Ultrasensitive upconverting nanoprobes for in situ imaging of drug-induced liver injury using miR-122 as the biomarker.

Drug-induced liver injury (DILI) is a frequent adverse drug reaction. The current clinical diagnostic methods are inadequate for accurate and early detection of DILI due to the lack of effective diagnostic biomarkers. Hepatocyte-specific miR-122 is released from injured hepatocytes promptly and its efflux is significantly correlated with the progression of DILI. Therefore, achieving precise in situ detection of miR-122 with high sensitivity is vital for early visualization of DILI. Herein, a new nanoprobe, consisting of miR-122 aptamer, upconversion nanoparticles (UCNPs) and Prussian blue nanoparticles (PBNPs) was introduced for the early and sensitive detection of DILI in situ. As the nanoprobes reached in the liver, miR-122 aptamer-based entropy-driven strand displacement (ESDR) signal amplification reaction was triggered and luminescence resonance energy transfer (LRET) between UCNPs and PBNPs was responded to achieve the high-fidelity detection of DILI. A negative correlation was observed between the intensity of upconversion luminescence (UCL) and the concentration of miR-122. UCL imaging conducted both in vivo and ex vivo indicated that a reduction in miR-122 concentration led to an increase in UCL intensity, revealing a precise state of DILI. The detection technique demonstrated a positive correlation between signal intensity and severity, offering a more straightforward and intuitive method of visualizing DILI.

Natural Product Baohuoside I Impairs the Stability and Membrane Location of MRP2 Reciprocally Regulated by SUMOylation and Ubiquitination in Hepatocytes.

Epimedii Folium (EF) is a botanical dietary supplement to benefit immunity. Baohuoside I (BI), a prenylated flavonoid derived from EF, has exhibited the cholestatic risk before. Here, the mechanism of BI on the stability and membrane localization of liver MRP2, a bile acid exporter in the canalicular membrane of hepatocytes, was investigated. The fluorescent substrate of MRP2, CMFDA was accumulated in sandwich-cultured primary mouse hepatocytes (SCH) under BI stimulation, followed by reduced membrane MRP2 expression. BI triggered MRP2 endocytosis associated with oxidative stress via inhibition of the NRF2 signaling pathway. Meanwhile, BI promoted the degradation of MRP2 by reducing its SUMOylation and enhancing its ubiquitination level. Co-IP and fluorescence colocalization experiments all proved that MRP2 was a substrate protein for SUMOylation for SUMO proteins. CHX assays showed that SUMO1 prolonged the half-life of MRP2 and further increased its membrane expression, which could be reversed by UBC9 knockdown. Correspondingly, MRP2 accumulated in the cytoplasm by GP78 knockdown or under MG132 treatment. Additionally, the SUMOylation sites of MRP2 were predicted by the algorithm, and a conversion of lysines to arginines at positions 940 and 953 of human MRP2 caused its decreased stability and membrane location. K940 was further identified as the essential ubiquitination site for MRP2 by an in vitro ubiquitination assay. Moreover, the decreased ubiquitination of MRP2 enhanced the SUMOylation MRP2 and vice versa, and the crosstalk of these two modifiers could be disrupted by BI. Collectively, our findings indicated the process of MRP2 turnover from the membrane to cytoplasm at the post-translational level and further elucidated the novel toxicological mechanism of BI.

Comprehensive analysis of JAZ family members in Ginkgo biloba reveals the regulatory role of the GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis.

Ginkgo biloba L., an ancient relict plant known as a 'living fossil', has a high medicinal and nutritional value in its kernels and leaves. Ginkgolides are unique diterpene lactone compounds in G. biloba, with favorable therapeutic effects on cardiovascular and cerebrovascular diseases. Thus, it is essential to study the biosynthesis and regulatory mechanism of ginkgolide, which will contribute to quality improvement and medication requirements. In this study, the regulatory roles of the JAZ gene family and GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis were explored based on genome and methyl jasmonate-induced transcriptome. Firstly, 18 JAZ proteins were identified from G. biloba, and the gene characteristics and expansion patterns along with evolutionary relationships of these GbJAZs were analyzed systematically. Expression patterns analysis indicated that most GbJAZs expressed highly in the fibrous root and were induced significantly by methyl jasmonate. Mechanistically, yeast two-hybrid assays suggested that GbJAZ3/11 interacted with both GbMYC2 and GbCOI1, and several GbJAZ proteins could form homodimers or heterodimers between the GbJAZ family. Moreover, GbMYC2 is directly bound to the G-box element in the promoter of GbLPS, to regulate the biosynthesis of ginkgolide. Collectively, these results systematically characterized the JAZ gene family in G. biloba and demonstrated that the GbCOI1/GbJAZs/GbMYC2 module could regulate ginkgolides biosynthesis, which provides a novel insight for studying the mechanism of JA regulating ginkgolide biosynthesis.

An integrated strategy combining network toxicology and feature-based molecular networking for exploring hepatotoxic constituents and mechanism of Epimedii Folium-induced hepatotoxicity in vitro.

Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.

Revealing active components and action mechanism of Fritillariae Bulbus against non-small cell lung cancer through spectrum-effect relationship and proteomics.

BACKGROUND: Fritillariae Bulbus (FB) is widely used as a traditional medicine for the treatment of lung meridian diseases. It has been proved that FB has good anti-non-small cell lung cancer (NSCLC) activity. However, the active components and potential mechanism are still not clear. PURPOSE: To reveal the bioactive components of FB against NSCLC and potential mechanism through spectrum-effect relationship and proteomics. METHOD: First, the FB extract was chemically profiled by UHPLC-QTOF-MS and the inhibitory effect of FB extract on A549 cell viability was evaluated by Cell Counting Kit-8 assay. Second, orthogonal-partial least squares-regression analysis was applied to screen potential active compounds through correlating the chemical profile with corresponding inhibitory effect. Third, the anti-NSCLC activities of potential active components were further investigated in terms of cell proliferation, cell cycle and cell apoptosis in vitro and tumor growth in vivo. Finally, proteomics was utilized to reveal the underlying anti-NSCLC mechanism. RESULTS: Six potential active components including verticine, verticinone, zhebeirine, ebeiedinone, yibeissine and peimisine were screened out by spectrum-effect relationship. Among them, zhebeirine showed higher inhibitory effect on A549 cell viability with IC50 value of 36.93 µM and dosage-dependent inhibition of A549 xenograft tumor growth in nude mice. Proteomics and western blotting assays indicated that zhebeirine could arrest cell cycle by down-regulating the expressions of CDK1, CDK2, Cyclin A2, Cyclin B2 and inhibiting the phosphorylation of p53. Moreover, the proteins participating in p53 signaling pathway including PCNA, 14-3-3σ, CHEK1 were significantly decreased, which suggested that zhebeirine affected cell cycle progression through p53 signaling pathway. CONCLUSION: This study not only provides scientific evidence to support the clinical application of FB against NSCLC, but also demonstrates that zhebeirine is a promising anti-NSCLC lead compound deserving further studies.

Baohuoside I inhibits FXR signaling pathway to interfere with bile acid homeostasis via targeting ER α degradation.

Epimedii folium (EF) is an effective herbal medicine in osteoporosis treatment, but the clinical utilization of EF has been limited due to potential hepatotoxicity. The previous studies identified that baohuoside I (BI), the main active component of EF, was relevant to EF-induced liver injury. However, the mechanisms of BI causing direct injury to hepatocytes remain unclear. Here, we reveal that BI inhibits FXR-mediated signaling pathway via targeting estrogen receptor α (ER α), leading to the accumulation of bile acids (BAs). Targeted bile acid analyses show BI alters the BA composition and distribution, resulting in impaired BA homeostasis. Mechanistically, BI induces FXR-dependent hepatotoxicity at transcriptional level. Additionally, ER α is predicted to bind to the FXR promoter region based on transcription factor binding sites databases and we further demonstrate that ER α positively regulates FXR promoter activity and affects the expression of target genes involved in BA metabolism. Importantly, we discover that ER α and its mediated FXR transcription regulation might be involved in BI-induced liver injury via ligand-dependent ER α degradation. Collectively, our findings indicate that FXR is a newly discovered target gene of ER α mediated BI-induced liver injury, and suggest BI may be responsible for EF-induced liver injury.