Improvement Effect And Mechanism Of Lactobacillus Casei And Inulin Supplementation Against Anti-tuberculosis Drug-induced Liver Injury In Mice

Objective:The application of anti-tuberculosis drugs is easy to cause liver damage,which adversely affects the compliance and results of antituberculosis therapy.So far,it has not been able to effectively solve anti-tuberculosis drug-induced liver injury(ATDILI),and it is urgent to find reliable and effective adjuvant therapy programs.Lactobacillus casaei(Lc)and inulin,as common probiotics and prebiotics,which have the health effects of regulating intestinal flora,anti-inflammatory,antioxidant,regulating immunity and metabolism.The aim of this study was to establish a mice model of liver injury induced by isoniazid and rifampicin,analyze and compare the improvement effects of Lactobacillus casei,inulin and their synbiotics,and explore the possible biological mechanism.Methods:(1)Effects of different doses of Lc on ATDILI in mice experiment:54 SPF male C57BL/6J mice were randomly divided into 6 groups(9 mice/group)after adaptive feeding for two weeks:normal control group(NC),isoniazid+rifampine model group(MOD),low-dose Lactobacillus casei(Lc)group(LLc),medium-dose Lc group(MLc)and high-dose Lc group(HLc)and positive control group(Sil).The mice in NC and MOD groups were given 0.9%normal saline for the first gavage,LLc,MLc and HLc were given 1×10~8 CFU,1×10~9 CFU and 5×10~9 CFU Lc,respectively,and the positive control group mice were given 100 mg/kg·bw silibinin.(2)On the basis of the above experiment,experiment two aimed to observe observe the effects of high-dose(5×10~9CFU/mice)Lc and inulin on ATDILI in mice:40 SPF male C57BL/6J mice were randomly divided into 5 groups(8 mice/group):normal control group(NC),isonazid+rifampicin model group(MOD),Lactobacillus casei group(Lc),inulin group(INU),and Lc+inulin group(LI).The mice were given intragastric administration twice a day.The mice in NC and MOD groups were given 0.9%normal saline for the first intragastric administration,and the Lc,INU and LI groups were given5×10~9 CFU Lc,600 mg/kg·bw inulin and their mixed solution,respectively.Two hours later,the NC group mice were given 0.5%sodium carboxymethyl cellulose intragastrically,and the mice in other groups were given the suspension of 150 mg/kg·bw isoniazid and 300 mg/kg·bw rifampicin.The intragastric dose was 10 m L/kg·bw for 8weeks.Mice were sacrificed 12 hours after the last gavage.Serum,liver,colon,cecum and their contents were collected and stored in a-80℃refrigerator.The contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and alkaline phosphatase(ALP)in serum were detected by automatic biochemical analyzer.Hematoxylin and eosin(HE)staining was performed on the colon and liver of mice.And the contents of superoxide dismutase(SOD),glutathione(GSH),glutathione peroxidase(GSH-px)and malondialdehyde(MDA)in liver homogenate were detected by chemical kit.ELISA kits were used to detect the contents ofβ-defentin 2(BD-2),secretory immunoglobulin(s Ig)A,lipopolysaccharide(LPS),interleukin(IL)-6 and tumor necrosisα(TNF-α).And 16S r RNA was used to detect intestinal flora in cecum contents.Transcriptome predicted altered relevant pathways in liver tissue.Real-time quantitative PCR(q RT-PCR)was used to detect liver mitogen-activated protein kinase(MAPK),protein kinase B(AKT),phosphatidylinositol3-kinase(PI3K),inhibitory subunit of NF kappa B alpha(IκBα)and NFκB m RNA expression.Spearman correlation analysis explored correlations between intestinal flora and other indicators.Results:(1)Ameliorative effects of different doses of Lc on ATDILI in mice:MOD group mice lobular structure disappeared,inflammatory cell infiltration,liver cell necrosis and other pathological changes occured.Compared with NC group,MOD mice liver index,serum ALP,AST and ALT levels,liver SOD,GSH,MDA content significantly increased.After intervention with different doses of Lc,only part of liver structure could be restored in LLc and MLc groups compared with MOD group.HLc group significantly repaired the damaged liver lobular structure,reduced the necrosis of liver cells,decreased serum ALP and liver SOD,GSH and MDA levels(P all<0.05).(2)Ameliorative effects of Lc and inulin on ATDILI in mice:The liver results of MOD group mice were similar to those of MOD group mice in Result(1).After Lc and/or inulin intervention,compared with MOD group,liver index,serum AST,ALT and ALP levels and liver SOD and MDA contents significantly decreased in Lc group(P all<0.05).Liver index and serum ALT and ALP levels decreased in INU group(P all<0.01),the levels of ALT,AST and ALP in serum and MDA content and GSH-px activity of liver in LI group were significantly decreased(P all<0.05).(3)Effects of Lc and inulin on intestinal barrier induced by anti-tuberculosis drugs in mice:The MOD group showed severe inflammatory infiltration and recess structure destruction in colon.After Lc and/or inulin intervention,compared with MOD group,the contents of BD-2 and TNF-αin colon,LPS content in serum and liver were significantly decreased in Lc group,the abundances of Desulfovibrio and Lactobacillus increased,the abundance of Bilophila decreased(P all<0.05).The content of colon s Ig A significantly increased in INU group(P<0.01),the content of TNF-αdecreased(P<0.05),the Shannon index of intestinal flora increased and the abundances of Akkermansia and Bilophila decreased(P<0.001),Desulfovibrio,Ligilactobacillus and Lactobacillus abundances increased(P all<0.05).The contents of BD-2,IL-6 and TNF-αin colon of LI group were significantly decreased,Desulfovibrio,Ligilactobacillus and Lactobacillus abundances increased and Bilophila abundance decreased(P all<0.05).(4)Explore the mechanism of Lc and inulin in improving ATDILI based on gut-liver axis:Compared with MOD group,137,249 and 143 genes were up-regulated and 117,217 and129 genes were down-regulated in Lc,INU and LI groups,respectively.There were significant differences between the three intervention groups and the MOD group in MAPK and PI3K-AKT signaling pathways.In addition,the results of q RT-PCR showed that the expression of AKT gene in Lc,INU and LI groups was significantly decreased compared with MOD group.In addition,the expression of NFκB decreased and IκBαincreased of downstream gene of MAPK signaling pathway in Lc group.The expression of NFκB gene in INU group was significantly decreased.And PI3K gene expression in LI group was significantly decreased.Spearman correlation analysis to test the relation of intestinal flora and serum,liver and intestinal indexes.The increase of Lactobacillus abundance was correlated with the decrease of IL-6 and TNF-αin colon,LPS and MDA,gene expresssions of MAPK and NFκB in liver and serum ALP(P all<0.05).Bilophila abundance was significantly positively correlated with colon BD-2,IL-6,TNF-α,serum ALT and liver MDA levels(P all<0.05).Conclusion:(1)ADILI occured in mice.High-dose Lc showed better improvement effect,and the effect was similar to that of Lc and inulin synbiotics,which might be related to restoring oxidative stress balance in liver.(2)Inulin alone or combined with Lc can significantly repair intestinal barrier,and its ability to regulate intestinal flora is superior to Lc alone intervention.(3)Lc,inulin and their synbiotics intervention may improve liver injury by regulating MAPK,PI3K-AKT and other signaling pathways,and intestinal barrier and regulation of intestinal flora is also related to it.This provides clues and evidence for clinical adjuvant treatment of ATDILI.