Lactobacillus Acidophilus Ameliorates Obesity In Mice And Its Mechanism

Objective:Obesity,a worldwide health concern,is associated with low-grade chronic inflammation and intestinal dysbiosis.Different probiotics have demonstrated beneficial effects on this disease,thus increasing the interest in the development of probiotic treatments.The aim of this study is to investigate the anti-obesity effects and mechanism of potential probiotic Lactobacillus acidophilus isolated from the porcine gut.Methods:Forty C57BL/6J male mice were randomly divided into normal chowdiet group(NCD),NCD-fed L.acidophilus-treated group(NCD+LA),high-fat diet group(HFD)and HFD-fed L.acidophilus-treated group(HFD+LA).NCD and NCD+LA groups were fed with low-fat diet,while HFD and HFD+LA groups were fed with high-fat diet.NCD+LA and HFD+LA groups were fed with Lactobacillus acidophilus(0.2ml,5×10~9CFU/day),and the NCD and HFD groups were treated with PBS(0.2 ml/day).Lactobacillus acidophilus intervened for 12 weeks,during which the weight,food intake and postprandial blood glucose of mice were recorded.The intestinal permeability experiment was carried out at the 11th week.After 12 weeks,the blood,liver,colon,fat,fur and feces of anesthetized mice were collected for detection.1.The liver weight and white fat weight of mice were recorded;Pathological sections of liver,epididymal fat,inguinal fat,brown fat and fur were stained with hematoxylin and eosin(HE).The liver was frozen and stained with Oil-Red O;Serum TC,TG,LDL-C and HDL-C were measured.2.The protein expression of TNF-α、IL-1β、IFN-γ和F4-80 in the liver,epididymal fat and colon of mice was detected by immunohistochemistry(IHC).3.The expression of proteins related to the NF-κB signaling pathway in the liver was detected by Western-blot.Plasma insulin and liver homogenate LPS concentrations were measured was by ELISA.Postprandial blood glucose(PBG)was measured by the glucometer.4.Real time PCR was used to detect the expression of genes related to lipid metabolism in brown fat,epididymal fat and liver of mice.5.The abundance of intestinal flora was sequenced by using 16S r RNA gene.6.Pathological sections and HE staining of mouse colon were performed.Immunofluorescence and real time PCR were used to detect the expression of mouse colonic tight junction protein and its gene.7.Faecal transplantation.Results:1.The results showed that compared with the normal group(NCD),the high-fat group(HFD)mice fed for 12 weeks resulted in a significant increase in body weight and liver weight,food efficiency ratio(FER:weight gain/food consumption),total white adipose tissue weight,epididymal white fat weight,adipocyte area and lipid deposition of hepatocytes.These indexes had no significant changes in NCD+La group mice,but in HFD+La group mice,body weight,liver weight,fat accumulation,food efficiency ratio and fur fat layer thickness were effectively reduced.In the liver,steatosis,balloon and inflammatory infiltration were significantly improved,and the lipid deposition in the liver were effectively reduced.After intragastric administration of Lactobacillus acidophilus,the TC and TG of mice in HFD+La group decreased significantly.2.The results of immunohistochemistry showed that after intragastric administration of Lactobacillus acidophilus,the expression level of TNF-αand IL-1βin HFD+LA group in the liver was significantly reduced and the infiltration of macrophages was improved.The expression level of TNF-α、IL-1βin Epididymal fat and TNF-α、IFN-γin colon decreased.3.LPS in liver homogenate of HFD mice increased significantly by ELISA.The expression of its receptor TLR4 and related inflammatory pathway protein NF-κB、IκB、P-IκB increased.The concentration of LPS in liver homogenate and the expression of related inflammatory pathway proteins decreased in HFD+LA group.Compared with HFD group,blood glucose in HFD+LA group decreased significantly and insulin resistance index improved.4.The results of Real time PCR showed that Lactobacillus acidophilus had different effects on the energy metabolism genes(such as UCP-1,CPT-1,PPAR-αAnd leptin),lipolytic genes(such as LPL and HSL),and lipid synthesis genes(such as PPAR)-γ、Fas and C/ebp-α)of different tissues.After intragastric administration of Lactobacillus acidophilus,the expression of UCP1 gene in brown fat of mice in HFD+LA group was significantly up-regulated.At the same time,HE staining showed that the area of brown fat cells in HFD+LA group increased significantly and the size of lipid droplets decreased significantly.The expression of PPAR-α、HSL in brown fat increased.In addition,after Lactobacillus acidophilus treatment,the expression of CPT-1,leptin and HSL in epididymal fat was significantly up-regulated,while Fas and PPAR-γwas significantly down-regulated.The expression of Leptin in liver increased and PPAR-γdecreased.5.The results of 16S r RNA gene sequencing in mouse feces showed that by calculating Chao index,ACE index,Simpson index and Shannon index,high-fat feeding significantly reduced the content of Intestinal flora in miceα-diversity.After treatment with Lactobacillus acidophilus,itsα-diversity was significantly restored.At the same time,Lactobacillus acidophilus treatment significantly increased the number of OTUs in high-fat fed mice.Principal component analysis(PCA)and principal coordinate analysis(PCo A)was also performed.The results showed that there was significant difference in the overall composition of intestinal flora among each group.HFD feeding profoundly affected the relative abundance of Firmicutes andFirmicutes/Bacteroidetes ratio compared with control NCD feeding.Under the HFD,L.acidophilus treatment decreased the relative abundance of Firmicutes and Firmicutes/Bacteroidetes ratio.In the top 15 families,a total of 11 families were significantly affected by the HFD rather than the NCD,which belonged to the phylum Bacteroidetes.Under the HFD,it was observed that some families(Bacteroidaceae,Desulfovibrionaceae,Rikenellaceae,Deferribacteraceae,Eggerthellaceae,Ruminococcaceae,Lachnospiraceae,Oscillospiraceae and Peptostreptococcaceae)significantly increased,whereas others(Erysipelotrichaceae and Bifidobacteriaceae)decreased drastically.Under the same situation,LA supplement remarkably increased Lactobacillaceae,Lachnospiraceae,Oscillospiraceae,Peptostreptococcaceae,Erysipelotrichaceae,and Bifidobacteriaceae,while decreased Bacteroidaceae,Desulfovibrionaceae,Deferribacteraceae,Eggerthellaceae and Ruminococcaceae to a great extent.The bacterial community structure with notable differences among the NCD group,the NCD-LA group,the HFD group,and the HFD-LA group was further analyzed by adopting the linear discriminant analysis(LDA)effect-size method(LEf Se).It was figured out that taxa in different levels had differential abundance in the four groups.Actinobacteriota,Actinobacteria,Bifidobacteriales,Lactobacillales,Bifidobacteriaceae,Lactobacillaceae,Lactobacillus,Bifidobacterium,B.pseudolongum,and L.johnsonii were vital and could be regarded as a biomarker in the HFD-LA group.L.acidophilus has a recovery effect on the increase of some specific microbiota in the HFD group.L.acidophilus treatment improves gut microbiota functions involving metabolism,immune response,and pathopoiesia.Under the HFD,L.acidophilus treatment increased aerobic and forming biofilm types,while dramatically decreased potentially pathogenic,anaerobic and stress tolerant ones.6.The results of intestinal permeability test showed that the content of FITC dextran in blood of HFD group increased significantly and the intestinal permeability of HFD group increased.The content of FITC dextran in blood of HFD+LA group decreased significantly.He staining showed that compared with HFD group,the inflammatory infiltration of colon in HFD+LA group was significantly reduced and the intestinal mucosa was restored.The results of immunofluorescence and Real time PCR showed that the expression of colonic tight junction proteins Intectin and Occludin in HFD+La group was significantly higher than that in HFD group.7.The results of fecal bacteria transplantation experiment showed that compared with obese mice receiving control fecal bacteria material,obese mice receiving Lactobacillus acidophilus intervention fecal bacteria material significantly reduced body weight,total white fat and liver weight.Conclusion:Lactobacillus acidophilus can improve lipopolysaccharide translocation,insulin resistance and low-grade systemic inflammation caused by high-fat diet;activate brown fat and regulate the expression of fat related genes by regulating intestinal flora and intestinal permeability to prevent and treat obesity.