Study On The Relationship Between Gut Microbiota Characteristics And Anti-tuberculosis Drug-induced Liver Injury

Objectives This study aimed to explore the dynamic changes of intestinal microflora when liver injury occurred in patients with pulmonary tuberculosis before?T1?and after treatment?T2?,and to analyze the relationship between intestinal flora and anti-tuberculosis drug-induced liver injury?ADLI?.The changes of intestinal flora and intestinal barrier function in liver injury model rats constructed by four first-line anti-tuberculosis drugs were analyzed.The screened gut bacteria were used to construct an intervention model to clarify its potential mechanism of action in anti-tuberculosis drug-induced liver injury.Methods First of all,we collected two stool samples of patients with diagnosed tuberculosis in the Infectious Disease Hospital in Hulunbeier,Inner Mongolia before?T1?and after treatment?T2?from October 2017 to October 2018 and then the patients were divided into liver injury group?ADLI group?and without liver injury group?Non_ADLI group?according to whether liver injury occurred within 3 to 4 weeks after treatment.The 16 S rDNA high-throughput sequencing was used to analyze the differences in intestinal microflora,the automatic biochemical analyzer was used to detect changes in liver function indicators and ELISA was also used to examine changes in serum TNF-? and IL-6 levels.Secondly,four anti-tuberculosis drugs including isoniazid?H?,rifampin?R?,pyrazinamide?Z?and ethambutol?E?were co-administered into rats to establish a rat liver injury model.Rats were randomly divided into the control group?group D,n=8?,the 15-day HRZE-administered group?HRZE_T1,n=8?and the 30-day HRZE-administered group?HRZE_T2,n=8?.Meanwhile,16 S rDNA high-throughput sequencing was conducted to explore the changes of intestinal microflora in the rat liver injury model,the automatic biochemical analyzer was used to determine the changes of serum ALT and AST,the HE staining was performed to observe liver histological changes,ELISA was used to detect TNF-? and IL-6 expression in serum,the fluorescein isothiocyanate-dextran tracer was used to detect the intestinal mucosa permeability,the limulus amebocyte lysate gel was used to detect changes in serum LPS expression,and RT-PCR as well as western blot were conducted to examine the mRNA and protein expression of Claudin-1,Claudin-3,Occludin and ZO-1 gene in the colon.Finally,we performed an in-depth study on Akkermansia bacteria related to liver injury found in both human and animal models.Interventional models were established by gavage of Akkermansia strains in rats every other day,and then several indicators in normal control group?group D?,HRZE combination medication group?HRZE group?and intervention group?HRZE_A.muc group?were analyzed,and the potential mechanism of Akkermansia strain's effect on liver injury caused by anti-tuberculosis drugs could be clarified.Results The dynamic comparison results of patients before and after treatment showed that both the ADLI group and the Non_ADLI group had the ability to decrease ? diversity?P<0.05?,while the ? diversity comparison did not show any difference?P> 0.05?.The LEfse analysis demonstrated that there were 50 and 59 taxa in ADLI and Non_ADLI groups showing statistically differences from T1 to T2?P> 0.05?.When comparing ADLI and Non_ADLI groups,there is no difference in both ? diversity and ? diversity during both T1 and T2?both P> 0.05?.LEfse data results showed that at T1,there were differences in 13 taxa?9 taxa were at the genus level?in both the ADLI and Non_ADLI groups while at T2,there were differences in 29 taxa?including 14 at genus level?.With further analysis of the correlation between differential changes in 14 genus at T2 and liver function indicators including ALT,AST,TNF-? and IL-6 expression,it could be seen that 7 genera were related to ALT,including Akkermansia,Clostridioides,Ruminiclostridium₅,Comamonas,unclassified_fDesulfovibrionaceae,Fusobacterium and Morganella.The analysis of the model with liver injury induced by HRZE four drugs indicated that compared with group D,? diversity decreased in HRZE_T1 and HRZE_T2 groups and at the same time,? diversity changed significantly.LEfse data revealed that compared with group D,the abundance of probiotics such as Lactobacillus and Akkermansia was significantly reduced in both HRZE_T1 and HRZE_T2 groups,while gram-negative bacteria such as Bacteroides and Enterorhabdus increased obviously.Meanwhile,serum LPS,IL-6 as well as TNF-? expression levels in HRZE_T1 group and HRZE_T2 group elevated significantly,intestinal permeability increased while the mRNA and protein expression levels of tight junction proteins like Occludin and ZO-1 in the colon decreased significantly when compared with group D.Results of the Akkermansia strain intervention model showed that in the HRZE group compared with group D,several indicators increased including the serum ALT and AST activity,MPO activity liver tissue,mRNA and protein expression of TNF-? and IL-6 in liver tissue,intestinal mucosa permeability and LPS expression in serum,while the sIgA content in the colon,the mRNA and protein expression of Occludin as well as ZO-1 were all decreased obviously.Compared with HRZE group,several indicators in HRZE_A.muc group were reduced like ALT and AST activity in serum,MPO activity in liver tissue,mRNA and protein expression of TNF-? and IL-6 in liver tissue,intestinal mucosal permeability and serum LPS level,while colon sIgA level,the mRNA and protein expression levels of Occludin as well as ZO-1 were elevated significantly.Conclusions 1 There are significant differences in the dynamic changes of intestinal microflora between ADLI patients and Non_ADLI patients before and after receiving anti-tuberculosis treatment.Compared with Non_ADLI patients,the alpha diversity of the intestinal microflora of ADLI patients decreased,but the structure of intestinal microflora did not change significantly.There were 7 genera found to be related to the occurrence of anti-tuberculosis-induced liver injury including Akkermansia,Clostridioides,unclassified_fDesulfovibrionaceae,Comamonas,Fusobacterium,Morganella and Ruminiclostridium₅;2 When the liver injury in rats was induced by HRZE drugs,the intestinal microflora would be disordered accompanied by damage to the intestinal mucosal barrier;3 The decreased abundance of Akkermansia was related to the occurrence of liver injury induced by anti-tuberculosis drugs and fortunately,oral supplementation to restore Akkermansia bacteria had the ability to relieve liver damage caused by anti-tuberculosis drugs.Figure 57;Table 13;Reference 279...