The Effects Of LFS-01 On DSS Induced Colitis In Mice

Objective: To evaluate the effects of the LFS-01 on DSS Induced colitis in mice,and lay a foundation for the drug development and utilization in the future.Methods:1.Makes the molds and the groupings.This experiment use C57 male mice were randomly divided into normal Control group,DSS group and LFS-01 group,10 mice in each group.Male mice were given a 3% DSS solution for 7 days via drinking to induce colitis.LFS-01 group administered orally daily with LFS-01 in 80mg/kg and in an administration volume of 0.2ml per mouse.2.Explore the LFS-01 inhibit the DSS induced colitis in mice.Sacrifice all mice in 8 day,measure the colon length and take photo for colon,observing inflammatory lesions,Evaluate overall score and DAI score.HE staining of the colon tissue of mice,histo-pathological score was performed to evaluate the injury of tissue.The levels of pro-inflammatory cytokine IL-17 and anti-inflammatory cytokine IL-22 levels in colon tissue of all mice were detected by ELISA.3.Explore the intestinal mucosa repair effect of LFS-01 on DSS induced colitis in mice.Immunofluorescence method was used to detect Occludin in colon tissue of mice in each group.Use RT-PCR to detect the changes of IL-17 RA,IL-17 RC,ACT1,Occludin,Claudin-1,ZO-1at m RNA level in colon tissue of mice in each group.Illustrated DSS induced colitis had serious damaged to intestinal mucosal barrier function,LFS-01 can repair the damage of intestinal mucosa barrier cause by DSS induced colitis.4.Explore the intestinal flora adjustment function of LFS-01 on DSS induced colitis in mice.Extract the DNA of colon contents in each group mice,use PCR-DGGE and rubber cutting sequencing to detect the changes in intestinal microbiota of mice in each group.Use Illuminate Hiseq PE250 16 Sr DNA sequencing analysis technology to comparison the intestinal flora in mice on biological taxonomy、Beta diversity analysis、Alpha diversity analysis and Species differences between group analysis.Results:1.LFS-01 inhibit DSS induced colitis in mice.After 7 days with DSS treatment,the body weight of model group mice significantly reduced(P < 0.05),all model group mice have blood in stool,spirits are drooping and deaths have begun.Control group no death,no bloody and all mice are active.LFS-01 group Without deaths occur and a few mice have slightly bloody in stool and Weight loss is not obvious,the most mice of LFS-01 group are active.In the control group(6.79±0.21CM)the colon length of mice significantly longer than the model group(4.36±0.25CM)(P<0.05).Disease Activity Index(DAI)of DSS model group mice is significantly higher than the control group(P<0.05),Colonic mucosa tissue injury is serious.In the LFS-01 group(5.2±0.25CM)the colon length of mice significantly longer than the model group(P<0.05).Disease scores decreased signifycantly(P<0.05),and the colon tissue injury have remit to some extent.The date of ELISA shows that the colon tissue IL-17 in LFS-01 group is increases slightly but no obvious difference with IBD model group(P>0.05),the IL-22 is also increases slightly but no obvious difference with IBD mice(P>0.05).In the colitis model group,colonic crypt damaged and have severe erosion and ulcer,and compared with normal control group the colon of mice significantly shorter.DAI score、 Tissue injury score and pathological score is higher than other groups.Illustrate modeling success.Compared with model group,colon length of LFS-01 group is longer,and DAI score、 Tissue injury score and pathological score is lower,Damage of colon tissue is lighter.2.Intestinal mucosa repair effect of LFS-01 on DSS induced colitis in mice.The result of Immunofluorescence: Occludin protein level of the colon tissue in Normal group and LFS-01 group significantly higher than the model group.According to the results of RT-PCR.In LFS-01 group,the m RNA level of IL-17 RA,IL-17 RC,ACT1,Occludin,Claudin-1,ZO-1 in colon tissue is Significantly higher than the model group(P<0.05),but in the model group,the m RNA level of IL-17 RA,IL-17 RC and Occludin is no no obvious difference with control group(P>0.05).3.Intestinal flora adjustment function of LFS-01 on DSS induced colitis in mice.Rubber cutting sequencing results show:Model group compared with normal group,conditioned pathogen Helicobacter pylori and Enterococcus were found;compared with the model group Control group and LFS-01 group have more normal flora such as Bacteroides,Land bacteria,and Clostridium.According to the results of 16 Sr DNA sequencing analysis about the colon contents in each group mice.Compared with Control group and LFS-01 group,the Number of bacterial species and bacterial flora diversity in the DSS group was obviously lower.The results of PCA shows that Control group and LFS-01 group is more closer,Show that LFS-01 group is more similar to Control group.The DSS group is far away Control group and LFS-01 group,indicate that the comparability coefficient betwee-n DSS group and Control group or LFS-01 group is low.Compared with Control group,in the DSS group,Bacteroidetes(phylum、order、 genus)、Clostridiales、Desulfovibrionales、Lactobacillus、Prevotellaceae-UCG-003、Lachnospiraceae and other bacteria significantly reduced,Proteobacteria(phylum、order、genus)、Deferribacteres(phylum、order)、Enterobacteriales、Campylobacterales、Erysipelotrichales、Burkholderiales、Escherichia-Shigella、Helicobacter、Allobaculum 、 Terrisporobacter 、 Mucispirillum and other bacteria increased significantly.In the DSS group,normal flora is reduced and Pathogens has increased.Compared with DSS group,in the LFS-01 group,Bacteroidetes(phylum、order、 genus)、Lactobacillus(phylum、order、genus)、Desulfovibrionales、Verrucomicrobiales、Prevotellaceae-UCG-003、Lachnospiraceae and other bacteria increased significantly,Proteobacteria(phylum 、 order 、 genus)、 Deferribacteres(phylum 、 order)、Enterobacteriales 、 Campylobacterales 、 Erysipelotrichales 、 Burkholderiales 、Escherichia-Shigella、Helicobacter、Allobaculum、Terrisporobacter、Mucispirillum and other bacteria significantly reduced.In the LFS-01 group,normal flora has increased,and Pathogens is reduced.Conclusions:1.After 7 days With 3% DSS solution free drinking,All mice of model group had colitis,All mice are bloody and weight dropped sharply and mice of model group occurs death,indicating that the model was successful.2.LFS-01 has obvious relieved DSS induced colitis in mice,and have obvious effect on repair the intestinal mucosa injury,and enhance the intestinal barrier function.3.To some extent,LFS-01 can adjustment bacterial flora disorder caused by colitis.,and increase the intestinal flora diversity,especially increase the intestinal normal flora.