Diosbulbin B Research Articles

Ameliorative effect of Schisandrol B against Diosbulbin B-induced hepatotoxicity via inhibiting CYP3A4-mediated bioactivation

Diosbulbin B (DBB), the major component isolated from herbal medicine Dioscorea bulbifera L. (DBL), can trigger severe hepatotoxicity. The previous studies demonstrated that DBB-induced hepatotoxicity is closely relevant to the bioactivation mediated by CYP3A4 and subsequent generation of adducts with cellular proteins. Schisandrol B (SchB), the primary lignan ingredient in herbal medicine Schisandra chinensis (SC), is able to inhibit CYP3A activity. The objective of this study is to investigate the protective effect of SchB against hepatotoxicity induced by DBB and to explore the underlying mechanism. Biochemical and histopathological analysis demonstrated that SchB exerted dose-dependent protective effect against DBB-induced hepatotoxicity. In vitro metabolism assay showed that the formation of pyrrole-glutathione (GSH) conjugates of DBB was inhibited by SchB in a concentration dependent manner, suggesting SchB inhibited the bioactivation of DBB in vitro. Pharmacokinetic studies demonstrated that SchB enhanced Cmax and AUCs of DBB in mouse blood and liver, resulting in accelerating the accumulation of DBB in the circulation. In addition, pretreatment with SchB alleviated DBB-induced hepatic GSH depletion, obviously facilitated the excretion of DBB in urine, and reduced the urinary excretion of DBB-GSH conjugates, indicating that SchB affected absorption, distribution, metabolism, and excretion (ADME) of DBB by inhibiting the bioactivation of DBB in vivo. In conclusion, our findings demonstrated the amelioration of SchB against DBB-induced hepatotoxicity was correlated with the inhibition of CYP3A4-mediated bioactivation of DBB. Thus, the findings indicated that SchB may serve as a potential candidate drug for the treatment of DBB intoxication.

New finding based on Comparative Toxicogenomics Database: Hepatic YY1 mediates drug-induced liver injury

BackgroundYY1 plays a crucial part in the onset and progression of numerous liver diseases, yet the significant contribution of YY1 to drug-induced liver injury (DILI) appears to have been underestimated by researchers. PurposeTo reveal the underlying role of YY1 in DILI. MethodThe compounds that interact with YY1 were queried in the Comparative Toxicogenomics Database (CTD), with the majority found to be hepatotoxic, which includes certain widely used drugs. Molecular docking and SPR characterized the robust binding of hepatotoxic compounds to YY1. The duty of YY1 in DILI was investigated in Diosbulbin B (DIOB), a recently identified hepatotoxic compound that tightly associates with YY1, and further validated on ANIT, LCA, APAP, and CDDP. Transcriptomic analysis disclosed the underlying mechanisms involved in DIOB-induced liver injury. RT-qPCR, immunohistochemistry, immunofluorescence, western blotting, and cellular transfection techniques were employed to validate the specific mechanism. ResultsAmong the 94 compounds affecting YY1 expression in the CTD, 59 compounds exhibited hepatotoxicity, showing close interactions with YY1 and almost consistent binding sites by molecular docking. The SPR validated the tough binding of several hepatotoxic compounds to YY1, including five FDA-approved hepatotoxic drugs. Mechanistically, the involvement of YY1 in DILI was uncovered through the cholestasis lens, mice hepatic YY1 was up-regulated by hepatotoxic DIOB and transcriptionally inhibited FXR and its downstream BSEP and MRP2 expression, initiating early in cholestatic liver injury and persisting to drive the progression of cholestasis. ANIT and LCA-induced model of cholestasis provided evidence for the hypothesis that YY1 frequently mediates drug induced cholestasis (DIC). APAP and CDDP indicated that YY1 may also be involved in hepatocellular and mixed type DILI. ConclusionYY1 widely mediated the development of DIC and also might be engaged in other types of DILI. YY1 presented a common target for hepatotoxic medications and the targeting of liver YY1 for drug development may offer a novel approach for managing DILI.

The anti-non-small cell lung cancer effect of Diosbulbin B: Targeting YY1 induced cell cycle arrest and apoptosis

BackgroundToxic components frequently exhibit unique characteristics and activities, offering ample opportunities for the advancement of anti-cancer medications. As the main hepatotoxic component of Dioscorea bulbifera L. (DB), Diosbulbin B (DIOB) has been widely studied for its anti-tumor activity at nontoxic doses. However, the effectiveness and mechanism of DIOB against non-small cell lung cancer (NSCLC) remains unclear. PurposeTo evaluate the anti-NSCLC activity of DIOB and to elucidate the specific mechanism of action. MethodThe effect of DIOB on NSCLCL in vitro was evaluated through CCK8, colony formation, and flow cytometry. The in vivo efficacy and safety of DIOB in treating NSCLC were assessed using various techniques, including HE staining, tunel staining, immunohistochemistry, and biochemical index detection. To understand the underlying mechanism, cell transfection, western blotting, molecular docking, cellular thermal shift assay (CESTA), and surface plasmon resonance (SPR) were employed for investigation. ResultsDIOB effectively hindered the progression of NSCLC both in vitro and in vivo settings at a no-observed-adverse-effect concentration (NOAEC) and a safe dosage. Specifically, DIOB induced significant G0/G1 phase arrest and apoptosis in A549, PC-9, and H1299 cells, while also notably inhibiting the growth of subcutaneous tumors in nude mice. Mechanistically, DIOB could directly interact with oncogene Yin Yang 1 (YY1) and inhibit its expression. The reduction in YY1 resulted in the triggering of the tumor suppressor P53, which induced cell cycle arrest and apoptosis in NSCLC cells by inhibiting the expression of Cyclin A2, B2, CDK1, CDK2, CDK4, BCL-2, and inducing the expression of BAX. In NSCLC cells, the induction of G0/G1 phase arrest and apoptosis by DIOB was effectively reversed when YY1 was overexpressed or P53 was knocked down. Importantly, we observed that DIOB exerted the same effect by directly influencing the expression of YY1-regulated c-Myc and BIM, particularly in the absence of P53. ConclusionFor the inaugural investigation, this research unveiled the anti-NSCLC impact of DIOB, alongside its fundamental mechanism. DIOB has demonstrated potential as a treatment agent for NSCLC due to its impressive efficacy in countering NSCLC.

Investigation of the principle of concoction by using the processing excipient Glycyrrhiza uralensis Fisch. juice to reduce the main toxicity of Dioscorea bulbifera L. and enhance its main efficacy as expectorant and cough suppressant

Ethnopharmacological relevanceDioscorea bulbifera L. (Rhizoma Dioscoreae Bulbiferae; RDB) is commonly used as an expectorant and cough suppressant herb but is accompanied by severe hepatotoxicity. Using the juice of auxiliary herbs (such as Glycyrrhiza uralensis Fisch. (Glycyrrhizae Radix et Rhizoma; GRR) juice) in concocting poisonous Chinese medicine is a conventional method to reduce toxicity or increase effects. Our previous study found that concoction with GRR juice provided a detoxifying effect against the major toxic hepatotoxicity induced by RDB, but the principle for the detoxification of the concoction is unknown to date. Aim of the studyThe principle of concoction was investigated by using the processing excipient GRR juice to reduce the major toxic hepatotoxicity of RDB, and the efficacy of RDB as an expectorant and cough suppressant was enhanced. Materials and methodsIn this study, common factors (RDB:GRR ratio, concocted temperature, and concocted time) in the concoction process were used for the preparation of each RDB concocted with GRR juice by using an orthogonal experimental design. We measured the content of the main toxic compound diosbulbin B (DB) and serum biochemical indicators and performed pathological analysis in liver tissues of mice to determine the best detoxification process of RDB concocted with GRR juice. On this basis, the biological mechanisms of target organs were detected by Western blot and enzyme-linked immunosorbent assay at the inflammation and apoptosis levels. Further, the effects of RDB on expectorant and cough suppressant with GRR juice were evaluated by the conventional tests of phenol red expectorant and concentrated ammonia-induced cough. Lastly, the major compounds in the GRR juice introduced to RDB concoction were determined. ResultsRDB concocted with GRR juice significantly alleviated DB content, serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels, and improved liver pathological damages. The best detoxification process was achieved by using an RDB:GRR ratio of 100:20 at 120 °C for 20 min. Further, RDB concocted with GRR juice down-regulated the protein levels of nuclear factor kappa B (NF-κB), cyclooxygenase 2 (COX-2), and Bcl-2 related X protein (Bax) in the liver and enhanced the expectorant and cough suppressant effects of RDB. Finally, liquiritin (LQ) and glycyrrhizic acid (GA) in the GRR juice were introduced to the RDB concoction. ConclusionConcoction with GRR juice not only effectively reduced the major toxic hepatotoxicity of RDB but also enhanced its main efficacy as an expectorant and cough suppressant, and that the rationale for the detoxification and/or potentiation of RDB was related to the reduction in the content of the main hepatotoxic compound, DB, the introduction of the hepatoprotective active compounds, LQ and GA, in the auxiliary GRR juice, as well as the inhibition of NF-κB/COX-2/Bax signaling-mediated inflammation and apoptosis.

Ferulic acid prevents Diosbulbin B-induced liver injury by inhibiting covalent modifications on proteins

Diosbulbin B (DIOB) has been reported to cause serious liver injury. However, in traditional medicine, DIOB-containing herbs are highly safe in combination with ferulic acid (FA)-containing herbs, suggesting potential neutralizing effect of FA on the toxicity of DIOB. DIOB can be metabolized to generate reactive metabolites (RMs), which can covalently bind to proteins and lead to hepatoxicity. In the present study, the quantitative method was firstly established for investigating the correlation between DIOB RM-protein adducts (DRPAs) and hepatotoxicity. Then, we estimated the detoxication effect of FA in combination with DIOB and revealed the underlying mechanism. Our data indicated that the content of DRPAs positively correlate with the severity of hepatotoxicity. Meanwhile, FA is able to reduce the metabolic rate of DIOB in vitro. Moreover, FA suppressed the production of DRPAs and decreased the serum alanine/aspartate aminotransferase (ALT/AST) levels elevated by DIOB in vivo. Thus, FA can ameliorate DIOB-induced liver injury through reducing the production of DRPAs.

Glycyrrhetinic acid ameliorates diosbulbin B-induced hepatotoxicity in mice by modulating metabolic activation of diosbulbin B.

Exposure to diosbulbin B (DBB), the primary component of the herbal medicine Dioscorea bulbifera L. (DB), can cause liver injury in humans and experimental animals. A previous study found DBB-induced hepatotoxicity was initiated by CYP3A4-mediated metabolic activation and subsequent formation of adducts with cellular proteins. The herbal medicine licorice (Glycyrrhiza glabra L.) is frequently combined with DB used in numerous Chinese medicinal formulas in an effort to protect against DB-elicited hepatotoxicity. Importantly, glycyrrhetinic acid (GA), the major bioactive ingredient in licorice, inhibits CYP3A4 activity. The study aimed to investigate the protection of GA against DBB-induced hepatotoxicity and the underlying mechanism. Biochemical and histopathological analysis showed GA alleviated DBB-induced liver injury in a dose-dependent manner. In vitro metabolism assay with mouse liver microsomes (MLMs) indicated that GA decreased the generation of metabolic activation-derived pyrrole-glutathione (GSH) conjugates from DBB. Toxicokinetic studies demonstrated that GA increased maximal serum concentration (Cmax ) and area under the serum-time curve (AUC) of DBB in mice. In addition, GA attenuated hepatic GSH depletion caused by DBB. Further mechanistic studies showed that GA reduced the production of DBB-derived pyrroline-protein adducts in a dose-dependent manner. In conclusion, our findings demonstrated that GA exerted protective effect against DBB-induced hepatotoxicity, mainly correlated with suppressing the metabolic activation of DBB. Therefore, the development of a standardized combination of DBB with GA may protect patients from DBB-induced hepatotoxicity.

Detoxification technology and mechanism of processing with Angelicae sinensis radix in reducing the hepatotoxicity induced by rhizoma Dioscoreae bulbiferae in vivo.

Rhizoma Dioscoreae Bulbiferae (RDB) was effective on relieving cough and expectorant but accompanied by severe toxicity, especially in hepatotoxicity. A previous study found that processing with Angelicae Sinensis Radix (ASR) reduced RDB-induced hepatotoxicity. However, up to now, the optimized processing process of ASR-processed RDB has not been explored or optimized, and the detoxification mechanism is still unknown. This study evaluated the detoxification technology and possible mechanism of processing with ASR on RDB-induced hepatotoxicity. The optimized processing process of ASR-processed RDB was optimized by the content of diosbulbin B (DB), the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and histopathological analysis. The processing detoxification mechanism was evaluated by detecting the antioxidant levels of nuclear factor E2 related factor 2 (Nrf2) and its downstream heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1), glutamylcysteine ligase catalytic subunit (GCLM), and the levels of downstream antioxidant factors of Nrf2. Besides, the antitussive and expectorant efficacy of RDB was also investigated. This work found that processing with ASR attenuated RDB-induced hepatotoxicity, which can be verified by reducing the levels of ALT, AST, and ALP, and reversing the pathological changes of liver histomorphology. And the optimized processing process of ASR-processed RDB is “processing at a mass ratio of 100:20 (RDB:ASR) and a temperature of 140°C for 10 min.” Further results corroborated that the intervention of processed products of ASR-processed RDB remarkably upregulated the Nrf2/HO-1/NQO1/GCLM protein expression levels in liver, and conserved antitussive and expectorant efficacy of RDB. The above findings comprehensively indicated that the optimized processing process of ASR-processed RDB was “processing at a mass ratio of 100:20 (RDB:ASR) and a temperature of 140°C for 10 min,” and the processing detoxification mechanism involved enhancing the level of Nrf2-mediated antioxidant defense in liver as a key target organ.

HMGB1 induced endothelial to mesenchymal transition in liver fibrosis: The key regulation of early growth response factor 1

BackgroundLiver fibrosis has been the focus and difficulty of medical research in the world and its concrete pathogenesis remains unclear. This study aims to observe the high-mobility group box 1 (HMGB1)-induced hepatic endothelial to mesenchymal transition (EndoMT) during the development of hepatic fibrosis, and further to explore the crucial involvement of Egr1 in this process. MethodsCarbon tetrachloride (CCl4), diosbulbin B (DB), N-acetyl-p-aminophenol (APAP) and bile duct ligation (BDL) were used to induce liver fibrosis in mice. Serum HMGB1 content, the occurrence of EndoMT and the production of extracellular matrix (ECM) in vitro and in vivo were detected by Western-blot. ResultsThe elevated serum HMGB1 content, the occurrence of EndoMT, the production of ECM and the activation of Egr1 were observed in mice with liver fibrosis induced by CCl4, DB, APAP or BDL. HMGB1 induced EndoMT and ECM production in human hepatic sinusoidal endothelial cells (HHSECs), and then HHSECs lost the ability to inhibit the activation of hepatic stellate cells (HSCs). The hepatic deposition of collagen, the increased serum HMGB1 content and hepatic EndoMT were further aggravated in Egr1 knockout mice. Natural compound silymarin attenuated liver fibrosis in mice induced by CCl4 via increasing Egr1 nuclear accumulation, decreasing serum HMGB1 content and inhibiting hepatic EndoMT. ConclusionEgr1 regulated the expression of HMGB1 that induced hepatic EndoMT, which plays an important role in the development of liver fibrosis.General significance:This study provides a novel therapeutic strategy for the treatment of liver fibrosis in clinic.

Diosbulbin B: An important component responsible for hepatotoxicity and protein covalent binding induced by Dioscorea bulbifera L.

BackgroundDioscorea bulbifera L. (DBL) is an herbal medicine used for the treatment of thyroid diseases and tumors in China. However, the hepatotoxicity of DBL limits its wide safe use. Diosbulbin B (DSB) is the most abundant diterpene lactone occurring in DBL. Numbers of studies showed that this furanoterpenoid plays an important role in DBL-induced liver injury and that DSB is metabolized to a cis-enedial intermediate which reacts with protein to form protein covalent binding and induces hepatotoxicity. PurposeThe present study aimed to define the association of DSB content in DBL with the severity of DBL hepatotoxicity to ensure the safe use of the herbal medicine in clinical practice and to determine the role of DSB in DBL-induced liver injury. MethodsChemical chromatographic fingerprints of DBL were established by UPLC-MS/MS. Their hepatotoxicity potencies were evaluated in vitro and in vivo. Metabolic activation of DSB was evaluated by liver microsomal incubation. Protein modification was assessed by mass spectrometry and immunostaining. ResultsThe contents of DSB in DBL herbs collected from 11 locations in China varied dramatically with as much as 47-fold difference. The hepatotoxicity potencies of DBL herbs were found to be proportional to the contents of DSB. Intensified protein adduction derived from the reactive metabolite of DSB was observed in mice administered DBL with high contents of DSB. ConclusionThe findings not only demonstrated that contents of DSB can be quite different depending on harvest location and special attention needs to pay for quality control of DBL but also suggest DSB is a key contributor for DBL-induced hepatotoxicity.

Development of a mechanism-based biomarker for Dioscorea bulbifera L. exposure and hepatotoxicity in rats

BackgroundDioscorea bulbifera L. (DBL) is a common herbal medicine where furanoterpenoid diosbulbin B (DSB) is a major component responsible for its hepatotoxicity. The metabolic oxidation of the furan moiety of DSB, resulting in covalent binding to hepatic protein, is considered to initiate its liver injury. PurposeWe aimed to develop a mechanism-based plasma protein adduction-based biomarker to determine DBL exposure and to predict the onset of hepatotoxicity induced by DBL. MethodsRats were intragastrically treated with DBL extract, and the plasma samples were collected. Plasma ALT and AST were measured with commercial kits. Plasma protein modification was determined by immunoblot assay. Assessment of DSB-induced protein adduction was achieved by LC-MS/MS analysis of complete proteolytic digestion of adducted protein to pyrroline derivative A4 using pronase enzyme. The structure of the resulting pyrroline derivatives was confirmed by NMR. ResultsPlasma protein of rats treated with DBL extract was covalently modified by the metabolite of DSB. Pyrroline derivative A4 was detected in proteolytic digestion of plasma obtained from rats administered DBL extract. The protein adduction elevated with the increase in the dosage of DBL extract. A detectable level of plasma was observed 10 days after withdrawal of DBL extract post 30-day continuous administration. In addition, the elevation trend of plasma ALT was found to be proportional to the accumulation trend of pyrroline derivative A4. ConclusionDSB-derived plasma protein adduction correlated well with the exposure of DBL in rats. The protein adduction may be used as a good biomarker for diagnosis of DBL-induced liver injury and a useful indicator for DBL medication plans.

Clarify the potential cholestatic hepatotoxicity components from Chinese Herb Medicine and metabolism’s role via hBSEP vesicles and S9/hBSEP vesicles

In this study, the inhibitory effect of components from Chinese Herb Medicine (CHMs) with potential hepatotoxicity was assessed by human bile salt export pump (hBSEP) vesicles with and without S9 metabolism. Sixty-three compounds from 22 hepatoxicity CHMs were selected as the test articles. In hBSEP vesicles, eighteen of them were found to have moderate or strong inhibitory effect towards BSEP. Further studies were performed to determine the IC50 values of strong inhibitors. For the compounds belong to CHMs reported to cause cholestasis and strong inhibitors defined in hBSEP vesicles, their relative transport activities of Taurocholic acid (TCA) were evaluated in hBSEP vesicles as well as hBSEP vesicles with S9 system (S9/hBSEP vesicles). The differences of their relative transport activities of TCA between the above two system were compared to reveal the net effect of metabolism on BSEP's activity. It was found that the inhibitory effect of Saikogenin A (SGA), Saikogenin D (SGD), Diosbulbin B (DB) and rhein were significantly increased; while the inhibitory effect of isobavachalcone, saikosaponin d and saikosaponin b2 were significantly decreased after S9 metabolizing. Identification of metabolic pathways suggested that CYP3A4 was responsible for aggravating inhibitory effect of SGA and SGD against BSEP.

Circular RNA circHECTD1 prevents Diosbulbin-B-sensitivity via miR-137/PBX3 axis in gastric cancer

BackgroundsGastric cancer (GC) is general disease in human digestive system with malignancy. Emerging findings indicated that hsa_circ_0031452 (circHECTD1) was strictly associated with carcinogenesis. Nevertheless, the role of circHECTD1 in drug-resistance still needed to be explained.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was employed to examine the expression profiles of circHECTD1, microRNA (miR)-137, and pre-leukemia transcription factor 3 (PBX3). The function of circHECTD1 in tumorigenesis was evaluated via xenograft tumor model. The IC50 of Diosbulbin-B (DB) was detected using Cell Counting Kit-8 (CCK8). Cell-cycle and apoptosis were reckoned by flow cytometry. Besides, western blot was administrated to reckon the levels of PBX3 and cell apoptotic indicators. Moreover, the interrelation between miR-137 and circHECTD1 or PBX3 was expounded by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull down assays.ResultsWe uncovered that circHECTD1 was ectopically up-regulated in GC tissues and cells. CircHECTD1 deficiency sensitized DB-treatment in DB-evoked AGS and HGC-27 cells. In vivo assay, circHECTD1 silencing led to the tumor reduction. Also, circHECTD1 served as miR-137 sponge in a sequence-complementary manner. Furthermore, transfection of miR-137 inhibitor markedly eliminated circHECTD1 absence-mediated promotion of DB-sensitivity in GC cells. Moreover, PBX3, a target of miR-137, play a DB-resistant role in GC cells. Fascinatingly, the deletion of PBX3 reversed the impact of miR-137 repression and circHECTD1 knockdown on DB-sensitivity in vitro.ConclusionsCircHECTD1 served as an oncogene by a novel miR-137/PBX3 axis, which might supply an underlying biomarker for the diagnosis and prognosis of GC management.

Metabonomic approaches investigate diosbulbin B-induced pulmonary toxicity and elucidate its underling mechanism in male mice.

Air Potato Yam is widely used in the treatment of many conditions such as cancer, inflammation, and goiter. Diosbulbin B (DIOB) is the primary active component of Air Potato Yam, and it exhibits anti-tumor and anti-inflammatory properties. The main purpose of this study was to determine the mechanism by which DIOB induces lung toxicity, using metabonomics and molecular biology techniques. The results showed that the lung toxicity induced by DIOB may occur because of a DIOB-induced increase in the plasma levels of long-chain free fatty acids and endogenous metabolites related to inflammation. In addition, treatment with DIOB increases the expression of the cyp3a13 enzyme, which leads to enhanced toxicity in a dose-dependent manner. The molecular mechanism underlying toxicity in mouse lung cells is the DIOB-mediated inhibition of fatty acid β-oxidation, partial glycolysis, and the TCA cycle, but DIOB treatment can also compensate for the low Adenosine triphosphate (ATP) supply levels by improving the efficiency of the last step of the glycolysis reaction and by increasing the rate of anaerobic glycolysis. Using metabonomics and other methods, we identified the toxic effects of DIOB on the lung and clarified the underlying molecular mechanism.

Low-dose Diosbulbin-B (DB) activates tumor-intrinsic PD-L1/NLRP3 signaling pathway mediated pyroptotic cell death to increase cisplatin-sensitivity in gastric cancer (GC)

BackgroundEmerging evidences suggests that Diosbulbin-B (DB) is effective to improve cisplatin (DDP)-sensitivity in gastric cancer (GC), but its molecular mechanisms were not fully delineated, and this study managed to investigate this issue.MethodsGenes expressions were determined by Real-Time qPCR and Western Blot at transcriptional and translational levels. Cell proliferation and viability were evaluated by cell counting kit-8 (CCK-8) and trypan blue staining assay. Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The Spheroid formation assay was used to evaluated cell stemness. The xenograft tumor-bearing mice models were established, and the tumors were monitored and the immunohistochemistry (IHC) was employed to examine the expressions and localization of Ki67 protein in mice tumor tissues.ResultsLow-dose DB (12.5 μM) downregulated PD-L1 to activate NLRP3-mediated pyroptosis, and inhibited cancer stem cells (CSCs) properties, to sensitize cisplatin-resistant GC (CR-GC) cells to cisplatin. Mechanistically, the CR-GC cells were obtained, and either low-dose DB or cisplatin alone had little effects on cell viability in CR-GC cells, while low-dose DB significantly induced apoptotic cell death in cisplatin treated CR-GC cells. In addition, low-dose DB triggered cell pyroptosis in CR-GC cells co-treated with cisplatin, which were abrogated by silencing NLRP3. Next, CSCs tended to be enriched in CR-GC cells, instead of their parental cisplatin-sensitive GC (CS-GC) cells, and low-dose DB inhibited spheroid formation and stemness biomarkers (SOX2, OCT4 and Nanog) expressions to eliminate CSCs in CR-GC cells, which were reversed by upregulating programmed death ligand-1 (PD-L1). Furthermore, we proved that PD-L1 negatively regulated NLRP3 in CR-GC cells, and low-dose DB activated NLRP3-mediated pyroptotic cell death in cisplatin treated CR-GC cells by downregulating PD-L1. Also, low-dose DB aggravated the inhibiting effects of cisplatin on tumorigenesis of CR-GC cells in vivo.ConclusionsCollectively, low-dose DB regulated intrinsic PD-L1/NLRP3 pathway to improve cisplatin-sensitivity in CR-GC cells, and this study provided alternative therapy treatments for GC.

Angelica sinensis polysaccharide (ASP) attenuates diosbulbin-B (DB)-induced hepatotoxicity through activating the MEK/ERK pathway

ABSTRACT Diosbulbin-B (DB) is a promising therapeutic drug for cancer treatment; however, DB-induced hepatotoxicity seriously limits its clinical utilization. Based on this, the present study investigated whether the Angelica sinensis extract, angelica sinensis polysaccharide (ASP), was effective to attenuate DB-induced cytotoxicity in hepatocytes. The primary hepatocytes were isolated from rats and cultured in vitro, which were subsequently treated with high-dose DB (100 μM) and ASP (12 μg/ml) to establish the DB-induced hepatotoxicity models. MTT assay and flow cytometry (FCM) were performed to evaluate cell viability, and the results showed that high-dose DB-induced cell apoptosis and inhibition of proliferation were reversed by co-treating cells with ASP, which were supported by our Western Blot assay data that ASP upregulated Cyclin D1 and CDK2 to abrogate high-dose DB-induced cell cycle arrest. In addition, ASP exerted its regulating effects on cell autophagy, and we found that ASP increased LC3B-II/I ratio and Atg5, but decreased p62 to activate the autophagy flux. Of note, the MEK/ERK pathway could be activated by ASP in the DB-treated hepatocytes, and the protective effects of ASP on high-dose DB-induced hepatocyte death were abolished by co-treating cells with the autophagy inhibitor (3-methyladenine, 3-MA) and MEK/ERK selective inhibitor (SCH772984). Moreover, blockage of the MEK/ERK pathway suppressed cell autophagy in the hepatocytes co-treated with ASP and high-dose DB. Taken together, this in vitro study illustrated that ASP activated the MEK/ERK pathway mediated autophagy to suppress high-dose DB-induced hepatotoxicity.

Evidence for Polyamine, Biogenic Amine, and Amino Acid Adduction Resulting from Metabolic Activation of Diosbulbin B.

Dioscorea bulbifera L. (DBL), a traditional Chinese medicine, is a well-known herb with hepatotoxicity, and the biochemical mechanisms of the toxic action remain unknown. Diosbulbin B (DSB), a major component of DBL, can induce severer liver injury which requires cytochrome P450-catalyzed oxidation of the furan ring. It is reported that a cis-enedial reactive intermediate resulting from metabolic activation of DSB can react with thiols and amines to form pyrrole or pyrroline derivatives. In this study, we investigated the interaction of the reactive intermediate with polyamines, biogenic amines, and amino acids involved in the polyamine metabolic pathway, including putrescine, spermidine, spermine, histamine, arginine, ornithine, lysine, glutamine, and asparagine. Seven DSB-derived amine adducts were detected in microsomal incubations supplemented with DSB and individual amines. Six adducts were observed in cultured rat primary hepatocytes after exposure to DSB. DSB was found to induce apoptosis and cell death in time- and concentration-dependent manners. Apparently, the observed apoptosis was associated with the detected amine adduction. The findings facilitate the understanding of the mechanisms of toxic action of DSB.

Systems Toxicology Approaches Reveal the Mechanisms of Hepatotoxicity Induced by Diosbulbin B in Male Mice.

Diosbulbin B (DIOB) is an effective component of air potato yam with antitumor and anti-inflammatory activities, and it is the main toxic component leading to hepatotoxicity. However, the mechanism of its hepatotoxicity remains unclear. In this study, we aimed to systematically elucidate the molecular action of DIOB on liver metabolic function through systems toxicology approaches. C57BL/6 mice were orally treated with DIOB (10, 30, 60 mg/kg) for 28 days, and the liver metabonomics and histopathology, molecular docking, mRNA expression levels, and activities of enzymes were analyzed. The results illustrated that DIOB could affect fatty acid and glucose metabolism, block the TCA cycle, and DIOB also could disorder bile acid synthesis and transport and promote the occurrence of hyperbilirubinemia. In addition, DIOB increased Cyp3a11 expression in a dose-dependent manner. Thus, these results provide new insights into the mechanism of hepatotoxicity caused by DIOB.

Downregulation of CircRNA CDR1as specifically triggered low-dose Diosbulbin-B induced gastric cancer cell death by regulating miR-7-5p/REGγ axis

Diosbulbin-B (DB) was the main compound of Dioscorea bulbifera L, which was widely used for cancer treatment in Asia. However, the hepatotoxicity induced by high-dose DB seriously limited its possibility using for gastric cancer (GC) treatment in clinic. In this study, we found that DB inhibited GC cells and hepatocytes cell viability in a dose- and time-dependent manner. Specifically, high-dose DB (50μM) significantly inhibited cell proliferation and promoted cell apoptosis, while low dose DB (12.5μM) had little effects on cell viability. Besides, high-dose DB (50μM) significantly decreased CircRNA CDR1as levels in gastric cancer cells instead of hepatocytes. Notably, knock-down of CircRNA CDR1as triggered low-dose DB (12.5μM) induced GC cell death, but had little effects on hepatocytes proliferation and apoptosis. Further results showed that CircRNA CDR1as increased REGγ expressions in GC cells by sponging miR-7-5p, and high-dose DB (50μM) increased miR-7-5p levels and inhibited REGγ expressions in GC cells instead of hepatocytes. In addition, either downregulated miR-7-5p or overexpressed REGγ reversed the promoting effects of downregulated CircRNA CDR1as on low-dose DB-induced GC cell death. Taken together, we concluded that knock-down of CircRNA CDR1as specifically promoted the cytotoxic effects of low-dose DB on GC cells instead of hepatocytes by regulating miR-7-5p/REGγ axis.

Development and application of a rapid and sensitive LC-MS/MS method for simultaneous quantification of six diterpene lactones in rats after oral administration of Rhizoma Dioscoreae Bulbiferae extract

Diterpene lactones have been considered as the main therapeutic and hepatotoxic constituents of Rhizoma Dioscoreae Bulbiferae in recent years. In this work, a simple, rapid and accurate LC-MS/MS method was established and validated to determine six diterpene lactones in rat plasma simultaneously, including Diosbulbin B (DIOB), Diosbulbin C (DIOC), Diosbulbin D (DIOD), Diosbulbin G (DIOG), Diosbulbin J (DIOJ) and Diosbulbin L (DIOL), after oral administration of Rhizoma Dioscoreae Bulbiferae extract. The six diterpene lactones, with the inclusion of two pairs of isomer (DIOB & D, DIOC & L), and Buspirone (internal standard, IS) were successfully separated using an XDB-C18 column with the gradient elution, consisting of water with 0.1% (v/v) FA and methanol with 0.1% (v/v) FA, under a flow rate of 0.50 mL/min in 5.8 min. Precursor-product ion transitions were optimized to be m/z 362.1 → 317.1, 363.1 → 207.1, 345.0 → 299.2, 364.3 → 347.0, 396.3 → 379.3, 363.2 → 345.1 and 386.3 → 122.2 for DIOB, DIOC, DIOD, DIOG, DIOJ, DIOL and buspirone at positive ion mode with an electrospray ionization source (ESI), respectively. The linearity ranges of this present method were 0.50 to 500 μg/L for DIOB, 20.0 to 20,000 μg/L for DIOC and 2.00 to 2000 μg/L for DIOD, DIOG, DIOJ and DIOL, respectively. And the LLOQs were as low as 0.20 μg/L for DIOB, 20.0 μg/L for DIOC and 2.00 μg/L for DIOB, D, G, J and L. The accuracy of each analyte was within the range of 95.8% to 101.0% and the precision was <11.3%. No matrix effect and carry over was observed, and the recovery of the six analytes ranged from 87.3% to 109% with the RSD <11.4% within the concentrations range. The validated method was further applied to the pharmacokinetics investigation of DIOB, DIOC, DIOD, DIOG, DIOJ and DIOL successfully after oral administration of Rhizoma Dioscoreae Bulbiferae extract at 1.53 g/kg in rats.

Diosbulbin B-Induced Mitochondria-Dependent Apoptosis in L-02 Hepatocytes is Regulated by Reactive Oxygen Species-Mediated Autophagy.

Aim: Diosbulbin B (DB) is a major diterpenoid compound found in Dioscorea bulbifera L, a traditional medicinal herb in China. Clinical reports have confirmed that Dioscorea bulbifera L. can induce significant hepatotoxicity. In this study, we showed that DB can induce mitochondria-dependent apoptosis and investigated the role of autophagy in DB-induced hepatotoxicity in L-02 hepatocytes. Methods: L-02 hepatocytes were treated with different concentrations of DB for 48 h, after which indicators of autophagy and apoptosis were measured. 3-Methyladenine (3-MA) and rapamycin (Rapa) were used as inhibitor and agonist of autophagy, respectively. Furthermore, the reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) was used in combination with DB to evaluate the relationship between ROS and autophagy. Results: L-02 cell viability was significantly decreased after treatment with DB for 48 h. Additionally, DB induced concentration-dependent apoptosis and autophagy and increased the activities of caspase-3, caspase-9, alanine aminotransferase (ALT), and aspartate transaminase (AST), and induced excessive leakage of lactate dehydrogenase (LDH). Inhibition of autophagy by 3-MA increased DB-induced apoptosis, resulting in aggravation of hepatotoxicity. Conversely, treatment with Rapa increased malondialdehyde (MDA) content and reduced superoxide dismutase (SOD) activity. Moreover, we found that DB treatment increased the level of intracellular ROS, decreased the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production, and caused abnormal opening of the mitochondrial permeability transition pore (mPTP), which were finally restored by the ROS scavenger NAC. Conclusions: Accumulation of ROS can induce mitochondria-dependent apoptosis and likely to play a key role in DB-induced hepatocellular injury. Activation of autophagy may inhibit apoptosis, but also reduces antioxidant capacity.