Integrative analysis of metabolomics and proteomics reveals mechanism of berberrubine-induced nephrotoxicity.

Berberrubine (BRB), a main metabolite of berberine, has stronger hypoglycemic and lipid-lowering activity than its parent form. We previously found that BRB could cause obvious nephrotoxicity, but the molecular mechanism involved remains unknown. In this study, we systematically integrated metabolomics and quantitative proteomics to reveal the potential mechanism of nephrotoxicity caused by BRB. Metabolomic analysis revealed that 103 significant- differentially metabolites were changed. Among the mentioned compounds, significantly upregulated metabolites were observed for phosphorylcholine, sn-glycerol-3-phosphoethanolamine, and phosphatidylcholine. The top three enriched KEGG pathways were the mTOR signaling pathway, central carbon metabolism in cancer, and choline metabolism in cancer. ERK1/2 plays key roles in all three metabolic pathways. To further confirm the main signaling pathways involved, a proteomic analysis was conducted to screen for key proteins (such as Mapk1, Mapk14, and Caspase), indicating the potential involvement of cellular growth and apoptosis. Moreover, combined metabolomics and proteomics analyses revealed the participation of ERK1/2 in multiple metabolic pathways. These findings indicated that ERK1/2 regulated the significant- differentially abundant metabolites determined via metabolomics analysis. Notably, through a cellular thermal shift assay (CETSA) and molecular docking, ERK1/2 were revealed to be the direct binding target involved in BRB-induced nephrotoxicity. To summarize, this study sheds light on the understanding of severe nephrotoxicity caused by BRB and provides scientific basis for its safe use and rational development.

Unraveling the enigma: Molecular mechanisms of berberrubine-induced nephrotoxicity reversed by its parent form berberine.

BACKGROUND: Berberine is an isoquinoline alkaloid that is extensively applied in the clinic due to its potential therapeutic effects on dysentery and infectious diarrhoea. Its main metabolite, berberrubine, a promising candidate for ameliorating hyperlipidaemia, has garnered more attention than berberine. However, our study revealed that berberrubine induces severe kidney damage, while berberine was proven to be safe. PURPOSE: Herein, we explored the opposite biological effects of these two compounds on the kidney and elucidated their underlying mechanisms. METHODS: First, integrated metabolomic and proteomic analyses were conducted to identify relevant signalling pathways. Second, a click chemistry method combined with a cellular thermal shiftassay, a drug affinity responsive target stability assay, and microscale thermophoresis were used to identify the direct target proteins. Moreover, a mutation experiment was performed to study the specific binding sites. RESULTS: Animal studies showed that berberrubine, but not berberine, induced severe chronic, subchronic, and acute nephrotoxicity. More importantly, berberine reversed the berberrubine-reduced nephrotoxicity. The results indicated that the cPLA2 signalling pathway was highly involved in the nephrotoxicity induced by berberrubine. We further confirmed that the direct target of berberrubine is the BASP1 protein (an upstream factor of cPLA2 signalling). Moreover, berberine alleviated nephrotoxicity by binding cPLA2 and inhibiting cPLA2 activation. CONCLUSION: This study is the first to revel the opposite biological effects of berberine and its metabolite berberrubine in inducing kidney injury. Berberrubine, but not berberine, shows strong nephrotoxicity. The cPLA2 signalling pathway can be activated by berberrubine through targeting of BASP1, while berberine inhibits this pathway by directly binding with cPLA2. Our study paves the way for studies on the exact molecular targets of herbal ingredients. We also demonstrated that natural small molecules and their active metabolites can have opposite regulatory roles in vivo through the same signalling pathway.

Chemometrics-based Chemical Analysis of Myrrh and Its Vinegar-processed Products by UPLC-MS/MS.

Myrrh is widely used in clinical practice but accompanied by obvious toxicity. According to traditional Chinese medicines theory, processing with vinegar can effectively reduce its toxicity. However, the detoxification processing technology of Myrrh and the corresponding mechanism have been unclear. The objective of this study is to systematically analyze the variation in chemical composition of raw Myrrh and its processed products using UPLC-Q-TOF-MS/MS coupled with chemometrics. A total of 75 compounds including 56 sesquiterpenoids, 2 diterpenoids, 15 triterpenoids and 2 other types were identified. Raw Myrrh and its processed products were divided into two major groups, and 14 chemical markers were selected out by principal component analysis and partial least square discriminant analysis. Additionally, the exact content of 5 representative chemical markers was determined to be significantly reduced after vinegar-processing by UPLC-QQQ-MS/MS. Moreover, multivariate statistical analysis and the quantitative results comprehensively indicated that the optimized processing method was processing at a ratio of 200 : 5 (Myrrh:vinegar). This research provides not only a reliable foundation for the study of Myrrh, but also a scientific reference for clinical use of this herb.

Elucidation of the relationship between evodiamine-induced liver injury and CYP3A4-mediated metabolic activation by UPLC-MS/MS analysis.

Evodiamine (EVD), which has been reported to cause liver damage, is the main constituent of Evodia rutaecarpa (Juss.) Benth and may be bioactivated into reactive metabolites mediated by cytochrome P450. However, the relationships between bioactivation and EVD-induced hepatotoxicity remain unknown. In this study, comprehensive hepatotoxicity evaluation was explored, which demonstrated that EVD caused hepatotoxicity in both time- and dose-dependent manners in mice. By application of UPLC-Q/TOF-MS/MS, two GSH conjugates (GM1 and GM2) derived from reactive metabolites of EVD were identified, in microsomal incubation systems exposed to EVD with glutathione (GSH) as trapping agents. CYP3A4 was proved to be the main metabolic enzyme. Correspondingly, the N-acetyl-L-cysteine conjugate derived from the degradation of GM2 was detected in the urine of mice after exposure to EVD. For the first time, the iminoquinone intermediate was found in EVD-pretreated rat bile by the high-resolution MS platform. Pretreatment with ketoconazole protected the animals from hepatotoxicity, decreased the protein expression of cleaved caspase-1 and -3, but increased the area under the serum-concentration-time curve of EVD in blood determined by UPLC-QQQ-MS/MS. Depletion of GSH by buthionine sulfoximine exacerbated EVD-induced hepatotoxicity. These results implicated that the CYP3A4-mediated metabolic activation was responsible for the observed hepatotoxicity induced by EVD.

PP2A interacts with KATANIN to promote microtubule organization and conical cell morphogenesis.

The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.

Quantitation of Diclofenac, Tolbutamide, and Warfarin as Typical CYP2C9 Substrates in Rat Plasma by UPLC-MS/MS and Its Application to Evaluate Linderane-Mediated Herb-Drug Interactions.

Linderane (LDR), the main active and distinctive component of L. aggregate, is a mechanism-based inactivator of CYP2C9 in vitro, indicating the occurrence of herb-drug interactions. However, little is known about the changes of the pharmacokinetic properties of the common clinical drugs as CYP2C9 substrates after coadministration with LDR. In this study, a selective and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method for the determination of diclofenac, tolbutamide, and warfarin as CYP2C9 substrates in rat plasma has been developed. Chlorzoxazone was employed as an internal standard (IS), and protein precipitation was used for sample preparation. Chromatographic separation was achieved on a UPLC BEH-C18 (2.1 × 50 mm, 1.7 µm) with 0.1% (v:v) formic acid in water (A) and acetonitrile (B) as the mobile phase with gradient elution. The total run time was only 3.8 min. MS analysis was performed under multiple reaction monitoring (MRM) with electron spray ionization (ESI) operated in the negative mode. The bioanalytical method was validated, and the selectivity, carryover effects, linearity, precision, accuracy, matrix effect, extraction recovery, and stability were acceptable. The validated method was then successfully applied for evaluating the potential pharmacokinetic interactions when LDR was used along with diclofenac, tolbutamide, and warfarin, respectively. Results showed that the C max of diclofenac in the treated group was 1287.82 ± 454.16 µg/L, which was about 5-fold of that in the control group (P < 0.01). The C max of tolbutamide in the treated group was 60.70 ± 10.70 mg/L, which was significantly decreased by about 25% when compared with the control group (P < 0.01). The V d of warfarin in the treated group was obviously increased, which was about 1.4-fold of that in the control group (P < 0.01).

Nephrotoxicity induced by natural compounds from herbal medicines - a challenge for clinical application.

Herbal medicines (HMs) have long been considered safe and effective without serious toxic and side effects. With the continuous use of HMs, more and more attention has been paid to adverse reactions and toxic events, especially the nephrotoxicity caused by natural compounds in HMs. The composition of HMs is complex and various, especially the mechanism of toxic components has been a difficult and hot topic. This review comprehensively summarizes the kidney toxicity characterization and mechanism of nephrotoxic natural compounds (organic acids, alkaloids, glycosides, terpenoids, phenylpropanoids, flavonoids, anthraquinones, cytotoxic proteins, and minerals) from different sources. Recommendations for the prevention and treatment of HMs-induced kidney injury were provided. In vitro and in vivo models for evaluating nephrotoxicity and the latest biomarkers are also included in this investigation. More broadly, this review may provide theoretical basis for safety evaluation and further comprehensive development and utilization of HMs in the future.

Highly Potent Inhibition of Tyrosinase by Mulberrosides and the Inhibitory Mechanism in Vitro.

Many stilbene glycosides can alleviate skin hyperpigmentation due to their inhibitory effect on tyrosinase. Mulberrosides in Morus alba L. are stilbene glycosides. In the present study, the inhibition of tyrosinase by five mulberrosides (S1-5), isolated from Morus alba L. was investigated and compared, and the inhibitory mechanism was explored. These five mulberrosides exhibited obvious inhibitory effects on tyrosinase only in a concentration-dependent manner, without time-dependence, indicating that they are reversible inhibitors of tyrosinase. S2, S1 and S5 inhibited tyrosinase activity with IC50 values of 28.93, 75.94 and 151.72 µM, respectively, and were more active than kojic acid (IC50 =169.13 µM). Kinetic studies revealed that S1, S2 and S4 were competitive inhibitors, while S3 and S5 were mixed inhibitors. Analysis of the fluorescent spectra showed that mulberrosides S1, S2 and S4 quenched the intrinsic fluorescence intensity of tyrosinase. A molecular docking study indicated that the interaction of tyrosinase with mulberrosides was reflected by compound scores as follows: S2>S5>S1>S3/S4>kojic acid, and hydroxy groups in the side chain of mulberrosides may play a crucial role in the binding of the enzyme. Our results suggest that mulberrosides in Morus alba L. could be further developed as whitening agents for enhanced performance against hyperpigmentation.

Drug-drug interactions induced by Linderane based on mechanism-based inactivation of CYP2C9 and the molecular mechanisms.

Linderane (LDR) is a main furan-containing sesquiterpenoid of the common herbal medicine Lindera aggregata (Sims) Kosterm. Our early study indicated that LDR led to mechanism-based inactivation (MBI) of CYP2C9 in vitro, implying possible drug-drug interactions (DDIs) in clinic. In the present study, influence of LDR on the pharmacokinetics of the corresponding hydroxylated metabolites of CYP2C9 substrates in rats was investigated. Pharmacokinetic studies revealed that pretreatment with LDR at 20 mg/kg for 15 days inhibited the metabolism of both tolbutamide and warfarin catalyzed by CYP2C9. As for 4-hydroxytolbutamide, the Cmax was decreased, the t1/2z was prolonged, and the Vz/F was increased, all with significant difference. As for 7-hydroxywarfarin, the AUC0-t/AUC0-∞ and CLz/F were significantly decreased and increased, respectively. Furthermore, the underlying molecular mechanisms based on MBI of CYP2C9 by LDR were revealed. Two reactive metabolites of LDR, furanoepoxide and γ-ketoenal intermediates were identified in CYP2C9 recombinant enzyme incubation systems. Correspondingly, covalent modifications of lysine and cysteine residues of CYP2C9 protein were discovered in the CYP2C9 incubation system treated with LDR. The formation of protein adducts exhibited obvious time- and dose-dependence, which is consistent with the trend of enzyme inhibition caused by LDR in vitro. In addition to the apoprotein of CYP2C9, the heme content was significantly reduced after co-incubation with LDR. These data revealed that modification of both apoprotein and heme of CYP2C9 by reactive metabolites of LDR led to MBI of CYP2C9, therefore resulting in the inhibition of biotransformation of CYP2C9 substrates to their corresponding metabolites in vivo.

Metabolic Activation and Covalent Protein Binding of Berberrubine: Insight into the Underlying Mechanism Related to Its Hepatotoxicity.

INTRODUCTION: Berberrubine (BRB), an isoquinoline alkaloid, is a major constituent of medicinal plants Coptis chinensis Franch or Phellodendron chinense Schneid. BRB exhibits various pharmacological activities, whereas exposure to BRB may cause toxicity in experimental animals. METHODS: In this study, we thoroughly investigated the liver injury induced by BRB in mice and rats. To explore the underlying mechanism, a study of the metabolic activation of BRB was conducted. Furthermore, covalent modifications of cysteine residues of proteins were observed in liver homogenate samples of animals after exposure to BRB, by application of an exhaustive proteolytic digestion method. RESULTS: It was demonstrated that BRB-induced hepatotoxicities in a time- and dose-dependent manner, based on the biochemical parameters ALT and AST. H&E stained histopathological examination showed the occurrence of obvious edema in liver of mice after intraperitoneal (i.p.) administration of BRB at a single dose of 100 mg/kg. Slight hepatotoxicity was also observed in rats given the same doses of BRB after six weeks of gavage. As a result, four GSH adducts derived from reactive metabolites of BRB were detected in microsomal incubations with BRB fortified with GSH as a trapping agent. Moreover, four cys-based adducts derived from reaction of electrophilic metabolites of BBR with proteins were found in livers. CONCLUSION: These results suggested that the formation of protein adducts originating from metabolic activation of BRB could be a crucial factor of the mechanism of BRB-induced toxicities.

Mechanism-based inactivation of cytochrome P450 enzymes by natural products based on metabolic activation.

Cytochrome P450 enzymes (P450 enzymes) are the most common and important phase I metabolic enzymes and are responsible for the majority of the metabolism of clinical drugs and other xenobiotics. Drug-drug interactions (DDIs) can occur when the activities of P450 enzymes are inhibited. In particular, irreversible inhibition of P450 enzymes may lead to severe adverse interactions, compared to reversible inhibition. Many natural products have been shown to be irreversible inhibitors of P450 enzymes. The risks for intake of naturally occurring irreversible P450 enzyme inhibitors have been rising due to the rapid growth of the global consumption of natural products. Irreversible inhibition is usually called mechanism-based inactivation, which is time-, concentration- and NADPH- dependent. Generally, the formation of electrophilic intermediates is fundamental for the inactivation of P450 enzymes. This review comprehensively classifies natural P450 enzyme inactivators, including terpenoids, phenylpropanoids, flavonoids, alkaloids, and quinones obtained from herbs or foods. Moreover, the structure - activity correlations according to the IC50 (or Ki) values reported in the literature as well as the underlying mechanisms based on metabolic activation are highlighted in depth.

Inhibition of CYP2C9 by natural products: insight into the potential risk of herb-drug interactions.

Due to the rapidly increasing global interest in the use of herbs, phytomedicines and other natural products as medical or complementary remedies, concerns about the clinical medication safety have drawn much attention worldwide. Particularly, many natural ingredients exhibit inhibitory effects on cytochrome P450 (CYP) enzymes, which are the most important Phase I metabolism enzymes in liver. CYP2C9 is one of the most abundant CYP enzymes and responsible for the metabolism of over 15% clinical drugs, including oral sulfonylurea hypoglycemics, nonsteroidal anti-inflammatory agents, selective cyclooxygenase-2 inhibitors, antiepileptics, angiotensin II receptor inhibitors and anticoagulants. Diclofenac (4'-hydroxylase) and tolbutamide (methylhydroxylation) are widely used as probe substrates for CYP2C9. To date, numerous natural products have been reported to have the capabilities of inhibiting the catalytic activity of CYP2C9 and further influencing the pharmacokinetic and pharmacodynamic behaviors of drugs that are mainly metabolized by CYP2C9, leading to potential herb-drug interactions. Moreover, some fatal adverse interactions may occur for drugs with a narrow therapeutic window when they are coadministered with a CYP2C9 inhibitor, especially irreversible inactivators. For the purpose of better understanding the interactions of natural products with CYP2C9, we comprehensively reviewed the characteristics of CYP2C9, the natural ingredients that inhibit CYP2C9, the related research approaches and strategies, the types of inhibition and the underlying mechanisms.

[Progress on application of terahertz time-domain spectroscopy in identification of traditional Chinese medicine].

With the increasing demand for traditional Chinese medicines(TCMs), the resources of TCMs are gradually rare, and the phenomenon that TCMs fake is a common occurrence. Consequently, we need to urgently improve identification technique of TCMs. Terahertz time-domain spectroscopy(THz-TDS) is a newly emerging spectroscopy technology that has been widely used in image, security inspection, biological detection, medicine, material technology, aerospace, oil exploration and other fields. Which is currently used as a simple and quick method to identify the origins, fake products, processed products and pesticide residues of TCMs, hence, it plays a significant role in supplementing and improving the quality control levels of TCMs. In this paper, the composition principle of THz-TDS and its advantages in authentification of TCMs have been summarized. Additionally, the current situation and application prospects of THz-TDS in the field of TCM identification have also been reviewed.

Arabidopsis IPGA1 is a microtubule-associated protein essential for cell expansion during petal morphogenesis.

Unlike animal cells, plant cells do not possess centrosomes that serve as microtubule organizing centers; how microtubule arrays are organized throughout plant morphogenesis remains poorly understood. We report here that Arabidopsis INCREASED PETAL GROWTH ANISOTROPY 1 (IPGA1), a previously uncharacterized microtubule-associated protein, regulates petal growth and shape by affecting cortical microtubule organization. Through a genetic screen, we showed that IPGA1 loss-of-function mutants displayed a phenotype of longer and narrower petals, as well as increased anisotropic cell expansion of the petal epidermis in the late phases of flower development. Map-based cloning studies revealed that IPGA1 encodes a previously uncharacterized protein that colocalizes with and directly binds to microtubules. IPGA1 plays a negative role in the organization of cortical microtubules into parallel arrays oriented perpendicular to the axis of cell elongation, with the ipga1-1 mutant displaying increased microtubule ordering in petal abaxial epidermal cells. The IPGA1 family is conserved among land plants and its homologs may have evolved to regulate microtubule organization. Taken together, our findings identify IPGA1 as a novel microtubule-associated protein and provide significant insights into IPGA1-mediated microtubule organization and petal growth anisotropy.