Effect Of Penehyclidine Hydrochloride On Intestinal HIF-1α Expression In Endotoxemia Of Neonatal Rats

Neonatal intestinal injury is a common disease in pediatrics, which is caused byinfection and ischemia and hypoxia induced by premature birth and asphyxia and soon. Severe infection often leads to critically ill children with gastrointestinal functionfailure. Gut plays a vital role in the process of development of systemicinflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome(MODS). Intestinal infection, ischemia and hypoxia can lead to intestinal mucosalbarrier function impaired, give rise to bacteria and endotoxin translocation, triggersepsis and septic shock, thus further aggravate the intestinal tissue damage, even canbe associated with remote organs injury. Lipopolysaccharide (LPS), a typicalendotoxin, can cause endotoxemia and lead to intestinal mucosal damage. Manystudies have found that hypoxia-inducible factor-1alpha (HIF-1α) is important in thepathological process of intestinal injury caused by endotoxin. HIF-1α could activateits downstream relative genes and promote inflammatory factors expression，thenmay aggravate the tissue injury in endotoxemia or ischemia-reperfusion injury.Penehyclidine hydrochloride (PHC) is an anticholinergic agent developedindependently by China. Studies have shown that penehyclidine hydrochloride has aprotective function of multiple organs in improving microcirculation and reducingcapillary wall permeability and lysosome release. To date, the protective function ofpenehyclidine hydrochloride in endotoxin-induced lung injury has been widelyconfirmed and recognized, and we also find that penehyclidine hydrochloride has agood effect of removing intestine convulsion and reducing the edema through a lot of clinical observations. Based on the above background, the research willinvestigate the effect of pretreatment with penehyclidine hydrochloride on HIF-1αexpression in LPS-induced intestinal injury in neonatal rats, and it’s intestinalprotection mechanisms will be discussed preliminarily.ObjectiveTo investigate the effect of pretreatment with penehyclidine hydrochloride onHIF-1α and other relative cytokins expression in LPS-induced intestinal injury inneonatal rats, and provide a new thought for clinical prevention and treatment ofneonatal intestinal injury.Materials and Methods1. Study ObjectSixty-nine healthy SPF Sprague-Dawley (SD) rats,7days old, provided byZhengzhou University laboratory animal center of Henan Province. Male and female,weighing18±2g.2. Experimental Methods2.1Animal groupsThe reaserch had two parts. The first part: thirty neonatal rats were randomlydivided into3groups,10for each group: Control group (group C), Intestinal injurymodel group (group L) and Penehyclidine hydrochloride group (group P). Wemeasured the wet/dry weight ratio of intestinal tissue, observed the morphologicalchanges of intestinal mucosa, and detected the expression of cytokins of intestinaltissue. The second part: thirty-nine neonatal rats were also randomly divided into3groups as part1,13for each group. We observed the ethological changes aftermodels prepared and calculate the rate of survivors in24hours of each group.2.2Experimental model preparationIntestinal injury model was administrated by intraperitoneal injection of LPS (E.coli055: B5, Sigma, USA)5mg/kg with the concentration of0.5mg/ml in group L.Thirty minutes before administration of LPS, first intraperitoneal injection of penehyclidine hydrochloride (Chengdu List Pharmaceutical Co., Ltd, China)2mg/kgwith the concentration of0.05mg/ml in group P. Intraperitoneal injection of normalsaline10ml/kg in group C. In the first part, at6hours post administration of LPS ornormal saline, we anesthetized the rats and got the samples. All the rats were putback to the cages with their mother rats after injection.2.3Intestinal tissue specimen collectionAfter inhalation of sevoflurane in an airtight glass container, we quicklycollected blood by heart puncture with a1ml syringe, stewing2hours, then4000rpmcentrifuged15min under4℃, got the supernate, and stored at-20℃for furtherdetections. Three centimeters above the ileocecus, we took7cm ileal tissue.4℃normal saline flushed the tissue3times, and filter paper blotted up the surfacemoisture. We divided the7cm ileal tissue into4parts, the upper1cm was used tomorphological observations under optical microscope; the middle upside2cm wasused to measure the wet weight and dry weight of the intestinal tissue; themiddle-lower2cm was used to prepare tissue homogenate; the lower2cm was usedfor RT-PCR analysis of HIF-1α mRNA expression in intestinal tissue.2.4Detection indexes2.4.1Calculation of intestinal tissue wet/dry weight ratioThe intestinal tissue was dried with filter paper and weighted to get the wetweight, then put into70℃oven for48hours to get the dry weight, then wecalculated the ratio.2.4.2Morphological observations under optical microscopeTo put the upper1cm intestinal tissue into4%paraformaldehyde, then into5μmparaffin sections and HE stain. We observed the ileal mucosal epithelial cellmorphology changes with400times.2.4.3Measurements of TNF-α, IL-6, HIF-1α, Gln, diamine oxidase (DAO)in intestinal tissue and DAO in serum by ELISAThe middle-lower2cm ileal tissue was weighted and prepared for tissuehomogenate.3000rpm centrifuged15min under4℃, got the supernate, and stored at-20℃for further detections. The operation was in strict accordance with the ELISAkits (R&D Company, USA) instructions. 2.4.4Analysis of HIF-1α mRNA-levels in intestinal tissue by RT-PCRThe lower2cm intestinal tissue was flushed by DEPC water and dried by filterpaper, then cooled by liquid nitrogen quickly, and stored at-80℃for furtherdetections. Through extraction of total RNA, reverse transcription of RNA,amplification of DNA and agarose gel electrophoresis, we tested the expression ofthe HIF-1α mRNA levels in the intestinal tissue.3. Statistic analysisData processing was performed by SPSS17.0. All data were presented as mean±standard deviation (x±s). One-Way ANOVA and Least Significant Differencetest (LSD-t) were used for statistical analysis.The inspection level is α=0.05. P<0.05indicates the difference is statistically significant.Results1. After administration of intraperitoneal injection LPS, within1hour, neonatalrats began to tremble with polypnea in both group L and group P. Between2and3hours after injection, the rats appeared cold, listless, indolent, and almost didn’t eat.Up to6hours, all the symptoms and signs of the rats were exacerbated, some ofthem even showed cyanotic lips. Apparently, the symptoms of group P wererelatively lighter than group L. Rats in group C were all normal.The rate of survivorsin24hours of each group were100%in group C,69.2%in group L and92.3%ingroup P. The death mainly occurred during12~24hours.2. Compared with group C, the W/D weight ratio was significantly higher ingroup L and group P (P<0.05); but the ratio was significantly lower in group P thanin group L (P<0.05).3. The results of optical microscope observations: in group C, the mucosalstructures were integrity, the epithelial cells were in alignment, and the morphologywere normal. In group L, we could see the villus cellular edema and the epithelialcells were disordered. In group P, the edema was all significantly attenuated and theepithelial cells were relatively orderly.4. Compared with group C, the content of TNF-α, IL-6, and HIF-1α in intestinal tissue were significantly higher in group L and group P (P<0.05), but the content ofGln was lower (P<0.05); compared with group L, the content of Gln was higher(P<0.05) in group P, but other indexes were significantly lower (P<0.05).5. Compared with group C, the content of DAO in intestinal tissue weresignificantly lower in group L and group P (P<0.05), but higher in serum (P<0.05);compared with group L, the tissue DAO content was higher (P<0.05) but serumDAO content was lower (P<0.05).6. Compared with group C, the expression of HIF-1α mRNA in intestinal tissuewere significantly higher in group L and group P (P<0.05); but it was lower in groupP compared with group L (P<0.05).ConclusionPretreatment with penehyclidine hydrochloride can obviously alleviate thevillus edema in ileal tissue of neonatal rats and the expressions of inflammatoryfactors. The mechanisms of the protective effects could be that pretreatment withpenehyclidine hydrochloride may down-regulate the expression of HIF-1α inintestinal tissue, reduce the inflammatory response, and protect the intestinalmucosal barrier functions.