Firm evidence for the detoxification of senecionine-induced hepatotoxicity via N-glucuronidation in UGT1A4-humanized transgenic mice.

Uridine diphosphate glucuronosyltransferase (UGT)1A4 is responsible for N-glucuronidation of tertiary amines but is a pseudogene in commonly used rodent models in toxicity and safety assessment. As a continuation of our investigation into the toxicity and safety assessment of pyrrolizidine alkaloid (PA)-containing herbs, we generated a UGT1A4-humanized (hUGT1A4) transgenic mouse model to systematically study the toxicity, metabolism network, and toxicokinetic characteristics of senecionine (a representative toxic PA) and compared with that in the wide-type controls in parallel. As results, senecionine-induced toxicity was significantly decreased as approved by mortality, pathology, and biochemistry assays in hUGT1A4 mice and cultured primary hepatocytes. More importantly N-glucuronidation adduct was exclusively identified in all the hUGT1A4 mice, liver microsomes, and cultured primary hepatocytes, yet absent in the wide-type controls. The variation in toxicokinetic characters was also observed between hUGT1A4 mice and the wide-type controls with a notably inhibition of the toxification metabolites, i.e., pyrrole-protein adducts, in hUGT1A4 mice. Conclusively, UGT1A4 plays an important role in detoxification of senecionine and the hUGT1A4 mouse model is promising for the pre-clinical evaluation of the efficacy and toxicity of tertiary amine agents in drug development and safety assessment.

Bear bile powder attenuates senecionine-induced hepatic sinusoidal obstruction syndrome in mice.

Hepatic sinusoidal obstruction syndrome (HSOS) via exposure to pyrrolizidine alkaloids (PAs) is with high mortality and there is no effective treatment in clinics. Bear bile powder (BBP) is a famous traditional animal drug for curing a variety of hepatobiliary diseases such as cholestasis, inflammation, and fibrosis. Here, we aim to evaluate the protective effect of BBP against HSOS induced by senecionine, a highly hepatotoxic PA compound. Our results showed that BBP treatment protected mice from senecionine-induced HSOS dose-dependently, which was evident by improved liver histology including reduced infiltration of inflammatory cells and collagen positive cells, alleviated intrahepatic hemorrhage and hepatic sinusoidal endothelial cells, as well as decreased conventional serum liver function indicators. In addition, BBP treatment lowered matrix metalloproteinase 9 and pyrrole-protein adducts, two well-known markers positively associated with the severity of PA-induced HSOS. Further investigation showed that BBP treatment prevents the development of liver fibrosis by decreasing transforming growth factor beta and downstream fibrotic molecules. BBP treatment also alleviated senecionine-induced liver inflammation and lowered the pro-inflammatory cytokines, in which tauroursodeoxycholic acid played an important role. What's more, BBP treatment also decreased the accumulation of hydrophobic bile acids, such as cholic acid, taurocholic acid, glycocholic acid, as well. We concluded that BBP attenuates senecionine-induced HSOS in mice by repairing the bile acids homeostasis, preventing liver fibrosis, and alleviating liver inflammation. Our present study helps to pave the way to therapeutic approaches of the treatment of PA-induced liver injury in clinics.

[Quantification of hepatotoxic pyrrolizidine alkaloid adonifoline in traditional Chinese medicine preparations containing Senecionis Scandentis Herba].

Pyrrolizidine alkaloids(PAs) are a kind of natural toxins, which can cause severe hepatotoxicity, pulmonary toxicity, genotoxicity, neurotoxicity, embryotoxicity and even death. Therefore, international organizations and countries such as World Health Organization have paid great attention to herbal medicines and preparations containing PAs. PAs are widely distributed in Chinese herb medicines and contained in hundreds of traditional Chinese medicine preparations. The content of adonifoline, the main PAs in Senecionis Scandentis Herba, shall be less than 0.004% in herbal medicines as described in Chinese pharmacopeia. However, there is no guidance in preparations which contain Senecionis Scandentis Herba. In this study, 14 preparations were analyzed by an UPLC-MS method. Among them, 8 preparations were found to contain adonifoline much higher than the content limits of such countries as Germany, Netherlands and New Zealand. And the highest contents of adonifoline were found in Qianbai Biyan Tablets and Qianbai Biyan Capsules, which are officially recorded in Chinese Pharmacopeia. The contents of adonifoline varied in different batches of the same preparations. The highest content was 156.10 µg·g~(-1) Qianbai Biyan Tablets, whose daily intake of adonifoline was up to 1 030.26 µg according to the recommended dosage of the preparation. Our results showed the potential risk of these preparations, and the content limit of adonifoline shall be inspected Chinese medicine preparations containing Senecionis Scandentis Herba.

Gynura Rhizoma containing pyrrolizidine alkaloids induces the hepatic sinusoidal obstruction syndrome in mice via upregulating fibrosis-related factors.

Recently, hepatic sinusoidal obstruction syndrome (HSOS) caused by herbal preparations containing pyrrolizidine alkaloids (PAs), such as Gynura Rhizoma (Tusanqi), has gained global attention. However, the lack of a reliable and reproducible animal model has greatly hampered mechanistic studies. Therefore, we aimed to establish a reproducible HSOS mouse model and investigate the hepatotoxic mechanism. The model was established by intragastrical administration of Gynura Rhizoma extract, i.e., 1.0 g extract/kg per day (equal to 16.7 g crude drug/kg per day based on extraction rate and 49.1 mg PA/kg per day based on the total PA content in the extract determined) for 40 successive days. Then, the mice were sacrificed, and their blood samples and livers were collected for analyses. Using hematoxylin-eosin (HE) and Masson staining, scanning electron microscopy imaging, clinical biomarkers, and other assays, we showed that the HSOS was successfully induced in our mouse model. Furthermore, we detected the key factors involved in liver fibrosis in the mice, revealing significantly increased hydroxyproline concentration; elevated expression of α-smooth muscle actin (α-SMA) and fibrosis-related genes such as Collagen-1, Collagen-3, Mmp2, Mmp13, Timp1, Timp3, and Activin, upregulated Smad3 phosphorylation, and increased serum TGF-ß levels. Moreover, pro-inflammatory cytokines, including Tnf-α, Il-1ß, and Il-6, were also increased in the model. All these results demonstrate the key roles of the TGF-ß-Smad3 and inflammatory signaling pathways in this Gynura Rhizoma-induced HSOS mouse model, suggesting that blockade of fibrosis and/or inflammation should be an effective treatment for HSOS.

Yinchenhao Decoction Alleviates Liver Fibrosis by Regulating Bile Acid Metabolism and TGF-ß/Smad/ERK Signalling Pathway.

Yinchenhao decoction (YCHD), comprising Yinchenhao (Artemisiae Scopariae Herba), Zhizi (Gardeniae Fructus) and Dahuang (Radix Rhei et Rhizoma), is widely used for treating various diseases. We aimed to investigate the bile acid metabolic mechanism of YCHD in dimethylnitrosamine (DMN)-induced liver fibrosis model. Rats received DMN (10 mg/kg, intraperitoneally) for four successive weeks for liver fibrosis induction and were treated with YCHD for the last 2 weeks. Histopathological analysis showed that YCHD prevented DMN-induced histopathological changes in liver tissues. Serum liver function in YCHD group improved. Ultraperformance liquid chromatography-mass spectrometry analysis showed that YCHD significantly restored both free and conjugated bile acid levels increased by DMN, to normal levels. RT-qPCR results showed that YCHD treatment upregulated the expression of genes related to bile acid synthesis, reabsorption, and excretion. Western blotting analysis showed that YCHD downregulated α-SMA, TGF-ß1, p-Smad3, and p-ERK1/2 expression in chenodeoxycholic acid (CDCA)-activated hepatic stellate cells (HSCs). The viability of CDCA-activated HSCs significantly increased after treatment with YCHD and PD98059 (an ERK inhibitor) compared to YCHD treatment alone. Our findings suggest that YCHD alleviated DMN-induced liver fibrosis by regulating enzymes responsible for bile acid metabolism. Additionally, it inhibits CDCA-induced HSC proliferation and activation via TGF-ß1/Smad/ERK signalling pathway.

[Systematical analysis of multiple components in drainage bear bile powder from different sources].

Bear Bile Powder contains bile acids, protein, amino acids, bilirubin and microelements and other compounds. Among them, the bile acids are the most active components. Currently, there are many studies on bile acids, but few reports on other components. Therefore, the purpose of this study is to carry out a systematical analysis of multiple components in drainage Bear Bile Powder from different sources. Bilirubin and protein were quantified by microplate spectrophotometer. The contents of bile acids and amino acids were determined by liquid chromatography coupled with mass spectrometry (LC-MS). The contents of microelements were determined by inductively coupled plasma mass spectrometry (ICP-MS) The result indicated that among 20 batches of bear bile powder from different sources there is high similarity (0.922-0.977). Tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) were the two most abundant components. The total contents of them were 41%-59% and met the current standard for quality control of bear bile powder. However, significant differences were found in their contents among samples from different sources. Besides, bilirubin, protein, amino acids and microelements also contributed to the differentiation of samples from different sources. The main components of bear bile powder from the different sources were with satisfactory similarity. But bile acids, bilirubin, protein, amino acids and microelements all contributed to the different among samples. Our present study provided a systematical approach for the better quality control and evaluation of bear bile powder.

Negative feedback regulation between microRNA let-7g and LOX-1 mediated hypoxia-induced PASMCs proliferation.

BACKGROUND: Pulmonary hypertension (PH) is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Our lately study demonstrated that let-7g inhibited hypoxia-induced proliferation of PASMCs via repressing c-myc-Bmi-1-p16 signaling pathway. However, the upstream of let-7g has not yet been fully defined. Previous studies have shown that LOX-1, a target of let-7g, could also regulate the expression of let-7g in human aortic endothelial cells. In this present study, we aimed to investigate whether there is a negative feedback regulation between microRNA let-7g and LOX-1 in hypoxia-induced proliferation of PASMCs. METHODS: SD Rats were exposed to hypoxia (10% O2, 3 weeks) to induce PH. HE staining was used to evaluate pulmonary artery remodeling. in situ hybridization and immunohistochemistry were performed to assess the expression and distribution of let-7g and LOX-1, respectively. MTS, EDU and flow cytometry were performed to evaluate PASMCs proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were conducted to assess the expression of let-7g, LOX-1, calpain-1,-2,-4, and OCT-1. RESULTS: The expression of let-7g was significantly down-regulated in pulmonary arteries of hypoxia-induced PH rats accompanied by pulmonary vascular remodeling, whereas let-7g mimic inhibited hypoxia-induced proliferation of PASMCs and up-regulation of LOX-1 expression. LOX-1 blocking reversed hypoxia-induced down-regulation of let-7g expression. Calpains, protein kinase C and OCT-1 were involved in negative feedback regulation between let-7g and LOX-1. CONCLUSION: Negative feedback regulation between let-7g and LOX-1 mediated hypoxia-induced proliferation of in PASMCs.

MicroRNA let-7g inhibited hypoxia-induced proliferation of PASMCs via G0/G1 cell cycle arrest by targeting c-myc.

AIMS: Pulmonary hypertension (PH) is a proliferative disorder characterized by enhanced proliferation and suppressed apoptosis of intrapulmonary vascular smooth muscle cells. Recently, network-based bioinformatics have identified let-7 family, a tumor suppressive microRNA, regulate multiple interacting targets relevant to PH. However, the role of let-7 in vascular homeostasis in PH remains unknown. Thus, we wanted to investigate the role of let-7 in hypoxia-induced PASMCs proliferation and the underlying mechanism in hypoxic pulmonary hypertension (HPH). MAIN METHODS: The male Sprague-Dawley (SD) rats were exposed to hypoxia (10% O2) for 21days to induce HPH. The expression of let-7 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. Primary rat PASMCs were exposed to hypoxia (3% O2). MTS and EDU were performed to evaluate PASMCs proliferation. The mRNA and protein expression of c-myc, Bmi-1 and p16 were determined by qRT-PCR and Western blotting, respectively. The functions of let-7g on PASMCs proliferation, c-myc, Bmi-1 and p16 expression were assessed by let-7g mimic and inhibitor transfection. KEY FINDINGS: Among let-7 family members, only let-7b and let-7g were significantly down-regulated in remodeled pulmonary artery in HPH rats. Furthermore, only let-7g level was decreased in hypoxic PASMCs. Either hypoxia or let-7g inhibitor stimulated proliferation of PASMCs, let-7g mimic inhibited hypoxia-induced PASMCs proliferation. C-myc was the target of let-7g in PASMCs. Transfect of let-7g mimic inhibited hypoxia-induced c-myc, Bmi-1 up-regulation and p16 down-regulation, which ultimately controls cell cycle progression. SIGNIFICANCE: Loss of inhibition on c-myc-Bmi-1-p16 signaling pathway by let-7g may lead to PASMCs proliferation and vascular remodeling in HPH.

Evaluation of hemostatic and anti-inflammatory activities of extracts from different Lagochilus species in experimental animals: comparison of different extractives and sources.

Different members of Lagochilus genus have been used in folkloric medicine to treat hemorrhages and inflammation. However, only a few species of them have received scientific attention supporting their efficacy. Here, the hemostatic and antiinflammatory activities of five Lagochilus species were determined and compared by using in vivo assays. The results showed that the extracts of Lagochilus lanatonodus and Lagochilus diacanthophyllus showed better hemostatic activities among five species. The high doses of L. lanatonodus extracts were able to shorten the values of thrombin time, activated partial thromboplastin time and prothrombin time in a rat model. Moreover, the extracts of L. lanatonodus and L. diacanthophyllus showed strong inhibitory effects on the acute phase of inflammation in both xylene-induced ear edema mouse model and carrageenan-induced paw edema rat model. In parallel, the treatment of these extracts modulated the expressions of those inflammatory parameters, that is, nitric oxide, prostaglandin E2 , inducible nitric oxide synthase, malondialdehyde and superoxide dismutase. L. lanatonodus and L. diacanthophyllus showed better hemostatic and antiinflammatory activities in several test models: these results therefore supported the folkloric utilization. L. lanatonodus was found to be the most active Lagochilus species.

Combination of UHPLC/Q-TOF-MS, NMR spectroscopy, and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans.

The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed "MS-NMR-MS" method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

A strategy for screening and identifying mycotoxins in herbal medicine using ultra-performance liquid chromatography with tandem quadrupole time-of-flight mass spectrometry.

The objective of this study was to develop an effective strategy for screening and identifying mycotoxins in herbal medicine (HM). Here, Imperatae Rhizoma, a commonly used Chinese herb, was selected as a model HM. A crude drug contaminated with fungi was analyzed by comparing with uncontaminated ones. Ultra-performance LC coupled to tandem quadrupole TOF-MS (UPLC-Q-TOF-MS) with collision energy function was applied to analyze different samples from Imperatae Rhizoma. Then, MarkerLynx(TM) software was employed to screen the excess components in analytes, compared with control samples, and those selected markers were likely to be the metabolites of fungi. Furthermore, each of the accurate masses of the markers obtained from MarkerLynx(TM) was then searched in a mycotoxins/fungal metabolites database established in advance. The molecular formulas with relative mass error between the measured and theoretical mass within 5 ppm were chosen and then applied to MassFragment(TM) analysis for further confirmation of their structures. With the use of this approach, five mycotoxins that have never been reported in HM were identified in contaminated Imperatae Rhizoma. The results demonstrate the potential of UPLC-Q-TOF-MS coupled with the MarkerLynx(TM) software and MassFragment(TM) tool as an efficient and convenient method to screen and identify mycotoxins in herbal materials and aid in the quality control of HM.

Radix Paeoniae Rubra and Radix Paeoniae Alba Attenuate CCl4-induced acute liver injury: an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) based metabolomic approach for the pharmacodynamic study of Traditional Chinese Medicines (TCMs).

Metabolomics has been frequently used in pharmacodynamic studies, especially those on traditional Chinese medicine (TCM). Radix Paeoniae Alba and Radix Paeoniae Rubra are popularly used in TCM, and both have hepatoprotective effects. In this study, a CCl(4)-induced acute liver injury rat model was established and confirmed by the observed serum aminotransferase activities. The metabolomics approach was applied to study the influence of Radix Paeoniae Alba and Radix Paeoniae Rubra on the metabolic changes in rats with acute liver injury. The partial least-squares-discriminant analysis (PLS-DA) of rat serum and their ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) fingerprints allowed discrimination of controlled, acute liver injury-model rats after administration of the two types of TCMs. The time-dependent PLS-DA plots showed that the changes in the metabolic patterns of the rats, which were administered with the TCMs, had stabilized within 2 h after they received the intraperitoneal CCl(4) injection. The results indicated the protective effect of TCMs against liver injury. Several potential biomarkers were detected and identified, which included creatine, deoxycholic acid, choline, 5-methylenetetrahydrofolate, folic acid, and glycocholic acid. The physiological significance of these metabolic changes was discussed.

Two new pyrrolizidine alkaloids from Senecio nemorensis.

Two new compounds, including petasinoside A and chloridate-[N-chloromethyl-hectorine], were isolated from Senecio nemorensis. The structures of these compounds were elucidated by spectroscopic methods including 1D and 2D NMR experiments.

Glucuronidation, a new metabolic pathway for pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) possess significant hepatotoxicity to humans and animals after metabolic activation by liver P450 enzymes. Metabolism pathways of PAs have been studied for several decades, including metabolic activation, hydroxylation, N-oxidation, and hydrolysis. However, the glucuronidation of intact PAs has not been investigated, although glucuronidation plays an important role in the elimination and detoxication of xenobiotics. In this study, PAs glucuronidation was investigated, and three important points were found. First, we demonstrated that senecionine (SEN)-a representative hepatotoxic PA-could be conjugated by glucuronic acid via an N-glucuronidation reaction catalyzed by uridine diphosphate glucuronosyl transferase in human liver microsomes. Second, glucuronidation of SEN was catalyzed not only by human but also other animal species and showed significant species differences. Rabbits, cattle, sheep, pigs, and humans showed the significantly higher glucuronidation activity than mice, rats, dogs, and guinea pigs on SEN. Kinetics of SEN glucuronidation in humans, pigs, and rabbits followed the one-site binding model of the Michaelis-Menten equation, while cattle and sheep followed the two-sites binding model of the Michaelis-Menten equation. Third, besides SEN, other hepatotoxic PAs including monocrotaline, adonifoline, and isoline also underwent N-glucuronidation in humans and several animal species such as rabbits, cattle, sheep, and pigs.

Identification of the UDP-glucuronosyltransferase isozyme involved in senecionine glucuronidation in human liver microsomes.

Senecionine (SEN) is a representative of the hepatotoxic pyrrolizidine alkaloids. Although phase I metabolism for cytochrome P450-mediated metabolic activation of SEN was investigated extensively, phase II metabolism for glucuronidation of this compound has not been investigated until now. In our present study, one unique glucuronidation product of SEN in human liver microsomes (HLMs) was identified as SEN N-glucuronide using an authentically synthesized product for which the structure was identified via (1)H and (13)C NMR analysis. Subsequently, kinetics indicated that SEN N-glucuronidation followed the typical Michaelis-Menten model and only one major isozyme participated in it. Finally, this isozyme was demonstrated to be UDP-glucuronosyltransferase (UGT) 1A4, with the direct evidence that recombinant UGT1A4 exhibited predominant and exclusive activity on SEN N-glucuronidation. This result was confirmed by other experiments including chemical inhibition by selective inhibitors and a correlation study between activities of SEN N-glucuronidation and various UGT isozymes. The exclusive role of UGT1A4 on SEN N-glucuronidation was strengthened additionally by its inhibitory kinetic study in which the selective inhibitor of UGT1A4 showed a similar inhibition pattern and K(i) values in both HLM and recombinant UGT1A4 systems. Because UGT2B10 activity failed to correlate with SEN N-glucuronidation in HLMs from 10 individuals, it was impossible for UGT2B10 to play an important role in this metabolism.

Determination of total retronecine esters-type hepatotoxic pyrrolizidine alkaloids in plant materials by pre-column derivatization high-performance liquid chromatography.

A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).

Quantitative analysis of total retronecine esters-type pyrrolizidine alkaloids in plant by high performance liquid chromatography.

Pyrrolizidine alkaloids (PAs) are alkaloids which typically contain a necine (7-hydroxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizidine) base unit, and they can be found in one third of the higher plants around the world. They are hepatotoxic, mutagenic and carcinogenic and pose a threat to human health and safety. A specific, quick and sensitive method is therefore needed to detect and quantify the PAs sometimes in trace amount in herbs, tea or food products. Based on high performance liquid chromatography with prior derivatization of the alkaloids using o-chloranil and Ehrlich's reagent, we report an improved method for quantitative analysis of the total amount of retronecine esters-type pyrrolizidine alkaloids (RET-PAs) in a plant extract. The total quantitation of RET-PAs is achieved because of a common colored retronecine marker, a 7-ethoxy-1-ethoxylmethyl retronecine derivative, is produced with all the different RET-PAs during the derivatization reaction. The chemical identity of the common retronecine marker was characterized on-line by positive mode electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The limit of detection using the improved method is 0.26 nmol mL(-1) and the limit of quantitation is 0.79 nmol mL(-1). The advantages of this method are much enhanced sensitivity in detection and quantitation, and, no restriction on the choice of RET-PA as a calibration standard. Application of the developed method to the quantitation of total RET esters-type PAs in Senecio scandens from different regions of China is also reported.