【作者】 谭家庆；

【导师】 丁春华；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2005， 硕士

【摘要】 目的:腹腔感染所致的感染性休克是一种临床常见的病理过程,发病急骤,并发症多,可累及机体多个器官,甚至导致多器官功能障碍综合征(multiple organ dysfunctionsyndrome,MODS)。而肺脏和肾脏是经常受累的器官,主要为内素素诱导诱导炎症介质失控性释放,从而引起机体器官的损伤,但其具体的作用机制尚未完全明了,氧化应激可能是肺和肾损伤的主要机制之一。已有研究表明,一氧化碳(carbon monoxide,CO)可抑制机体炎症因子的产生,对抗氧自由基损伤,但对盲肠结扎穿孔(cecal ligation andpuncture,CLP)导致感染性休克时肺和肾损伤是否也具有保护作用尚有待探讨。本研究使用CLP 的方法建立感染性休克模型,通过应用血红素氧合酶(heme oxygenase,HO)诱导剂氯血红素(Hemin)和抑制剂锌原卟啉(Zinc protoporphyrin,ZnPP)调节HO-1 活性来控制内源性CO 的生成,探讨内源性CO 对肺和肾损伤的保护作用及其可能机制。方法:实验用雄性Sprague Dawley(SD)大鼠,体重200～250 克,随机分为四组,每组24 只。(1)假手术组:腹腔注射25%乌拉坦4mg/kg 麻醉,行腹部正中切口,于盲肠根部穿线而不结扎,逐层缝合;(2)CLP 组:开腹后结扎盲肠根部,在肠系膜相对的盲肠浆膜面用18 号针头穿刺两个孔(相距1 厘米),逐层缝合;(3)CLP+Hemin 组:手术前10min 经舌下静脉注射Hemin(10mg/kg),其余操作同CLP 组;(4)CLP+ZnPP 组:手术前10min 舌下静脉注射ZnPP(10mg/kg),其余操作同CLP 组。各组分别于模型成立后2h、4h、6h 从右颈静脉插管至右心房,分离左颈静脉插管至主动脉根部,采集血液分别代表出肺血(Out-going pulmonaryblood,OPB)和入肺血(In-flowing pulmonary blood,IPB),以检测其碳氧血红蛋白(carboxyhemoglobin,COHb)水平,以此代表CO 含量,经颈静脉取血液标本后继续放血处死动物,取肺、肾组织标本,将血液标本离心后取上清液,将肺、肾组织标本制备匀浆,以检测血液及肺、肾组织中的超氧化物歧化酶(superoxidedismutase,SOD)活性、丙二醛(malondialdehyde,MDA)含量。另外留取肺、肾组织标本,光镜观察其病理学改变,免疫组织化学染色观察其HO-1 的表达及分布。数据用均数±标准差(x±s)表示。单因素方差分析进行组间比较,有显著差异者用最小显著差法进行两两比较,以p<0.05 为有显著差异。结果:各组不同时间点OPB 和IPB 的COHb 含量和血液及肺、肾组织匀浆的SOD 活性、MDA 含量。①与同时间点Sham 组相比,CLP 组2h、4h、6hOPB 和IPB 的COHb含量及其差值(OPB-IPB)均显著升高(P 均<0.05),且相邻时间点间也有显著差异(P 均<0.05),并呈递增趋势,血液及肺、肾组织匀浆的SOD 活性显著降低(P 均<0.05 或0.01),相邻时间点间也有显著差异(P 均<0.05 或0.01),呈递减趋势,而MDA 含量则显著升高(P 均<0.05 或0.01),呈递增趋势;②与同时间点CLP 组相比,CLP+Hemin 组2h、更多还原

【Abstract】 Objective: Endotoxemia results from intestinalinfection is a kind of familiar pathological process. Itdevelops quickly and has many complications. It can involvemultiple organ or lead to multiple organ dysfunctionsyndrome(MODS) sometimes. Lung and kidney are ofteninvolved, mainly because endotoxin induces inflammationmediators to result in uncontrollable inflammation. Itsdetailed mechanism is unclear and oxidative injury isprobably one of the main mechanisms. Studies showed thatendogenous carbon monoxide(CO) could inhibit theproduction of inflammation mediator and alleviate theoxidative injury . However, whether CO can protect lung andkidney from injuries induced by cecal ligation andpuncture(CLP) deserves studying. In this study, septic shockwas induced by operation of CLP. Controlling the product ofCO by using the donor(Hemin) of heme oxygenase-1 (HO-1)and inhibitor(Zina protoporphyrin-Ⅸ,ZnPP) to regulate theactivity of HO-1, we evaluated the protective effect on lungand kidney of CO.Method: By using a Sprague-Dawley(SD) rat model of CLP,96 rats were randomly divide into four groups. ①the Sham group:only traversed a string at cecal root, did not ligate and puncture;②CLP group:treated by the operation of CLP;③CLP+Hemin(COdonor) group: Hm (10mg/kg, 10mg/ml, iv)was injected 10minbefore CLP operation; ④CLP+ZnPP(the specific HO-1inhibitor)group: ZnPP(10mg/kg, 10mg/ml, iv) was injected 10minbefore CLP operation. Parameter was observed respectively 2h, 4h,6h after CLP operation. Carpbxyhemoglbin (COHb) level inin-flowing pulmonary blood(IPB) and out-going pulmonaryblood(OPB) were detected. The level of COHb represented COcontent. The malondialdehyde(MDA) content and the activity ofsuperoxide dismutase(SOD) in blood、lung and kidney weredetected using test kits. The pathomorphological changes in thelung and kidney were observed under light microscope andimmunohistochemical technique were used to detect expression ofHO-1 protein in the lung and kidney after CLP. The results were asfollows:Level of COHb in OPB and IPB、content of MDA andactivity of SOD in blood、lung and kidney: Compared withSham group at corresponding time points, level of COHb inOPB、IPB and numerical value of (OPB-IPB) of CLP groupincreased significantly (P<0.05), and showed a tendency ofincreasing significantly with time (P<0.05). Activity of SODin blood、lung and kidney decreased significantly(P<0.05 or0.01), and showed a tendency of decreasing significantlywith time(P<0.05 or 0.01).While content of MDA in blood、lung and kidney increased significantly(P<0.05 or 0.01), andshowed a tendency of increasing with time;Compared withCLP group in corresponding time points, level of COHb inOPB 、IPB and numerical value of (OPB-IPB) ofCLP+Hemin group increased significantly(P<0.05),andshowed a tendency of increasing significantly withtime(P<0.05 or 0.01).Activity of SOD in blood、lung andkidney increased significantly (P<0.01), and showed atendency of increasing significantly with time(P<0.05 or0.01). While content of MDA in blood、lung and kidneydecreased significantly(P<0.05 or 0.01), and showed atendency of decreasing with time; Compared with CLPgroup in corresponding time points, level of COHb in OPB、IPB and numerical value of (OPB-IPB) of CLP+ZnPP groupdecreased significantly(P<0.05), and showed a tendency ofdecreasing with time. Activity of SOD in blood、lung andkidney decreased(P<0.05 or 0.01), and showed a tendency ofdecreasing with time. While content of MDA in blood、lungand kidney increased significantly(P<0.05 or 0.01), andshowed a tendency of decreasing significantly with time(P<0.05 or 0.01).The light microscope results showed that the destroiedstructure of alveolus, diffuse infiltration and migration ofacute inflammatory cells. Partition of alveolus wascongestive and dropsical. There were hyperemia、edema anddiffuse infiltration of acute inflammatory cells in glomerulus.And there was tumefaction and exfoliation on renal tubulesepithelium cells. Red blood cells could be seen in interstitialparts. The injuries in CLP+Hemin group at 2h、4h and 6hwere lighter than that of CLP group at corresponding timepoints. The injuries of CLP+ZnPP group at 2h、4h and 6hwas severer than CLP group on corresponding time.Immunohidtochemical studies showed:①Lung: In Shamgroup, there was no brown positive signals of HO-1;in CLPgroup, Positive signals were obvious and mainly located incytoplasm of macarophage of alveoli、epithelial cells ofalveoli and airway、vascular smooth muscle cells; Positivesignals in CLP+Hemin group were more obvious than that ofCLP group; In CLP+ZnPP group, positive signals wereweaker than that of CLP group. ②Kidney: In Sham group,there was mild brown positive signals of HO-1;The positivesignals in CLP group were more obvious than that of Shamgroup and mainly located in cytoplasm of endothelial cells ofcollecting tube、Henle’s loops and glomerular capillary loops;The positive signals in CLP+Hemin group were moreobvious than that of CLP group, especially in glomerularcapillary loops and renal tubules. The signals in CLP+ZnPPgroup is weakened compared with CLP group atcorresponding time points, mainly in glomerular capillaryloops and renal tubules.Conclusion:①CLP caused septic shock in rats andresulted in the acute injury of lung and kidney. The model of更多还原