The Research On The Mechanism Of The Protective Effect Of Montmorillonite Combined With Probiotics On The Intestinal Mucosa In Suckling Mice Infected Human Rotavirus

Objective:The model of diarrhea with rotavirus was established by rotavirus infection in newborn mice.On this basis,the effects of montmorillonite combined with probiotics on intestinal mucosa in mice infected with rotavirus were observed.And the effects of montmorillonite combined with probiotics on intestinal mucosal barrier were explored.Methods:1 Human rotavirus were cultured with MA104 cells?herb fetus kidney cells?and cell culture infective dose 50%(CCID₅₀)was used to detect the virulence of RV.180 Kunming suckling mice were randomly divided into five groups:20 in the groupwith control,40 in the group with virus,40 in the group with montmorillonite treatment,40 in group with montmorillonite combined with probiotics treatment,and 40 in the group with probiotic treatment.The mice in experimental group were given 0.2ml of RV(10^-3.167CCID₅₀),and the mice in control group were given 0.2ml of the virus-free medium.From the second day,the suckling mice which after successful infetion was given 0.1ml of montmorillonite in montmorillonite treatment group,and 1 time/day for 5 consecutive days;the suckling mice which after successful infetion was given 0.1ml of Saccharomyces boulardii sachets after given 0.1ml of montmorillonite in montmorillonite combined with probiotics treatment group,and 1 time/day for 5 consecutive days;the suckling mice which after successful infetion were given 0.1ml of Saccharomyces boulardii sachets in probiotic treatment group,and 1 time/day for 5 consecutive days;And the virus group was not given treatment after successful infetion.The clinical manifestations,traits of feces,diarrhea rate,and body weight were recorded daily.The feces of suckling mice were collected on the second,fourth,sixth and tenth days after successful infetion.We detect RV in feces by ELISA.The 16SrDNA PCR sequencing method was used to analyze the gene copy numbers of Bifidobacteria?B?and E.coli?E?in feces,and the B/E values were calculated at the same time;10 suckling mice in the experimental group and 5 suckling mice in the control group were randomly killed,the small intestine tissue in suckling mice were collected,the level of secretory IgA?sIgA?was detected by ELISA,the samples of blood were collected from the the aorta of abdominal,and the content of diamine oxidase?DAO?in serum was detected by ELISA.Taken another small intestine,and the pathological changes of the intestinal mucosa were observed with a light microscope.2 SPSS 21.0 software was used for statistical analysis.Results:1 Human rotavirus of Wa strain can successfully infected Kunming suckling mice and cause symptoms of diarrhea.2 The suckling mice whice fed the virus were suffer from mental fatigue,decreased activity,poor appetite,dry skin,and diarrhea compared in the control group.The clinical symptoms of suckling mice were improved after different treatment,although the diarrhea rate is lower than that in the virus group,there is no statistical difference?P>0.05?.There was no statistical difference in weight of suckling mice at different times points?P>0.05?.3 The test results of intestinal flora showed that disturbances in the intestinal microflora of the suckling mice.About Bifidobacteria:On the second day,the number of bifidobacteria in the experimental groups were lower than that in the control group?P<0.05?.On the 4th day,the number of bifidobacteria in the treatment groups were higher than that in the virus group,but there was no statistical difference in each treatment groups?P>0.05?.On the 6th day,the number of bifidobacteria in the combined treatment group was significantly higher than that in the single treatment group?P<0.05?;On the 10th day,the number of bifidobacteria in the virus group was still lower than that in the control group?P<0.05??About E.coli:The number of intestinal E.coli had increased in the experimental group at different time points,But there was no statistical difference compared with the control group?P>0.05?.About B/E:On the second day,the values of B/E of the suckling mice in experimental groups were smaller than those in control group?P<0.05?.On the 4th day,the values of B/E in treatment groups were higher than that in the virus group,but there was no statistical difference in each treatment group?P>0.05?.On the 6th day,the values of B/E of the combined treatment group was larger than that in single treatment group?P<0.05?.On the 10th day,the values of B/E in the virus group was still lower than that in the control group?P<0.05??4 The results of sIgA in intestine showed that on the second day,the concentration of sIgA in the intestine in experimental group was lower than that in the control group?P<0.05?.On the 4th day,the sIgA in the treatment groups were higher than that in the virus group,but there was no statistical difference in each treatment groups?P>0.05?;On the 6th day,the sIgA in the treatment group was higher than that in single treatment group?P<0.05?.On the 10th day,the sIgA in the virus group was still lower than that in the control group?P<0.05?.5 The results of concentrations of DAO in serum showed that on the second day,the DAO in experimental groups were higher than those in the control group on the second day of RV?P<0.05?;On the 4th day,the concentrations of DAO in the treatment groups were lower than that in the virus group,but there was no statistical difference in each treatment groups?P>0.05?;On the 6th day,the DAO in the combined treatment group was lower than that in the single treatment groups?P<0.05?.On the 10th day,the DAO in the virus group was still higher than that in the control group?P<0.05?.6 The pathological changes in intestinal mucos obsered with Light microscopeshowedthatthesmallintestine mucosalvilli were morphologically and structurally intact,and no damage was observed in the control group;But the mucosa of small intestine of the suckling mice in the experimental group were damaged and shed,epithelial cells with edema,some cells became to vacuolization,and some cells were necrotic.Conclusions:1.The RV of Human of Wa strain can successfully infect 4-day-old suckling Kunming mice,and the diarrhea model was successful;2.The imbalance of intestinal microflora was occurred in the early stage ofsuckling mice after RV-infected,which showed that the number of bifidobacteria had decreased and the number of E.coli didn't changed with significantly,but the valueof B/E had decreased.3.The concentration of sIgA in the intestine of suckling mice after RV-infected showed a trend change,that is,the concentration of sIgA in the early stage had decreased,and then it were increased gradually.It were peaked on the 4th to 6th day after the virus was infected,and it was almost normal on the 10th day.4.The concentrationof DAO in the blood of suckling mice after infected with RV had increased with different extents,and the serious damage was became on 4th to 6th days.The it was almost normal on 10th day.5.Montmorillonite combined with probiotics is the best treatment for enteritis of RV.Montmorillonite combined with probiotics can promote the secretion of sIgA of intestinal and improve the immune function of the body's.And it can protect the integrity of intestinal mucosa and improve intestinal permeability.And improve the intestinal microecology of rotavirus enteritis mice,rapidly increase the number of beneficial bacteria in the intestine,regulate intestinal flora structure,and correct the intestinal microecological balance as soon as possible.