Study On Hepatotoxicity Of Zhixue Capsules Based On Intestinal Flora And Metabonomics Technology

Herb induces liver injury（HILI）,the occurrence of liver injury related to herbal medicines,specifically direct or indirect liver damage caused by herbal medicines or natural drugs and preparations.It is often an important reason for the failure of the development of herb-related preparations and their withdrawal from the market or restriction of use after marketing,and is also one of the main factors inducing liver diseases^[1],and liver injury caused by herbs or their preparations has received widespread attention in China^[2],so it is important to correctly understand the mechanism of action of herbal liver injury.The drug composition of Zhixue Capsules includes Dictamni Cortex and Sophorae Flavescentis Radix,which appear in Sophorae Flavescentis Radix San（Taiping Sheng Hui Fang）and have the function of clearing heat and detoxifying,drying dampness and relieving itching,cooling blood and stopping bleeding,and have good efficacy for hemorrhoids with blood in the stool,anal pain and constipation.Zhixue Capsules were approved for marketing in 2003,and were eventually discontinued and recalled in 2008 due to the many reports of adverse reactions to liver injury.Research on them can help understand the mechanisms of adverse reactions in Chinese medicine to guide the development of new Chinese medicine and safe clinical use.The relationship between intestinal flora dysbiosis and liver disease has received increasing attention in recent years,and intestinal flora is an important factor affecting liver metabolism.Studies have shown that the severity of liver injury can be reduced by regulating intestinal microecology^[3-6] In this study,we investigated the mechanism of hemorrhoid capsules’ hepatotoxicity in rats through the toxicological study of intestinal flora and metabolomics,which was funded by the National Natural Science Foundation of China（81274181） from the basic theory of intestine-hepatic absorption and metabolism axis of oral drugs.1 Objective of the studyTo observe the hepatotoxic effects of hemorrhoid capsules in Wistar rats,and to explore the hepatotoxicity and mechanism of action of hemorrhoid capsules in rats from the basic physiological theory of the intestinal-liver absorption and metabolism axis of orally administered drugs,using intestinal flora and me tabolomics.2 Research methods2.1 Confirmatory toxicity testIn this study,the original formula of Zhixue Capsules was prepared as the test drug,and Wistar rats without specific pathogens（SPF grade）were used as the test subjects.Different concentration doses of toxicity tests were established in the low dose group（4.125g of raw drug/kg）,medium dose group（8.25g of raw drug/kg）and high dose group（16.5g of raw drug/kg）,and the blank control group with the same volume of deionized water was used as the control.All drugs were administered by gavage simulating oral administration.According to the results of the previous experiments,the administration time was set at 4 weeks,and the activity,mental status and abnormalities of the rats were observed and recorded,and the body weight and food intake of the rats were checked at a fixed time every week.After 4 weeks of administration,serum and liver of rats in the blank control group and low,medium and high dose groups of Zhixue Capsule were collected for serum biochemistry and histopathology to evaluate the hepatotoxicity of Zhixue Capsule.2.2 Intestinal flora studyRat feces were collected,DNA was extracted,and the V3-V4 variable region of rat fecal 16S rDNA was amplified by PCR,and the rat feces were sequenced using Illumina high-throughput sequencing technology platform.The statistical analysis of sequencing was carried out,focusing on the analysis of the distribution structure,diversity characteristics and differential flora of intestinal flora in rats with Zhixue capsule hepatotoxicity by α diversity,β diversity and comparison of differences between groups.2.3 Metabolomics analysisThe sera from the blank control group and the rats in the high dose group administered with Zhixue capsules were subjected to ultra performance liquid chromatography-quadrupole orbital ion trap mass spectrometry（UPLC-Q-Exactive-MS）,and the results were subjected to multivariate statistical analysis to identify the differential metabolites for further analysis of metabolic pathways and to reveal the potential mechanism of action of Zhixue capsules hepatotoxicity.3 Study results3.1 Hepatotoxicological study①The hepatic coefficients of Zhixue Capsules were changed and the liver weight increased after 28 days of administration in each dose group.Specifically,the liver weight and liver coefficient of rats increased with increasing dose.The difference was statistically significant（P<0.05）in the middle dose group and high dose group of Zhixue Capsules compared with the blank control group.The difference was statistically significant（P<0.05）.The hepatic pathological changes showed slight to mild hepatocellular steatosis in the liver of rats in each dose group,mainly manifested by tiny vacuoles in the hepatocytes of the hepatic lobules in bands 1 and 2（peripheral and intermediate bands）;compared with the blank control group,the number of hepatocellular steatosis in the liver of animals in each dose group of Zhixue Capsule increased,the pathological score increased and the degree of lesions tended to be aggravated.3.2 Intestinal flora studyThe feces of rats with Zhixue capsule hepatotoxicity were subjected to high-throughput sequencing,and a total of 3210293 valid sequences were obtained for analysis,with an average length of 417bp and 1913 OUT clusters,including 28 at the phylum level,65 at the order level,152 at the order level,269 at the family level,567 at the genus level and 956 at the species level.The composition of rat intestinal flora at the phylum level was mainly Bacteroidota,Firmicutes,Campilobacterota,Proteobacteria,and Actinobacteriota.α-diversity analysis showed that after drug administration The Sobs index,Chao index and Ace index increased,while the Shannon index and Simpson index did not change significantly.28 days after the administration of low,medium and high dosesto Wistar rats,the richness of the flora increased and the relative content of the flora changed,mainly in the increase of the relative content of the phylum Actinobacteriota and the decrease of the relative content of the phylum Thick-walled bacteria and Campylobacter.By LEFse analysis,the different groups were Lanchnospiraceae＿NK4A136＿group,Ruminococcus,Allobaculum,Faecalibaculum,Parabacteroides) and other 10 strains of bacteria.3.3 Metabolomics studyThe differences in serum metabolomics between the hemorrhoid capsule high-dose group and the blank control group were analyzed using UPLC-Q-Exactive-MS technique,and the data were downscaled by PCA analysis and OPLS-DA to screen the differential metabolites with VIP value>1P value<0.05.A total of 51 differential metabolites were obtained,of which 29 potential differential metabolites were identified in the positive ion mode and Twenty-four potential differential metabolites were identified in negative ion mode,and 10-gingerol and indoleacetic acid were identified in both positive and negative ion mode.The differential metabolites included multiple carnitines,sphingosine,taurocholic acid,and taurodeoxycholic acid.The related pathways involved 16 path ways including sphingolipid metabolism,valine,leucine and isoleucine biosynthesis,taurine and hypotaurine metabolism.4 Conclusion of the studyThe original formula of hemorrhoidal capsule was continuously given to Wistar rats for 28 days,and the rats showed hepatotoxicity,the severity of which was positively correlated with the administered dose.The richness of intestinal microbial species in rats increased after administ ration,and the composition of bacterial flora changed,decreasing the proportion of the thick-walled phylum and the phylum of Bacillus mimicus,which may be related to gastric tumorigenic bacteria and Trichoderma spiralis.Hepatotoxicity may be related to the decrease in the content of butyric acid-producing bacteria and the limitation of short-chain fatty acid synthesis and thus 3 hepatotoxicity may be related to the decrease of butyric acid-producing bacteria content and the limitation of short-chain fatty acid synthesis,thus affecting energy and lipid metabolism.