The Mechanism Research Of Enteral Nutrition Effect On Intestinal Barrier Function In Patients With AIDS

Part I Research on the change of intestinal barrier function in AIDS Objective To stuy the changes of AIDS patients’ intestinal mechanical barrier、 biological barrier and immune barrier function, which helps explore the influence and mechanism of AIDS.Methods Use culture method to Determinate Bifidobacterium spp., Lactobacillus group, Escherichia coli, Enterococcus faecalis.Fecal samples bacterial genome DNA were extracted and quantified by real-time fluorescence quantitative PCR to analyze bacterial amounts.According bacteria cfiA gene sequence of B.fragilis designed specific primers;16S rRNA gene sequence of Bifidobacterium designed specific primers; rrnA-16S gene sequence of Clostridium designed specific primers; cfxA gene sequence of Bacteroides designed specific primers; gyrB gene sequence of B.thetaiotaomicron Fusobacterium designed specific primers; ddl gene sequence of Enterococcus faecalis and feces Enterococcu designed specific primers; swp84 gene sequence of C. difficile designed specific primers; mob gene sequence of B.uniformis designed specific primers; 16S rRNA gene sequence of Lactobacillus, Escherichia coli B.thetaiotaomicron and Fusobacterium designed specific primers; rpoB gene sequence of B.ovatus、B.distasoni and B.vulgatus designed specific primers; BoNT gene sequence of C.botulinum designed specific primers; Collected the feces of healthy volunteers(n=50) and patients with AIDS (n=50), Fecal samples bacterial genome DNA were extracted and quantified by fluorescence quantitative PCR,which analysis bacterial amounts of two groups.To observe the changes of intestinal mechanical barrier function in patients with AIDS by ELISA.Collected the blood of healthy volunteers(n=50) and patients with AIDS (n=50), we check the level of Diamine oxidase(DAO), D-lactic acid (D-LA), lipopolysaccharide (LPS) intestinal fatty acid.binding protein (iFABP)、anti-endotoxin core antibody (EndoCab) and soluble CD 14 (sCD14)from plasma. To analyse the difference of the index of intestinal mechanical barrier function in health group and AIDS patients group.To observe the changes of intestinal immune barrier function in patients with AIDS by ELISA. Collected the blood of healthy volunteers(n=50) and patients with AIDS (n=50), we check the level of secretory immune globulin A (sIgA), interleukin-12 (IL-12)、 interleukin-17(IL-17)、Tumor necrosis factor (TNF-α)、interleukin-1β (IL-1β)、 interleukin-6 (IL-6)、interleukin-10(IL-10)、FAS and FASL from plasma. To analyse the difference of the index of intestinal immune barrier function in health group and AIDS patients group. To analyse the CD4+,CD8+, CD4+/CD8+T cell’s difference of intestinal immune barrier function in patients with AIDS by flow cytometry. Determination of triceps skinfold thickness(TSF), arm circumference(AC), total lymphocyte count(TLC), body mass index(BMI), serum prealbumin(PALB), albumin(ALB), hemoglobin(Hb), analyze the nutritional status of patients with AIDS.Results In normal control group and the level of Lactobacillus group (7.38 ± 0.44 vs 3.54±0.36), Bifidobacterium spp. (8.63±0.71 vs 4.48±0.27) in AIDS patients group were significantly lower than healthy controls (P<0.05), whereas Enterococcus faecalis (5.72±0.38 vs 6.52±0.61)、Escherichia coli (5.35±0.52 vs 6.51±0.62) were significantly higher than normal control group (P<0.05) by use of culture methods. The level of Lactobacillus group(7.35±0.46; 5.12±0.48) and Bifidobacterium spp. (9.14±0.72 vs 5.74±0.58) in AIDS patients group were significantly lower than normal control group (P<0.05), whereas Enterococcus faecalis(5.85±0.55; 7.08±0.72)、Escherichia coli (5.77±0.43 vs 7.58±0.78) and feces Enterococcus (5.24±0.54; 7.38±0.76), Fusobacterium (7.95±0.83; 8.79± 0.64)、Bacteroides(7.67±0.85; 8.66±0.58)、Clostridium (4.55±0.38; 6.13±0.24)、 B.distasonis(4.22±1.21; 5.54±1.46)、B.fragilis (3.25±0.48; 4.06±0.58)、 B.vulgatus(2.93+0.30; 4.49±1.22)、B.uniformis(5.58±0.85; 6.72±0.83) B.ovatus(5.76±1.02; 7.07±1.65)、B.thetaiotaomicron(5.02±0.51; 6.42±1.34)、 C.botulinum (2.78±1.14; 5.38±0.45)、C. difficile(1.65±0.26;3.45±0.47) and F. necrosis(2.32±0.26;6.34±0.62)、were significantly higher than those of normal control group(P<0.05)by real-time fluorescence quantitative PCR.The level of DAO (0.97±0.18 u/ml vs 1.43±0.28 u/ml)、D-LA(25.92±3.25 umol/L vs 38.92±5.89 umol/L)、LPS(32.82±5.28 ng/ml vs 66.68±7.85 ng/ml)、iFABP(99.12±10.56 pg/ml vs 156.22±18.78 pg/ml)、sCD14)(0.43±0.03 ug/mL vs 0.74±0.22 ug/mL)、 EndoCAb(20.36±0.88 MMU/L vs 24.33±2.34 MMU/L)were significantly higher than those of healthy controls(P<0.05).The level of sIgA(62.4±14.21pg/mL vs 27.56 ±1.43 pg/mL)、IL-12(2.74±0.35pg/mL vs 0.72±0.32 pg/mL)、IL-17(20.28± 4.83pg/mL vs 5.75±0.97pg/mL)、TNF-α:(11.4±4.12pg/mL vs 6.61±1.32 pg/mL)、IL-1β(23.2±3.21pg/mL vs 7.03±0.60pg/mL)、IL-6:(10.86±4.15ng/L vs 1.13±0.47ng/L)、CD4+:(40.75±4.73 vs 30.52±4.52)、CD8+:(23.72±3.72 vs 14.75±1.58)、CD4+/CD8+T cell:(1.86±0.83 vs 1.26±0.23), TSF:(11.28 ±1.82mm vs 7.08±0.17mm),AC:(24.82±3.28cm vs 20.75±2.78cm),TLC: (1.79±0.54 vs 0.78±0.12), BMI:(22.8±0.58 vs 17.12±0.23),PALB: (180.5±20.5 vs 110.5±13.2), ALB:(38.5±3.85 vs 27.25±2.87), Hb: (123.35±13.83 vs 98.28±10.12),in AIDS patients group were significantly lower than those of healthy controls(P<0.05),whereas,IL-10(37.6±10.5ug/L vs 79.5± 13.2 ug/L).FAS:(0.33±0.12 pg/mL；1.21±0.13 pg/mL)and FASL:(42.21±5.14 pg/mL;82.49±6.72 pg/mL)in AIDS patients group were significantly higher than those of normal control group(P<0.05).Conclusions AIDS patients intestinal microflora have obviously changed.The probiotics disorders. bacterial.endotoxin translocation and intestinal microflora of AIDS patients were confusion.Intestinal mechanical barrier and immune function was significantly impaired.Nutritional status and immunity levels were significantly reduced in AIDS patients.PartⅡ The mechanism research of Enteral Nutrition effect on intestinal barrier function in patients with AIDSObjective To analysize the AIDS patients’ intestinal microflora after 4 weeks the treatment of enteral nutrition, which helps study the influence of intestinal barrier function. To provide practical significance and new theoretical basis for the clinical treatment of AIDS and research the mechanism.Methods The AIDS patients(n=50) drink enteral nutrition.Collected the feces after nutrition treatment for 4 weeks. Use culture methods to Determinate Escherichia coli, Bifidobacterium spp., Enterococcus faecalis and Lactobacillus group, from Fecal samples Bacterial genome DNA were extracted and quantified by real-time fluorescence quantitative PCR. Collected the blood of patients with AIDS (n=50) after nutrition treatment for 4 weeks, we check the level of Diamine oxidase(DAO), D-lactic acid (D-LA), lipopolysaccharide (LPS) intestinal fatty acid binding protein (iFABP)、anti-endotoxin core antibody (EndoCab)、soluble CD 14 (sCD14)from plasma. To analyse the difference of the index of intestinal mechanical barrier function in AIDS patients group before and after 4 weeks the treatment of enteral nutrition. we check the level of secretory immune globulin A (sIgA), interleukin-12 (IL-12)、 interleukin-17(IL-17)、Tumor necrosis factor (TNF-α)、interleukin-1β (IL-1β)、 interleukin-6 (IL-6)、and interleukin-10 (IL-10)、FAS and FASL from plasma. To analyse the difference of the index of intestinal immune barrier function in AIDS patients group before and after 4 weeks the treatment of enteral nutrition. To analyse the CD4+,CD8+, CD4+/CD8+T cell’s difference of intestinal immune barrier function in patients with AIDS after EN by flow cytometry. Determination of triceps skinfold thickness(TSF), arm circumference(AC), total lymphocyte count(TLC), body mass index(BMI), serum prealbumin(PALB), albumin(ALB), hemoglobin(Hb), analyze the nutritional status of patients with AIDS after EN.Results After the nutrition treatment for 4 weeks, the level of Lactobacillus group (3.54±0.36 vs 6.76±1.26)、Bifidobacterium spp. (4.48±0.27 vs 7.15±0.72) were significantly higher than before the nutrition treatment (P<0.05), whereas Enterococcus faecalis (6.52±0.61 vs 4.82±0.42) and Escherichia coli (6.51±0.62 vs 5.23±0.55) were significantly lower than before the nutrition treatment (P<0.05) by use culture methods.After the nutrition treatment for 4 weeks,the Ievel of Lactobacillus group(5.12±0.48 vs 7.86±1.13)and Bifidobacterium spp.(5.74±0.58 vs 8.25±1.27)in AIDS patients group were significantly lower than before the nutrition treatment(P<0.05),whereas Enterococcus faecalis(7.08±0.72 vs 4.78± 0.82).Escherichia coli(7.58±0.78 vs 6.21±1.07)and feces Enterococcus(7.38± 0.76 vs 6.13±0.87),Fusobacterium(8.79±0.64 vs 7.62±0.54)、Bacteroides(8.66± 0.58 vs 7.55±0.44)、Clostridium(6.13±0.24 vs 4.32±0.38)、B.distasonis(5.54± 1.46 vs 4.33±1.22)、B.fragilis(4.06±0.58 vs 3.14±0.42)、B.vulgatus(4.49±1.22 vs 3.21±0.46)、B.uniformis(6.72±0.83 vs 5.67±0.62).B、ovatus(7.07±1.65 vs 5.85 ±1.21)、B.thetaiotaomicron(6.42±1.34 vs 5.12±0.63)、C.botulinum(5.38±0.45 vs 2.47±0-34)、C.difficile(3.45±0.47 vs 1.43±0.24)and F.necrosis(6.34±0.62 vs 3.57±0.46)were significantly lowet than before the nutrition treatment(P<0.05)by real-time fluorescence quantitative PCR.The level of DAO(1.43±0.28u/ml vs 1.23 ±0.13 u/ml)、D.LA(38.92±5.89umol/L vs 30.44±4.81 umol/L)、LPS(66.68 ±7.85ng/ml vs 51.03±5.26 ng/ml).iFABP(156.22±18.78pg/ml vs 120.75± 15.55pg.ml)、sCD14(0.74±0.22ug/mL vs 0.58±0.24ug/mL)、EndoCAb(24.33 ±2.34MMU/L vs 22.18±2.15MMU/L)were significantly lower than before the nutrition treatment(P<0.05).The level of sIgA(27.56±1.43 pg/mL；43.30±3.16 pg/mL).IL-12(0.72±0.32pg/mL；1.53±0.27pg/mL).IL-17(5.75±0.97pg/mL;8.79 ±0.36pg/mL)、TNF-α:(6.61±1.32 pg/mL;8.77±1.22 pg/mE)、IL-1β(7.03± 0.60pg/mL;9.36±1.48pg/mL)、IL-6:(1.13±0.47ng/L vs 2.72±0.52ng/L)、were significantly higher than before the nutrition treatment(P<0.05),whereas IL-10(79.5 ±13.2 ug/L;48.3±11.2 ug/L)、FAS(1.21±0.13pg/mL；0.52±0.12pg/mL)and FASL (82.49±6.72pg/mL；49.61±4.76pg/mL);CD4+:(30.52±4.52 vs 38.25±3.82)、 CD8+:(14.75±1.58 vs 21.57±3.14)、CD4+/CD8+T cell:(1.26±0.23 vs 1.78± 0.42),TSF:(7.08±0.17mmvs 10.06±1.28mm),AC:(20.75±2.78cm vs 23.28 ±2.35cm),TLC:(0.78±0.12 vs 1.62±0.25),BMI:(17.12±0.23 vs 21.62±2.26), PALB:(110.5±13.2 vs 165.32±17.25),ALB:(27.25±2.87 vs 32.24±3.32), Hb:(98.28±10.12 vs 110.21±11.15)were significantly higher than before the nutrition treatment(P<0.05).Conclusion Enteral nutrition can rise AIDS patients’ intestinal probiotics, reduce opportunity pathogenic bacteria, significantly improve the intestinal microflora of AIDS patients. Enteral nutrition can repair the intestinal mechanical and immune barrier function, Can improve their nutritional status and immune function of AIDS patients. To provide practical significance、new theoretical basis for the clinical treatment of AIDS and research the mechanism.