Clinical Study Of Qingxia Therapy On Intestinal Endotoxemia Of Cirrhotic Patients With Infection And Its Mechanism On Anti-hepatic Injury Induced By Lipopolysaccharide/D-galactosamine

Part I Clinical study of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with infection and the intervention of endotoxin release in the process ofantibiotic treatmentBackgroundCirrhotic patients with bacteria infection are mostly accompanied with intestinalendotoxemia which always aggravates liver injury and induces various complications.Therefore,there is important clinical significance in preventing and treating intestin-al endotoxemia in cirrhotic patients with bacteria infection.Anti-infective therapy isessential to the cirrhotic patients with bacteria infection,but the release of endotoxincaused by antibiotic treatment makes it more difficult in curing those patients.Thereare no specific ways in treating intestinal endotoxemia presently,especially in interve-ning the release of endotoxin in the process of antibiotic treatment.It is proved bymany researches that on the basis of western medicine,the curative effect could beimproved if combined with traditional Chinese medicine. And treatment based on syndrome differentiation is an ideal program because of the complicated pathogenesisof endotoxemia.We concluded the etiology and patheogenesis of intestional endotoxe-mia as internal heat toxin、stasis and toxin intertwine and yangming fu-viscera excess.So,we proposed QingXia therapy to treat the intestinal endotoxemia of cirrhotic pati-ents with bacteria infection as a new way and discuss its interventional effect inendotoxin release during antibiotic treatment.ObjectiveTo discuss the effect of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with bacteria infection and its interventional effect in endotoxin releaseduring antibiotic treatment.Methods90cases of cirrhotic patients with bacteria infection collected from October2010to January2013in the division of Traditional Chinese Medicine and infectiousdiseases of our hospital were randomly divided into three groups:study group I、studygroup II and control group.30cases in each group respectively.All patients werebeing treated by routine comprehensive therapy(stay in bed、anti-infection、protectliver and lower transaminase、normalize gallbladder to cure jaundice、diuresis、regulate intestinal flora、supplement the human blood albumin、supplement freshfrozen plasma,etc) for2weeks,the antibiotic for all cases was cefoperazone-/sulbactam(2g bid ivdrip).Cases in group I were administered QingXia decoction14days successively rom the first day besides routine comprehensive therapy, Cases instudy group II were administered QingXia decoction7days successively from theeighth day.None QingXia decoction was administered in control group,then observethe effect of QingXia therapy on patients’ clinical syndromes and signs、liver function、coagulation function、LPS、TNF-α、IL-6.Results1. Clinical syndromes and signs in patients of three groups were significantly improved after2weeks of treatment（P<0.05）, Between study group and controlgroup there were significant differences in reducing abdominal distension、stool stemnode and maintaining defecate unobstructed (P <0.05).2.Blood routin parameters、liver function and coagulation function in patients ofthree groups were significantly improved after2weeks of treatment（P<0.05）,butcompared with control group,study groups were better in reducing serum bilirubin（P<0.01）。3.After2weeks of treatment,the child-pugh classification was improved and theascitic fluid was reduced significantly in patiens of three groups（P<0.050.01）,butthere were no statistically differences among three groups.4. After2weeks of treatment, ascitic WBC and PMN in patients of three groupswere significantly reduced（P<0.05）, but there were no statistically differences amongthree groups.5.Serum LPS、TNF-α、IL-6were significantly increased in study group II andcontrol group after one week of treatmen（tP<0.05）,then they all significantly reducedat the end of treatment （P<0.05）,but compared with control group,study groupswere better in reducing serum LPS （P<0.01）。6.Child-pugh classification in cirrhotic patients with bacteria infection ispositively correlated with serum LPS、TNF-α、IL-6(P<0.01)。Conclusions1.QingXia therapy can significantly improve the clinical syndromes and signs、liver function and coagulation function of cirrhotic patients with bacteria infection onthe basis of routine comprehensive treatment of western medicine.2.QingXia therapy can intervene the release of endotoxin and inflammatorymediators in the process antibiotic treatment which improve the curative effect.3.The intervention of the release of endotoxin by QingXia therapy may related tothe inhibition of inflammatory mediators release.4. Child-pugh classification in cirrhotic patients with bacteria infection is positively correlated with serum LPS、TNF-α、IL-6.Part II Mechanism of QingXia therapy on hepatocyte apoptosis inrats with hepatic injury induced by lipopolysaccharide/D-galactosamineBackgroundEndotoxin can induce hepatic injury through cell apoptosis,so it is important toprevent hepatocyte apoptosis that induced by LPS.It was proved by some researchesthat traditional Chinese medicine can inhibit hepatocyte apoptosis that induced byLPS,In part I of this paper,we found that QingXia therapy can significantly improvethe clinical syndromes and signs、liver function、coagulation function and decreaseserum LPS、TNF-α、IL-6of cirrhotic patients with bacteria infection,and reduce LPSrelease during antibiotic treatment,so we regard that QingXia therapy could alleviatethe endotoxic hepatic injury.But the exact mechamism remains unclear,therefore,inthis section,we use apoptosis as a breakthrough point to research the mechanism ofQingXia therapy in rats with hepatic injury induced by lipopolysaccharide/D-galactosamine.and presently we see no report about the the anti-apoptosismechanism of QingXia therapy.ObjectiveTo study the anti-apoptotic mechanism of QingXia therapy on hepatocyte apoptosisinduced by lipopolysaccharide/D-galactosamineMethods40SD rats were randomly divided into three groups:control group(10), QingXiagroup(15) and model group（15）. Except control group,QingXia group and modelgroup were intraperitoneal injected D-GalN(400mg/kg body weight) plusLPS(50μg/kg body weight) once to build acute hepatic injury model induced by LPS/D-GalN.Rats in QingXia group were intragastric gavaged3days before LPS/D‐GalN intraperitoneal injection by QingXia decoction (2ml/100g body weight twice a day)for three days,after the model was built, QingXia decoction was intragastric gavagedper12hours,a total of2times. Control group and model group were intragastricgavaged by the same amount of saline.12hours after the last intragastric gavage ofQingXia group,we took the arterial blood and liver tissue of each rat for liverfunction examination and liver tissue pathological examination,Also,hepatocyteapoptosis index was detected by TUNEL,mRNA and protein expressions ofcaspase3,Bcl-2,Bax, TNF-αwere detected by RT-qPCR and western blotting.Results1.Serum ALT,AST,TBIL and PT were significantly increased in QingXia groupand model group than in control group（P<0.01,0.05）after the model was made.ButQingXia decoction can decreased serum ALT,AST,TBIL in rats,and the differenceswere significant compared with model group（P<0.01,0.05）。2. The examination of liver tissue pathology shows that no obvious abnormitywas found in control group, mild swelling、scattered necrosis and haemorrhage ofrats hepatocyte were found in QingXia group.In model group, there are markedlyswollen、 ballooning change、 obvious necrosis、inflammatory cell infiltration、punctate and splinter hemorrhage in the hepatocyte of model group.3.When detected by TUNEL,only minute quantity of apoptotic hepatocytes werefound in control group,apoptotic hepatocytes were more in QingXia group than thosein control group(P<0.01),in model group,apoptotic hepatocytes were much more thanthose in control and QingXia group(P<0.01),which mostly concentrated around thecentral wein.4.After RT-qPCR detection,compared with control group and QingXiagroup,mRNA expressions of caspase3、Bax were significantly increased and Bcl-2mRNA expression was significantly decreased in model group (P<0.05).TNF-αmRNA expression in model group was higher than that in controlgroup(P<0.01),but there was no statistical difference when compared with QingXiagroup. 5.When detected by western-blot,protein expressions of Bax and caspase3inmodel group were significantly higher than those in control group and QingXia group(P<0.05),but Bcl-2protein expression was lower(P<0.05), TNF-αprotein expressionin model and QingXia group was higher than that in control group（P<0.05），but therewas no significant difference between QingXia group and model group.Conclusions1.Acute hepatic injury model in rat can be successfully duplicated by usingLPS/D-GalN and the death rates of rats can be maintained in a relatively lowlevel,which is beneficial to the follw-up study.2. QingXia therapy can ameliorate the liver function of rats with hepatic injuryinduced by LPS/D-GalN.It can also alleviate hepatocytes necrosis and inflammatorycell infiltration.3.Hepatocytes apoptosis rates in rats with hepatic injury can be decreased byusing QingXia therapy.4.The mechanism of anti-apoptosis of QingXia therapy is that it down-regulatedthe expressions of caspase3and Bax, up-regulated the expression of Bcl-2,andadjusted the balance of Bcl-2/Bax.