【作者】 蒋华；

【导师】 周珉；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2017， 博士

【摘要】 研究背景脓毒症是感染引起的全身炎症反应综合征,可以进一步发展为感染性休克、多器官功能障碍综合征,是重症患者死亡的最主要原因之一。在脓毒症发病和发展的整个过程中,肝脏清除细菌和内毒素,以及产生炎症介质和炎性因子作用能对人体产生有效保护作用,但是肝脏也会成为失控的炎症反应袭击的潜在靶器官。脓毒症引起肝损伤在以前被认为是严重疾病的终末期,主要表现为转氨酶升高和黄疸。但是现在许多研究发现脓毒症初期就会出现症状不显著的肝功能障碍。虽然脓毒症并不经常并发肝损伤,但是一旦并发肝损伤,病情已经到了严重阶段。因此,尽早诊断和尽早治疗脓毒症肝损伤,对防治脓毒症有极其重要意义。研究目的1)通过临床研究初步明确脓毒症肝损伤患者的主要证候分布。2)运用犀角地黄汤治疗脓毒症肝损伤患者,评估其疗效和对预后的影响。3)通过盲肠结扎穿刺术造模大鼠实验研究,探讨犀角地黄汤对脓毒症肝损伤的作用机制。4)为将来临床开展大规模多中心前瞻性研究提供可靠依据。研究方法1)检索关于脓毒症肝损伤和犀角地黄汤的相关文献,为运用犀角地黄汤治疗脓毒症肝损伤提供理论依据。2)收集200例脓毒症肝损伤患者肝功能指标和中医症状体征等四诊信息,采用聚类分析的方法评估症状体征,结合卫气营血辨证,归纳中医的证候分类,获得脓毒症肝损伤患者的证候分布规律,并通过分析各类别患者的肝功能指标和预后来评估不同证候患者的病情严重程度。3)采用随机对照方法观察犀角地黄汤治疗30例脓毒症肝损伤患者的疗效,评估治疗后3天、7天犀角地黄汤对肝功能指标、APACHEⅡ评分、SOFA评分和28天累积生存率的影响。4)运用盲肠结扎穿刺(CLP)法模拟脓毒症肝损伤大鼠模型,以病理改变作为肝损伤的依据。设预先喂养犀角地黄汤的造模大鼠为试验组(CLP+XJDHT组),对照组为普通造模大鼠(CLP组)观察犀角地黄汤在不同时间点对脓毒症肝损伤大鼠一般情况、血清肝功能指标、血清氧化应激指标、血清炎性因子浓度和对肝组织STAT1和STAT3信号通路的影响。研究结果1)通过文献研究发现脓毒症适合于卫气营血辨证论治,而脓毒症肝损伤往往处于血分证,适合使用犀角地黄汤治疗。2)共收集了 200例脓毒症肝损伤患者的中医症状体征信息,通过聚类分析,共得出了三类证候。经专家共识,表现为血分证的患者有106例,表现为营分证的患者有64例,表现为卫分证/气分证的患者有30例。对三类患者肝功能指标和28天累积生存率进行分析,血分证患者的AST、ALT和TBIL水平明显高于卫分证/气分证和营分证(P<0.05)。血分证患者的28天累计生存率显著低于卫分证/气分证、营分证(P<0.01)。3)60例脓毒症肝损伤患者随机分为犀角地黄汤组和对照组,经过治疗后发现犀角地黄汤能改善脓毒症肝损伤患者的ALT、AST和TBIL水平(P<0.05),降低APACHE Ⅱ评分及SOFA评分(P<0.05),而28天生存率没有差异。4)盲肠结扎穿刺法造模脓毒症大鼠,以病理图片为依据判定脓毒症肝损伤模型造模成功。CLP 组术后 6h,12h,24h 时点存活率分别为:81.25%、45.83%、37.50%,CLP+XJDHT组相对应的存活率分别为:87.50%、54.16%、43.75%,两组没有统计学差异。5)CLP+XJDHT组大鼠血清ALT、AST水平在24h时点显著低于CLP组(P<0.01),而TBIL水平没有统计学差异。6)在12h时点CLP+XJDHT组大鼠肝脏SOD活力值高于CLP组(P<0.05),在24h时点CLP+XJDHT组大鼠肝脏SOD活力值显著高于CLP组(P<0.01)。在12h时点,CLP+XJDHT组大鼠肝组织MDA水平显著低于CLP组(P<0.01);在24h时点CLP+XJDHT组大鼠肝组织iNOS水平显著低于CLP组(P<0.01)。7)造模后CLP组的TNF-α、IL-1β、IL-6和IL-8水平均在6h达到高峰,然后逐步回落。而CLP+XJDHT组的IL-1β浓度6h达到高峰,而TNF-α、IL-6和IL-8水平的高峰出现在12h左右。12h时点CLP+XJDHT组大鼠血清TNF-α、IL-1β和IL-8均低于CLP组(P<0.05)。24h 时点 CLP+XJDHT 组大鼠血清 IL-6、IL-8 高于于 CLP 组(P<0.05)。而两组血清IL-4和IL-10高峰均出现在24h时点。6h时点CLP+XJDHT组大鼠血清IL-4和 IL-10 均低于 CLP 组(P<0.05 或P<0.01)。12h、24h 时点 CLP+XJDHT 组大鼠血清 IL-4 和 IL-10 均高于 CLP 组(P<0.01)。8)两组大鼠肝脏组织STAT1mRNA和STAT3mRNA的表达没有差异。两组大鼠肝组织STAT1和STAT3白水平没有差异。发现CLP术后6h、12h、24h,CLP组大鼠的p-STAT1平明显高于CLP+XJDHT组(P<0.01),发现CLP术后6h、12h,CLP组大鼠的p-STAT3水平高于 CLP+XJDHT 组(P<0.05)。研究结论1)证候聚类分析提示脓毒症肝损伤患者适合使用卫气营血辨证的证候分型治疗,临床上以血分证居多,病情最危重。2)谷丙转氨酶和谷草转氨酶在脓毒症肝损伤初期更加敏感,总胆红素的改变在时间上相对延后。3)犀角地黄汤能改善脓毒症肝损伤患者肝功能指标及危重病评分,不影响预后。4)犀角地黄汤可以改善脓毒症肝损伤大鼠的部分肝功能指标。5)临床和实验结果显示脓毒症早期的总胆红素变化不明显,不宜纳入脓毒症肝损伤的早期诊断依据。6)犀角地黄汤改善脓毒症肝损伤的可能机制:减轻氧化应激状态和提高抗氧化能力;减少促炎因子释放,增加抗炎因子释放,调控失衡的炎症反应,在脓毒症早期能干预STAT1和STAT3的信号通路。更多还原

【Abstract】 Research BackgroundSepsis is the systemic inflammatory response syndrome caused by infection,which can be further developed into septic shock and multiple organ dysfunction syndrome.Sepsis is one of the major causes of death in critically ill patients.In the process of sepsis,removing bacteria and producing inflammatory mediators of liver can produce effective defense for the body,but liver will also become a potential target organ attacked by uncontrolled inflammation response.Historically,liver injury happened in the end-stage of sepsis,mainly with clinical manifestation of elevated transaminase and jaundice.But many studies now show liver dysfunction appeared in the early stage of sepsis.Although sepsis is not often associated with liver damage,the condition has reached a critical stage when liver injury is initiated.Therefore,early detection and early treatment of liver injury is important for the prevention and treatment of sepsis.Objective1)To investigate the distribution of main traditional Chinese medicine(TCM)syndromes in sepsis patients with liver injury.2)To use Xijiaodihuang Decoction in the treatment of sepsis in patients with liver injury,to assess efficacy and influence on prognosis.3)Animal models were made by cecal ligation and puncture in rats,and to explore the mechanism of Xijiaodihuang Decoction on liver injury of sepsis.4)To provide a reliable basis for the future clinical development of large-scale multi center prospective study.Method1)To retrieve relevant literatures about sepsis with liver injury and Xijiaodihuang decoction in order toprovide a theoretical basis for the application of Xijiaodihuang Decoction in the treatment of sepsis with liver injury.2)To collected liver parameters and TCM syndromes in two hundreds sepsis patients with liver injury.To use the method of cluster analysis to evaluate symptoms and signs,combined with the theory of wei qi ying xue,and to sum up the classifications of TCM syndrome.To evaluate the relation between the classifications of TCM syndrome and the severity of the disease through the analysis of various categories of patients the index of liver function and prognosis.3)To observe the curative effect of Xijiaodihuang Decoction in the treatment of thirty sepsis patients with liver injury in 3rd,7th day after treatment.The observing indexes are including liver function parameters,APACHE II score,SOFA score and the cumulative survival rate of 28 days.4)The rat model of sepsis was made by cecal ligation and puncture(CLP),and the pathological picture was used as the standard for evaluating liver injury.Experimental group(CLP+XJDHT group)of rats was pre-feeded Xijiaodihuang Decoction,while the control group was normal rats(CLP group).To observe the general conditon,liver function parameters of serum,oxidative stress index of liver,various concentrations of inflammatory factors of serum and STAT1 and STAT3 signal pathway of liver in both groups at 6h,12h,24h.Result1)Through the literature research we found that the treatmeng of sepsis can be treated on base of the theory of wei qi ying xue syndrome.Sepsis patients with liver injury are often in Xuefen syndrome,and are suitable for the use of Xijiaodihuang decoction.2)A total of 200 cases of hepatic injury in septic patients were collected,and three kinds of syndromes were obtained by cluster analysis.After experts’ consensus,there were 106 patients with Xuefen syndrome,and 64 patients with Yingfen syndrome,and there were 30 patients with Wei syndrome/Qi syndrome.The liver function parameters and cumulative survival rate of 28 days were analyzed in three groups.The levels of AST,ALT and TBIL in patients with Xue syndrome were higher than those of Wei syndrome/Qi syndrome and Yingfen syndrome(P<0.05).The cumulative survival rate of patients with Xuefen syndrome in 28 days was significantly lower than that of Wei syndrome/Qi syndrome(P<0.01).3)60 cases of sepsis patients with liver injury were randomly divided into Xijiaodihuang Decoction group and control group.After treatment of Xijiaodihuang decoction,ALT,AST and TBIL level were decreased(P<0.05).The APACHE II score and the SOFA score were decreased too(P<0.05).But the 28 day survival rate had no difference in two groups.5)The rat model of sepsis was induced by cecal ligation and puncture,and the model of liver injury was successfully evaluated by pathological pictures.The survival rates of CLP group on 6h,12h and 24h after CLP were 81.25%,45.83%and 37.50%,respectively,while the corresponding survival rates in CLP+XJDHT group were 87.50%,54.16%and 43.75%,respectively.There was no statistical difference between the two groups.6)The level of serum ALT and AST in CLP+XJDHT group was significantly lower than that in CLP group at 24h(P<0.01),but there was no statistical difference in the level of TBIL.7)The SOD activity in liver of rats in group CLP+XJDHT was higher than that in group CLP on 12h(P<0.05),and the SOD activity in group CLP+XJDHT was higher than that in group CLP on 24h(P<0.01).The MDA level in CLP+XJDHT group was significantly lower than that in CLP group on 12h(P<0.01).The iNOS level in CLP+XJDHT group was significantly lower than that in group CLP on 24h(P<0.01).7)After modeling,the concentration of TNF-α,IL-1β IL-6 and IL-8 in CLP group reached the peak at 6h,and then gradually declined.The concentration of IL-1β in CLP+XJDHT group reached the peak at 6h,while the peak of TNF-α,IL-6 and IL-8 appeared around 12h.The concentration of TNF-α,IL-1β,IL-8 in CLP+XJDHT group were lower than those in the CLP group at 12h(P<0.05).The concentration of IL-6 and IL-8 in CLP+XJDHT group were higher than those in the CLP group at 24h(P<0.05).The peak concentration of IL-4 and IL-10 both appeared at 24h in two groups.The concentration of IL-4 and IL-10 in CLP+XJDHT group were lower than those in CLP group at 6h(P<0.05 or P<0.01).The concentratin of IL-4 and IL-10 levels in CLP+XJDHT group were higher than those in group CLP at 12h and 24h(P<0.01).8)There was no difference in the expression of STAT1mRNA and STAT3 mRNA in the liver tissues between the two groups.There were no differences in protein expression of STAT1 and STAT3 in two groupss.It was found that the level of p-STATl in CLP group was significantly higher than that in CLP+XJDHT group at 6h,12h,24h(P<0.01),and the level of p-STAT3 in CLP group was higher than that in group CLP+XJDHT at 6h,12h(P<0.05).Conclusion1)Cluster analysis of TCM syndromes suggests that sepsis patients with liver injury are suitable for the treatment on syndrome differentiation of Wei Qi Ying Xue theory,and the majority of them are Xuefen syndrome,and the condition of Xuefen syndrome is the most critical.2)The level of alanine aminotransferase and aspartate aminotransferase are sensitive indexes in estimating liver injury,the level of total bilirubin changes later.3)Xijiaodihuang decoction can improve the liver function,critical illness scores of sepsis patients with liver injury,but can not improve the prognosis.4)Xijiaodihuang decoction can improve parts of the liver function parameters in sepsis rats with liver injury.5)The clinical and experimental results show that the level of total bilirubin does not changes obviously in early stage of sepsis,so it is not suitable for the early diagnosis of hepatic injury due to sepsis.6)The mechanism of Xijiaodihuang Decoction improving liver injury may including:reducing oxidative stress and improving antioxidant capacity,decreasing proinflammatory cytokine and increasing inflammatory cytokine release for mediating uncontrolled inflammatory state,and interfering with signaling pathway of STAT1 and STAT3 in early stage of sepsis.更多还原