| The traditional Chinese medicine Gardeniae Fructus(“Zhi Zi”in Chinese,ZZ in brief)is the dried mature fruit of Gardenia jasminoides Ellis of Rubiaceae family,which has the efficacy of discharging fire,eliminating vexation,reducing fever,causing diuresis,and cooling blood to remove apthogentic heat and so on.In recent years,there have been a growing number of literature reports that ZZ and its main active component geniposide,which cause liver injury when used in large quantities or for a long time.In Chinese medicine clinical,ZZ usually forms Zhi-Zi-Chi Decoction(ZZCD)with Semen Sojae Praeparatum(“Dandouchi”in Chinese,DDC in brief)compatibility,and on this basis is added flavor,making up different compound uses.DDC is a fermented processed product of mature seeds of soybean Glycine max(L.)Merr from the family of Fabaceae,which is bitter and cold in nature and has the effects of relieving symptoms,removing irritation,promoting depression and detoxifying.While there is a growing number of literature reporting hepatotoxicity,there are few studies suggesting ZZCD leads to liver injury.The preliminary research work of our group found that the metabolite of geniposide in the intestine,genipin,is the direct material basis that leads to the hepatotoxicity of ZZ,and the hepatotoxicity of genipin is significantly greater than that of geniposide.While the hepatotoxicity of ZZ decreases significantly when it is combined with DDC.DDC can increase the abundance of probiotic bacteria and reduce the abundance of conditionally pathogenic bacteria to maintain intestinal flora homeostasis by regulating the structural and metabolic characteristics of the intestinal flora,while exerting hepatoprotective effects by regulating the expression of nuclear factor-erythroid 2-related factor 2(Nrf2)protein in rat liver tissues through the regulation of butyric acid content in the intestine.The results showed that the expression of Nrf2 protein in rat liver tissues could play a role in liver protection.Based on the above results,we screened a range of butyric acid-producing bacteria from the rat cecum contents,and further screened the activity of butyric acid-producing bacteria using the active substances in ZZCD,and finally found that Lactobacillus rhamnosus was more active among the butyric acid-producing bacteria.Combining these findings,we hypothesized that Lactobacillus rhamnosus could antagonize the hepatotoxicity induced by genipin and exert hepatoprotective effects.Therefore,in this study,the hepatoprotective effect of Lactobacillus rhamnosus was first demonstrated by using genipin-induced liver injury rats as a model;after that,metabolomics and ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS/MS)techniques were used to reveal the small molecule mechanism of the action of Lactobacillus rhamnosus.Finally,the expression of key targets in the Kelch like epichlorohydrin associated protein 1(Keap1)/Nrf2 pathway and its upstream and downstream regulated proteins related to oxidative stress injury in rat liver was investigated to explore how Lactobacillus rhamnosus alleviates the mechanism of genipin-induced liver injury through oxidative stress pathway.The details of the study are as follows:1.In order to clarify whether Lactobacillus rhamnosus can improve the hepatotoxicity induced by genipin,this study firstly gave rats genipin by gavage at a dose of 100 mg/kg to establish an acute liver injury model,and used the body weight,alanine aminotransferase(ALT),aspartate aminotransferase(AST)and Hematoxylin-eosin(HE)staining of liver tissue sections.The results showed that gavage administration of 100 mg/kg caused significant decrease in body weight and increased ALT and AST in SD rats,and the liver tissue sections showed that genipin could produce significant liver injury in rats;Lactobacillus rhamnosus suspension was subsequently given to liver-injured rats by gavage at a dose of 2×10~8 CFU.Significant improvements in body weight,biochemical parameters of liver function,and liver tissue sections were found,indicating that Lactobacillus rhamnosus could reverse genipin-induced liver injury.This part of work demonstrated that Lactobacillus rhamnosus could alleviate the liver tissue damage induced by genipin in rats from three perspectives:body weight,liver indexes and pathological sections,which provided a reference for further development of Lactobacillus rhamnosus and the study of detoxification mechanism of ZZ.2.In order to investigate its effect on metabolic profiles in rat liver tissues and to explore the mechanism of Lactobacillus rhamnosus to ameliorate the hepatic injury caused by genipin,we used a non-targeted metabolomics approach based on ultra performance liquid chromatography-quadrupole-exactive orbitrap technique to analyze the differential metabolites and differential metabolic pathways in rat liver tissues.The peak areas were extracted and corrected using 2,6-dihydroxybenzoic acid as an internal standard.The liver metabolic profiles of rats were analyzed by multivariate analysis such as principal component analysis and supervised orthogonal partial least squares discriminant analysis,and it was found that the liver metabolic profiles between the blank control group and the model group,and between the model group and the bacteria-giving group were significantly distinguished,and the bacteria-giving group was more biased toward the blank control group.50 endogenous metabolites with variable projection importance(VIP)>1.5 and P ANOVA<0.05 were further screened as differential metabolites.By analyzing the metabolic pathways involved in the differential metabolites,it was found that Lactobacillus rhamnosus exerts hepatoprotective effects mainly related to tryptophan metabolism,arginine biosynthesis,and arginine and proline metabolic pathways.This result provides an important substance bases and scientific basis for further searching the biomarkers of hepatic injury caused by genipin and exploring the molecular mechanism of Lactobacillus rhamnosus exerting its anti-hepatic injury effect,also provides implications for safer clinical use of ZZ.3.In order to clarify the specific mechanisms of the effects of Lactobacillus rhamnosus on tryptophan metabolism and arginine-related metabolic pathway in the liver,this study was conducted by UHPLC-MS/MS technique,using exogenous substance 2,6-dihydroxybenzoic acid as internal standard,and semi-quantitative study was conducted around 15 endogenous small molecules in tryptophan metabolic pathway and arginine-related metabolic pathway which are closely related to the development mechanism of liver injury.Firstly,the established UHPLC-MS/MS semi-quantitative method was examined,and the results showed that the specificity,precision,detection limit and stability of the method were good,indicating that the established semi-quantitative analytical method could obtain stable and reliable results.The results of the semi-quantitative analysis of the target metabolites in rat liver showed that Lactobacillus rhamnosus was able to modulate the effects of tryptophan(Trp),quinolinic acid(QA),melatonin,arginine(Arg),proline,ornithine,glutamic acid,aspartic acid,5-hydroxytryptophan(5-HT)and citrulline in the liver of rats with liver injury induced by genipin.In addition,in this study,plasma samples were collected from healthy volunteers and patients with liver injury,and plasma levels of Trp,Kyn and other endogenous small molecules were analyzed by semi-quantitative analysis,and it was found that compared with healthy volunteers,plasma levels of Trp,Kyn,Kyn/Trp,NAS and glutamine were significantly higher in patients with liver injury;the levels of QA,Arg and glutamine were significantly downregulated;liver The levels of 3HAA,5-HT,5-HIAA and aspartate were not significantly different in patients with liver injury compared with healthy volunteers,although there was a trend of upregulation;melatonin and proline were not significantly different.The results in this part suggest that Lactobacillus rhamnosus antagonizes hepatic injury caused by genipin and exerts hepatoprotective effects,which may be related to its regulation of the levels of metabolites in amino acid metabolism-related pathways such as Trp,Kyn,and 3HAA.4.Several substances in the tryptophan pathway and arginine-related metabolic pathway have been closely linked to the development of liver injury.The Keap1/Nrf2 pathway is an important signaling pathway for the body to cope with oxidative stress.Combining the previous research results of our group,we hypothesized that Lactobacillus rhamnosus could exert hepatoprotective effects by modulating the Keap1/Nrf2 pathway in the organism.We therefore investigated the relative gene expression of key targets in the Keap1/Nrf2 pathway and their upstream and downstream regulatory proteins associated with oxidative stress injury in rat liver tissue,and measured the expression of the Keap1 protein using the Western Blot technique.The results demonstrated that Lactobacillus rhamnosus was able to indirectly regulate the expression of Keap1 and Nrf2 by increasing the expression of protein kinase B(AKT),extracellular-signal-regulated kinases(ERK)5,SQSTM1,c-Jun n-terminal kinase(JNK)and other genes to reduce the ubiquitination of Keap1 against Nrf2 and enhance the antioxidative capacity.Lactobacillus rhamnosus can also directly down-regulate the gene expression of Keap1 and up-regulate the expression of Nrf2 gene,thus affecting the expression of antioxidant genes such as Glutathione peroxidase 4(GPx4),NADPH:quinone oxidoreductase 1(NQO1)in the downstream pathway of Keap1/Nrf2,which exert their hepatoprotective effects.In conclusion,this study verified the protective effect of Lactobacillus rhamnosus on the hepatic injury caused by genipin,which provides a reference for the further development of Lactobacillus rhamnosus and the study of the detoxification mechanism of ZZCD.A non-targeted metabolomics approach was used to analyze the metabolic profile in the rat liver and reveal the protective mechanism of Lactobacillus rhamnosus against the toxicity of genipin.A semi-quantitative analytical method based on UHPLC-MS/MS was established to study the metabolic levels of the key targets in the tryptophan metabolism,arginine biosynthesis,and arginine and proline metabolic pathways,elucidating the small molecule mechanism of hepatoprotective effects of Lactobacillus rhamnosus.RT-q PCR and Western blot techniques were used to investigate the key targets in the Keap1/Nrf2 pathway in rat liver and their upstream and downstream regulated proteins and relative expression of genes associated with oxidative stress injury.We found that Lactobacillus rhamnosus could improve Nrf2 gene expression,reduce its ubiquitination,and further regulate the expression of antioxidant genes such as GPx4 and NQO1 to exert hepatoprotective effects through multiple pathways,revealing the mechanism by which Lactobacillus rhamnosus exerts hepatoprotective effects through oxidative stress pathways.This study provides an important draw for elucidating the molecular mechanism by which DDC decreases ZZ hepatotoxicity by regulating intestinal microorganisms,and provides an important draw for improving the clinical drug safety of ZZ containing compounds,and provides an avenue for further development of Lactobacillus rhamnosus as a novel approach to ameliorate Drug-induced liver injury. |
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