【作者】 陈仲清；

【导师】 赵克森； 古妙宁；

【作者基本信息】 南方医科大学 ， 麻醉学（专业学位）， 2014， 博士

【摘要】 多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)是重症监护病房(intensive care unit, ICU)患者发病和死亡的一个重要原因。MODS的成年患者在ICU中的发生率为11%～40%,在我国归因于MODS的总死亡率超过了60%。目前对于MODS的共识认为它是一种连续的生理紊乱状态,但对于这种状态的发病机制尚未完全明了。现有观点认为MODS与炎症反应、自由基生成增加和肠道菌群移位等有关。缺血再灌注损伤和严重脓毒症是MODS最常见的致病原因之一。MODS的治疗主要集中在支持性治疗,如增加氧输送,营养支持等。开发一种以减少氧化应激和调节炎症反应为靶点的药物可能是一种具有前景的研究方向。虎杖苷(3,4’,5-三羟基芪-3-葡萄糖甙,Polydatin,PD)是单晶体药物,它是从中国传统中药虎杖分离出来的(图1)。我们实验组以往工作表明,在严重失血性休克大鼠模型中,PD能通过减少大鼠动脉血管平滑肌细胞和神经元线粒体损伤发挥作用,这些作用与减少机体氧化应激水平有关。本实验拟通过复制大鼠失血性休克和盲肠结扎穿刺(cecal ligation puncture, CLP)二次打击模型,研究PD是否具有治疗MODS大鼠的作用以及它作用的可能机制。本研究主要分为两个步骤进行：1.MODS模型复制和PD最佳给药剂量探索；2.PD作用于MODS大鼠的机制探讨。第一部分MODS模型复制和PD最佳给药剂量探索方法：实验分为正常对照组(sham组)、MODS后生理盐水组(normal saline,NS)组和MODS后PD给药组,其中PD组又分为不同的给药剂量组(15mg/kg,30mg/kg,45mg/kg,60mg/kg大鼠体重)。sham组大鼠仅行基本外科操作,其余组大鼠在以上外科操作基础上进行失血性休克和CLP双重打击,随后立即进行给药(图2)。NS组给药为0.3mlNS×3次(CLP操作完成后立即开始,之后每6小时1次),PD组给药为不同剂量的PD溶解于0.3mlNS中通过股静脉10分钟内缓慢推注。建模治疗后各组大鼠(每组8只)撤管并缝合伤口,回笼,室温下自由饮食饮水。观察各组生存时间(每小时观察1次,以72小时为观察终点),计算各组72小时的生存率。为评估MODS诱导24小时后大鼠造模是否成功,抽取静脉血测定血清生化指标(BUN,CR-MB,TBIL,LDH,CK,ALT and AST)和动脉血气。随后立即颈椎脱臼法处死大鼠,常规固定肾、肺和肝组织,苏木精-伊红(hematoxylin-eosin staining,HE)染色行病理分析。采用SPSS13.0软件录入数据,Kaplan-meier法进行各组生存时间分析,One-way ANOVA方法比较各组平均数±标准差,LSD-t法检验多重样本均数。1.各组给药后生存时间观察和最佳PD给药剂量sham组8只大鼠在24h、36h和48h三个观察时间点均存活。MODS处理的各组均有大鼠死亡。其中MODS+NS组前36小时仅有2只大鼠存活,48小时内全部死亡,72h观察时间段的中位数生存时间是29h。MODS+PD各给药组中PD45mg/kg组效果最优,8只大鼠在前36小时均存活,48小时时间内仅有1只死亡,72小时的中位数生存时间为49小时。由此初步确立PD45作为以后的给药剂量。2.生化指标测定和血气分析和sham相比,MODS+NS组大鼠血生化值均显著增高且两组间比较差别具有统计学意义(P<0.05).MODS+NS组的血气参数,包括pH值、血乳酸、碱剩余和HC03-均较sham组均有增高且两组间差值具有统计学意义(P<0.05)。同时PD各给药组中45mg/Kg组对于MODS各脏器的生化指标和血气分析改善作用最佳且明显优于MODS+NS组。3.病理鉴定sham组大鼠各脏器均未出现明显病理变化。然而,在MODS+NS组中,肾、肝和肺的病理均出现了病变。部分近端肾小管上皮细胞出现浊肿、变性、细胞部分缺失、管腔狭窄,有的管腔伴有少量血栓沉积；部分肝中央静脉缩小,肝窦扩张充血,肝细胞胞浆呈混浊肿胀,许多肝细胞出现气球样变或退行性变；肺间质水肿和扩大,部分肺泡萎陷,血管和间质周围有许多炎症细胞浸润。PD45mg/Kg给药组均有病变减轻。结论1.大鼠MODS模型诱导24小时后,肝脏、肾脏、心脏、肺脏功能均出现了损伤,肝、肾和肺的病理学结果支持了功能学的变化。2.以血生化指标增高倍数和病理的结果作为判定标准,大鼠MODS建模24小时后NS组肝脏和肾脏的功能出现了衰竭,说明MODS模型复制成功。3.结合MODS发生率、中位数生存时间、生化和血气指标的测定,45mg/kg的PD单次静脉给药是最合适的剂量。第二部分PD对MODS大鼠的治疗作用和机制探索方法实验分为sham组,MODS+NS组和MODS+PD组。sham组和MODS+NS组的操作同前,MODS+PD组大鼠每次给药为等容积的PD(45mg/Kg)。为评估MODS诱导24小时后大鼠多脏器情况(每组8只)。血清生化指标(BUN, CR, TBIL, LDH, CK, ALT,AST)、动脉血气分析、晚期氧化蛋白产物(advance oxidative protein products,AOPPs)和炎症因子(IL-1, IL-6, TNF-α)分别被测定。之后颈椎脱臼法处死大鼠,取肾、肺和肝组织常规固定HE染色,免疫组化法测定凋亡相关蛋白(Bcl-2、Bax、Cytochrome C)和ELISA法测定肾和肝匀浆的caspase-3活性(每组6-8只)。采用SPSS13.0软件录入数据,Kaplan-meier法进行各组生存时间分析,One-way ANOVA方法比较各组平均数±标准差,LSD-t法检验多重样本均数。结果1.PD对于MODS大鼠模型整体生存时间的影响sham组的大鼠48小时均存活。MODS+PD组和MODS+NS组的中位数生存时间为分别为49小时和29小时,PD处理后实验大鼠的中位数生存时间明显延长。2.PD对MODS大鼠模型主要血液生化指标和动脉血气的影响和MODS+NS组相比,MODS+PD组所有的血生化值均显著降低(图3)。血气参数包括pH值、血乳酸、碱剩余和HC03-在MODS+PD组均有改善,以上参数两组比较均具有统计学意义(P<0.05)。3.PD对MODS大鼠的病理的影响在MODS+NS组中,肾、肝和肺的病理均出现了病变。部分近端肾小管上皮细胞出现浊肿、变性、细胞部分缺失、管腔狭窄,有的管腔伴有少量血栓沉积；部分肝中央静脉缩小,肝窦扩张充血,肝细胞胞浆呈混浊肿胀,许多肝细胞出现气球样变或退行性变；肺间质水肿和扩大,部分肺泡萎陷,血管和间质周围有许多炎症细胞浸润。与NS组比较,PD45mg/Kg给药组肾脏、肝脏和肺脏的病理损伤均有减轻。4.PD对MODS的大鼠血清AOPPs水平的影响MODS+NS组血清AOPPs明显增高,PD治疗组血清AOPPs浓度显著下降。较sham组比较,两组的增高率分别为77.4%和33.5%,两组间比较具有统计学意义(F=7.919,P<0.05)。5.PD对MODS大鼠肾脏、肝脏和肺脏组织凋亡相关蛋白表达的影响与MODS+NS组相比,MODS+PD组在肾脏和肝脏组织胞质细胞色素C免疫组化染色的相对灰度值均显著减少。各组肺组织蛋白表达之间无显著差异。(两组在肾脏和肝脏组织的相对值分别是110%vs30.2%,F=70.4,P<0.01；40%vs11.2%,F=22.4,P<0.05)。与MODS+NS组相比,MODS+PD组在肾脏和肝脏组织Bax蛋白免疫组化染色的相对灰度值均显著减少。各组肺组织蛋白表达之间无显著差异。(两组在肾脏和肝脏组织的相对值分别是67.1%vs24.2%,F=110.7,P<0.01；49.6%vs34.1%,F=67.4,P<0.05)。与MODS+NS组相比,MODS+PD组在肾脏和肝脏组织Bcl-2免疫组化染色的相对灰度值均显著增加。各组肺组织蛋白表达之间无显著差异。(两组在肾脏和肝脏组织的相对灰度值在肾脏和肝脏分别是9.9%vs31.4%,F=21.1,P<0.01；18.3%vs33.3%,F=4,P<0.05)。同sham组相比,MODS+NS组和MODS+PD组中caspase-3活性增高明显。然而,MODS+PD组的caspase-3的活性显著低于MODS+NS组且两组间比较具有统计学意义。(肾脏52.1%vs148%,F=44.5,P<0.01；肝脏39.8%vsl23.8%,F=27.9,P<0.01)。6.PD对MODS大鼠血清炎症细胞因子水平的影响同sham组相比,MODS+NS组和MODS+PD组的三种细胞因子水平均升高。然而,这些指标在MODS+PD组明显低于MODS+NS组,且两组间比较均具有统计学意义(IL-6：12倍vs8倍,F=33.6,P<0.01；TNF-α:13倍vs10倍,F=77.5,P<0.05;IL-1：8倍vs5倍,F=41,P<0.05)。结论PD改善了MODS大鼠多个器官(肝、肾和肺)的功能和整体生存时间。它的作用机制可能与抑制氧化应激、降低炎症反应和抑制激活线粒体介导的凋亡途径等有关。更多还原

【Abstract】 Multiple organ dysfunction syndrome (MODS) is a major cause of morbidity and mortality in patients of intensive care units (ICUs). The incidence of MODS in adult patients in ICUs ranges from11%to40%, while the overall mortality rate due to MODS in China is over60%. MODS is now recognized as a continuum of physiologic derangements and the main pathogenesis of this condition is not yet fully understood. Currently, the consensus opinion holds that MODS is linked to a combination of inflammation, increased generation of free radicals, intestinal kinetic alterations and so on. Ischemia-reperfusion injury and severe sepsis are among the most common causative factors. Currently, therapy for MODS is largely supportive, such as recovery of oxygen delivery, nutritional support, and so on. More effective methods and drugs are urgently required for the prevention and treatment of MODS, and drugs which could reduce oxidative stress and regulate the inflammatory response may be a promising approach.Polydatin (PD;3,4’,5-trihydroxystibene-3-monoglucoside) is a monocrystalline drug isolated from the traditional Chinese herb Polygonum cuspidatum (Fig1). Our previous work demonstrated that PD can protect arterial smooth muscle and neuronal cells against mitochondrial dysfunction after severe ischemia-reperfusion injury in hemorrhagic shock rats; and these effects were related to reducing levels of oxidative stress. Recently, PD was reported to reduce the inflammatory response in various disease models. Here, we investigated the effects and potential molecular mechanisms of PD action, specifically in the kidney, liver and lung, using a two-hit model of MODS induced by traumatic hemorrhagic shock and cecal ligation puncture (CLP).The study is divided into two steps:1. MODS model reproduction and optimal dose of PD exploration;2.The effect of PD on MODS rats and its possible mechanisms investigation. The first part MODS model reproduction and the optimal dose of PD explorationMethodsThe experiment was divided into two major groups, namely normal control group and model group. Model group was divided into normal saline (NS) group and polydatin administration group, the control group which received only basic surgical procedures. The animals in the model group experienced hemorrhagic shock (the mean blood pressure was reached to45-50mmHg within10min by hemorrhaging and maintained in this level in the next50min, then all the shed blood was infused) and cecal ligation puncture as "two hit" on the basis on the basic surgical procedures mentioned above(Fig2). Then NS was administered (performed after CLP operation,0.3ml a time, every6hours×3.See diagram:Figure2).In MODS+PD group, the PD with different doses was dissolved in the same volume of physiological saline and was administered by intravenous injection within10minutes. After the model was set up and drug administration, one part of the rats (n=8, a total of6sub-groups:sham group, MODS+NS group, MODS+PD15mg group, MODS+PD30mg group, MODS+PD45mg group, MODS+PD60mg group) whose tube was withdrawn and suture wounds, were returned to their individual cage for survival time observation. All the rats were given freedom of drinking water at room temperature (observe per hour until the end point at72h) and72-hour survival was calculated. To evaluate whether the MODS was successfully induced at24hours, serum biochemical parameters (BUN, CR, TBIL, LDH, CK, ALT and AST) and arterial blood gases were measured separately (6-8in each group). Also kidney, lung and liver tissue pathology were acquired immediately using conventional fixed, HE staining after the animals were sacrificed by cervical dislocation. All datas were input in SPSS13.0software, survival time analysis was study by Kaplan-meier method, One-way ANOVA was used to compare the mean+SD of dates among groups, LSD-t was selected to test multiple samples.Results1. Effects of PD on overall survival and the optimal dose of PD on MODS ratsEight rats in control group (normal group) at three time points (24h,36h and48h) were alive. However, a part of MODS rats died. Only two rats survived in MODS+NS group at time point of36h and all8rats died within48h. The median survival time is29h in72h observation period. MODS+PD group(PD45mg/kg BW) was established as the most optimaldose among PD various subgroups, while eight rats still survived after36hours, only one died within48hours. The median survival time was49hours. The median survival time in72hours in MODS+NS group and MODS+PD group differed statistically (log rank test, Chi-Square=19.641, P<0.01=. Single dose of45mg/kg PD was selected as the best optimaldose in the experiments followed.2. Biochemical parameters and blood gas analysis of MODS rats.Compared with control group rats, all the blood biochemical values in MODS+NS group were increased significantly (control group vs MODS+NS group:ALT48.6u/L vs200.4u/L; AST76u/L vs545.5u/L; LDH205u/L vs417.6u/L; TBIL 0.4μmol/L vs2.7μmol/L; BUN8.3μmol/L vs16.2μmol/L; Cr22.3μmol/L vs37.2μmol/L; CK-MB268.8u/L vs820.2u/L, all P<0.05=. In MODS+NS group, The blood gas analysis parameters, including pH, base excess and HCO3-were all increased markedly than the values in the control group and the difference between the two groups was statistically significant (P<0.05=.Meanwhile, comparing to other subgroups, single dose of45mg/Kg in PD group on MODS treatment showed the best improvement of biochemical markers of various organs and blood gas analysis.3. Multiple organs pathology of MODS ratsNo significant abnormalities were observed in the control group. However, pathological changes in the kidney, liver and lung were observed in the MODS+NS group. In the kidney, a number of proximal tubular epithelial cells displayed cloudy swelling and degeneration, and some regions of the proximal tubular epithelium were absent. Additionally, the proximal tubular lumen was narrowed, with deposition of small thrombi. In the liver of the MODS+NS group, a number of hepatic central veins were narrowed with sinusoidal dilatation and congestion, the cytoplasm of the hepatocyte was cloudy and swollen, and many hepatocytes had ballooned or degenerated. In the lung tissue of the MODS+NS group, pulmonary interstitial edema and broadening leading to alveolar collapse was observed, and numerous inflammatory cells had infiltrated around the blood vessels and interstitium.Conclusion1. After24hours of MODS induction, liver, kidney, heart, lung functions were damaged in varying degrees. And the liver, kidney and lung function changes supported the pathology results.2. Combine the results of biochemical and pathological analysis, liver and kidney function failure can be determined after MODS induction24hours later, MODS model was (liver, kidney) reproduced successfully.3. Combine the results of survival time, biochemical indicators and blood gas measurement, a single dose of45mg/kg intravenous administration of polydatin is the most appropriate dose. The second part:the therapeutic effect of polydatin on MODS rats and its mechanism explorationMethodsThe experiment rats were divided into control group, MODS+NS group and MODS+PD group. The procedures in control group and MODS+NS group were the same as in the first part. In MODS+PD group,45mg/Kg body weight of rats with equal volume of NS was adopted in this study. In order to assess the multiple organ condition of rats24hours after MODS induction (6-8in each group), serum biochemical parameters (BUN, CR, TBIL, LDH, CK-MB, ALT and AST), arterial blood gases, advanced oxidation protein products and inflammatory cytokines were measured, respectively. Meanwhile, after being killed by cervical dislocation, HE staining, apoptosis-related proteins (Bcl-2, Bax, Cytochrome C) by immunohistochemical staining in multiple organs (liver, kidney and lung) and tissue homogenates (liver and kidney) for caspase-3activity were all assayed (n>3in each group). All datas were input in SPSS13.0software, survival time analysis was study by Kaplan-meier method, One-way ANOVA was used to compare the mean+SD of dates among groups, LSD-t was selected to test multiple samples.Results1. Effects of PD on overall survival in a rat model of MODSAll of the rats in the control group survived. The median survival times for the MODS+PD and MODS+NS groups were50.5±8.72h and32±6.41h, respectively.2. Effects of PD on major blood biochemical parameters and arterial blood gases in MODSThe values for all blood biochemical parameters(Bun, CR, TBIL, LDH, CK, ALT and AST) were significantly higher in the MODS+NS group compared to the corresponding values in the control group (48.6u/L vs200.4u/L in ALT;76u/L vs545.5u/L in AST;205u/L vs417.6u/L in LDH;0.4μmol/L vs2.7μmol/L in TBIL;8.3μmol/L vs16.2μmol/L in BUN;22.3μmol/L vs37.2μmol/L in Cr;268.8u/L vs 820.2u/L in CK. all P<0.05,respectively.). Additionally, the changes of blood gas parameters in MODS+NS group, including pH, Lac, Base and HCO3-were ameliorated by treatment with PD in the MODS+PD group (all P<0.05).3. Effects of PD on organ pathological changes in MODSNo significant abnormalities were observed in the control group. However, pathological changes in the kidney, liver and lung were observed in the MODS+NS group. In the kidney, a number of proximal tubular epithelial cells displayed cloudy swelling and degeneration, and some regions of the proximal tubular epithelium were absent. Additionally, the proximal tubular lumen was narrowed, with deposition of small thrombi. In the liver of the MODS+NS group, a number of hepatic central veins were narrowed with sinusoidal dilatation and congestion, the cytoplasm of the hepatocyte was cloudy and swollen, and many hepatocytes had ballooned or degenerated. In the lung tissue of the MODS+NS group, pulmonary interstitial edema and broadening leading to alveolar collapse was observed, and numerous inflammatory cells had infiltrated around the blood vessels and interstitium. Compared to the MODS+NS group, administration of PD to the MODS+PD group alleviated the severity of the pathological changes observed in the kidney, liver and lung.4. Effect of PD on the serum levels ofAOPPsin MODSCompared to the MODS+NS group, the serum levels of AOPPs were significantly reduced in the MODS+PDgroup (P<0.05). While the levels of AOPPs was44.73μM in the MODS+NS group, which was reduced by24%in MODS+PD group(F=7.919, P<0.05)5. Effects of PD on the expression of apoptosis-related proteins in organs during MODSLight positive cytochrome C staining was observed in the control group, with more intense positive cytochrome C staining observed in the kidney and liver of the MODS+NS and MODS+PD groups. No significant differences in cytochrome C staining were observed among the lung tissues in the three groups. The relative grey values for cytochrome C were significantly higher in the kidney and liver of the MODS+NS group than that in the MODS+PD group (110%vs.30.2%, F=70.4, P <0.01and40%vs.11.2%, F=22.4, P<0.05, respectively).Light positive staining for Bax was observed in the kidney and liver of the control group, with more intense positive staining observed in the kidney and liver of both the MODS+NS and MODS+PD groups. No significant difference of the expression of Bax in the lung tissues was observed among the three groups. The relative grey intensity values for Bax in the kidney and liver were higher in the MODS+NS group than the MODS+PD group (67.1%vs.24.2%in kidney, F=110.7, P<0.01;49.6%vs.34.1%in liver,, F=67.4, P<0.05).Positive Bcl-2staining was observed in the kidney and liver of the control group, with a lower staining intensity in the kidney and liver of both the MODS+NS group and MODS+PD group. No significant differences in Bcl-2lung tissues staining were found among the three groups. However, the relative grey values for Bcl-2in the kidney and liver were higher in the MODS+NS group than the MODS+PD group (31.4%vs.9.9%in kidney, F=21.1, P<0.01;33.3%vs.18.3%in liver, F=4, P<0.05; Fig.8B).Additionally, caspase-3activity was assayed in kidney and liver homogenates24h after the induction of MODS using an ELISA. Compared to the control group, caspase-3activity was higher in the MODS+NS group and MODS+PD group. However, the caspase-3activity of the MODS+NS group was significantly higher than the MODS+PD group (148%vs.52.1%in kidney, F=44.5, P<0.01;123.8%vs.39.8%in liver, F=27.9, P<0.01)6. Impact of PD on the serum levels of inflammatory cytokines in MODSCompared to the control group, the levels of all three cytokines were higher in the MODS+NS group. However, compared to MODS+NS group, the levels of all three cytokines were significantly reduced the MODS+PD group (IL-6: approximately12-fold vs.8-fold, F=33.6, P<0.01; TNF-α:13-fold vs.10-fold, F=77.5, P<0.05; IL-1:8-fold vs.5-fold, F=41, P<0.05.)Conclusion PD prolonged overall survival time and improved functions of organs (liver, kidney and lung) in MODS rats.The mechanism of PD on MODS may be related to its inhibition of oxidative stress, reducing inflammation and inhibiting the activation of apoptosis pathway mediated by mitochondria etc.更多还原