.We had to find another way to cure HPS.Tea polyphenol(TP)act as an antioxidant,scavenging reactive and immunoregulation.In addition,it has been found to modulate cytokine expression related to inflammation.For example,TP has been shown to decrease the expression of tumor necrosis factor-a and to inhibit tyrosine phosphorylation of PDGF receptor.In another side,TP could restrain the bacteria such as staphylococci,colibacillary and so on.Therefore,we can investigate the effect of TP on HPS.ObjectiveEffects of tea polyphenol on the lung of rats with hepatopulmonary syndrome Materials and MethodsRats model of HPS were induced by ligating the bile duct to result in the biliary cirrhosis.Thirty male SD rats were randomly divided into three groups with 10 animals in each one:sham operation group(rats were isolated the bile duct and intragastrically administrated 1ml NS),HPS model group(rats were ligated the bile duct and administrated as the way in the former group)and TP group (rats were ligated the bile duct and intragastrically administrated 250mg/kg TP).After 6 weeks the samples were taken to detect parameters as the way as in partâ…¡.ResultsIn TP group rats,portal pressure,lung Wet-to-Dry Weight Ratio,endotoxin contents of plasma were significantly decreased in compared with those in HPS group.So did the MPO active,content of MDA,TNF-a,TGF-β,1L-1β,PDGF-BB,NO,and ET-1 in the serum,BALF and lung homogenate. The mRNA levels of PDGF-BB,TNF-a,ET-1 and ICAM-1 in the lung were impaired in TP group in comparison with HPS group.The protein expression of TGF-β,PDGF-BB,ET-1,ED-1 were also lower in TP group than that of HPS group.But all parameters mentioned above were higher in HPS group than that in sham operation group.Particularly,the plasma endotoxin in HPS group was ten times higher than that in sham operation group.But the content of MPO,MDA,ET-1 did not show significant difference in the lung homogenate between the TP group and control group.There was more collagen in the lung of HPS group than TP group.For the endotoxin sensitivity,as expected,sham operation group were highly resistant to endotoxin with 40%mortality at 10 mg/kg of LPS,i.v.,and no death at 1 mg/ kg or lower doses of LPS.In contrast,LPS doses as low as 10 mg/kg caused significant mortality in BDL group.Furthermore,the mortality in the levofloxacin group rats was lower than HPS rats.The collagen was also less in levofloxacin group than that in HPS rats.The lamellar bodies were more destroyed and vacuolizationed in the HPS group in comparison with TP group.DisccussionIntestinal bacterial overgrowth and bacterial translocation would lead to endotoxemia and act ivated macrophage which would induce the production of cytokines involves activation of the re dox to release oxygen radicals.The action of LPS on cells to induce ubiquitous nuclear transcr iption factor nuclear factor KB(NF-KB)which activates the genes involved in the inflammator y and immune response in the lung.LPS also triggers the production of reactive oxygen and n itrogen species which may cause lipid peroxidation and disturb the integrity of cellular membra nes.TP maybe reduced the endotoxin by the way of rescontraction of bateria propagation.EGCG,which was the main composition of tea polyphenol,could suppress the activity of the receptor of PDGF,furthermore reduced the function of PDGF.In addition,it could inhibit t he mRNA expression of TGF-β,TNF-a,and IL-1β.By those ways,all mentioned cytokines,whi ch played an important role in lung fibrosis,were decreased both in serum and lung.So the P ulmonary injury was palliated.TP was sources of reactive oxygen species and its ability of antioxidation was many times a s Vitamin C and E.To clear the oxygen radical would reduced the cell injury and decreased t he inflammation factors.TP not only suppressed the activity of lipoxygenase,cytochrome P450 and so on,also boosted that of antiperoxidase such as glutathion peroxidase and NAPDH-redox enzyme.By these ways TP would removed the metabolic toxicity of NO and suppressed the a ctivity of iNOs.EGCG inhibits the phosphorylation process of tryrosine to reduce the signal tra nsduction pathway of NF-κB.EGCG suppresses receptor type protein tyrosine kinase(PTK)mu ch more than that of non-receptor type PTK(protein kinase C,protein kinase A and PP60v-src), such decreasing the activity of iNOs and further decreasing the concentration of NO.P44/p42 MAP activity was inhibited and the mRNA expression of COX-1/2 was suppressed by EGCG t o reduce the expression of ET-1 and ET-A receptor.Pulmonary injury can be reduced by the way of decreasing ET-1 in the lung.Conclusion1 TP inhibits bacteria overgrowth to decrease endotoxin in HPS rats. 2 TP removes the oxygen radicals to alleviate pulmonary injury in HPS rats.3 TP reduces the levels of TNF-a,IL-1β,TGF-β,PDGF,ET-1 and NO to protect the lung in HPS rats.