Preventive And Therapeutic Effects Of Probiotics Combined With Prebiotics On The Non-alcoholic Fatty Liver Disease In Mice Model And Related Molecular Mechanism

IntroductionThe incidence of nonalcoholic fatty liver disease(NAFLD)is constantly rising with the improvement of living standards,which has become a global public problem.It became the world’s most common liver disease in adult and children,which had impacted on people’s health and quality of life seriously.Although the morbidity of NAFLD is much high,the specific pathogenesis is still unclear.Moreover,there is no effective treatment until now.Therefore,it is much urgent to develop the effective treatment for patients suffering NAFLD.NAFLD patients usually accompany with obesity,diabetes and insulin resistance,but not all obese people will develop into NAFLD.The intestinal microbiota and barrier may play a key role in the pathogenesis of NAFLD.We found intestinal dysbacteriosis and intestinal barrier dysfunction appeared both in animal model and human model.Once the intestinal mucosal barrier destroyed,the intestinal bacteria and its products could invade the liver through the enteric liver axis and caused a series of immune and inflammatory reactions,which further lead to NAFLD,obesity,type 2 diabetes and other metabolic diseases.Intestinal barrier is the body’s first line of defense to resist the intestinal harmful substances into body circulation.The integrity of intestinal is especially important to maintain the stability of the internal environment.The tight junction of intestinal epithelium was the main guarantee to maintain the integrity of intestinal barrier function,which is the key factor of cell permeability.When intestinal mucosal permeability increase,intestinal mucosal epithelial cell barrier would be destroyed,and a large number of intestinal bacteriaand their products could enter the body circulation.Therefore,maintaining the integrity of the intestinal mucosa barrier could reduce the risk of intestinal bacteria and their products entering into the body circulation,which would be the key step to prevent and treat the NAFLD.Many studies confirmed that probiotics could improve the intestinal mucosal permeability by regulating the intestinal microbiota,which further reduce the incidence of high fat diet induced metabolic syndrome.Moreover,probiotics could inhibit the accumulation of fat in the liver and liver inflammation,also inhibit insulin resistance to delay the development of NAFLD.Fructooligosaccharides is a water-soluble dietary fiber.Although it can not be directly digested and absorbed by the human body,it could promote the intestinal bacteroides,Lactobacillus proliferation.It could regulate the expression of fat-producing enzyme gene and reduce the source of liver fatty acid synthesis,which further resultin the reduction of liver triglycerides,liver fat content and serum cholesterol levels.Although some studies had confirmed that probiotics and Fructooligosaccharides(FOS)could improve blood lipids,and prevent NAFLD,it is still unknown that if Lactobacillus paracasei N1115 can prevent and treat the NAFLD.It is also unclear whether application of Lactobacillus paracasei N1115 combined with FOS together as a symbiotic formulation could enhance the NAFLD preventing effect.In summary,we hypothesized that combining Lactobacillus paracasei N1115 and FOS could improve the preventive effects on NAFLD,which may be related to their role in the regulation of intestinal mucosal barrier integrity.To further validate this hypothesis,we treat HFD induced NAFLD mice with Lactobacillus paracasei N1115 and FOS to test their symbiotic formulation effectPart I Co-administration of Lactobacillus paracasei N1115 and FOS effecton the nonalcoholic fatty liver disease in high fat diet induced NAFLD mice model Objectives:Lactobacillus paracasei N1115 combined with FOS were used to treat NAFLD mice,then the blood lipid,blood glucose and liver fat deposition in NAFLD mice were further analyzed.This will validate if Lactobacillus paracasei N1115 and FOS can reduce the lipid level and inbit insulin resistance,which further play a role inprevention and treatment of NAFLD in mice model.Materials and Methods:1.Male C57 mice were randomly divided into 5 experimental groups.Group 1was received a normal diet(ND).Group 2 was received a high-fat diet(HFD)to establish NAFLD models.Group 3 was received HFD containing L(HFD+L)(2.2×109 CFU/mL).Group 4 was received HFD containing FOS(HFD+FOS)(4g/kg per day).Group 5 was received HFD containing L and FOS.All mice in the experimental group were sacrificed after 16 weeks of continuous intervention.2.The general condition,body weight,Lee’s index and liver index in all group were evaluated.3.The level of blood lipid,blood glucose and insulin resistance were measured in each group.Liver pathological alteration was observed by HE and Sudan Ⅲstaining.Results:1.The NAFLD mice model was built by feeding HFD for 16 consecutive weeks.2.All group mice vital signs were stable,compared with other group.The level of body weight,Lee’s index and liver index of HFD Group increased significantly(P 0.05).In the three intervention groups,the levelof body weight,Lee’s index and liver index decreased greatly(P 0.05).Lactobacillus paracasei N1115,FOS and their combination could significantly reduce the level of body weight,Lee’s index and liver index(P 0.05).3.Compared with other groups,the serum level of TC,TG,LDL,GLU and insulin resistance of HFD Group increased significantly(P 0.05),but HDL decreased significantly(P 0.05).From the HE and Sudan Ⅲ staining,the hepatic steatosis and scattered chronic inflammatory cells were seen in the infiltration.Compared with the Group HFD,the serum TC,TG,LDL and insulin resistance of Group HFD+FOS,Group HFD+L and HFD+FOS+L decreased,while serum HDL increased(P 0.05).While the liver steatosis and inflammatory cell infiltration of the other three intervention groups were improved.Conclusions:Lactobacillus paracasei N1115 and FOS could reduce the blood lipid,blood glucose and insulin resistance in high fat diet induced C57 mice,which may prevent NAFLD in C57 mice.Part II Co-administration of Lactobacillus paracasei N1115 and FOS reduce inflammation in NAFLD mice model Objectives:Inflammatory response played an important role in the pathogenesis of NAFLD.Therefore,based on the first part of the study,we further studied whether Lactobacillus paracasei N1115,FOS and their combination could reduce inflamm-ation in high fat diet inducedNAFLD mice model.Materials and Methods:1.The level of serum endotoxin(LPS),diamine oxidase(DAO)were detected;serum and liver TNF-α,IL-1β,IL-6 and IFN-γ were measured.2.The m RNA level of TLR4、NF-kb、InsR、IRS-1 in mouse liver were analyzed by qPCR Results:1.Compared with other group,the serum level of LPS,DAO,serum and hepatic TNF-α,IL-1β,IL-6 and IFN-γ level of HFD Group were significantly increased than those in the other experiment groups(P 0.05).Compared with the HFD Group,the serum LPS,DAO,serum and hepatic TNF-α,IL-1β,IL-6 and IFN-γ level of Group HFD+FOS,Group HFD+L and HFD+FOS+L decreased(P 0.05).2.Compared with the Group HFD,the expression levels of TLR4,NF-kb mRNA in liver of Group HFD+L,Group HFD+FOS and HFD+FOS+L down-regulated,while InsR,IRS-1 mRNA in liver up-regulated(P 0.05).Conclusions:Lactobacillus paracasei N1115,FOS and combination therapy could reduce the level of LPS in the blood circulation,inhibit the activation of LPS/TLR4 signaling pathway and reduce the release of TNF-α,IL-1β,IL-6 and IFN-γ in liver,and reduce liver inflammatory reaction of NAFLD induced by the high fat diet.At the same time,the reduction of LPS and inflammatory factors made InsR,IRS-1 mRNA expression increased in liver,thereby reducing the body’s insulin resistance,which further regulated the blood lipid level and played a role in improving NAFLD in mice model.Part III Lactobacillus paracasei N1115 combined with FOS improve the intestinal mucosal barrier of mice with NAFLD Objectives:Intestinal mucosal barrier function played an important role in the pathogenesis of NAFLD.Therefore,based on the previous two-part studies,we further validate the effects of Lactobacillus paracasei N1115,FOS and their combination on intestinal barrier integrity.Materials and Methods:1.The intestinal mucosa pathogenesis was observed by HE staining.2.The mRNA level of tight-junction proteins Claudin-1 and Occludin in the intestine were analyzed by qPCR.3.The expression level of tight-junction proteins Claudin-1,Occludin and p38,p-p38 in intestinal were analyzed by Western-Bloting.Results:1.The intestinal mucosa of mice in HFD group showed defect,fracture,glandular destruction,irregular arrangement and structural disorder from mouse intestinal biopsy.Through the intervention of Lactobacillus paracasei N1115,FOS and their combinati-on,the arrangement of intestinal mucosa in every intervention group was more regular,neat in morphology.2.Compared with the high fat diet group,the mRNA and protein level of Claudin-1,Occludin in the intestine of the mice were significantly increased by the intervention of Lactobacillus paracasei N1115,FOS and combination therapy(P0.05),and also the integrity of the intestinal mucosa increased;3.The Lactobacillus paracasei N1115,FOS and their combination can inhibit phosphorylation of p38 protein.Conclusions:Lactobacillus paracasei N1115,FOS and combined application could increasethe expression of Claudin-1 and Occludin by inhibiting the phosphorylation of intestinal p38,which further improve the intestinal mucosal barrier integrity.Combined with the second part,we find that with the improvement of intestinal mucosal barrier integrity,LPS reduced in the bowel.The LPS-TLR4 signaling pathway was also inhibited in the liver.The release of liver inflammatory factor and insulin resistance were also reduced,which further improved the NAFLD.