| Background and ObjectiveSepsis is an acquired disease caused by combined action of multiple factors, and canbe caused by factors such as congenital stress, cell injury and organ dysfunction. It is acommon complication after operations such as infection, burn injury, severe trauma,surgical major surgery, obstetric complications, poisoning and cardiopulmonaryresuscitation (CPR), and is the leading cause of death for ICU. Fatality rate of the disease isas high as30%to70%, while its incidence rate is still rising at an annual rate of1.5%to8%. Sepsis most often affects the intestine, and when the intestinal damage happened, itspermeability increase and intestinal barrier function is impaired. So the intestinal bacteriaand toxins can be absorbed into the circulatory system and cause bacterial translocation, asa result, systemic inflammatory response syndrome (SIRS) is aggravated and out of control.It can induce multiple organ dysfunction syndrome (MODS) when it is serious, and evenendanger the patient lives. Many studies have shown that decreased intestinal functionclosely associated with the intestinal barrier. The intestinal barrier consists of mechanicalbarrier, chemical barrier, immune barrier and biological barriers. Among that function ofmechanical barrier and immune barrier is a common indicator for judging the intestinalbarrier function, and mechanical barrier refers to the complete intestinal mucosal epithelialstructure which closely connects with each other. Intestinal mucosal defensins-5(RD-5)and intestinal trilobites factor family-3(TFF-3) protein are common indicator proteinsfor judging the intestinal immune barrier function. As we all know, TNF-α, IL-6andHMGB1are the most important proinflammatory factors in occurrence and development ofsepsis, affecting the recovery of intestinal function in sepsis seriously, and even causewhole body multiple organ damage. Above active factors can promote inflammation, andchange of vascular tone and increase vascular permeability through specific G proteinreceptor. In addition, these cytokines form closely interactive relationship between each other, and can form "inflammatory cascade effect". Recently, many studies showed thatTNF-α, IL-6and HMGB1are closely related to the severity and prognosis of sepsis. So,observing indicators such as TNF-α, IL-6, and HMGB1ia of great significance for theprevention and treatment of sepsis.Ulinastatin (Ulinastatin) is a kind of glycoprotein separated and purified from urine ofhealthy adult male, which can restrain the activity of various proteolytic enzymes, and it isa protease inhibitor with broad spectrum (Ulinary trypsin inhibitor, UTI). In addition, it hasfunctions such as inhibiting release of lysosomal enzymes, inhibiting production ofmyocardial depressant factors, stabilizing lysosomal membrane, eliminating oxygen freeradicals and inhibiting release of inflammatory mediators.Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GSHR).Most of Ghrelin are secreted by X/A like cells at the bottom of the stomach, while the otherpart is from the small intestine. It can be combined with GHSR in vivo, and promote releaseof growth hormone dose-dependently, and then mediate its biological activity through theGH/IGF-1shaft. Numerous studies have shown that Ghrelin can obviously promotegastrointestinal peristalsis and accelerate gastric emptying. For paralytic ileus caused bysepsis, Ghrelin also has a good curative effect. At the same time, Ghrelin can obviouslyimprove formation of gastrointestinal ulcers,tissue hyperemia and changes of capillarypermeability, also reduce the concentration of blood TNF-α in rats withischemia-reperfusion.Although studies have showed that UTI and GHL all have improvement action toviscera dysfunction in sepsis, specific studies for intestinal dysfunction of sepsis are less,and the corresponding mechanism is unclear. According to the important role of intestinalfunction played in occurrence and development of sepsis, this study used rats withendoxemia as sepsis model, and effect of UTI and GHL on intestinal barrier function,motor function and systemic and local intestinal inflammation were observed, hoping toprovide theoretical and experimental basis for treatment of intestinal dysfunction in sepsis.Methods60adult male SD were randomly divided into5groups by random number method (n=12), CON group (control group), LPS group (endotoxin group), ulinastatin group (UTIgroup), Ghrelin group (GHL groups), UTI+Ghrelin group (UTI+GHL group). Rats were given with3%pentobarbital sodium (35mg/kg) for celiac anesthesia. After anesthesia ineach group, intraperitoneal injection of endotoxin (Lipopolysaccharide, LPS,15mg/kg)was used to construct endoxemia model, and CON group was given intraperitonealinjection with equal dose of saline instead of endotoxin. After10min of LPS injection, UTIgroup was given intraperitoneal injection of UTI150000U/kg, GHL group was givenintraperitoneal injection of Ghrelin25mu g/kg, UTI+GHL group was givenintraperitoneal injection of UTI150000U/kg+Ghrelin25ug/kg, while CON and LPSgroup were given equal dose of saline. After12h drugs wer administrated again. After12h and24h,6rats of each experimental group were put to death, using methods such asHE staining and RealTime-PCR, small intestine dextran blue-2000(BD-2000) propulsionrate and ELISA to observe intestinal barrier function, small intestinal motor function andchanges of inflammatory mediators such as TNF-α, IL-6and HMGB1of rats in eachgroup.ResultsResults of HE staining showed that UTI+GHL group and UTI group can reduce theileum structural damage to different degrees compared with LPS, and UTI+GHL groupreduced more obviously (P <0.05). UTI group and UTI+GHL group enhanced expressionlevels of RD-5and TFF3mRNA significantly compared with LPS group (P <0.05), andUTI+GHL group increased significantly (P <0.05). GHL group and UTI+GHL groupincreased small intestinal motor function significantly compared with LPS group, and UTI+GHL group increased significantly (P <0.05). UTI+GHL group, UTI group and LPSgroup all can decrease expression of inflammation factors such as TNF-α, IL-6andHMGB1significantly compared with LPS group (P <0.05), and similar results were foundin the intestinal mucosal tissue.ConclusionFirstly, this study confirmed that combined use of UTI and Ghrelin can significantlyimprove the immune function of intestinal endothelial cells, protect the intestinal mucosalbarrier, effectively alleviate the symptoms of sepsis, slow the progression of sepsiscondition by observing the damage degree of intestinal mucosal microstructure in sepsisand changes of expression levels of RD-5and TFF3mRNA. Secondly, combined use ofUTI and Ghrelin can effectively reduce the excessive activation and release of inflammatory factors in systemic and intestinal mucosa of sepsis rats to improve theintestinal function. Damage of structure and function of intestinal barrier certainly lead toattenuation of intestinal motor function. Therefore, although Ghrelin having directprotection to the intestinal mucosa of sepsis was not observed in this study, it canstrengthen the protection effect of UTI to intestinal mucosa by improving intestinal motorfunction. Analysis according to the experimental results showed that combined use of UTIand GHL could significantly inhibit systemic inflammatory response and intestinal tissuelocal inflammatory response of endoxemia, and to improve the intestinal barrier functionenhance the intestinal motor function. |
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