Investigating the Immune Basis of Green Tea Extract Induced Liver Injury in Healthy Donors Expressing HLA-B*35:01.

Epigallocatechin-3-O-gallate (EGCG) is the major component of green tea extract, commonly found in dietary supplements, and has been associated with immune-mediated liver injury. The purpose of this study was to investigate the immunogenicity of EGCG in healthy donors expressing HLA-B*35:01, and characterize EGCG responsive T-cell clones. We have shown that EGCG can prime peripheral blood mononuclear cells and T-cells from donors with and without the HLA-B*35:01 allele. T-cell clones were CD4+ve and capable of secreting Th1, Th2, and cytolytic molecules. These data demonstrate that EGCG can activate T-cells in vitro, suggesting a significant role in the pathogenesis of green tea extract induced liver injury.

In vitro and in vivo analysis of metabolites involved in the TCA cycle and glutamine metabolism associated with cisplatin resistance in human lung cancer.

Elucidating the dysregulated metabolic pathways in cancer cells and their relevance to cisplatin resistance could yield new insights into cancer therapy. We previously reported that eight metabolites involved in the tricarboxylic acid (TCA) cycle and glutamine metabolism were associated with platinum-based chemotherapy efficacy in human lung cancer. Here, we investigated the metabolic differences upon cisplatin treatment in lung cancer in vitro and in vivo. A simple and partially validated standard addition method was applied for the quantification of five metabolites involved in the TCA cycle and glutamine metabolism using amide hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The present study investigated the levels of these biomarkers in A549 cells and the cisplatin-resistant A549-DDP cells, as well as in the plasma before and after cisplatin treatment in A549 xenograft mice. Levels of five metabolites, including 2-hydroxyglutaric acid (2-HG), α-ketoglutarate (α-KG), succinate, glutamine, and glutamate, showed a decreasing trend in A549-DDP cells. In addition, 2-HG and glutamine were the most significantly altered metabolites in cisplatin-treated A549 xenograft mice. These data indicate that the TCA cycle and glutamine metabolism play important roles in cisplatin-based chemotherapy resistance in lung cancer. Our results provide a new angle for exploring the molecular mechanisms underlying cisplatin resistance.

Evaluating the protective effects of individual or combined ginsenoside compound K and the downregulation of soluble epoxide hydrolase expression against sodium valproate-induced liver cell damage.

Sodium valproate (SVP) is one of the most commonly prescribed antiepileptic drugs. However, SVP is known to induce hepatotoxicity, which limits its clinical application for treating various neurological disorders. Previously, we found that ginsenoside compound K (G-CK) demonstrated protective effects against SVP-induced hepatotoxicity by mitigating oxidative stress and mitochondrial damage, as well as downregulating the expression of soluble epoxide hydrolase (sEH) in rats. This study aimed to assess the effect of G-CK on SVP-induced cytotoxicity in human hepatocytes (L02 cell line), as well as the effect of the downregulation of sEH expression on both the hepatotoxicity of SVP and the hepatoprotective effects of G-CK. We observed that G-CK significantly ameliorated the decrease of cell viability, elevated ALT, AST and ALP activities, significant oxidative stress, and loss of mitochondrial membrane potential induced by SVP in L02 cells. G-CK also inhibited the SVP-mediated upregulation of sEH expression. Transfection of the L02 cells with siRNA-sEH led to a partial improvement in the L02 cytotoxicity caused by SVP by mitigating cellular oxidative stress without recovering the reduced mitochondrial membrane potential. Furthermore, the combination of siRNA-sEH and G-CK had better inhibitory effects on the SVP-induced changes of all detection indices except mitochondrial membrane potential than G-CK alone. Together, our results demonstrated that the combination of siRNA-sEH and G-CK better suppressed the SVP-induced cytotoxicity in L02 cells compared to either G-CK or siRNA-sEH alone.

The hepatotoxicity of Polygonum multiflorum: The emerging role of the immune-mediated liver injury.

Herbal and dietary supplements (HDS)-induced liver injury has been a great concern all over the world. Polygonum multiflorum Thunb., a well-known Chinese herbal medicine, is recently drawn increasing attention because of its hepatotoxicity. According to the clinical and experimental studies, P. multiflorum-induced liver injury (PM-DILI) is considered to be immune-mediated idiosyncratic liver injury, but the role of immune response and the underlying mechanisms are not completely elucidated. Previous studies focused on the direct toxicity of PM-DILI by using animal models with intrinsic drug-induced liver injury (DILI). However, most epidemiological and clinical evidence demonstrate that PM-DILI is immune-mediated idiosyncratic liver injury. The aim of this review is to assess current epidemiological, clinical and experimental evidence about the possible role of innate and adaptive immunity in the idiosyncratic hepatotoxicity of P. multiflorum. The potential effects of factors associated with immune tolerance, including immune checkpoint molecules and regulatory immune cells on the individual's susceptibility to PM-DILI are also discussed. We conclude by giving our hypothesis of possible immune mechanisms of PM-DILI and providing suggestions for future studies on valuable biomarkers identification and proper immune models establishment.

A conjunctive lipidomic approach reveals plasma ethanolamine plasmalogens and fatty acids as early diagnostic biomarkers for colorectal cancer patients.

Background: Colorectal cancer (CRC) represents a third leading cause of cancer-related death worldwide. The reliable diagnostic biomarkers for detecting CRC at early stage is critical for decreasing the mortality.Method: A conjunctive lipidomic approach was employed to investigate the differences in plasma lipid profiles of CRC patients (n = 101) and healthy volunteers (n = 52). Based on UHPLC-Q-TOF MS and UHPLC-QQQ MS platforms, a total of 236 lipids were structurally detected. Multivariate data analysis was conducted for biomarkers discovery.Results: A total of 11 lipid species, including 1 Glycerophosphoethanolamine (PE), 3 ethanolamine plasmalogens (PlsEtn), 1 plasmanyl glycerophosphatidylethanolamine (PE-O), 3 fatty acids (FFA), 1 Fatty acid ester of hydroxyl fatty acid (FAHFA) and 2 Diacylglycerophosphates (PA) were identified to distinguish the CRC patients at early stage from healthy controls. In addition, these potential lipid biomarkers achieved an estimated AUC=0.981 in a validation set for univariate ROC analysis.Conclusion: By combining Q-TOF MS and QQQ MS analysis, the 11 lipids exhibited good performance in differentiating early-stage CRC and healthy control. This study also demonstrated that lipidomics is a powerful tool in discovering new potential biomarkers for cancer diagnosis.

HLA-B*35:01 Allele Is a Potential Biomarker for Predicting Polygonum multiflorum-Induced Liver Injury in Humans.

Polygonum multiflorum (PM) is a well-known Chinese herbal medicine that has been reported to induce inflammation-associated idiosyncratic liver injury. This study aimed to identify the genetic basis of susceptibility to PM-drug-induced liver injury (PM-DILI) and to develop biological markers for predicting the risk of PM-DILI in humans. The major histocompatibility complex (MHC) regions of 11 patients with PM-DILI were sequenced, and all human leukocyte antigen (HLA)-type frequencies were compared to the Han-MHC database. An independent replication study that included 15 patients with PM-DILI, 33 patients with other DILI, and 99 population controls was performed to validate the candidate allele by HLA-B PCR sequence-based typing. A prospective cohort study that included 72 outpatients receiving PM for 4 weeks was designed to determine the influence of the risk allele on PM-DILI. In the pilot study, the frequency of HLA-B*35:01 was 45.4% in PM-DILI patients compared with 2.7% in the Han Chinese population (odds ratio [OR], 30.4; 95% confidence interval [CI], 11.7-77.8; P = 1.9 × 10-10 ). In the independent replication study and combined analyses, a logistic regression model confirmed that HLA-B*35:01 is a high-risk allele of PM-DILI (PM-DILI versus other DILI, OR, 86.5; 95% CI, 14.2-527.8, P = 1.0 × 10-6 ; and PM-DILI versus population controls, OR, 143.9; 95% CI, 30.1-687.5, P = 4.8 × 10-10 ). In the prospective cohort study, an asymptomatic increase in transaminase levels was diagnosed in 6 patients, representing a significantly higher incidence (relative risk, 8.0; 95% CI, 1.9-33.2; P < 0.02) in the HLA-B*35:01 carriers (37.5%) than in the noncarriers (4.7%). Conclusion: The HLA-B*35:01 allele is a genetic risk factor for PM-DILI and a potential biomarker for predicting PM-DILI in humans.

Leucovorin ameliorated methotrexate induced intestinal toxicity via modulation of the gut microbiota.

Methotrexate (MTX) is a widely used therapeutic agent for the treatment of cancer and autoimmune diseases. However, its efficacy is often limited by adverse effects, such as intestinal toxicity. Although treatment with leucovorin (LV) is the most common method to reduce the toxic effects of MTX, it may also compromise the therapeutic effects of MTX. The gut microbiome has been reported to be associated with the intestinal toxicity of MTX. In this study, the intestinal damage of MTX was ameliorated by treatment with LV. Moreover, the population, diversity, and principal components of the gut microbiota in MTX-treated mice were restored by treatment with LV. The only element of the gut microbiota that was significantly changed after treatment with LV was Bifidobacterium, and supplementation with Bifidobacterium longum ameliorated MTX-induced intestinal damage. In conclusion, our results suggest that the balance and the composition of gut microbiota have an important role in the LV-mediated protection against MTX-induced intestinal toxicity. This work provides foundation of data in support of a new potential mechanism for the prevention of MTX-induced intestinal toxicity.

The biotransformation of Bupleuri Radix by human gut microbiota.

1. Bupleuri Radix (BR) is a herbal medicine traditionally used orally in oriental countries, which inevitably comes into contact with the intestinal microbiota. However, whether gut microbiota contribute to the biotransformation of BR, and/or the formation of pharmacologically active compounds remains unknown.2. In this study, the main saikosaponins (SAPs) of Bupleurum (including saikosaponin a, b1, b2, c, d, f, h) and BR extract (BRE) were individually incubated with human fecal suspensions (HFS), and metabolic time courses of SAPs and their metabolites by human gut bacteria were systematically characterized.3. Deglycosylation and dehydration were the main metabolic pathways identified for SAPs including newly investigated saikosaponin f (SSf) and saikosaponin h (SSh); dehydration had not been reported previously. A total of 19 dehydrated and deglycosylated metabolites of SAPs were detected and characterized, and 10 of them were newly identified. Moreover, SAPs of BRE were found to be deglycosylated to prosaikogenins. In addition, 13 metabolic pathways related to human gut microbiota were identified for phytochemicals of BRE except for SAPs. Gut microbiota may play a significant role in the biotransformation of BR in humans.

The pharmacogenetics of natural products: A pharmacokinetic and pharmacodynamic perspective.

Natural products have represented attractive alternatives for disease prevention and treatment over the course of human history and have contributed to the development of modern drugs. These natural products possess beneficial efficacies as well as adverse efffects, which vary largely among individuals because of genetic variations in their pharmacokinetics and pharmacodynamics. As with other synthetic chemical drugs, the dosing of natural products can be optimized to improve efficacy and reduce toxicity according to the pharmacogenetic properties. With the emergence and development of pharmacogenomics, it is possible to discover and identify the targets/mechanisms of pharmacological effects and therapeutic responses of natural products effectively and efficiently on the whole genome level. This review covers the effects of genetic variations in drug metabolizing enzymes, drug transporters, and direct and indirect interactions with the pharmacological targets/pathways on the individual response to natural products, and provides suggestions on dosing regimen adjustments of natural products based on their pharmacokinetic and pharmacogenetic paratmeters. Finally, we provide our viewpoints on the importance and necessity of pharmacogenetic and pharmacogenomic research of natural products in natural medicine's rational development and clinical application of precision medicine.

LC-MS-based lipid profile in colorectal cancer patients: TAGs are the main disturbed lipid markers of colorectal cancer progression.

Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. Emerging evidence has shown that lipid metabolism plays important roles in the occurrence and progression of CRC. The identification of potential biomarkers for CRC progression is critical for precise diagnosis and treatment. Therefore, the aim of this study is to explore the potential lipid markers in relation to CRC progression. The plasma of patients with stage I/II CRC (n = 20) and stage III/IV CRC (n = 20) was collected. Lipidomic screening was performed by ultrahigh-performance liquid chromatography-mass spectrometry. After multivariate data analysis, including orthogonal partial least squares discriminant analysis, determination of the fold change, and the Mann-Whitney U test, eight lipid species with altered levels with p < 0.05 and fold change greater than 2 were selected as potential lipid biomarkers. Compared with patients with early-stage CRC, patients with advanced-stage CRC showed significantly higher levels of cholesteryl ester (20:4) and some triglycerides with a saturated fatty acid chain and a lower level of fatty acid ester of hydroxy fatty acid 27:1 (9:0-18:1) in plasma. Furthermore, the receiver operating characteristic including these potential lipid biomarkers yielded a sensitivity of 85% and specificity of 80% for separation of early-stage CRC patients from advanced-stage CRC patients. In all, this is the first report showing that the levels of triglycerides, the major contents of lipid droplets, increase in plasma of advanced-stage CRC patients compared with early-stage CRC patients. These data indicate that lipid droplets may be target organelles for the study of CRC progression and treatment. Graphical abstract.

Comparative pharmacokinetic study on three formulations of Astragali Radix by an LC-MS/MS method for determination of formononetin in human plasma.

Astragali Radix (AR) is a widely used traditional Chinese medicine for healing the cardiovascular, liver and immune systems. Recently, superfine pulverizing technology has been applied to developing novel formulations to improve bioavailability of the active constituents in herbs, such as ultrafine granular powder of AR. In this study, a universal and sensitive quantitative method based on LC-MS/MS was employed for determining formononetin, the main flavonoid in AR, in human plasma for comparative pharmacokinetics of three oral formulations of AR. Formononetin and IS (quercetin) were extracted by ethyl acetate from human plasma and were separated on a C18 column with a mobile phase consisting of acetonitrile and 0.1% formic acid. Positive-ion electrospray-ionization mode was applied in mass spectrometric detection. The quantitative method was validated with regards to selectivity, linearity, accuracy and precision, matrix effect, extraction recovery and stability, and was applied to comparing the pharmacokinetics of ultrafine granular powder (UGP), ultrafine powder (UP) and traditional decoction pieces (TDP) of AR after oral administration. The peak concentration and areas under the concentration-time curve of formononetin in UGP and UP were significantly higher than those of TDP. UGP and UP could significantly improve the bioavailability of AR in human compared with TDP after oral administration.

Pharmacokinetic and pharmacodynamic evidence for developing an oral formulation of octreotide against gastric mucosal injury.

Among the somatostatin analogues, octreotide (OCT) is the most commonly used in clinic via intravenous or subcutaneous injection to treat various diseases caused by increased secretion of growth hormone, gastrin or insulin. In order to assesse the feasibility of developing oral formulations of OCT, we conducted systematical pharmacokinetic and pharmacodynamic analyses of OCT in several animal models. The pharmacokinetic studies in rats showed that intragastric administration of OCT had extremely low bioavailability (<0.5%), but it could specifically distribute to the gastric mucosa due to the high expression of somatostatin receptor 2 (SSTR2) in the rat stomach. The pharmacodynamic studies revealed that intragastric administration of OCT dose-dependently protected against gastric mucosal injury (GMI) in mice with WIRS-induced mouse gastric ulcers, which were comparable to those achieved by intravenous injection of OCT, and this effect was markedly attenuated by co-administration of CYN-154806, an antagonist of SSTR2. In pyloric ligation-induced ulcer mice, we further demonstrated that OCT significantly reduced the secretion of gastric acid via down-regulating the level of gastrin, which was responsible for the protective effect of OCT against GMI. Overall, we have provided pharmacokinetic and pharmacodynamic evidence for the feasibility of developing an oral formulation of OCT. Most importantly, the influence of SSTR2 on the pharmacokinetics and pharmacodynamics of OCT suggested that an oral formulation of OCT might be applicable for other clinical indications, including neuroendocrine neoplasms and pituitary adenoma due to the overexpression of SSTR2 on these tumor cells.

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC-MS/MS: application to a real-time uptake study for Schisandra Lignan Extract.

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis. In the present study, a robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C18 column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]+ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2-1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra- and inter-day precision was <15% and the accuracy (relative error) ranged from -15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time-dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.

[Low serum testosterone level does not predict bone metastasis of prostate cancer].

OBJECTIVE: To evaluate the role of the serum testosterone level as an independent predictor of bone metastasis of prostate cancer. METHODS: This study included 165 male patients with prostate cancer confirmed by biopsy. The patients were aged 58－78 (66.6±5.3) years and none had received androgen-deprivation therapy, chemotherapy or radiotherapy previously. We obtained the baseline clinical data from the patients, including prostate biopsy Gleason scores and the levels of serum prostate-specific antigen (PSA), total testosterone (TT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), alkaline phosphatase (ALP) and prolactin. According to the results of bone scanning, we divided the patients into a bone metastasis and a non-bone metastasis group and screened out the differential factors by univariate analysis and the independent predictor of bone metastasis using the multivariate non-conditional logistic regression model. RESULTS: Univariate analysis showed no statistically significant differences between the bone metastasis and non-bone metastasis groups in age (P = 0.126) or the levels of serum LH (P = 0.930), FSH (P = 0.763) and E2 (P = 0.256), but that the former had remarkably higher Gleason scores (P < 0.01), total PSA (P <0.01) and ALP (P <0.01) but a lower TT level than the latter (P = 0.013). According to the results of multivariate logistic regression analysis, serum ALP (P <0.01, OR = 1.018 ï¼»1.011－1.026ï¼½) and total PSA (P <0.01, OR = 1.029 ï¼»1.015－1.044ï¼½) could be regarded as independent predictors of bone metastasis of prostate cancer but not low serum TT (P = 0.531, OR = 0.999 ï¼»0.996－1.002ï¼½) or biopsy Gleason score (P = 0.898, OR = 0.787 ï¼»0.412－1.9559ï¼½). CONCLUSIONS: The low level of serum testosterone is closely associated with but not an independent predictor of bone metastasis of prostate cancer.

Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies.

This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid-liquid extraction with n-butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C18 column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one-step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ≤ ± 15) and precision (% RSD ≤ 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II.

Evaluation of liquid chromatography-ion trap-time of flight hybrid mass spectrometry on the quantitative analysis for ginsenosides.

It is ideal and desirable for a single instrument to meet the requirement of both qualitative and quantitative analysis of complicated components in pharmacokinetic research for herbal medicine. Liquid chromatography combined with hybrid ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF) was recently confirmed as a very powerful tool for the qualitative analysis of both target and nontarget components in herbal medicines. The present study was designed to investigate the feasibility of LCMS-IT-TOF on quantitative analysis of ginsenosides in biological matrices. A simple liquid-liquid extraction procedure was followed by injection of the extracts onto a C18 column with gradient elution and detection based on LCMS-IT-TOF system in negative scan mode. The developed method was validated with respect to the limit of quantification, linear dynamic range, precision, accuracy, matrix effects and stabilities. All the results suggested that the presently developed method was sufficiently sensitive and robust enough to simultaneously monitor 15 ginsenosides with diverse properties and a large range of concentration differences. Therefore, this method would be expected to be highly useful for comprehensive studies of ginsenosides in complicated matrix.

2,3,5,4'- tetrahydroxystilbene-2-O-ß-D- glucopyranoside (TSG)-Driven immune response in the hepatotoxicity of Polygonum multiflorum.

ETHNOPHARMACOLOGICAL RELEVANCE: 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside (TSG) as the primary constituent of Polygonum multiflorum Thumb. (PM) possesses anti-oxidative, antihypercholesterolemic, anti-tumor and many more biological activities. The root of PM has been used as a tonic medicine for thousands of years. However, cases of PM-induced liver injury are occasionally reported, and considered to be related to the host immune status. AIM OF THE STUDY: The primary toxic elements and specific mechanisms PM causing liver damage are still not thoroughly clear. Our study aimed to investigate the influences of TSG on the immune response in idiosyncratic hepatotoxicity of PM. MATERIALS AND METHODS: The male C57BL/6 mice were treated with different doses of TSG and the alterations in liver histology, serum liver enzyme levels, proportions of T cells and cytokines secretion were evaluated by hematoxylin and eosin (HE), RNA sequencing, quantitative real time polymerase chain reaction (qRT-PCR), Flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA), respectively. Then, primary spleen cells from drug-naive mice were isolated and cultured with TSG in vitro. T cell subsets proliferation and cytokines secretion after treated with TSG were assessed by CCK8, FCM and ELISA. In addition, mice were pre-treated with anti-CD25 for depleting regulatory T cells (Tregs), and then administered with TSG. Liver functions and immunological alterations were analyzed to evaluate liver injury. RESULTS: Data showed that TSG induced liver damage, and immune cells infiltration in the liver tissues. FCM results showed that TSG could activate CD4+T and CD8+T in the liver. Results further confirmed that TSG notably up-regulated the levels of inflammatory cytokines including TNF-α, IFN-γ, IL-18, perforin and granzyme B in the liver tissues. Furthermore, based on transcriptomics profiles, some immune system-related pathways including leukocyte activation involved in inflammatory response, leukocyte cell-cell adhesion, regulation of interleukin-1 beta production, mononuclear cell migration, antigen processing and presentation were altered in TSG treated mice. CD8+T/CD4+T cells were also stimulated by TSG in vitro. Interestingly, increased proportion of Tregs was observed after TSG treatment in vitro and in vivo. Foxp3 and TGF-ß1 mRNA expressions were up-regulated in the liver tissues. Depletion of Tregs moderately enhanced TSG induced the secretion of inflammatory cytokines in serum. CONCLUSIONS: Our findings showed that TSG could trigger CD4+T and CD8+T cells proliferation, promote cytokines secretion, which revealed that adaptive immune response associated with the mild liver injury cause by TSG administration. Regulatory T cells (Tregs) mainly sustain immunological tolerance, and in this study, the progression of TSG induced liver injury was limited by Tregs. The results of our investigations allow us to preliminarily understand the mechanisms of PM related idiosyncratic hepatotoxicity.

HLA-B*35:01-mediated activation of emodin-specific T cells contributes to Polygonum multiflorum thunb. -induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: HLA-B*35:01 has been identified as a risk allele for Polygonum multiflorum Thunb.-induced liver injury (PMLI). However, the immune mechanism underlying HLA-B*35:01-mediated PMLI remains unknown. AIM OF THE STUDY: To characterize the immune mechanism of HLA-B*35:01-mediated PMLI. MATERIALS AND METHODS: Components of P. multiflorum (PM) bound to the HLA-B*35:01 molecule was screened by immunoaffinity chromatography. Both wild-type mice and HLA-B*35:01 transgenic (TG) mice were treated with emodin. The levels of transaminases, histological changes and T-cell response were assessed. Splenocytes from emodin-treated mice were isolated and cultured in vitro. Phenotypes and functions of T cells were characterized upon drug restimulation using flow cytometry or ELISA. Emodin-pulsed antigen-presenting cells (APCs) or glutaraldehyde-fixed APCs were co-cultured with splenocytes from emodin-treated transgenic mice to detect their effect on T-cell activation. RESULTS: Emodin, the main component of PM, could non-covalently bind to the HLA-B*35:01-peptide complexes. TG mice were more sensitive to emodin-induced immune hepatic injury, as manifested by elevated aminotransferase levels, infiltration of inflammatory cells, increased percentage of CD8+T cells and release of effector molecules in the liver. However, these effects were not observed in wild-type mice. An increase in percentage of T cells and the levels of interferon-γ, granzyme B, and perforin was detected in emodin-restimulated splenocytes from TG mice. Anti-HLA-I antibodies inhibited the secretion of these effector molecules induced by emodin. Mechanistically, emodin-pulsed APCs failed to stimulate T cells, while fixed APCs in the presence of emodin could elicit the secretion of T cell effector molecules. CONCLUSION: The HLA-B*35:01-mediated CD8+ T cell reaction to emodin through the P-I mechanism may contribute to P. multiflorum-induced liver injury.

Gut microbiota affects sensitivity to immune-mediated isoniazid-induced liver injury.

Isoniazid (INH) is a highly effective single and/or combined first-line anti-tuberculosis (anti-TB) therapy drug, and the hepatotoxicity greatly limits its clinical application. INH-induced liver injury (INH-DILI) is a typical immune-mediated idiosyncratic drug-induced liver injury. Existing mechanisms including genetic variations in drug metabolism and immune responses cannot fully explain the differences in susceptibility and sensitivity to INH-DILI, suggesting that other factors may be involved. Accumulating evidence indicates that the development and severity of immune-mediated liver injury is related to gut microbiota. In this study, INH exposure caused liver damage, immune disregulation and microbiota profile alteration. Depletion of gut microbiota ameliorated INH-DILI, and improved INH-DILI-associated immune disorder and inflammatory response. Moreover, hepatotoxicity of INH was ameliorated by fecal microbiota transplantation (FMT) from INH-treated mice. Notably, Bifidobacterium abundance was significantly associated with transaminase levels. In conclusion, our results suggested that the effect of gut microbiota on INH-DILI was related to immunity, and the difference in INH-DILI sensitivity was related to the structure of gut microbiota. Changes in the structure of gut microbiota by continuous exposure of INH resulted in the tolerance to liver injury, and probiotics such as Bifidobacterium might play an important role in INH-DILI and its "adaptation" phenomenon. This work provides novel evidence for elucidating the underlying mechanism of difference in individual's response to INH-DILI and potential approach for intervening anti-TB drug liver injury by modulating gut microbiota.

A Joint Technology Combining the Advantages of Capillary Microsampling with Mass Spectrometry Applied to the Trans-Resveratrol Pharmacokinetic Study in Mice.

Mice are the most frequently used animals in pharmacokinetic studies; however, collecting series of blood samples from mice is difficult because of their small sizes and tiny vessels. In addition, due to the small sample size, it is problematic to perform high required quantification. Thus, present work aims to find an effective strategy for overcoming these challenges using trans-resveratrol as a tool drug. Based on the idea of a joint technology, the capillary microsampling (CMS) was chosen for blood sample collection from mice after delivery of trans-resveratrol (150 mg/kg) by gavage, and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of trans-resveratrol and its main metabolites. All the mouse blood samples were exactly collected by CMS without obvious deviation. This provided credible samples for subsequent quantitative analysis. The HPLC-MS/MS method was found to be sensitive, accurate, and repeatable, and the pharmacokinetic parameters for all analytes were comparable with those reported in previous studies. However, the present joint technology offers the advantages of less animal damage, easy for sample preparation, and improved reliability. It has overcome some of the major limitations revealed in previous pharmacokinetic studies in mice and therefore provides a more effective option for future studies.