清下法治疗肝硬化并感染患者肠源性内毒素血症的临床研究及其抗内毒素性肝损伤机制探讨
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摘要
第一部分清下法治疗肝硬化合并感染患者肠源性内毒素血症及其干预抗生素治疗过程中内毒素释放的临床研究
研究背景
肝硬化合并感染患者多伴有肠源性内毒素血症，内毒素血可加重肝损害并可导致各种并发症，因此，防治肝硬化并感染患者肠源性内毒素血症具有重要临床意义。针对肝硬化合并感染患者，抗感染治疗是必要的治疗手段，而抗生素治疗过程中，可以引起内毒素的释放，因而加重肝硬化合并感染患者内毒素血症治疗的难度。而目前西医对肝硬化并感染患者病肠源性内毒素血症尚无特效治疗手段尤其是如何干预抗生素治疗过程中内毒素的释放，是亟需解决的问题，研究已证实，在西医治疗基础上，加用中医药，可明显提高慢性肝病合并肠源性内毒素血症患者的疗效，由于内毒素血症中医病机复杂，因而采取辩证论治是较理想的方案，通过分析肝硬化合并感染患者肠源性内毒素血症的病因病机，将其归纳为热毒内蕴，瘀毒互结，阳明腑实。故提出采用清下法治疗肝硬化并感染患者肠源性内毒素血症，以期为肝硬化肠源性内毒素血症提供新的治疗手段，并探讨其对抗生素治疗过程中内毒素释放的干预作用。
研究目的
探讨清下法对肝硬化合并感染患者肠源性内毒素血症的影响及其对抗生素治疗过程中内毒素释放的干预作用。
研究方法
选取2010年10月至2013年1月中山大学附属第三医院中医肝病专科及感染科肝炎后肝硬化合并感染患者90例为研究对象，随机分为研究1组、研究2组及对照组各30例。所有患者均采用常规综合治疗（卧床休息、抗感染、保肝降酶、利胆退黄、利尿、调节肠道菌群等，低白蛋白者补充人血白蛋白，凝血功能明显异常者补充新鲜冰冻血浆）2周，所有患者所用抗生素均为头孢哌酮钠/舒巴坦钠（静脉滴注，每天2次，每次2g，疗程为2周）。其中，研究1组在常规综合治疗基础上，从第1天开始服用清下中药，连服14日，研究2组在常规综合治疗一周后，加用清下中药，连服7日，对照组仅采取常规综合治疗，不加用任何中药治疗。
结果
1.治疗2周后，三组患者的临床症状及体征均有明显改善（P<0.05），但研究组在改善患者腹胀、便秘及保持大便通畅方面疗效较对照组更佳（P<0.05）。
2.治疗2周后，三组患者血常规、肝功能、凝血功能各项指标均有明显改善(P<0.01)，但研究组在降低血清TBIL方面较对照组更佳（P<0.01）。
3.治疗2周后，三组患者child-pugh分级得到明显改善、腹水亦有明显减轻（P<0.05，0.01），但各组间比较无统计学差异。
4.治疗2周后，三组患者腹水白细胞、中性粒细胞均明显降低（P<0.05），但各组间比较无统计学差异。
5.研究二组及对照组在治疗第一周结束后血LPS、TNF-α、IL-6均较治疗前明显升高（P<0.05）,而研究一组无升高，在治疗两周后，三组患者血LPS、TNF-α、IL-6均较治疗前明显降低（P<0.05）,但研究组在降低血LPS方面疗效更佳(P<0.01)。
6．Child-pugh分级与血清LPS、TNF-α、IL-6之间均存在显著正相关(P<0.01)。
结论
1.在西医综合治疗的基础上，加用清下中药，可明显改善患者临床症状及体征、肝功能及凝血功能。
2.清下法可干预抗生素治疗过程中内毒素及炎症介质释放。
3.清下法干预抗生素治疗过程中的内毒素释放可能与其抑制炎症介质的产生有关。
4.肝硬化并感染患者child-pugh分级与血LPS、TNF-α、IL-6呈明显正相关
第二部分清下法抗内毒素性肝损伤大鼠肝细胞凋亡机制探讨
研究背景
内毒素可通过细胞凋亡机制造成肝损伤，防治内毒素所致的肝细胞凋亡，显得尤为重要。研究已证实，中医药可抑制内毒素所致的肝细胞凋亡，本论文第一部分研究发现，清下法可减轻肝硬化合并感染患者肠源性内毒素血症的临床症状及体征，可明显改善患者的肝功，降低患者血LPS及TNF-α、IL-6水平，且可减少抗生素治疗过程中血LPS的释放，因LPS的肝损害作用已经被明确，故考虑清下法可减轻肝硬化患者的内毒素性肝损害，但确切机制尚未明确，故本部分以细胞凋亡机制为切入点，研究清下法对内毒素性肝损害的保护机制，而且目前尚未发现关于清下法抗内毒素导致的肝细胞凋亡的报道。
研究目的
通过动物实验，探讨清下法抗内毒素性肝细胞凋亡的机制.
研究方法
40只大鼠随机分为正常对照组10只，清下组15只、模型组15只,除正常对照组外，清下组及模型组均腹腔内注射D-GalN（400mg/kg）+脂多糖LPS（50μg/kg），造成急性内毒素性肝损伤模型，清下组于造模前3天灌胃清下中药，按2ml/100g灌胃,每日2次，连续3d，造模后灌胃清下中药，每12小时一次，共2次。正常对照组和模型组灌等量生理盐水。在清下组最后一次灌胃清下中药12小时后，取大鼠动脉血及肝组织以备肝功能、肝组织病理检查及采用TUNEL法检查肝细胞凋亡指数，RT-qPCR及western blot法检测caspase3、Bcl-2、Bax、TNF-α的基因及蛋白表达。
结果
1.造模后，清下组及模型组血ALT、AST、TBIL及PT均有升高，与对照组比较有显著差异（P<0.05,0.01）,但清下组血ALT、AST、TBIL水平较模型组明显降低，与模型组比较有显著差异（P<0.01,0.05）。
2.肝组织病理检查，对照组大鼠肝组织病理无明显异常，清下组大鼠肝细胞轻度肿胀，仅见散在的坏死及出血点，而模型组大鼠肝细胞明显肿胀及气球样变，肝细胞可见明显坏死及炎性细胞浸润。
3.经Tunel检测，对照组大鼠肝细胞仅见极少量细胞凋亡，清下组大鼠肝细胞凋亡数较对照组增多(P<0.01)，模型组可见较多凋亡细胞，多集中在中央静脉周边，与对照组及清下组相比均有显著差异（P<0.01，0.05）。
4.通过RT-qPCR检测，模型组大鼠肝组织组Caspase3、Bax mRNA表达量明显升高，Bcl-2mRNA表达则明显降低，与对照组及清下组比较具有显著差异（P<0.05），而清下组Caspase3mRNA表达量则较模型组明显降低、Bcl-2mRNA表达量较模型组明显升高（P<0.05）,模型组及清下组TNF-αmRNA表达量均升高，其中模型组与对照组相比具有显著差异（P<0.05），但与清下组比较无显著差异。
5.经western-blot检测，模型组Bax、Caspase3蛋白表达量明显高于对照组及清下组（P<0.05），Bcl-2蛋白表达量则较对照组及清下组明显降低（P<0.05），模型组及清下组TNF-α蛋白表达量均较对照组高（P<0.05），但模型组与清下组之间比较无显著差异。
结论
1:采用脂多糖联合D-氨基半乳糖(LPS/D-GalN)可成功复制急性内毒素性肝损伤大鼠模型，且可将造模过程中及造模后的动物死亡率维持在较低水平，有利于后续的研究。
2：清下法可改善内毒素性肝损伤大鼠肝功能，肝组织病理检查显示其可明显减轻肝细胞的坏死及炎性细胞浸润。
3：清下法可降低内毒素性肝损伤大鼠肝细胞凋亡率。
4：清下法主要通过降低caspase家族中关键凋亡执行酶caspase3的表达，从而阻断caspase级联反应，通过上调Bcl-2蛋白家族中Bcl-2的表达，降低Bax的表达以及调控Bcl-2、Bax之间的平衡达到抑制肝细胞凋亡的目的。
Part I Clinical study of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with infection and the intervention of endotoxin release in the process ofantibiotic treatment
Background
Cirrhotic 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.
Objective
To discuss the effect of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with bacteria infection and its interventional effect in endotoxin releaseduring antibiotic treatment.
Methods
90cases 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.
Results
1. 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)。
Conclusions
1.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-galactosamine
Background
Endotoxin 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.
Objective
To study the anti-apoptotic mechanism of QingXia therapy on hepatocyte apoptosisinduced by lipopolysaccharide/D-galactosamine
Methods
40SD 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.
Results
1.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.
Conclusions
1.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.
研究背景
肝硬化合并感染患者多伴有肠源性内毒素血症，内毒素血可加重肝损害并可导致各种并发症，因此，防治肝硬化并感染患者肠源性内毒素血症具有重要临床意义。针对肝硬化合并感染患者，抗感染治疗是必要的治疗手段，而抗生素治疗过程中，可以引起内毒素的释放，因而加重肝硬化合并感染患者内毒素血症治疗的难度。而目前西医对肝硬化并感染患者病肠源性内毒素血症尚无特效治疗手段尤其是如何干预抗生素治疗过程中内毒素的释放，是亟需解决的问题，研究已证实，在西医治疗基础上，加用中医药，可明显提高慢性肝病合并肠源性内毒素血症患者的疗效，由于内毒素血症中医病机复杂，因而采取辩证论治是较理想的方案，通过分析肝硬化合并感染患者肠源性内毒素血症的病因病机，将其归纳为热毒内蕴，瘀毒互结，阳明腑实。故提出采用清下法治疗肝硬化并感染患者肠源性内毒素血症，以期为肝硬化肠源性内毒素血症提供新的治疗手段，并探讨其对抗生素治疗过程中内毒素释放的干预作用。
研究目的
探讨清下法对肝硬化合并感染患者肠源性内毒素血症的影响及其对抗生素治疗过程中内毒素释放的干预作用。
研究方法
选取2010年10月至2013年1月中山大学附属第三医院中医肝病专科及感染科肝炎后肝硬化合并感染患者90例为研究对象，随机分为研究1组、研究2组及对照组各30例。所有患者均采用常规综合治疗（卧床休息、抗感染、保肝降酶、利胆退黄、利尿、调节肠道菌群等，低白蛋白者补充人血白蛋白，凝血功能明显异常者补充新鲜冰冻血浆）2周，所有患者所用抗生素均为头孢哌酮钠/舒巴坦钠（静脉滴注，每天2次，每次2g，疗程为2周）。其中，研究1组在常规综合治疗基础上，从第1天开始服用清下中药，连服14日，研究2组在常规综合治疗一周后，加用清下中药，连服7日，对照组仅采取常规综合治疗，不加用任何中药治疗。
结果
1.治疗2周后，三组患者的临床症状及体征均有明显改善（P<0.05），但研究组在改善患者腹胀、便秘及保持大便通畅方面疗效较对照组更佳（P<0.05）。
2.治疗2周后，三组患者血常规、肝功能、凝血功能各项指标均有明显改善(P<0.01)，但研究组在降低血清TBIL方面较对照组更佳（P<0.01）。
3.治疗2周后，三组患者child-pugh分级得到明显改善、腹水亦有明显减轻（P<0.05，0.01），但各组间比较无统计学差异。
4.治疗2周后，三组患者腹水白细胞、中性粒细胞均明显降低（P<0.05），但各组间比较无统计学差异。
5.研究二组及对照组在治疗第一周结束后血LPS、TNF-α、IL-6均较治疗前明显升高（P<0.05）,而研究一组无升高，在治疗两周后，三组患者血LPS、TNF-α、IL-6均较治疗前明显降低（P<0.05）,但研究组在降低血LPS方面疗效更佳(P<0.01)。
6．Child-pugh分级与血清LPS、TNF-α、IL-6之间均存在显著正相关(P<0.01)。
结论
1.在西医综合治疗的基础上，加用清下中药，可明显改善患者临床症状及体征、肝功能及凝血功能。
2.清下法可干预抗生素治疗过程中内毒素及炎症介质释放。
3.清下法干预抗生素治疗过程中的内毒素释放可能与其抑制炎症介质的产生有关。
4.肝硬化并感染患者child-pugh分级与血LPS、TNF-α、IL-6呈明显正相关
第二部分清下法抗内毒素性肝损伤大鼠肝细胞凋亡机制探讨
研究背景
内毒素可通过细胞凋亡机制造成肝损伤，防治内毒素所致的肝细胞凋亡，显得尤为重要。研究已证实，中医药可抑制内毒素所致的肝细胞凋亡，本论文第一部分研究发现，清下法可减轻肝硬化合并感染患者肠源性内毒素血症的临床症状及体征，可明显改善患者的肝功，降低患者血LPS及TNF-α、IL-6水平，且可减少抗生素治疗过程中血LPS的释放，因LPS的肝损害作用已经被明确，故考虑清下法可减轻肝硬化患者的内毒素性肝损害，但确切机制尚未明确，故本部分以细胞凋亡机制为切入点，研究清下法对内毒素性肝损害的保护机制，而且目前尚未发现关于清下法抗内毒素导致的肝细胞凋亡的报道。
研究目的
通过动物实验，探讨清下法抗内毒素性肝细胞凋亡的机制.
研究方法
40只大鼠随机分为正常对照组10只，清下组15只、模型组15只,除正常对照组外，清下组及模型组均腹腔内注射D-GalN（400mg/kg）+脂多糖LPS（50μg/kg），造成急性内毒素性肝损伤模型，清下组于造模前3天灌胃清下中药，按2ml/100g灌胃,每日2次，连续3d，造模后灌胃清下中药，每12小时一次，共2次。正常对照组和模型组灌等量生理盐水。在清下组最后一次灌胃清下中药12小时后，取大鼠动脉血及肝组织以备肝功能、肝组织病理检查及采用TUNEL法检查肝细胞凋亡指数，RT-qPCR及western blot法检测caspase3、Bcl-2、Bax、TNF-α的基因及蛋白表达。
结果
1.造模后，清下组及模型组血ALT、AST、TBIL及PT均有升高，与对照组比较有显著差异（P<0.05,0.01）,但清下组血ALT、AST、TBIL水平较模型组明显降低，与模型组比较有显著差异（P<0.01,0.05）。
2.肝组织病理检查，对照组大鼠肝组织病理无明显异常，清下组大鼠肝细胞轻度肿胀，仅见散在的坏死及出血点，而模型组大鼠肝细胞明显肿胀及气球样变，肝细胞可见明显坏死及炎性细胞浸润。
3.经Tunel检测，对照组大鼠肝细胞仅见极少量细胞凋亡，清下组大鼠肝细胞凋亡数较对照组增多(P<0.01)，模型组可见较多凋亡细胞，多集中在中央静脉周边，与对照组及清下组相比均有显著差异（P<0.01，0.05）。
4.通过RT-qPCR检测，模型组大鼠肝组织组Caspase3、Bax mRNA表达量明显升高，Bcl-2mRNA表达则明显降低，与对照组及清下组比较具有显著差异（P<0.05），而清下组Caspase3mRNA表达量则较模型组明显降低、Bcl-2mRNA表达量较模型组明显升高（P<0.05）,模型组及清下组TNF-αmRNA表达量均升高，其中模型组与对照组相比具有显著差异（P<0.05），但与清下组比较无显著差异。
5.经western-blot检测，模型组Bax、Caspase3蛋白表达量明显高于对照组及清下组（P<0.05），Bcl-2蛋白表达量则较对照组及清下组明显降低（P<0.05），模型组及清下组TNF-α蛋白表达量均较对照组高（P<0.05），但模型组与清下组之间比较无显著差异。
结论
1:采用脂多糖联合D-氨基半乳糖(LPS/D-GalN)可成功复制急性内毒素性肝损伤大鼠模型，且可将造模过程中及造模后的动物死亡率维持在较低水平，有利于后续的研究。
2：清下法可改善内毒素性肝损伤大鼠肝功能，肝组织病理检查显示其可明显减轻肝细胞的坏死及炎性细胞浸润。
3：清下法可降低内毒素性肝损伤大鼠肝细胞凋亡率。
4：清下法主要通过降低caspase家族中关键凋亡执行酶caspase3的表达，从而阻断caspase级联反应，通过上调Bcl-2蛋白家族中Bcl-2的表达，降低Bax的表达以及调控Bcl-2、Bax之间的平衡达到抑制肝细胞凋亡的目的。
Part I Clinical study of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with infection and the intervention of endotoxin release in the process ofantibiotic treatment
Background
Cirrhotic 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.
Objective
To discuss the effect of QingXia therapy on intestinal endotoxemia of cirrhoticpatients with bacteria infection and its interventional effect in endotoxin releaseduring antibiotic treatment.
Methods
90cases 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.
Results
1. 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)。
Conclusions
1.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-galactosamine
Background
Endotoxin 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.
Objective
To study the anti-apoptotic mechanism of QingXia therapy on hepatocyte apoptosisinduced by lipopolysaccharide/D-galactosamine
Methods
40SD 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.
Results
1.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.
Conclusions
1.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.
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