抑制肠源性内毒素改善大鼠极限肝切除术预后的三重作用及机制
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摘要
背景和目的：肝切除术仍是临床治疗肝胆管肿瘤的最有效办法。然而对于肝脏多发或者巨大肿瘤，常须扩大手术切除范围。大范围的肝切除术将导致一定数量的患者由于预留肝体积不足和感染等并发症发生率的增高，其预后和肿瘤切除率均受到严重影响。以往研究者多将目光集中在如何充分利用肝脏再生潜能，加快肝再生速度以尽早恢复功能性肝体积和肝脏功能。然而，越来越多的证据表明，对于大范围的肝切除术而言，肝切除术后早期过快的肝再生将无益于肝脏组织结构的重建和预后。因此，对于较大范围的肝切除术，必须通过对残留肝脏细胞的充分保护，提供有利于肝脏多种细胞协调增殖的环境，防止肝脏免受细菌和代谢产物等物质的损伤，才是避免发生肝功能衰竭、保证肝细胞增殖和肝脏组织结构重建、改善术后肝功能和预后的关键。虽然对于肝切除术后感染并发症已经有了足够的认识和有效的预防、治疗方法，然而，临床上对于抑制肠源性内毒素LPS（lipopolysaccharide）在大范围肝切除术中的重要性并未给予充分重视，肝脏切除范围的大小也没有成为衡量是否预防性应用抗生素的标准。LPS作为革兰氏阴性细菌内毒素的主要成分，能够通过介导炎症因子大量产生、增加肠粘膜通透性、降低血胆屏障功能等严重影响肝切除术后患者肝功能的恢复和预后。而在大范围肝切除情况后，小肝综合征、胆汁淤积致和门脉高压的发生，使LPS生成、吸收增加，清除减少，又进一步促使肝脏和全身发生炎症反应的发生，最终使机体进入恶性循环状态，并很快导致肝功能衰竭和MODS。如能通过预防性应用抗生素以控制肠道菌群、减少LPS的产生和吸收，或将有利于改善大范围肝切除术后患者的肝功能和预后，并提高对于肝脏多发肿瘤和巨大肿瘤的手术切除率。此外，抑制肠源性LPS的产生对大范围肝切除术预后的保护作用及机制并不完全清楚。
在本实验研究中，我们通过建立大鼠不同范围肝切除术的标准化模型，探讨不同范围肝切除术，特别是大范围肝切除术（90％）后，大鼠血LPS和肝功能指标等的变化。并于通过大鼠90％肝切除术围手术期预防性抗生素（庆大霉素）的使用，抑制肠源性内毒素的产生，观察术后大鼠血浆LPS水平变化，以及在LPS变化基础上发生的包括肝脏血胆屏障、肠粘膜屏障、细胞增殖和凋亡等的一系列病理生理改变，进一步了解抑制肠源性LPS对大范围肝切除术后病理生理改变的影响及可能机制，以求能够通过预防性抗生素的应用，抑制肠源性内毒素，进而改善大范围肝切除术患者的肝功能和预后，提高肿瘤切除率。
方法：
1.利用显微外科技术，建立大鼠不同范围肝切除术标准化模型。所有SD大鼠肝切除术均在乙醚麻醉下经固定、消毒、开腹后进行。70％肝切除术（PH），按顺序切除左外叶、中叶；85％肝切除是在70％肝切除基础上，再先后切除尾状叶和右下叶；90％肝切除（极限肝切除术）则是在70％肝切除基础上，再行切除右下叶和右上叶；假手术组作为对照组，仅在乙醚麻醉下开腹后立即关闭腹腔。为了尽可能缩小差异，所有入肝管道均采用显微外科技术于肝门部分离，套线结扎后离断；除左外叶以外，所有肝静脉均利用解剖位置采用肝实质内缝扎的方法离断。盲肠结扎穿刺术（CLP）用以作为测定血浆LPS水平时的阳性对照。
2.实验中首先探讨了不同肝切除范围大鼠病理生理和肝脏功能改变的差异。为了研究抑制肠源性LPS对大鼠90％肝切除术后肝功能和生存率等的影响，我们选择对革兰氏阴性菌有较强抑制作用的硫酸庆大霉素作为治疗组用药。又由于庆大霉素具有口服不易吸收的特性，给药途径我们采用插管灌胃的方式，以明确抑制胃肠道内LPS的产生。硫酸庆大霉素5mg／ml，1ml，分别于大鼠90％切除术前1天、术后即刻、术后1天，三次插管灌胃，共计15mg。
3.为了同时测定静脉血和门脉血血浆LPS水平，对于实验动物血液标本的采集我们均采用乙醚麻醉下原腹腔切口开腹后，分别经下腔静脉和门静脉取血，同时留取组织标本后，乙醚过量吸入的方法处死大鼠。
4.我们运用免疫荧光、western blot和Real－time PCR等实验方法测定肝组织和肠粘膜紧密连接蛋白Occludin的表达，探讨90％肝切除术后血胆屏障和肠粘膜屏障功能；并通过EGF、TLR－4、PCNA、SOCS－3、Caspase3、iNOS等因子的测定，探讨抑制肠源性内毒素LPS对大鼠90％肝切除（极限肝切除）术后肝细胞增殖、凋亡的影响和机制。
结果：
1.成功建立SD大鼠不同范围肝脏切除标准化模型。
2.与其它组别相比大鼠90％肝切除组术后肝功能障碍更为明显，表现为血清ALT、AST和TBIL水平明显升高，ALB水平明显降低。通过比较90％肝切除组大鼠与庆大霉素治疗组大鼠肝功能我们发现：1.庆大霉素治疗组大鼠术后1天、2天血ALT、AST和TBIL水平均明显高于Sham组，ALB水平明显低于Sham组；2.治疗组术后1－3天大鼠血清ALT、AST和TBIL水平均明显低于90％肝切除组（P≤0.01）；3.治疗组术后1天血清ALB与90％肝切除组相比并无显著差异，术后2天和术后3天明显高于90％肝切除组（＃P<0.01、＊P<0.05），两组术后5天与Sham组相比基本恢复正常。表示庆大霉素治疗明显改善大鼠90％肝切除术后肝功能。同时治疗组大鼠术后生存率明显高于90％肝切除组，治疗组术后7天生存率为60％，而SH组仅为20％。
3.鲎试剂法检测大鼠血浆LPS水平，以CLP术后大鼠血LPS水平作为阳性对照。CLP组术后门静脉及下腔静脉血LPS水平较其余各组均显著升高（＃P<0.01）；PH组与Sham组相比无显著差异；90％肝切除组大鼠术后门脉及静脉血LPS水平明显升高（＃P<0.01）；治疗组大鼠术后LPS水平也较术前明显升高（P≤0.01），然而该组大鼠与90％肝切除组大鼠血LPS水平相比，术后1天治疗组大鼠门脉血LPS明显低于90％肝切除组（＊P<0.05），而静脉血LPS两组间并无显著差异，术后2天，治疗组大鼠门静脉和下腔静脉血LPS水平均明显低于90％肝切除组（＃P<0.01）。
4.免疫荧光和Western Blot检测肝脏及肠道紧密连接蛋白Occludin蛋白表达水平。结果发现90％肝切除后肝脏及肠道Occludin表达明显下调，提示血胆屏障和肠粘膜屏障功能障碍。与血胆屏障功能障碍伴随发生的是血TBA水平明显升高。与此相比，治疗组大鼠肝脏及肠道Occludin表达均明显上调，血TBA水平也明显下降。Occludin表达水平与EGF mRNA表达水平在不同范围肝切除比较中呈正相关，即随切除范围的扩大，EGF mRNA和Occludin表达水平均下降，而庆大霉素抑制肠源性LPS所引起的Occludin表达上调也伴随着肝组织EGF mRNA表达的上调。抑制肠源性内毒素通过上调EGF的表达，而维持肝脏及肠道紧密连接Occludin的表达，进而改善血胆屏障功能和肠黏膜屏障功能。
5.通过对肝组织PCNA的检测发现，90％肝切除术后早期发生了过激的细胞增殖反应，并影响到后期细胞的进一步增殖，庆大霉素抑制LPS减缓了术后早期的肝细胞增殖并有利于肝脏进一步的再生反应，表现为术后1天肝组织PCNA表达与90％肝切除相比明显下调，而术后2天即表现出较强的PCNA表达。这与LPS受体TLR－4及其下游肝细胞增殖抑制因子SOCS－3表达水平有关。90％肝切除术后TLR－4表达首先下调，意味着肝脏对LPS敏感性下降，TLR－4信号通路下游抑制性因子SOCS－3延迟表达，直到TLR－4表达上调（术后2天），SOCS－3才开始大量表达，进而抑制了肝细胞的进一步增殖。庆大霉素抑制LPS，能够上调90％肝切除术后1天TLR－4表达水平，引起肝脏诸如IL－6等炎症因子的大量产生，进而诱导SOCS－3表达上调，以控制早期过激的肝细胞增殖反应，随后SOCS－3表达的下调，使肝细胞得以有序持续增殖，直至恢复正常体积。
6.90％肝切除还伴有明显的细胞凋亡反应，表现为20kDa大小左右的Caspase3活化片段表达的加强。这与LPS升高所引起的iNOS表达显著上调有关，90％肝切除术后肝组织iNOS mRNA表达显著上调。庆大霉素抑制肠源性LPS能够降低iNOS的表达水平，使Caspase3活化下调，凋亡反应减弱。即，90％肝切除大鼠肝组织iNOS mRNA表达明显上调，这将引起NO的大量产生，由于NO的产量与凋亡相关因子表达密切相关，NO的大量产生引起促凋亡因子的激活，表现为Caspase3活化加强，最终细胞凋亡明显加强。而抑制LPSneng shi能使大鼠肝组织iNOS mRNA表达与90％肝切除组相比明显下调，Caspase3活化也受到抑制，凋亡反应减弱。
结论：
1.深入研究不同范围肝切除术后肝脏、肠粘膜等器官脏器发生的病理生理变化和机制必须建立在不同范围标准化肝切除模型的基础上。由于目前报道的大鼠肝切除术，特别是大鼠90%肝切除术，其手术操作方式复杂且多种多样，并无统一标准，其后续研究也都建立在各自所建肝切除模型之上，导致研究所获得的包括肝脏功能和生存率等结果均有很大差别。这也是导致对于不同范围肝切除大鼠术后的病理生理过程和具体机制研究较少的原因之一。为此，必须首先通过规范化和标准化大鼠不同范围肝切除手术方式和方法，建立了标准化的大鼠不同范围肝切除模型，以避免对大鼠进行不同范围肝切除术时由于手术操作方式的不同所造成的差异，使我们能够以此为模型探讨不同范围肝切除术后大鼠肝脏和机体发生的病理生理改变和可能的相关机制，进一步深入认识临床上不同范围肝切除患者术后可能发生的病理生理过程。研究中我们主要利用血管铸型和显微外科技术，精确、详细且具体的对手术方式加以规范，以尽可能的减少误差。根据实验室前期大鼠肝脏血管铸型实验结果，在充分了解了大鼠门静脉、肝动脉、肝静脉和胆管系统等重要解剖结构后，严格遵守操作原则，规范手术方法和步骤，特别是通过采用显微外科技术，精确的对大鼠肝门部和肝实质内门静脉和肝静脉等重要结构进行处理，尽可能的缩小了手术操作和方法对于不同范围肝切除产生的差异，成功建立和规范了大鼠不同范围肝切除标准化模型的方法，为后续研究奠定了基础。模型稳定性通过肝切除术后肝功能指标加以验证。
2.大鼠不同范围标准化肝切除模型方法建立成功，各实验组样本之间差异小，且肝脏功能恶化程度以及术后血内毒素水平均与切除范围相关。该部分结果表明，随着肝切除范围的扩大，ALT、AST、TBIL和ALB等肝功能指标恶化更为明显。在70％和80％肝切除情况下，虽然肝功能指标与假手术组相比确有明显改变，但由于大鼠肝脏强大的再生和解毒能力，肝功能指标术后迅速开始好转。而90％肝切除大鼠除了其肝功能指标恶化更为明显外，其好转趋势也较70％肝切除和85％肝切除组明显减慢，甚至术后短期内很难恢复至正常水平。预示90％肝切除大鼠肝脏自身恢复不良，或毒性物质没有解毒，仍在一段时间内对肝脏造成持续损伤。另外，通过对比大鼠不同范围肝切除术术后血浆内毒素（LPS）水平，我们发现：更大范围的肝切除术或极限肝切除术（90％肝切除）将导致血LPS水平明显升高，这与肝切除术后肝功能的损伤呈正相关，肝脏以及肠道粘膜屏障功能的损伤与血LPS水平的升高互为因果，且均由肝切除范围的扩大所致。
3.围手术期预防性应用庆大霉素能够降低大鼠90％肝切除术后血浆LPS水平，同时改善术后肝脏功能，有利于肝功能的恢复和预后。通过比较90％肝切除组大鼠与在围手术期预防性应用庆大霉素的治疗组大鼠血浆LPS水平，我们发现：庆大霉素治疗能够显著降低术后大鼠血浆LPS水平，在此基础上，大鼠术后肝脏功能恶化程度也明显好转，且恢复较快。最终明显改善90%肝切除大鼠术后生存率。
4.肠道紧密连接蛋白Occludin表达水平的显著降低是大鼠极限肝切除术后肠粘膜损伤的特征性表现，临床上可能能够以此作为判断肝脏切除范围是否达到极限水平，是否需要预防性应用抗生素抑制肠道细菌及其毒性产物以改善预后的依据。
90％肝切除大鼠肠道及肝脏紧密连接蛋白Occludin表达明显下调，预示着其肝脏血胆屏障和肠粘膜屏障功能障碍，90％肝切除术后血胆酸（TBA）水平的上升和血LPS水平的大幅升高，分别为血胆屏障和肠粘膜屏障功能损伤的表现。围手术期预防性应用庆大霉素，抑制肠源性细菌及其毒性产物LPS和炎症因子等，明显上调了肝脏和肠道Occludin的表达，意味着肝血胆屏障和肠粘膜屏障功能得以改善。研究中虽然发现了不同范围肝切除术后，Occludin表达水平的改变与EGF mRNA表达相关或部分相关，庆大霉素抑制肠源性LPS即能够上调EGF mRNA表达，又能上调Occludin的表达，但EGF与Occludin的表达是否存在着直接联系，尚须进行更为深入的研究。
5.围手术期预防性应用庆大霉素灌胃能够通过调节肝脏TLR－4mRNA和蛋白表达，从而改变肝脏对肠源性LPS的敏感性，调动细胞增殖反馈抑制相关因子，延缓细胞过激增殖。
大鼠90％肝切除术后，肝细胞增殖过激，表现为术后PCNA表达“先增后降”的趋势，即，术后第1天明显上调，而术后2天明显下调，其过激的肝细胞增殖不利于肝脏的再生。这一现象的发生与不同范围肝切除术后肝脏LPS受体－TLR－4表达水平有关。TLR－4信号通路下游的IL－6等炎症因子的大量产生，能够诱导抑制性因子SOCS－3的表达，即反馈抑制过激的肝细胞增殖的效果减弱。然而，作为LPS受体复合物之一的TLR－4表达在90%肝切除术后明显下调，意味着肝脏对LPS反应性下降，因此未能引起下游IL－6等炎症因子的大量产生，而在术后2天，TLR－4表达明显上调，使得IL－6等大量产生，诱导SOCS－3的表达，进而抑制了肝细胞的进一步增殖。然而，预防性应用庆大霉素降低血LPS水平，通过上调术后1天TLR－4表达，并介导SOCS－3表达，发挥控制细胞增殖的作用，此后（术后2天）治疗组大鼠肝脏TLR－4表达的下调，降低了SOCS－3表达水平，肝组织PCNA蛋白表达也较90％肝切除组显著上调，提示肝脏再生良好。
6.围手术期预防性应用庆大霉素灌胃能够通过调节肝脏iNOS mRNA的表达的量，以降低NO产量，从而抑制细胞凋亡。预防性应用庆大霉素抑制血LPS水平能够显著降低肝脏iNOS表达，进而调节肝内NO产量，使其发挥抑制凋亡的作用，结果表明，Caspase3活化明显降低。而90％肝切除组大鼠iNOS表达明显上调，并伴随Caspase3活化增强。
Background and objective:
Background and Objectives: liver resection is still the most effective treatment forhepatobiliary malignancies. However, for multiple or huge tumors, aggressive hepatectomymight be inevitable. Extended liver resection would doubtlessly increase the incidence ofinfection and other complications due to insufficient liver remnant, effecting the prognosisand curative rate after hepatectomy. Previously, most researchers have been devoted toexplore the potential regenerative capacity of the liver as well as its acceleration to retainsufficient functional volume postoperatively. However, accumulating data hasdemonstrated that it is the excessive regeneration after aggressive hepatectomy thathindered proper structure restoration. Therefore, for extended liver resection, it is necessaryto protect the residual hepatocytes, regulate the micro－environment to promote hepatocyteproliferation, and prevent liver damage from bacteria and/or their metabolites. Liver failurecan thusly be avoided, and hepatocyte proliferation can be guaranteed to ensure structurereconstruction and improve prognosis. Although there have been sufficient knowledge andeffective prevention, treatment method for postoperative infection as a complication forliver resection, in practice, inhibition of intestinal LPS (lipopolysaccharide) was never paidenough attention, and application of antibiotics in prevention has never been standardized.LPS is the main component of the gram－negative bacterial endotoxin, it induce a largenumber of inflammatory cytokines, increase intestinal permeability, reduce blood－bilebarrier function, etc. thereby seriously affect recovery and prognosis. After extendedhepatectomy, small for size syndrome, cholestasis and portal hypertension would give riseto increased LPS generation, absorption and decreased clearance, which further aggregateliver and systemic inflammatory response, and eventually lead to liver failure and MODS. Application of prophylactic antibiotics in the control of intestinal flora to reduce theproduction and absorption of LPS, might help to improve prognosis of aggressivehepatectomy. In addition, the mechanism of prognosis improvement by inhibiting LPS isnot entirely clear.
In this experiment, we established standardized model of rat hepatectomy in differentextend, investigate blood－LPS and other indicators after different resection extend,especially subtotal hepatectomy (90％). And we applied perioperative antibiotics(gentamicin) in rats after90％hepatectomy, examined inhibition of LPS, as well as aseries of pathophysiological changes including liver blood bile barrier, mucosal barrier, cellproliferation and apoptosis, to further understand the mechanism of effect induced by LPSinhibition.
Method:
1. Using microsurgical techniques to establish standardized model of rat hepatetomy.All SD rat liver resection were operated after disinfection, laparotomy and under etheranesthesia. In70％hepatectomy (PH), left lateral lobe and mid lobe were removed, in85％hepatectomy, the caudate lobe and right lower lobe were removed in addition to PH;90％liver resection (subtotal hepatectomy) the right lower lobe and right upper lobe wereremoved in addition to PH; sham group were used as control. All hilar are dissected usingmicrosurgical techniques and ligated trasected under microscope; all hepatic vein weretransected after intra－parenchyma suture ligation. Cecal ligation and puncture (CLP) isused as a positive control in the determination of plasma LPS level.
2. Firstly, the experiment investigated pathophysiological changes after hepatectomyof different extend. In order to study the effect of inhibiting enterogenous LPS rats on90％liver resection liver function and survival, we chose a strong inhibitory effect on Gram－negative bacteria, gentamicin sulfate medication as the treatment group. Gentamicin is notabsorbed if applied orally, we used cannula gavage as method of administration.Gentamicin sulfate5mg/ml,1ml was administrated, in rats one day before the resection,immediate postoperative, and one day after subtotal hepatectomy, a total of15mg.
3. For simultaneous determination of venous and portal blood plasma LPS level, ratswere operated under ether anesthesia to take the inferior vena cava and portal vein blood,and then sacrificed via ether overdose inhalation.
4. We implemented immunofluorescence, western blot and Real－time PCR todetermine liver tissue and intestinal mucosa expression of Occludin expression toinvestigate the90％liver resection blood－bile barrier and mucosal barrier function; andby determination of EGF, TLR－4, SOCS－3, PCNA, Caspase3, iNOS factordetermination to explore the mechanism of inhibiting LPS on rats with90％hepatectomy.
Results:
1. SD rat model with different extend of liver resection were successfully establishedand standardized.
2.With the expansion of extend of resection, postoperative liver function deteriorate.After90％hepatectomy, serum ALT, AST were significantly higher, especially the firstthree days, then gradually recovered.70％and85％hepatectomy rats postoperative7days survival rate was100％,90％hepatectomy was20％.
3. The Limulus test detects rat inferior vena cava and portal blood plasma LPS foundthat, after cecal ligation and puncture (CLP) rat1－2天plasma LPS level weresignificantly elevated compared with the rest of the groups; after90％hepatectomy, portalblood and inferior vena cava LPS was significantly higher than that of70％hepatectomyrats. Treatment group demonstrated significantly decreased blood LPS levels and at thesame time improved postoperative liver function and survival (60％).
4. We also found that after90％hepatectomy, Occludin expression decreasedsignificantly both in liver tissue and in intestinal mucosa, compared with sham－operatedrats, indicating blood bile barrier and the intestinal mucosal barrier dysfunction. SerumTBA was significantly higher in90％hepatectomy than70％hepatectomy, indicating thatthe blood－bile barrier function was severely impaired. In treatment group, liver tissueOccludin expression was significantly higher, immunofluorescence revealed less damagedliver and intestinal mucosa structure, serum TBA levels were also significantly lower thanthe90％hepatectomy rats.
5. EGF mRNA expression levels was related to the extend of liver resection,90％hepatectomy resulted in EGF mRNA down－regulation, while treatment resulted insignificant EGF up－regulation, which may be related to Occludin expression. Inhibition ofLPS would up－regulate EGF expression, and maintain liver and intestinal tight junctions Occludin expression, thus improving the the blood bile barrier function and intestinalbarrier function.
6.90％liver resection induced over－response of cell proliferation, which is relatedto TLR－4－mediated decreased sensitivity to LPS. In90％hepatectomy, postoperativefirst day, TLR－4mRNA and protein expression in liver tissue were significantly lowerthan the70％hepatectomy group, and lower than that of the treatment group. Treatmentresulted in increased TLR－4level, thereby inhibited liver regeneration. Post－operative1day, PCNA protein expression in liver tissue of treated rats was significantly lower than the90％hepatectomy rats, indicating effective control of excessive hepatocyte proliferation.
7. In90％hepatectomy group, iNOS mRNA expression was significantly increased,leading to Caspase3activation and significant apoptosis. INOS mRNA expression intreatment group was significantly down－regulated with suppreesed Caspase3activation.
Conclusion:
1. SD rat model with different extend of liver resection were successfully establishedand standardized. With the expansion of resection extend, liver function indicators andpathological findings showed more server structural and functional damaged.90％hepatectomy resulted in elevated plasma LPS level, and the use of prophylactic antibiotics,significantly decresed plasma LPS level and improved liver function and survival rates after90％liver resection.
2. Inhibition of enterogenous LPS by upregulating liver tissue expression of EGF andOccludin, improve blood bile barrier functions and structure of the intestinal mucosalbarrier.
3. LPS inhibition would upregulate TLR－4, to increase the sensitivity of liver to LPS,which would lead to a substantial increase in IL－6and other cytokines, and then thedownstream feedback inhibition factor SOCS－3. Excessive proliferation of liver cellscould be inhibited, the liver cells would proliferate in an more organized manner.
4.Inhibition of enterogenous LPS inhibit liver cell apoptosis by down－regulatingiNOS expression, and reducing Caspase3activation.
在本实验研究中，我们通过建立大鼠不同范围肝切除术的标准化模型，探讨不同范围肝切除术，特别是大范围肝切除术（90％）后，大鼠血LPS和肝功能指标等的变化。并于通过大鼠90％肝切除术围手术期预防性抗生素（庆大霉素）的使用，抑制肠源性内毒素的产生，观察术后大鼠血浆LPS水平变化，以及在LPS变化基础上发生的包括肝脏血胆屏障、肠粘膜屏障、细胞增殖和凋亡等的一系列病理生理改变，进一步了解抑制肠源性LPS对大范围肝切除术后病理生理改变的影响及可能机制，以求能够通过预防性抗生素的应用，抑制肠源性内毒素，进而改善大范围肝切除术患者的肝功能和预后，提高肿瘤切除率。
方法：
1.利用显微外科技术，建立大鼠不同范围肝切除术标准化模型。所有SD大鼠肝切除术均在乙醚麻醉下经固定、消毒、开腹后进行。70％肝切除术（PH），按顺序切除左外叶、中叶；85％肝切除是在70％肝切除基础上，再先后切除尾状叶和右下叶；90％肝切除（极限肝切除术）则是在70％肝切除基础上，再行切除右下叶和右上叶；假手术组作为对照组，仅在乙醚麻醉下开腹后立即关闭腹腔。为了尽可能缩小差异，所有入肝管道均采用显微外科技术于肝门部分离，套线结扎后离断；除左外叶以外，所有肝静脉均利用解剖位置采用肝实质内缝扎的方法离断。盲肠结扎穿刺术（CLP）用以作为测定血浆LPS水平时的阳性对照。
2.实验中首先探讨了不同肝切除范围大鼠病理生理和肝脏功能改变的差异。为了研究抑制肠源性LPS对大鼠90％肝切除术后肝功能和生存率等的影响，我们选择对革兰氏阴性菌有较强抑制作用的硫酸庆大霉素作为治疗组用药。又由于庆大霉素具有口服不易吸收的特性，给药途径我们采用插管灌胃的方式，以明确抑制胃肠道内LPS的产生。硫酸庆大霉素5mg／ml，1ml，分别于大鼠90％切除术前1天、术后即刻、术后1天，三次插管灌胃，共计15mg。
3.为了同时测定静脉血和门脉血血浆LPS水平，对于实验动物血液标本的采集我们均采用乙醚麻醉下原腹腔切口开腹后，分别经下腔静脉和门静脉取血，同时留取组织标本后，乙醚过量吸入的方法处死大鼠。
4.我们运用免疫荧光、western blot和Real－time PCR等实验方法测定肝组织和肠粘膜紧密连接蛋白Occludin的表达，探讨90％肝切除术后血胆屏障和肠粘膜屏障功能；并通过EGF、TLR－4、PCNA、SOCS－3、Caspase3、iNOS等因子的测定，探讨抑制肠源性内毒素LPS对大鼠90％肝切除（极限肝切除）术后肝细胞增殖、凋亡的影响和机制。
结果：
1.成功建立SD大鼠不同范围肝脏切除标准化模型。
2.与其它组别相比大鼠90％肝切除组术后肝功能障碍更为明显，表现为血清ALT、AST和TBIL水平明显升高，ALB水平明显降低。通过比较90％肝切除组大鼠与庆大霉素治疗组大鼠肝功能我们发现：1.庆大霉素治疗组大鼠术后1天、2天血ALT、AST和TBIL水平均明显高于Sham组，ALB水平明显低于Sham组；2.治疗组术后1－3天大鼠血清ALT、AST和TBIL水平均明显低于90％肝切除组（P≤0.01）；3.治疗组术后1天血清ALB与90％肝切除组相比并无显著差异，术后2天和术后3天明显高于90％肝切除组（＃P<0.01、＊P<0.05），两组术后5天与Sham组相比基本恢复正常。表示庆大霉素治疗明显改善大鼠90％肝切除术后肝功能。同时治疗组大鼠术后生存率明显高于90％肝切除组，治疗组术后7天生存率为60％，而SH组仅为20％。
3.鲎试剂法检测大鼠血浆LPS水平，以CLP术后大鼠血LPS水平作为阳性对照。CLP组术后门静脉及下腔静脉血LPS水平较其余各组均显著升高（＃P<0.01）；PH组与Sham组相比无显著差异；90％肝切除组大鼠术后门脉及静脉血LPS水平明显升高（＃P<0.01）；治疗组大鼠术后LPS水平也较术前明显升高（P≤0.01），然而该组大鼠与90％肝切除组大鼠血LPS水平相比，术后1天治疗组大鼠门脉血LPS明显低于90％肝切除组（＊P<0.05），而静脉血LPS两组间并无显著差异，术后2天，治疗组大鼠门静脉和下腔静脉血LPS水平均明显低于90％肝切除组（＃P<0.01）。
4.免疫荧光和Western Blot检测肝脏及肠道紧密连接蛋白Occludin蛋白表达水平。结果发现90％肝切除后肝脏及肠道Occludin表达明显下调，提示血胆屏障和肠粘膜屏障功能障碍。与血胆屏障功能障碍伴随发生的是血TBA水平明显升高。与此相比，治疗组大鼠肝脏及肠道Occludin表达均明显上调，血TBA水平也明显下降。Occludin表达水平与EGF mRNA表达水平在不同范围肝切除比较中呈正相关，即随切除范围的扩大，EGF mRNA和Occludin表达水平均下降，而庆大霉素抑制肠源性LPS所引起的Occludin表达上调也伴随着肝组织EGF mRNA表达的上调。抑制肠源性内毒素通过上调EGF的表达，而维持肝脏及肠道紧密连接Occludin的表达，进而改善血胆屏障功能和肠黏膜屏障功能。
5.通过对肝组织PCNA的检测发现，90％肝切除术后早期发生了过激的细胞增殖反应，并影响到后期细胞的进一步增殖，庆大霉素抑制LPS减缓了术后早期的肝细胞增殖并有利于肝脏进一步的再生反应，表现为术后1天肝组织PCNA表达与90％肝切除相比明显下调，而术后2天即表现出较强的PCNA表达。这与LPS受体TLR－4及其下游肝细胞增殖抑制因子SOCS－3表达水平有关。90％肝切除术后TLR－4表达首先下调，意味着肝脏对LPS敏感性下降，TLR－4信号通路下游抑制性因子SOCS－3延迟表达，直到TLR－4表达上调（术后2天），SOCS－3才开始大量表达，进而抑制了肝细胞的进一步增殖。庆大霉素抑制LPS，能够上调90％肝切除术后1天TLR－4表达水平，引起肝脏诸如IL－6等炎症因子的大量产生，进而诱导SOCS－3表达上调，以控制早期过激的肝细胞增殖反应，随后SOCS－3表达的下调，使肝细胞得以有序持续增殖，直至恢复正常体积。
6.90％肝切除还伴有明显的细胞凋亡反应，表现为20kDa大小左右的Caspase3活化片段表达的加强。这与LPS升高所引起的iNOS表达显著上调有关，90％肝切除术后肝组织iNOS mRNA表达显著上调。庆大霉素抑制肠源性LPS能够降低iNOS的表达水平，使Caspase3活化下调，凋亡反应减弱。即，90％肝切除大鼠肝组织iNOS mRNA表达明显上调，这将引起NO的大量产生，由于NO的产量与凋亡相关因子表达密切相关，NO的大量产生引起促凋亡因子的激活，表现为Caspase3活化加强，最终细胞凋亡明显加强。而抑制LPSneng shi能使大鼠肝组织iNOS mRNA表达与90％肝切除组相比明显下调，Caspase3活化也受到抑制，凋亡反应减弱。
结论：
1.深入研究不同范围肝切除术后肝脏、肠粘膜等器官脏器发生的病理生理变化和机制必须建立在不同范围标准化肝切除模型的基础上。由于目前报道的大鼠肝切除术，特别是大鼠90%肝切除术，其手术操作方式复杂且多种多样，并无统一标准，其后续研究也都建立在各自所建肝切除模型之上，导致研究所获得的包括肝脏功能和生存率等结果均有很大差别。这也是导致对于不同范围肝切除大鼠术后的病理生理过程和具体机制研究较少的原因之一。为此，必须首先通过规范化和标准化大鼠不同范围肝切除手术方式和方法，建立了标准化的大鼠不同范围肝切除模型，以避免对大鼠进行不同范围肝切除术时由于手术操作方式的不同所造成的差异，使我们能够以此为模型探讨不同范围肝切除术后大鼠肝脏和机体发生的病理生理改变和可能的相关机制，进一步深入认识临床上不同范围肝切除患者术后可能发生的病理生理过程。研究中我们主要利用血管铸型和显微外科技术，精确、详细且具体的对手术方式加以规范，以尽可能的减少误差。根据实验室前期大鼠肝脏血管铸型实验结果，在充分了解了大鼠门静脉、肝动脉、肝静脉和胆管系统等重要解剖结构后，严格遵守操作原则，规范手术方法和步骤，特别是通过采用显微外科技术，精确的对大鼠肝门部和肝实质内门静脉和肝静脉等重要结构进行处理，尽可能的缩小了手术操作和方法对于不同范围肝切除产生的差异，成功建立和规范了大鼠不同范围肝切除标准化模型的方法，为后续研究奠定了基础。模型稳定性通过肝切除术后肝功能指标加以验证。
2.大鼠不同范围标准化肝切除模型方法建立成功，各实验组样本之间差异小，且肝脏功能恶化程度以及术后血内毒素水平均与切除范围相关。该部分结果表明，随着肝切除范围的扩大，ALT、AST、TBIL和ALB等肝功能指标恶化更为明显。在70％和80％肝切除情况下，虽然肝功能指标与假手术组相比确有明显改变，但由于大鼠肝脏强大的再生和解毒能力，肝功能指标术后迅速开始好转。而90％肝切除大鼠除了其肝功能指标恶化更为明显外，其好转趋势也较70％肝切除和85％肝切除组明显减慢，甚至术后短期内很难恢复至正常水平。预示90％肝切除大鼠肝脏自身恢复不良，或毒性物质没有解毒，仍在一段时间内对肝脏造成持续损伤。另外，通过对比大鼠不同范围肝切除术术后血浆内毒素（LPS）水平，我们发现：更大范围的肝切除术或极限肝切除术（90％肝切除）将导致血LPS水平明显升高，这与肝切除术后肝功能的损伤呈正相关，肝脏以及肠道粘膜屏障功能的损伤与血LPS水平的升高互为因果，且均由肝切除范围的扩大所致。
3.围手术期预防性应用庆大霉素能够降低大鼠90％肝切除术后血浆LPS水平，同时改善术后肝脏功能，有利于肝功能的恢复和预后。通过比较90％肝切除组大鼠与在围手术期预防性应用庆大霉素的治疗组大鼠血浆LPS水平，我们发现：庆大霉素治疗能够显著降低术后大鼠血浆LPS水平，在此基础上，大鼠术后肝脏功能恶化程度也明显好转，且恢复较快。最终明显改善90%肝切除大鼠术后生存率。
4.肠道紧密连接蛋白Occludin表达水平的显著降低是大鼠极限肝切除术后肠粘膜损伤的特征性表现，临床上可能能够以此作为判断肝脏切除范围是否达到极限水平，是否需要预防性应用抗生素抑制肠道细菌及其毒性产物以改善预后的依据。
90％肝切除大鼠肠道及肝脏紧密连接蛋白Occludin表达明显下调，预示着其肝脏血胆屏障和肠粘膜屏障功能障碍，90％肝切除术后血胆酸（TBA）水平的上升和血LPS水平的大幅升高，分别为血胆屏障和肠粘膜屏障功能损伤的表现。围手术期预防性应用庆大霉素，抑制肠源性细菌及其毒性产物LPS和炎症因子等，明显上调了肝脏和肠道Occludin的表达，意味着肝血胆屏障和肠粘膜屏障功能得以改善。研究中虽然发现了不同范围肝切除术后，Occludin表达水平的改变与EGF mRNA表达相关或部分相关，庆大霉素抑制肠源性LPS即能够上调EGF mRNA表达，又能上调Occludin的表达，但EGF与Occludin的表达是否存在着直接联系，尚须进行更为深入的研究。
5.围手术期预防性应用庆大霉素灌胃能够通过调节肝脏TLR－4mRNA和蛋白表达，从而改变肝脏对肠源性LPS的敏感性，调动细胞增殖反馈抑制相关因子，延缓细胞过激增殖。
大鼠90％肝切除术后，肝细胞增殖过激，表现为术后PCNA表达“先增后降”的趋势，即，术后第1天明显上调，而术后2天明显下调，其过激的肝细胞增殖不利于肝脏的再生。这一现象的发生与不同范围肝切除术后肝脏LPS受体－TLR－4表达水平有关。TLR－4信号通路下游的IL－6等炎症因子的大量产生，能够诱导抑制性因子SOCS－3的表达，即反馈抑制过激的肝细胞增殖的效果减弱。然而，作为LPS受体复合物之一的TLR－4表达在90%肝切除术后明显下调，意味着肝脏对LPS反应性下降，因此未能引起下游IL－6等炎症因子的大量产生，而在术后2天，TLR－4表达明显上调，使得IL－6等大量产生，诱导SOCS－3的表达，进而抑制了肝细胞的进一步增殖。然而，预防性应用庆大霉素降低血LPS水平，通过上调术后1天TLR－4表达，并介导SOCS－3表达，发挥控制细胞增殖的作用，此后（术后2天）治疗组大鼠肝脏TLR－4表达的下调，降低了SOCS－3表达水平，肝组织PCNA蛋白表达也较90％肝切除组显著上调，提示肝脏再生良好。
6.围手术期预防性应用庆大霉素灌胃能够通过调节肝脏iNOS mRNA的表达的量，以降低NO产量，从而抑制细胞凋亡。预防性应用庆大霉素抑制血LPS水平能够显著降低肝脏iNOS表达，进而调节肝内NO产量，使其发挥抑制凋亡的作用，结果表明，Caspase3活化明显降低。而90％肝切除组大鼠iNOS表达明显上调，并伴随Caspase3活化增强。
Background and objective:
Background and Objectives: liver resection is still the most effective treatment forhepatobiliary malignancies. However, for multiple or huge tumors, aggressive hepatectomymight be inevitable. Extended liver resection would doubtlessly increase the incidence ofinfection and other complications due to insufficient liver remnant, effecting the prognosisand curative rate after hepatectomy. Previously, most researchers have been devoted toexplore the potential regenerative capacity of the liver as well as its acceleration to retainsufficient functional volume postoperatively. However, accumulating data hasdemonstrated that it is the excessive regeneration after aggressive hepatectomy thathindered proper structure restoration. Therefore, for extended liver resection, it is necessaryto protect the residual hepatocytes, regulate the micro－environment to promote hepatocyteproliferation, and prevent liver damage from bacteria and/or their metabolites. Liver failurecan thusly be avoided, and hepatocyte proliferation can be guaranteed to ensure structurereconstruction and improve prognosis. Although there have been sufficient knowledge andeffective prevention, treatment method for postoperative infection as a complication forliver resection, in practice, inhibition of intestinal LPS (lipopolysaccharide) was never paidenough attention, and application of antibiotics in prevention has never been standardized.LPS is the main component of the gram－negative bacterial endotoxin, it induce a largenumber of inflammatory cytokines, increase intestinal permeability, reduce blood－bilebarrier function, etc. thereby seriously affect recovery and prognosis. After extendedhepatectomy, small for size syndrome, cholestasis and portal hypertension would give riseto increased LPS generation, absorption and decreased clearance, which further aggregateliver and systemic inflammatory response, and eventually lead to liver failure and MODS. Application of prophylactic antibiotics in the control of intestinal flora to reduce theproduction and absorption of LPS, might help to improve prognosis of aggressivehepatectomy. In addition, the mechanism of prognosis improvement by inhibiting LPS isnot entirely clear.
In this experiment, we established standardized model of rat hepatectomy in differentextend, investigate blood－LPS and other indicators after different resection extend,especially subtotal hepatectomy (90％). And we applied perioperative antibiotics(gentamicin) in rats after90％hepatectomy, examined inhibition of LPS, as well as aseries of pathophysiological changes including liver blood bile barrier, mucosal barrier, cellproliferation and apoptosis, to further understand the mechanism of effect induced by LPSinhibition.
Method:
1. Using microsurgical techniques to establish standardized model of rat hepatetomy.All SD rat liver resection were operated after disinfection, laparotomy and under etheranesthesia. In70％hepatectomy (PH), left lateral lobe and mid lobe were removed, in85％hepatectomy, the caudate lobe and right lower lobe were removed in addition to PH;90％liver resection (subtotal hepatectomy) the right lower lobe and right upper lobe wereremoved in addition to PH; sham group were used as control. All hilar are dissected usingmicrosurgical techniques and ligated trasected under microscope; all hepatic vein weretransected after intra－parenchyma suture ligation. Cecal ligation and puncture (CLP) isused as a positive control in the determination of plasma LPS level.
2. Firstly, the experiment investigated pathophysiological changes after hepatectomyof different extend. In order to study the effect of inhibiting enterogenous LPS rats on90％liver resection liver function and survival, we chose a strong inhibitory effect on Gram－negative bacteria, gentamicin sulfate medication as the treatment group. Gentamicin is notabsorbed if applied orally, we used cannula gavage as method of administration.Gentamicin sulfate5mg/ml,1ml was administrated, in rats one day before the resection,immediate postoperative, and one day after subtotal hepatectomy, a total of15mg.
3. For simultaneous determination of venous and portal blood plasma LPS level, ratswere operated under ether anesthesia to take the inferior vena cava and portal vein blood,and then sacrificed via ether overdose inhalation.
4. We implemented immunofluorescence, western blot and Real－time PCR todetermine liver tissue and intestinal mucosa expression of Occludin expression toinvestigate the90％liver resection blood－bile barrier and mucosal barrier function; andby determination of EGF, TLR－4, SOCS－3, PCNA, Caspase3, iNOS factordetermination to explore the mechanism of inhibiting LPS on rats with90％hepatectomy.
Results:
1. SD rat model with different extend of liver resection were successfully establishedand standardized.
2.With the expansion of extend of resection, postoperative liver function deteriorate.After90％hepatectomy, serum ALT, AST were significantly higher, especially the firstthree days, then gradually recovered.70％and85％hepatectomy rats postoperative7days survival rate was100％,90％hepatectomy was20％.
3. The Limulus test detects rat inferior vena cava and portal blood plasma LPS foundthat, after cecal ligation and puncture (CLP) rat1－2天plasma LPS level weresignificantly elevated compared with the rest of the groups; after90％hepatectomy, portalblood and inferior vena cava LPS was significantly higher than that of70％hepatectomyrats. Treatment group demonstrated significantly decreased blood LPS levels and at thesame time improved postoperative liver function and survival (60％).
4. We also found that after90％hepatectomy, Occludin expression decreasedsignificantly both in liver tissue and in intestinal mucosa, compared with sham－operatedrats, indicating blood bile barrier and the intestinal mucosal barrier dysfunction. SerumTBA was significantly higher in90％hepatectomy than70％hepatectomy, indicating thatthe blood－bile barrier function was severely impaired. In treatment group, liver tissueOccludin expression was significantly higher, immunofluorescence revealed less damagedliver and intestinal mucosa structure, serum TBA levels were also significantly lower thanthe90％hepatectomy rats.
5. EGF mRNA expression levels was related to the extend of liver resection,90％hepatectomy resulted in EGF mRNA down－regulation, while treatment resulted insignificant EGF up－regulation, which may be related to Occludin expression. Inhibition ofLPS would up－regulate EGF expression, and maintain liver and intestinal tight junctions Occludin expression, thus improving the the blood bile barrier function and intestinalbarrier function.
6.90％liver resection induced over－response of cell proliferation, which is relatedto TLR－4－mediated decreased sensitivity to LPS. In90％hepatectomy, postoperativefirst day, TLR－4mRNA and protein expression in liver tissue were significantly lowerthan the70％hepatectomy group, and lower than that of the treatment group. Treatmentresulted in increased TLR－4level, thereby inhibited liver regeneration. Post－operative1day, PCNA protein expression in liver tissue of treated rats was significantly lower than the90％hepatectomy rats, indicating effective control of excessive hepatocyte proliferation.
7. In90％hepatectomy group, iNOS mRNA expression was significantly increased,leading to Caspase3activation and significant apoptosis. INOS mRNA expression intreatment group was significantly down－regulated with suppreesed Caspase3activation.
Conclusion:
1. SD rat model with different extend of liver resection were successfully establishedand standardized. With the expansion of resection extend, liver function indicators andpathological findings showed more server structural and functional damaged.90％hepatectomy resulted in elevated plasma LPS level, and the use of prophylactic antibiotics,significantly decresed plasma LPS level and improved liver function and survival rates after90％liver resection.
2. Inhibition of enterogenous LPS by upregulating liver tissue expression of EGF andOccludin, improve blood bile barrier functions and structure of the intestinal mucosalbarrier.
3. LPS inhibition would upregulate TLR－4, to increase the sensitivity of liver to LPS,which would lead to a substantial increase in IL－6and other cytokines, and then thedownstream feedback inhibition factor SOCS－3. Excessive proliferation of liver cellscould be inhibited, the liver cells would proliferate in an more organized manner.
4.Inhibition of enterogenous LPS inhibit liver cell apoptosis by down－regulatingiNOS expression, and reducing Caspase3activation.
引文
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