Tri－effect On Improving Prognosis Of Subtotal Hepatectomy Through Inhibition Of Intestinal Endotoxin In Rats

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.