Study Of The Effect Of L-Arginine On Gut Barrier Function In Severe Abdominal Infection

Objective:Ⅰ: To research the change of intestinal mucosal pathology and the mucosal damage index, the number of intestinal epithelial apoptotic cells and the apoptosis index, the serum nitric oxide(NO) level and inducible nitric oxide synthase (iNOS) level of the Wistar mice with severe abdominal infection by replicating the Wistar mice severe abdominal infection model, studying the change of the intestinal mucosal pathology, the change of the intestinal epithelial apoptotic cells, the systemic inflammation response syndrome by NO mediated, to clarify the effect of L-arginine in mice with severe abdominal infection.Ⅱ: To explore the effect of L-arginine supplemented PN on gut barrier function in severe abdominal infection patients, the proportion of complications and hospital period, SIRS score grade, serum C-reactive protein (CRP) levels, plasma endotoxin and plasma D-lactate levels and serum nanobacteria (NB) were observed, discuss the mechanism of gut barrier disfunction in severe abdominal infection, and clarify the effect of L-arginine on gut barrier function in patients with severe abdominal infection.Methods:Ⅰ: A total of 18 Wistar rats were divided into two groups randomly:①CLP group (n=9): rats were underwent cecal ligation plus puncture (CLP) to replicate sever abdominal infection model;②ARG group (n=9) 300mg/kg of L-Argine were injected in abdomen after CLP. Blood and small intestine tissue sample were taken at 24h after CLP, detect the serum level of NO and iNOS by spectrophotometric method. The changes of intestinal mucosal pathology were observed through optics and evaluation of mucosa damage index. The intestinal epithelial apoptotic cells in both groups were observed through optics microscope by using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) methods.Ⅱ: A total of 40 patients with severe abdominal infection who were in Nanfang hospital between May 2005 to Jan 2006 were randomly devided into general therapy group (n=20) and L-arginine treatment group (n=20). In L-Arginine treatment Group 0.3g(kg.d) of arginine hydrochloride added intravenously guttae base on conventional therapy, and 15 patients without abdominal infection were observed as control group. The whole arginine treatment lasted for five days. The Proportion of complications and hospital period were observed and compared in both groups. SIRS score grade, serum C-reactiv protein (CRP) levels were carried out on day before and at the 4th day after operation. Plasma endotoxin and plasma D-lactate levels in blood were detected to acquaint the gut barrier function. The nanobactera were observed though optics microscope after staininged by sodium alizarinsulfonate and observed though scanning electron microscope (SEM), the positive rate of nanobactera in blood were carry out.The experimental data was test with paired T test, Pearson Chi-Square test by Statistical Package for the Social Science 10.0(SPSS 10.0). The test levelα=0.05, a P value of＜0.05 was considered significant.Results:Ⅰ: The Wistar mice severe abdominal infection model:(1) Serum NO level: the serum NO level of CLP group and ARG group were (87.2±16.7umol/L vs 79.9±18.0umol/L, t=0.881, P=0.391), and no significant difference were observed between the two groups. (2) Serum iNOS level: the serum iNOS level of the two groups were (44.4±6.6 vs 30.6±7.4t=4.157, P=0.001), the serum iNOS level of CLP group were higher than that of ARG group significantly. (3) Intestinal mucosa pathology: the intestinal mucosal damage were observed in both CLP group and ARG group, hydropic small intestine mucous membrane were observed, mucosal blood capillary were congested, neutrophile granulocyte infiltrated in mucous membrane, and some districts desquamated. the mucosa injury index were(4.18±0.38 Vs 3.62±0.49, t=2.673, P=0.017), the mucosa injury index in ARG group were lower than that of CLP group significantly. (4) Intestinal mucosal cell apoptosis: the intestinal mucosal cell apoptosis were observed in both CLP group and ARG group, the caryon of apoptotic cells were brown and pycnosis. The number of apoptotic cells of CLP groups were significantly higher than that of L-Arg group at 24h after operation, The apoptosis index were (14.94±1.83 vs 12.64±2.20, t=2.414, P=0.028).Ⅱ. The patients with severe abdominal infection: 1. The general data of patients: The mean age of L-Arginine treatment group and general therapy group were(36.55±17.55 vs 37.45±9.54,). The mean age (t=0.201, P=0.842), sexual constituent ratio (x~2=0.114, P=0.736) and protopathy constituent ratio (x~2=0.210, P=0.995) of the two groups were compared and there was no significant differenc between them. 2. Clinical data of patients: The proportion of complications of L-Arginine treatment group and general therapy group were (20% vs 15%, x~2=0.173, P=0.677), postoperative hospital period of the two groups were (14.45±17.83d vs 13.75±17.10d, t=0.127, P=0.900), and no significant differenc were been found. 3. Gut barrier function: the plasma endotoxin level of L-Arginine treatment group when being hospitalized and 4d days after operation were (0.33±0.11 kEu/L vs 0.18±0.10 kEu/L, t=8.360, P＜0.001) and that of general therapy group were (0.31±0.09 kEu/L vs 00.27±0.11 kEu/L, t=3.457, P=0.003). The plasma D-lactate level of L-Arginine treatment group when being hospitalized and 4d days after operation were (4.13±0.79 mg/L vs 2.77±0.75 mg/L, t=9.240, P＜0.001) and that of general therapy group were (4.20±0.71 mg/L vs 3.50±0.79 mg/L, t=5.376, P＜0.001). Compared with general therapy group, the plasma endotoxin level and D-lactate level of L-Arginine treatment group were lower significantly (F=13.478, P=0.001) at the 4th day after operation. 4. Systemic inflammation response data of patients: SIRS score grade of L-Arginine treatment group when being hospitalized and 4d days after operation were (2.65±0.75 vs 0.85±0.99, t=13.077, P＜0.001) and that of general therapy group were (2.40±0.68 vs 1.55±1.00, t=6.474, P＜0.001). Compared with general therapy group, the SIRS score grade of L-Arginine treatment group were lower significantly (F=25.417, P＜0.001) at the 4th day after operation. Serum CRP levels of the two group were (27.99±21.81 mg/L vs 48.18±22.23 mg/L H=5.927, P=0.015), the serum CRP level of L-Arginine treatment group were significantly lower. 5. The nanobacteria in blood: stained by sodium alizarinsulfonate, the nanobacteria were observed though optics microscope, it present orange granule alike. In SEM photograph, the nanobacteria is ball alike, the diameter of nanobacteria is about 80～500nm. the positive rate of nanobacteria in blood of severe abdominal infection patients were 65.2%, and patients without abdominal infection were 13.3% (x~2=9.886, P=0.002). The positive rate of nanobacteria in blood of severe abdominal infection patients significantly higher than that of patients without abdominal infection.Conclusions:Ⅰ: L-Arginine could depress the serum level of iNOS, release the intestinal mucosal injury and may inhibite the apoptosis of intestinal epithelial cells in severe abdominal infection rats.Ⅱ: Gut barrier dysfunction could be observed in severe abdominal infection patients. L-arginine can reduce the systematic inflammatory reaction and protect gut barrier function in several abdominal patients. Nanobacteria translocation may result in positive rate of nanobacteria in blood in severer abdominal infection patients.