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.

The efficacy and toxicity of antineoplastic antimetabolites: Role of gut microbiota.

The incidence and mortality of cancer are rapidly growing all over the world. Nowadays, antineoplastic antimetabolites still play a key role in the chemotherapy of cancer. However, the interindividual variations in the efficacy and toxicity of antineoplastic antimetabolites are nonnegligible challenges to their clinical applications. Although many studies have focused on genetic variation, the reasons for these interindividual variations have still not been fully understood. Gut microbiota is reported to be associated with the efficacy and toxicity of antineoplastic antimetabolites. In this review, we summarize the interaction of antineoplastic antimetabolites on gut microbiota and the influences of shifted gut microbiota profiles on the efficacy and toxicity of antineoplastic antimetabolites. The factors affecting the efficacy and toxicity of antineoplastic antimetabolites via gut microbiota are also discussed. In addition, we present our viewpoints that regulating the gut microbiota may increase the efficacy and decrease the toxicity of antineoplastic antimetabolites. This will help us better understand the new mechanism via gut microbiota and promote individualized use of antineoplastic antimetabolites.

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.

Panax notoginseng saponins prevent colitis-associated colorectal cancer development: the role of gut microbiota.

Gut microbiota dysbiosis is a risk factor for colorectal cancer (CRC) in inflammatory bowel disease (IBD). In this study, the effects of Panax notoginseng saponins (PNS) on colitis-associated CRC progression were evaluated on an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model. In vivo, PNS significantly relieved AOM/DSS-induced colon tumorigenesis and development by reducing the disease activity index (DAI) scores and colon tumor load. The 16S rRNA data of fecal samples showed that the microbiome community was obviously destructed, while PNS could recover the richness and diversity of gut microbiota. Especially, PNS could increase the abundance of Akkermansia spp. which was significantly decreased in model group and negatively correlated with the progression of CRC. Moreover, ginsenoside compound K (GC-K) was evaluated on the effects of human CRC cells, which was the main bio-transformed metabolite of PNS by gut microbiota. Our data showed that PNS played important role in the prevention of the progression of CRC, due to their regulation on the microbiome balance and microbial bio-converted product with anti-CRC activity.

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.

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.

Chemo-Preventive Potential of Falcarindiol-Enriched Fraction from Oplopanax elatus on Colorectal Cancer Interfered by Human Gut Microbiota.

Oplopanax elatus (Nakai) Nakai is an oriental herb, the polyyne-enriched fraction of which (PEFO) showed anticolorectal cancer (anti-CRC) effects. Other concomitant components, which are inevitably bio-transformed by gut microbiota after oral administration, might be interfere with the pharmacodynamics of polyynes. However, the influence of human gut microbiota on molecules from O. elatus possessing anticancer activity are yet unknown. In this study, the compounds in PEFO and PEFO incubated with human gut microbiota were analyzed and tentatively identified by HPLC-DAD-QTOF-MS. Two main polyynes ((3S,8S)-falcarindiol and oplopandiol) were not significantly decomposed, but some new unknown molecules were discovered during incubation. However, the antiproliferative effects of PEFO incubated with human gut microbiota for 72 h (PEFO I) were much lower than that of PEFO on HCT-116, SW-480, and HT-29 cells. Furthermore, PEFO possessed better anti-CRC activity in vivo, and significantly induced apoptosis of the CRC cells, which was associated with activation of caspase-3 according to the Western-blot results (P<0.05). These results suggest anticolorectal cancer activity of polyynes might be antagonized by some bio-converted metabolites after incubation with human gut microbiota. Therefore, it might be better for CRC prevention if the polyynes could be orally administrated as purified compounds.

The role of PEG3 in the occurrence and prognosis of colon cancer.

PURPOSE: Imprinted genes are often identified as key players in the etiology and prognosis of many tumors; however, the role they play in colon cancer remains unclear. Along with the development of big data analysis came the discovery of a wealth of genetic prognostic factors, like microsatellite instability for colon cancer, which need to be taken into consideration when evaluating new biomarkers for the disease. METHODS: We systematically mined public databases to find recurrence free survival (RFS)-related imprinted genes for colon cancer patients on the mRNA level by univariate and multivariate survival analyses. We then investigated the association of methylation status and microRNA expression of the targeted imprinted genes with survival rate of colon cancer patients. Lastly, in a clinical study we used qRT-PCR and immunohistochemistry to quantify mRNA and protein expression of the imprinted genes that related to RFS in our bioinformatics screening, respectively, in 20 tumor tissues compared to paired adjacent tissues. RESULTS: The results show that paternally expressed gene 3 (PEG3) is the only imprinted gene related to colon cancer patient prognosis on the mRNA level in our datasets, and high mRNA expression of PEG3 is associated with a poor prognosis. Furthermore, the methylation beta value of cg13960339, as well as the expression of 4 microRNAs, negatively correlated with PEG3 mRNA level and were correlated with the prognosis of colon cancer patients. Moreover, the expression of PEG3 mRNA in colon cancer is significantly lower, but PEG3 protein expression is significantly higher compared to that in normal tissues. CONCLUSION: PEG3 is likely associated with the progression and prognosis of colon cancer.

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.

Harmine enhances type H vessel formation and prevents bone loss in ovariectomized mice.

Recently, researchers identified a distinct vessel subtype called type H vessels that couple angiogenesis and osteogenesis. We previously found that type H vessels are reduced in ovariectomy (OVX)-induced osteoporotic mice, and preosteoclasts are able to secrete platelet-derived growth factor-BB (PDGF-BB) to stimulate type H vessel formation and thereby to promote osteogenesis. This study aimed to explore whether harmine, a ß-carboline alkaloid, is capable of preventing bone loss in OVX mice by promoting preosteoclast PDGF-BB-induced type H vessel formation. METHODS: The impact of harmine on osteoclastogenesis of RANKL-stimulated RAW264.7 cells was verified by gene expression analysis and tartrate-resistant acid phosphatase (TRAP) staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to test PDGF-BB production by preosteoclasts. A series of angiogenesis-related assays in vitro were performed to assess the pro-angiogenic effects of the conditioned media from RANKL-stimulated RAW264.7 cells treated with or without harmine. Meanwhile, the role of PDGF-BB in this process was determined. In vivo, OVX mice were intragastrically administrated with harmine emulsion or an equal volume of vehicle. 2 months later, bone samples were collected for µCT, histological, immunohistochemical and immunofluorescent analyses to evaluate bone mass, osteogenic and osteoclastic activities, as well as the numbers of type H vessels. Bone marrow PDGF-BB concentrations were assessed by ELISA. RESULTS: Exposure of RANKL-stimulated RAW264.7 cells to harmine enhanced the formation of preosteoclasts and the production of PDGF-BB. Harmine augmented the ability of RANKL-stimulated RAW264.7 cells to promote angiogenesis of endothelial cells, whereas the effect was blocked by PDGF-BB inhibition. In vivo, the oral administration of harmine emulsion to OVX mice resulted in enhanced trabecular bone mass and osteogenic responses, increased numbers of preosteoclasts, as well as reduced numbers of osteoclasts and fat cells. Moreover, OVX mice treated with harmine exhibited higher levels of bone marrow PDGF-BB and much more type H vessels in bone. CONCLUSION: Harmine may exert bone-sparing effects by suppression of osteoclast formation and promotion of preosteoclast PDGF-BB-induced angiogenesis.

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 of a novel sectional multiple filtering scheme for rapid screening and classifying metabolites of ziyuglycoside II in rat liver and excreta specimen based on high-resolution mass spectrometry.

Ziyuglycoside II, one of the major effective ingredients of Sanguisorba officinalis L., had various pharmacological activities including anticancer, anti-inflammation and anti-oxidation, etc. Better understanding of the pharmacology and toxicology of ziyuglycoside II requires the detailed elucidation of its biologic fates in vivo. Herein, the metabolic fate of ziyuglycoside II in rats was investigated based on liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). To accelerate and simplify the process of metabolite identification from complicated biological matrix, the sectional multiple filtering (SMF) scheme was designed according to the relationship among the molecular weight (MW), mass defect (MD) and retention time (tR) of the metabolites. SMF-I (MW: 700-850Da, MD: 0.40-0.45Da, tR: 4.0-10.0min), SMF-II (MW: 550-700Da, MD: 0.30-0.40Da, tR: 6.0-14.0min) and SMF-III (MW: 400-550Da, MD at 0.25-0.35Da, tR at 9.5-16.0min) were built and utilized to screen phase II conjugations and phase I redox metabolites and deglycosylated derivatives, respectively. As a result, dozens of metabolites, including glucuronic conjugates, hydroxylation, oxidization, dehydration and deglycosylation products, were rapidly discovered, classified and structural identified in rat urine and feces based on SMF scheme and accurate MS(1)/MS(2) information. Obviously, the SMF technique showed superior efficiency and selectivity in ziyuglycoside II metabolite identification. More importantly, SMF would find its extensive application in, but not limited to, the metabolic study for single drug or homologous compounds in traditional Chinese medicine.

The metabolic and pharmacokinetic studies for HM-3 in rats based on LC-Q-TOF/MS and LC-MS/MS combing a convenient biological sample processing method.

In this contribution, the metabolic and pharmacokinetic characteristics for the therapeutic peptide HM-3 were investigated using LC-Q-TOF/MS and LC-MS/MS systems combing a fast biological sample processing method. According to the accurate MS(1) and MS(2) data generated by LC-Q-TOF/MS, a total of 6 metabolites in rats were detected and tentatively identified as the degradation products which formed by successive loss of amino acid from HM-3. The structures of the 2 main metabolites (M1 and M2) were confirmed by comparing the chromatographic and mass spectrographic characteristics with the corresponding synthetic standards. Then, an absolute quantitative analysis method based on LC-MS/MS system was built and fully validated with respect to linearity, sensitivity, accuracy, precision, matrix effect, stability, etc. The results indicated that HM-3, M1 and M2 were linear in peak area ratios over the concentration range of 0.5-200.0ng/mL with a correlation coefficient>0.99. The intra-day and inter-day precisions (RSD%) were less than 15%, and the accuracy was below 10% in terms of RE%. The validated method was then successfully applied to the studies of preclinical pharmacokinetics for HM-3.