| Objective:Liver injury is one of the most common adverse reactions of Polygonum multiflorum Thunb.(PMT)which was reported recently.It is a common method to study the toxic mechanism of liver injury induced by PMT from cell or molecular level.But most studies in these years have showed that there was a great correlation between gut microbiota and the occurrence and development of liver disease.However,only a few studies were focused on the relationship between gut microbiota and PMT induced liver injury recently.Therefore,based on the model our group established previously of PMT induced liver injury activated by lipopolysaccharide(LPS),then taken the Illumina high-throughput sequencing and Real-time quantitative PCR(qRT-PCR)to analysis the changes on gut microbiota of PMT induced liver injury,to exploring the toxic mechanism and providing a theoretical basis for more reasonable and scientific feeding of PMT.Methods:References on the model our group established previously,taken all the rats divided into control group(group C),LPS group(group L),LPS+Acetaminophen(group LA),PMT group(group P)and LPS+PMT group(group LP)randomly,and 6 rats in each group.There were 4 time points,which were the 2nd hour,the 14 th hour,the 5th day and the8 th day.At the 2nd hour,all the rats divided into 2 groups: the rats were injected LPS or without LPS,so there were only group C and group L at this time point.Otherwise,there were 5 groups at the other 3 time points.Then,group L,group LA and group LP were injected 0.004 g/Kg LPS via tail vein,and the group C and group P were injected the salineat the same way.After 2 hours,group LA were i.g 0.625 g/Kg APAP,group P and group LP were i.g 12g/Kg PMT,and taken it once a day for 7 days,recorded the body weight of the rats.Then taken the blood and the liver sample of the rats’ at the 4 different time points.The blood sample for detecting the value of ALT,AST and LPS,the liver sample for recording the weight and histopathological examination.At the same time,taken the fecal sample of group C,group L,group P and group LP at 4 different time points,then exacted the DNA from gut microbiota,and taken PCR amplification after DNA with high quality.Then sequenced the V4 region of the 16 S rDNA by Illumina high-throughput sequencing,testing the α and β diversity,the relative abundance of gut microbiota,the LEfSE analysis to identified the biomarkers.Taken the qRT-PCR to tested and verified the quantitative results of the biomarkers finally.Results:2 hours after LPS activated,compared to group C,the value of serum ALT,AST and LPS were obviously increased in group L,and the body weight obviously dropped and the liver was swollen slightly.At the 8th day,compared to group C,there were no abnormal conditions in group P,the value of ALT,AST and LPS in group L have no difference with group C,and weight again,the histopathological examination of liver showed that some microgranuloma were existed.Compared to group L,the value of ALT,AST and LPS in group LA were obviously increased and the body weight gain was inhibited.Moreover the histopathological examination of liver found that multiple liver parenchyma were scattered with microgranulima and lymphocyte infiltration.Compared to group L,body weight of group LP was obviously dropped,and the liver histopathological examination showed that multiple liver parenchyma were scattered with microgranulima and existed mild degeneration of liver cells.The results showed that the liver injury model induced by PMT have been established,which is able to study further.Analysis the 54 feces samples of each group at 4 different time point by high-throughput sequencing,we obtained 3429945 raw database,3402523 clean tags,3177193 effective tags,803105345 nt base,the average base are 14872321.2 nt and the average tags are 253 bp.α diversity analysis showed that,rarefaction cure means an reasonable database and species accumulation curve means enough samples among the feces sample,and it is able to further study.The Simpson index,Chao1 index and ACE index had no difference in this study,while the Shannon index showed that after 7 days feeding,the diversity of gut microbiota of group LP had increased.β diversity analysis showed that the PCA,PCoA had no difference in gut microbiota at different time point,while based on weighted unifrac distance unweighted pair-group method with arithmetic(UPGA)analysis showed that there was a difference among them.Moreover,the LEfSE analysis showed that there were some differences in gut microbiota between group LP and the other groups at different time point.The result detected by Illumina high-throughput sequencing and qRT-PCR indicated that,at the 14 th hour,compared to group C,Enterobacteriaceae among group L and group LP had significantly increased.As the PMT administration times increasing,compared to group C and group L,the abundance of Lachnospiraceae and Enterococcaceae in group LP were increased,while Lactobacillus decreased.It is indicated that there was a gut microbiota imbalance in group LP at the 8th day to some extent.Conclusion:1.The liver injury model induced by PMT had established,which is able to study and analysis further.2.The diversity of gut microbiota of this model was increased,LPS+PMT feeding promoted the abundance of Lachnospiraceae and Enterococcaceae,whereas,the abundance of Lactobacillus was suppressed.It is indicated that there was a mild gut microbiota imbalance among rats in group LP.3.The Illumina high-throughput sequencing and qRT-PCR applied in quantitative analysis showed a good consistency,which the former obtained more information about microbiota and had a greater advantage than the later. |
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