PPARα suppresses low-intensity-noise-induced body weight gain in mice: the activated HPA axis plays an critical role.

BACKGROUND: As the second most risky environmental pollution, noise imposes threats to human health. Exposure to high-intensity noise causes hearing impairment, psychotic disorders, endocrine modifications. The relationship among low-intensity noise, obesity and lipid-regulating nuclear factor PPARα is not yet clear. METHODS: In this study, male wild-type (WT) and Pparα-null (KO) mice on a high-fat diet (HFD) were exposed to 75 dB noise for 12 weeks to explore the effect of low-intensity noise on obesity development and the role of PPARα. 3T3-L1 cells were treated with dexamethasone (DEX) and sodium oleate (OA) to verify the down-stream effect of hypothalamic-pituitary-adrenal (HPA) axis activation on the adipose tissues. RESULTS: The average body weight gain (BWG) of WT mice on HFD exposed to noise was inhibited, which was not observed in KO mice. The mass and adipocyte size of adipose tissues accounted for the above difference of BWG tendency. In WT mice on HFD, the adrenocorticotropic hormone level was increased by the noise challenge. The aggravation of fatty liver by noise exposure occurred in both mouse lines, and the transport of hepatic redundant lipid to adipose tissues were similar. The lipid metabolism in adipose tissue driven by HPA axis accorded with the BWG inhibition in vivo, validated in 3T3-L1 adipogenic stem cells. CONCLUSION: Chronic exposure to low-intensity noise aggravated fatty liver in both WT and KO mice. BWG inhibition was observed only in WT mice, which covered up the aggravation of fatty liver by noise exposure. PPARα mediates the activation of HPA axis by noise exposure in mice on HFD. Elevated adrenocorticotropic hormone (ACTH) promoted lipid metabolism in adipocytes, which contributed to the disassociation of BWG and fatty liver development in male WT mice. Summary of PPARα suppresses noise-induced body weight gain in mice on high-fat-diet. Chronic exposure to low-intensity noise exposure inhibited BWG by PPARα-dependent activation of the HPA axis.

Aspirin Caused Intestinal Damage through FXR and ET-1 Signaling Pathways.

Aspirin is a non-steroidal, anti-inflammatory drug often used long term. However, long-term or large doses will cause gastrointestinal adverse reactions. To explore the mechanism of intestinal damage, we used non-targeted metabolomics; farnesoid X receptor (FXR) knockout mice, which were compared with wild-type mice; FXR agonists obeticholic acid (OCA) and chenodeoxycholic acid (CDCA); and endothelin-producing inhibitor estradiol to explore the mechanisms of acute and chronic intestinal injuries induced by aspirin from the perspective of molecular biology. Changes were found in the bile acids taurocholate acid (TCA) and tauro-ß-muricholic acid (T-ß-MCA) in the duodenum, and we detected a significant inhibition of FXR target genes. After additional administration of the FXR agonists OCA and CDCA, duodenal villus damage and inflammation were effectively improved. The results in the FXR knockout mice and wild-type mice showed that the overexpression of endothelin 1 (ET-1) was independent of FXR regulation after aspirin exposure, whereas CDCA was able to restore the activation of ET-1, which was induced by aspirin in wild-type mice in an FXR-dependent manner. The inhibition of ET-1 production could also effectively protect against small bowel damage. Therefore, the study revealed the key roles of the FXR and ET-1 pathways in acute and chronic aspirin-induced intestinal injuries, as well as strategies on alleviating aspirin-induced gastrointestinal injury by activating FXR and inhibiting ET-1 overexpression.

A CDAHFD-induced mouse model mimicking human NASH in the metabolism of hepatic phosphatidylcholines and acyl carnitines.

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of a cluster of conditions associated with lipid metabolism disorders. Ideal animal models mimicking the human NASH need to be explored to better understand the pathogenesis. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has recently been used to induce the NASH model, but the advantages are not established. NASH models were induced using the well-known traditional methionine- and choline-deficient (MCD) diet for 5 weeks and the recently used CDAHFD for 3 weeks. Liver phenotypes were analyzed to evaluate the differences in markers related to NASH. Lipidomics and metabolism analyses were used to investigate the effects of dietary regimens on the lipidome of the liver. The CDAHFD induced stronger NASH responses than the MCD, including lipid deposition, liver injury, inflammation, bile acid overload and hepatocyte proliferation. A significant difference in the hepatic lipidome was revealed between the CDAHFD and MCD-induced NASH models. In particular, the CDAHFD reduced the hepatic levels of phosphatidylcholines (PCs) and acylcarnitines (ACs), which was supported by the metabolism analysis and in line with the tendency of human NASH. Pathologically, the CDAHFD could effectively induce a more human-like NASH model over the traditional MCD. The hepatic PCs, ACs and their metabolism in CDAHFD-treated mice were down-regulated, similar to those in human NASH.

Celastrol as an intestinal FXR inhibitor triggers tripolide-induced intestinal bleeding: Underlying mechanism of gastrointestinal injury induced by Tripterygium wilfordii.

BACKGROUND: Tripterygium wilfordii has been widely used for the treatment of rheumatoid arthritis, which is frequently accompanied by severe gastrointestinal damage. The molecular mechanism underlying the gastrointestinal injury of Tripterygium wilfordii are yet to be elucidated. METHODS: Transmission electron microscopy, and pathological and biochemical analyses were applied to assess intestinal bleeding. Metabolic changes in the serum and intestine were determined by metabolomics. In vivo (time-dependent effect and dose-response) and in vitro (double luciferase reporter gene system, DRATs, molecular docking, HepG2 cells and small intestinal organoids) studies were used to identify the inhibitory role of celastrol on intestinal farnesoid X receptor (FXR) signaling. Fxr-knockout mice and FXR inhibitors and agonists were used to evaluate the role of FXR in the intestinal bleeding induced by Tripterygium wilfordii. RESULTS: Co-treatment with triptolide + celastrol (from Tripterygium wilfordii) induced intestinal bleeding in mice. Metabolomic analysis indicated that celastrol suppressed intestinal FXR signaling, and further molecular studies revealed that celastrol was a novel intestinal FXR antagonist. In Fxr-knockout mice or the wild-type mice pre-treated with pharmacological inhibitors of FXR, triptolide alone could activate the duodenal JNK pathway and induce intestinal bleeding, which recapitulated the pathogenic features obtained by co-treatment with triptolide and celastrol. Lastly, intestinal bleeding induced by co-treatment with triptolide and celastrol could be effectively attenuated by the FXR or gut-restricted FXR agonist through downregulation of the duodenal JNK pathway. CONCLUSIONS: The synergistic effect between triptolide and celastrol contributed to the gastrointestinal injury induced by Tripterygium wilfordii via dysregulation of the FXR-JNK axis, suggesting that celastrol should be included in the quality standards system for evaluation of Tripterygium wilfordii preparations. Determining the mechanism of the FXR-JNK axis in intestinal bleeding could aid in the identification of additional therapeutic targets for the treatment of gastrointestinal hemorrhage diseases. This study also provides a new standard for the quality assessment of Tripterygium wilfordii used in the treatment of gastrointestinal disorders.

Metabolomics Reveals Gut Microbiota Contribute to PPARα Deficiency-Induced Alcoholic Liver Injury.

Incidence and mortality rates of alcoholic liver disease (ALD) is one of the highest in the world. In the present study, we found that the genetic knockout nuclear receptor the peroxisome proliferator-activated receptor α (PPARα) exacerbated ALD. Lipidomics of the liver revealed changed levels of lipid species encompassing phospholipids, ceramides (CM), and long-chain fatty acids in Ppara-null mice induced by ethanol. Moreover, 4-hydroxyphenylacetic acid (4-HPA) was changed as induced by ethanol in the metabolome of urine. Moreover, the phylum level analysis showed a decrease in the level of Bacteroidetes and an increase in the level of Firmicutes after alcohol feeding in Ppara-null mice, while there was no change in wild-type mice. In Ppara-null mice, the level of Clostridium_sensu_stricto_1 and Romboutsia were upregulated after alcohol feeding. These data revealed that PPARα deficiency potentiated alcohol-induced liver injury through promotion of lipid accumulation, changing the metabolome of urine, and increasing the level of Clostridium_sensu_stricto_1 and Romboutsia. 4-HPA could improve ALD in mice by regulating inflammation and lipid metabolism. Therefore, our findings suggest a novel approach to the treatment of ALD: focusing on the gut microbiota and its metabolites. Data are available via ProteomeXchange (PXD 041465).

Parabacteroides distasonis ameliorates hepatic fibrosis potentially via modulating intestinal bile acid metabolism and hepatocyte pyroptosis in male mice.

Parabacteroides distasonis (P. distasonis) plays an important role in human health, including diabetes, colorectal cancer and inflammatory bowel disease. Here, we show that P. distasonis is decreased in patients with hepatic fibrosis, and that administration of P. distasonis to male mice improves thioacetamide (TAA)- and methionine and choline-deficient (MCD) diet-induced hepatic fibrosis. Administration of P. distasonis also leads to increased bile salt hydrolase (BSH) activity, inhibition of intestinal farnesoid X receptor (FXR) signaling and decreased taurochenodeoxycholic acid (TCDCA) levels in liver. TCDCA produces toxicity in mouse primary hepatic cells (HSCs) and induces mitochondrial permeability transition (MPT) and Caspase-11 pyroptosis in mice. The decrease of TCDCA by P. distasonis improves activation of HSCs through decreasing MPT-Caspase-11 pyroptosis in hepatocytes. Celastrol, a compound reported to increase P. distasonis abundance in mice, promotes the growth of P. distasonis with concomitant enhancement of bile acid excretion and improvement of hepatic fibrosis in male mice. These data suggest that supplementation of P. distasonis may be a promising means to ameliorate hepatic fibrosis.

Tripterygium wilfordii protects against an animal model of autoimmune hepatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii tablets (TWT) is widely used to treat autoimmune diseases such as rheumatoid arthritis. Celastrol, one main active ingredient in TWT, has been shown to produce a variety of beneficial effects, including anti-inflammatory, anti-obesity, anti-cancer, and immunomodulatory. However, whether TWT could protect against Concanavalin A (Con A)-induced hepatitis remains unclear. THE AIM OF THE STUDY: This study aims to investigate the protective effect of TWT against Con A-induced hepatitis and elucidate the underlying mechanism. MATERIALS AND METHODS: Metabolomic analysis, pathological analysis, biochemical analysis, qPCR and Western blot analysis and the Pxr-null mice were used in this study. RESULTS: The results indicated that TWT and its active ingredient celastrol could protect against Con A-induced acute hepatitis. Plasma metabolomics analysis revealed that metabolic perturbations related to bile acid and fatty acid metabolism induced by Con A were reversed by celastrol. The level of itaconate in the liver was increased by celastrol and speculated as an active endogenous compound mediating the protective effect of celastrol. Administration of 4-octanyl itaconate (4-OI) as a cell-permeable itaconate mimicker was found to attenuate Con A-induced liver injury through activation of the pregnane X receptor (PXR) and enhancement of the transcription factor EB (TFEB)-mediated autophagy. CONCLUSIONS: Celastrol increased itaconate and 4-OI promoted activation of TFEB-mediated lysosomal autophagy to protect against Con A-induced liver injury in a PXR-dependent manner. Our study reported a protective effect of celastrol against Con A-induced AIH via an increased production of itaconate and upregulation of TFEB. The results highlighted that PXR and TFEB-mediated lysosomal autophagic pathway may offer promising therapeutic target for the treatment of autoimmune hepatitis.

Down-regulation of hepatic CLOCK by PPARα is involved in inhibition of NAFLD.

This work aimed to investigate the role of nuclear factor peroxisome proliferator-activated receptor α (PPARα) in modification of circadian clock and their relevance to development of nonalcoholic fatty liver disease (NAFLD). Both male wild-type (WT) and Pparα-null (KO) mice treated with high-fat diet (HFD) were used to explore the effect of PPARα and lipid diet on the circadian rhythm. WT, KO, and PPARα-humanized (hPPARα) mice were treated with PPARα agonist fenofibrate to reveal the hPPARα dependence of circadian locomotor output cycles kaput (CLOCK) down-regulation. The mouse model and hepatocyte experiments were designed to verify the action of PPARα in down-regulating CLOCK and lipid accumulation in vivo and in vitro. Strongest NAFLD developed in mice fed 45%HFD, and it was inhibited in WT mice. The activity rhythm of WT mice was found to be different from that of the KO mice on normal diet and HFD. The core circadian factor CLOCK was down-regulated by HFD in both WT and KO mice in the liver, not in the hypothalamus. More interestingly, hepatic CLOCK was down-regulated by basal PPARα and activated PPARα in dose dependence of fenofibrate. Accordingly, CLOCK down-regulation dependent of PPARα activity was involved in inhibition of lipid metabolism in hepatocytes. Down-regulation of hepatic CLOCK by basal PPARα contributes to tolerance against development of NAFLD. Inhibition of CLOCK by activated PPARα is involved in inhibition of NAFLD by PPARα agonists. KEY MESSAGES: • PPARα inhibited NAFLD development induced by HFD. • PPARα mediated modifications of circadian rhythm and the hepatic circadian factor CLOCK in NAFLD models. • Down-regulation of hepatic CLOCK by basal PPARα contributed to tolerance against development of NAFLD. • Inhibition of CLOCK by activated PPARα was involved in therapeutic actions against fatty liver diseases by PPARα agonists.

Comparative Metabolomic Profiling of the Metabolic Differences of Δ9-Tetrahydrocannabinol and Cannabidiol.

More than one hundred cannabinoids have been found in cannabis. Δ9-Tetrahydrocannabinol (THC) is the recognized addictive constituent in cannabis; however, the mechanisms underlying THC-induced toxicity remain elusive. To better understand cannabis-induced toxicity, the present study compared the metabolic pathways of THC and its isomer cannabidiol (CBD) in human and mouse liver microsomes using the metabolomic approach. Thirty-two metabolites of THC were identified, including nine undescribed metabolites. Of note, two glutathione (GSH) and two cysteine (Cys) adducts were found in THC's metabolism. Molecular docking revealed that THC conjugates have a higher affinity with GSH and Cys than with the parent compound, THC. Human recombinant cytochrome P450 enzymes, and their corresponding chemical inhibitors, demonstrated that CYP3A4 and CYP1B1 were the primary enzymes responsible for the formation of THC-GSH and THC-Cys, thus enabling conjugation to occur. Collectively, this study systematically compared the metabolism of THC with the metabolism of CBD using the metabolomic approach, which thus highlights the critical role of metabolomics in identifying novel drug metabolites. Moreover, this study also facilitates mechanistic speculation in order to expand the knowledge of drug metabolism and safety.

Metabolomics reveals the role of PPARα in Tripterygium Wilfordii-induced liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium glycosides tablets (TGT) and Tripterygium wilfordii tablets (TWT) have been used to treat autoimmune diseases clinically, however, the side effects of TWT are higher than TGT, especially for hepatotoxicity. THE AIM OF THE STUDY: This study aims to determine the mechanism of TWT-induced liver injury. MATERIALS AND METHODS: We performed metabolomic analysis of samples from mice with liver injury induced by TGT and TWT. Ppara-null mice were used to determine the role of PPARα in TWT-induced liver injury. RESULTS: The results indicated that TWT induced the accumulation of medium- and long-chain carnitines metabolism, which was associated with the disruption of PPARα-IL6-STAT3 axis. PPARα agonists fenofibrate could reverse the liver injury from TWT and TP/Cel, and its protective role could be attenuated in Ppara-null mice. The toxicity difference of TWT and TGT was due to the different ratio of triptolide (TP) and celastrol (Cel) in the tablet in which TP/Cel was lower in TWT than TGT. The hepatotoxicity induced by TP and Cel also inhibited PPARα and upregulated IL6-STAT3 axis, which could be alleviated following by PPARα activation. CONCLUSIONS: These results indicated that PPARα plays an important role in the hepatotoxicity of Tripterygium wilfordii, and PPARα activation may offer a promising approach to prevent hepatotoxicity induced by the preparations of Tripterygium wilfordii.

Polyamine metabolism links gut microbiota and testicular dysfunction.

BACKGROUND: Male fertility impaired by exogenous toxins is a serious worldwide issue threatening the health of the new-born and causing infertility. However, the metabolic connection between toxic exposures and testicular dysfunction remains unclear. RESULTS: In the present study, the metabolic disorder of testicular dysfunction was investigated using triptolide-induced testicular injury in mice. We found that triptolide induced spermine deficiency resulting from disruption of polyamine biosynthesis and uptake in testis, and perturbation of the gut microbiota. Supplementation with exogenous spermine reversed triptolide-induced testicular dysfunction through increasing the expression of genes related to early and late spermatogenic events, as well as increasing the reduced number of offspring. Loss of gut microbiota by antibiotic treatment resulted in depletion of spermine levels in the intestine and potentiation of testicular injury. Testicular dysfunction in triptolide-treated mice was reversed by gut microbial transplantation from untreated mice and supplementation with polyamine-producing Parabacteroides distasonis. The protective effect of spermine during testicular injury was largely dependent on upregulation of heat shock protein 70s (HSP70s) both in vivo and in vitro. CONCLUSIONS: The present study linked alterations in the gut microbiota to testicular dysfunction through disruption of polyamine metabolism. The diversity and dynamics of the gut microbiota may be considered as a therapeutic option to prevent male infertility. Video Abstract.

Discovery of quality markers in Rubus Chingii Hu using UPLC-ESI-QTOF-MS.

Raspberry, the fruit of Rubus Chingii Hu, has been used as a traditional Chinese medicine (TCM) to nourish kidney and strengthen Yang-qi. In order to determine the quality of raspberry, the quality markers (Q-markers) of raspberry that can improve renal function were investigated using UPLC-ESI-QTOF-MS in this study. The results of serum pharmacochemistry indicated that six components rutin, ellagic acid, kaempferol-3-rutinoside, astragalin, tiliroside, and goshonoside F5 in raspberry were absorbed into rat blood. The HEK293 cells treated with cisplatin were used to evaluate the kidney-protecting activity of these absorbed components. All these components could markedly inhibit cell damage induced by cisplatin and restore the levels of malondialdehyde (MDA) and catalase (CAT) in the cells, suggesting that these components may be the Q-markers of raspberry. More importantly, except for ellagic acid, other five Q-markers in raspberries from Dexing of Jiangxi province were higher than those from most of other areas. It is well known that Dexing raspberry is the Dao-di herbs raspberry used in the clinic of Chinese Medicine, demonstrating that these components could be used as Q-markers of raspberry. This study provides a reliable and valuable method for quality evaluation of raspberry.

FXR activation prevents liver injury induced by Tripterygium wilfordii preparations.

Tripterygium glycosides tablets (TGT) and Tripterygium wilfordii tablets (TWT) are the preparations of Tripterygium wilfordii used to treat rheumatoid arthritis (RA) in the clinic, but the hepatotoxicity was reported frequently. This study aimed to determine the potential toxicity mechanism of liver injury induced by the preparations of Tripterygium wilfordii in mice.Here, we performed metabolomic analysis, pathological analysis and biochemical analysis of samples from mice with liver injury induced by TGT and TWT, which revealed that liver injury was associated with bile acid metabolism disorder. Quantitative real-time PCR (QPCR) and western blot indicated that the above changes were accompanied by inhibition of farnesoid X receptor (FXR) signalling.Liver injury from TWT could be alleviated by treatment of the FXR agonist obeticholic acid (OCA) via activation of the FXR to inhibit the c-Jun N-terminal kinase (JNK) pathway and improve bile acid metabolism disorder by activating bile salt export pump (BSEP) and organic solute-transporter-ß (OSTB). The data demonstrate that FXR signalling pathway plays a key role in T. wilfordii-induced liver injury, which could be alleviated by activated FXR.These results indicate that FXR activation by OCA may offer a promising therapeutic opportunity against hepatotoxicity from the preparations of T. wilfordii.

Discovery and validation of quality markers of Fructus Aurantii against acetylcholinesterase using metabolomics and bioactivity assays.

Fructus Aurantii is a traditional medicated diet in East Asia. To determine the underlying chemical markers responsible for the quality and efficacy of Fructus Aurantii, a sensitive metabolomic method was applied to distinguish Fructus Aurantii in Jiangxi Province from other two geographical locations (Hunan Province and Chongqing City) in China. In the present study, multivariate analyses were adopted to compare chemical compositions in 21 batches of Fructus Aurantii samples. Among three geographical origins, 23 differential compounds were structurally identified. Serum pharmacochemistry exhibited that 22 components could be detected in rat serum. Six differential and absorbed components were selected as six potential markers. Statistical analysis revealed that the content of six markers varied widely in three origins of Fructus Aurantii. Six differential and absorbed components were evaluated further by biological activity. Neohesperidin, naringin, and meranzin showed inhibitory effect on acetylcholinesterase that regulates gastrointestinal motility in vitro and in silico, suggesting that these three components may be determined as the active biomarkers of Fructus Aurantii. These findings demonstrate the potential of biomarkers for identification and quality control of Fructus Aurantii.

Fenofibrate reverses liver fibrosis in cholestatic mice induced by alpha-naphthylisothiocyanate.

Cholestatic liver fibrosis occurs in liver injuries accompanied by inflammation, which develops into cirrhosis if not effectively treated in early stage. The aim of the study is to explore the effect of fenofibrate on liver fibrosis in chronic cholestatic mice. In this study, wild-type (WT) and Pparα-null (KO) mice were dosed alpha-naphthylisothiocyanate (ANIT) diet to induce chronic cholestasis. Induced liver fibrosis was determined by pathological biomarkers. Then fenofibrate 25 mg/kg was orally administrated to mice twice/day for 14 days. Serum and liver samples were collected for analysis of biochemistry and fibrosis. In WT mice, cholestatic biomarkers were increased by 5-8-fold and the expression of tissue inhibitors of metalloproteinases 1 (TIMP-1), Monocyte chemoattractant protein 1 (MCP-1), Collagen protein I (Collagen I) was increased by more than 10-fold. Fenofibrate significantly downgraded the biochemical and fibrotic biomarkers. In Western blot analysis, levels of collagenI and alpha-smooth muscle actin (α-SMA) were strongly inhibited by fenofibrate. In KO mice, liver fibrosis was induced successfully, but no improvement after fenofibrate treatment was observed. These data showed low-dose fenofibrate reverses cholestatic liver fibrosis in WT mice but not in KO mice, suggesting the dependence of therapeutic action on peroxisome proliferator-activated receptor alpha (PPARα). The study offers an additional therapeutic strategy for cholestatic liver fibrosis in practice.

Inhibition of inflammation by SP600125 in cholestatic liver injury is dependent on the administration­based exposure profile.

SP600125 is a classic inhibitor of c­Jun­N­terminal kinase (JNK) that is widely used in numerous medicinal studies, but its administration regimen has yet to be optimized. In the present study, intraperitoneal (i.p.) and intragastric (i.g.) injections of 15 mg/kg SP600125 was performed in mice to compare the inhibitory effect against JNK signalling in cholestasis induced by α­naphthylisothiocyanate (ANIT). SP600125 at a dose of 15 mg/kg administered by i.p. substantially decreased ANIT­induced liver injury as observed by biochemical and histopathological examinations. The adaptation of bile acid synthesis was inhibited in the A­SP­i.p. group compared with that in the A­SP­i.g. group, as indicated by the expression analysis of CYP7A1 and CYP8B1. The transcription of the pro­inflammatory factors IL­6, IL­1ß, ICAM­1 and IL­10 supported the differential toxic responses. Western blot analysis revealed that JNK signalling activated by ANIT was inhibited more markedly in the A­SP­i.p. group than in the A­SP­i.g. group. The peak concentration and the AUC0­24 of SP600125 in the A­SP­i.p. group were 5­fold and 1.56­fold higher, respectively, compared with those in the A­SP­i.g. group. These data indicated that i.p. administration of SP600125 produced a high plasma exposure profile, which directly determined its efficacy of blocking the JNK signalling. This effect of SP600125 on the JNK pathway may provide an optimized design for future in vivo investigations.

Cholestatic models induced by lithocholic acid and α­naphthylisothiocyanate: Different etiological mechanisms for liver injury but shared JNK/STAT3 signaling.

α­naphthylisothiocyanate (ANIT) is used to induce intrahepatic cholestasis and it is frequently used for investigations into the disease mechanism. The lithocholic acid (LCA) cholestatic model has also been extensively used in various studies; however, to the best of our knowledge, a comparative study determining the hepatotoxic mechanisms induced by these two models has not been previously conducted. In the present study, ICR mice were treated with ANIT or LCA to induce cholestatic liver injury. Biochemical analysis was used to determine the serum. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acid (TBA) levels, and histopathological assessment was used to examine the liver tissue. Metabolomic analysis was used for the serum biomarker identification. Reverse transcription­quantitative PCR analysis and western blotting were used to analyze the inflammation biomarkers. The serum metabolome of the ANIT group clustered away from of the LCA group, which was demonstrated by the different modifications of the BA components. ALP level was found to be preferentially increased in the ANIT group from 24 to 48 h. Total BA levels was only increased in the ANIT group at 24 h. In contrast, AST and ALT activity levels were preferentially increased in the LCA group. The bile ducts in the hepatic tissues of the ANIT group were observed to be severely dilated, whereas the presence of edematous hepatocytes around the necrotic lesions and neutrophil infiltration were identified in the LCA group. The expression levels of cholesterol 7α­hydroxylase and sterol 12α­hydroxylase genes were significantly downregulated in the ANIT group compared with the LCA group, where a stronger adaptation of BA metabolism was supported by major differences in the concentration of the BA components. Despite the aforementioned etiological differences in the cholestasis induced by each treatment, the activation of the JNK/STAT3 signaling pathway was similar between the two cholestatic models. In conclusion, these data suggested that the liver injury induced by ANIT may be cholestatic, while the liver injury caused in the LCA model may be hepatocellular. Moreover, the downstream cholestatic liver injury in both models was indicated to be mediated by the JNK/STAT3 signaling pathway.

Gut microbiota protects from triptolide-induced hepatotoxicity: Key role of propionate and its downstream signalling events.

As a potential drug for treating inflammatory, autoimmune diseases and cancers, triptolide (TP) is greatly limited in clinical practice due to its severe toxicity, particularly for liver injury. Recently, metabolic homeostasis was vitally linked to drug-induced liver injury and gut microbiota was established to play an important role. In this study, we aimed to investigate the functions of gut microbiota on TP-induced hepatotoxicity using metabolomics in mice. Here, predepletion of gut microbiota by antibiotic treatment strikingly aggravated liver injury and caused mortality after treated with a relatively safe dosage of TP at 0.5 mg/kg, which could be reversed by gut microbial transplantation. The loss of gut microbiota prior to TP treatment dramatically elevated long chain fatty acids and bile acids in plasma and liver. Further study suggested that gut microbiota-derived propionate contributed to the protective effect of gut microbiota against TP evidenced by ameliorative inflammatory level (Tnfa, Il6 and Cox2), ATP, malondialdehyde and hepatic histology. Supplementing with propionate significantly decreased the mRNA levels of genes involved in fatty acid biosynthesis (Srebp1c, Fasn and Elovl6), resulting in the decreased long chain fatty acids in liver. Moreover, TP restricted the growth of Firmicutes and led to the deficiency of short chain fatty acids in cecum content. In conclusion, our study warns the risk for TP and its preparations when antibiotics are co-administrated. Intervening by foods, prebiotics and probiotics toward gut microbiota or supplementing with propionate may be a clinical strategy to improve toxicity induced by TP.

Therapeutic action against chronic cholestatic liver injury by low-dose fenofibrate involves anti-chemotaxis via JNK-AP1-CCL2/CXCL2 signaling.

BACKGROUND: Fenofibrate was reported to be beneficial for cholestasis in combination with ursodeoxycholic acid. However, its therapeutic action as single therapy for chronic cholestasis and the underlying mechanism are not known. METHODS: In the present study, wild-type (WT) mice were administered a 0.05% ANIT diet to mimic chronic cholestatic liver injury. Mice were dosed fenofibrate 25 mg/kg twice every day for 10 days to investigate the therapeutic action of fenofibrate on chronic cholestatic liver injury. Ppara-null (KO) mice were used to explore PPARα's role in the therapeutic outcome. RESULTS: Fenofibrate, administered at 25 mg/kg twice daily, substantially reversed ANIT-induced chronic cholestatic liver injury shown by biochemical and pathological end points. The modifications of bile acid metabolism were found to be adaptive responses. The JNK-AP1-CCL2/CXCL2 axis was activated in all the mice administered ANIT which developed chronic cholestatic liver injury. But it was substantially decreased by fenofibrate in WT mice rather than that in KO mice. CONCLUSIONS: Low-dose fenofibrate reversed chronic cholestatic liver injury in mice. The therapeutic action was dependent on PPARα activation and occurred by inhibiting chemotaxis via the JNK-AP1-CCL2/CXCL2 signaling. These data provided an exciting basis for optimization of therapeutic fenofibrate regimen in the clinic. Additionally, they suggested anti-chemotaxis of low-dose fenofibrate in single therapy to treat cholestatic liver diseases.

Metabolic Activation of Elemicin Leads to the Inhibition of Stearoyl-CoA Desaturase 1.

Elemicin is a constituent of natural aromatic phenylpropanoids present in many herbs and spices. However, its potential to cause toxicity remains unclear. To examine the potential toxicity and associated mechanism, elemicin was administered to mice for 3 weeks and serum metabolites were examined. Enlarged livers were observed in elemicin-treated mice, which were accompanied by lower ratios of unsaturated- and saturated-lysophosphatidylcholines in plasma, and inhibition of stearoyl-CoA desaturase 1 (Scd1) mRNA expression in liver. Administration of the unsaturated fatty acid oleic acid reduced the toxicity of 1'-hydroxylelemicin, the primary oxidative metabolite of elemicin, while treatment with the SCD1 inhibitor A939572 potentiated its toxicity. Furthermore, the in vitro use of recombinant human CYPs and chemical inhibition of CYPs in human liver microsomes revealed that CYP1A1 and CYP1A2 were the primary CYPs responsible for elemicin bioactivation. Notably, the CYP1A2 inhibitor α-naphthoflavone could attenuate the susceptibility of mice to elemicin-induced hepatomegaly. This study revealed that metabolic activation of elemicin leads to SCD1 inhibition in liver, suggesting that upregulation of SCD1 may serve as potential intervention strategy for elemicin-induced toxicity.