Ursolic acid reduces hepatocellular apoptosis and alleviates alcohol-induced liver injury via irreversible inhibition of CASP3 in vivo.

Alcoholic liver disease (ALD) is one of the pathogenic factors of chronic liver disease with the highest clinical morbidity worldwide. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, has shown many health benefits including antioxidative, anti-inflammatory, anticancer, and hepatoprotective activities. We previously found that UA was metabolized in vivo into epoxy-modified UA containing an epoxy electrophilic group and had the potential to react with nucleophilic groups. In this study we prepared an alkynyl-modified UA (AM-UA) probe for tracing and capturing the target protein of UA from liver in mice, then investigated the mode by which UA bound to its target in vivo. By conducting proteome identification and bioinformatics analysis, we identified caspase-3 (CASP3) as the primary target protein of UA associated with liver protection. Molecule docking analysis showed that the epoxy group of the UA metabolite reacted with Cys-163 of CASP3, forming a covalent bond with CASP3. The binding mode of the UA metabolites (UA, CM-UA, and EM-UA) was verified by biochemical evaluation, demonstrating that the epoxy group produced by metabolism played an important role in the inhibition of CASP3. In alcohol-treated HepG2 cells, pretreatment with the UA metabolite (10 µM) irreversibly inhibited CASP3 activities, and subsequently decreased the cleavage of PARP and cell apoptosis. Finally, pre-administration of UA (20-80 mg· kg-1 per day, ig, for 1 week) dose-dependently alleviated alcohol-induced liver injury in mice mainly via the inhibition of CASP3. In conclusion, this study demonstrates that UA is a valuable lead compound for the treatment of ALD.

Quantitative Proteomics Combined with Affinity MS Revealed the Molecular Mechanism of Ginsenoside Antitumor Effects.

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 µM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.

Active fragments-guided drug discovery and design of selective tropane alkaloids using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry coupled with virtual calculation and biological evaluation.

Tropane alkaloids (TAs), rich in the plant of Physochlaina infundibularis Kuang, which is named Huashanshen (HSS) in China, showed good effects on types of spasms. However, no data were collected to explore the relationship between the specificity for muscarinic receptor subtypes and the structures of these TAs. To address this issue, an extracted ion chromatogram (EIC) strategy using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on the fragmentation behavior of the TA standards was established to rapidly capture the varied TAs from HSS. Based on the provided structural information of diagnostic ions or neutral loss, 29 TAs were efficiently profiled, especially some trace ingredients. In additional, via virtual validation combined with molecular dynamic simulation, approximately a dozen alkaloids were found with high selectivity for muscarinic receptors. In additional, N-acetyl convolicine was chosen for selectivity evaluation of M2 or M3 receptors through the use of a dual-luciferase reporter assay system at the cellular level and an ACh-induced constricted strip test in vitro. After summarizing the active fragments and the structure-activity relationship (SAR) information, a new modified TA that takes advantage of both the high affinity and high selectivity for M3 receptors was proposed and evaluated successfully. This study provided an effective approach for the discovery and design of natural products based on highly selective drugs by UPLC-Q/TOF-MS coupled with virtual calculation and biological evaluation. Graphical Abstract Active fragments-guided strategy for selective inhibitors from HSS.

Biodistribution of arctigenin-loaded nanoparticles designed for multimodal imaging.

BACKGROUND: Tracking targets of natural products is one of the most challenging issues in fields ranging from pharmacognosy to biomedicine. It is widely recognized that the biocompatible nanoparticle (NP) could function as a "key" that opens the target "lock". RESULTS: We report a functionalized poly-lysine NP technique that can monitor the target protein of arctigenin (ATG) in vivo non-invasively. The NPs were synthesized, and their morphologies and surface chemical properties were characterized by transmission electron microscopy (TEM), laser particle size analysis and atomic force microscopy (AFM). In addition, we studied the localization of ATG at the level of the cell and the whole animal (zebrafish and mice). We demonstrated that fluorescent NPs could be ideal carriers in the development of a feasible method for target identification. The distributions of the target proteins were found to be consistent with the pharmacological action of ATG at the cellular and whole-organism levels. CONCLUSIONS: The results indicated that functionalized poly-lysine NPs could be valuable in the multimodal imaging of arctigenin.

Sign Learning Kink-based (SiLK) Quantum Monte Carlo for molecular systems.

The Sign Learning Kink (SiLK) based Quantum Monte Carlo (QMC) method is used to calculate the ab initio ground state energies for multiple geometries of the H2O, N2, and F2 molecules. The method is based on Feynman's path integral formulation of quantum mechanics and has two stages. The first stage is called the learning stage and reduces the well-known QMC minus sign problem by optimizing the linear combinations of Slater determinants which are used in the second stage, a conventional QMC simulation. The method is tested using different vector spaces and compared to the results of other quantum chemical methods and to exact diagonalization. Our findings demonstrate that the SiLK method is accurate and reduces or eliminates the minus sign problem.

Screening and identification of Caulis Sinomenii bioactive ingredients with dual-target NF-κB inhibition and ß2- AR agonizing activities.

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti-inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with a dual-target bioactive screening assay for anti-inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF-κB inhibitors were identified, including laudanosoline-1-O-xylopyranose, 6-O-methyl-laudanosoline-1-O-glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL-6 and IL-8 assays confirmed the anti-inflammatory effects of these potential NF-κB inhibitors, in which laudanosoline-1-O-d-xylopyranose and menisperine were revealed as novel NF-κB inhibitors. Among the seven identified alkaloids, three potential ß2 -adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of ß2 -adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF-κB inhibitors but also as potential ß2 -adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual-bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain-induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the ß2 -adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.

An integrated global chemomics and system biology approach to analyze the mechanisms of the traditional Chinese medicinal preparation Eriobotrya japonica - Fritillaria usuriensis dropping pills for pulmonary diseases.

BACKGROUND: Traditional Chinese medicine (TCM) herbal formulae provide valuable therapeutic strategies. However, the active ingredients and mechanisms of action remain unclear for most of these formulae. Therefore, the identification of complex mechanisms is a major challenge in TCM research. METHODS: This study used a network pharmacology approach to clarify the anti-inflammatory and cough suppressing mechanisms of the Chinese medicinal preparation Eriobotrya japonica - Fritillaria usuriensis dropping pills (ChuanbeiPipa dropping pills, CBPP). The chemical constituents of CBPP were identified by high-quality ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), and anti-inflammatory ingredients were selected and analyzed using the PharmMapper and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatics websites to predict the target proteins and related pathways, respectively. Then, an RNA-sequencing (RNA-Seq) analysis was carried out to investigate the different expression of genes in the lung tissue of rats with chronic bronchitis. RESULTS: Six main constituents affected 19 predicted pathways, including ursolic acid and oleanolic acid from Eriobotrya japonica (Thunb.) Lindl. (Eri), peiminine from Fritillaria usuriensis Maxim. (Fri), platycodigenin and polygalacic acid from Platycodon grandiflorum (Jacq.) A. DC. (Pla) and guanosine from Pinellia ternata (Thunb.) Makino. (Pin). Expression of 34 genes was significantly decreased after CBPP treatment, affecting four therapeutic functions: immunoregulation, anti-inflammation, collagen formation and muscle contraction. CONCLUSION: The active components acted on the mitogen activated protein kinase (MAPK) pathway, transforming growth factor (TGF)-beta pathway, focal adhesion, tight junctions and the action cytoskeleton to exert anti-inflammatory effects, resolve phlegm, and relieve cough. This novel approach of global chemomics-integrated systems biology represents an effective and accurate strategy for the study of TCM with multiple components and multiple target mechanisms.

3,5-Dimethyl-8-methoxy-3,4-dihydro-1H-isochromen-6-ol Attenuates Ulcerative Colitis via Targeting NLRP3 in Mice.

3,5-Dimethyl-8-methoxy-3,4-dihydro-1H-isochromen-6-ol (DMD) is a polyketide compound obtained from the endophytic fungus Penicillium sp. HJT-A-10 of Rhodiola tibetica. R. tibetica is a nourishing food and also used in traditional Chinese medicine and Xizang medicine. In dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice, DMD significantly alleviated the pathological symptom of UC. Network pharmacology studies have shown that nucleotide-binding domain-like receptor family pyrin domain containing (NLRP) 3 is the primary target protein of DMD associated with anti-UC. In molecular biology studies, DMD suppressed the activation of NLRP3 and decreased the expression of downstream inflammatory proteins and pro-inflammatory cytokines in UC. The finding was further verified in knockout mice. DMD lost the effect of attenuating DSS-induced UC in NLRP3-/- mice. In conclusion, this study demonstrates that DMD reduces inflammatory response and balances the barrier integrity to attenuate UC via targeting NLRP3, and DMD is a potential natural agent or dietary supplement for attenuating UC.

Pollution characteristics, source appointment and environmental effect of oxygenated volatile organic compounds in Guangdong-Hong Kong-Macao Greater Bay Area: Implication for air quality management.

Same as other bay areas, the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) is also suffering atmospheric composite pollution. Even a series of atmospheric environment management policies have been conducted to win the "blue sky defense battle", the atmospheric secondary pollutants (e.g., O3) originated from oxygenated volatile organic compounds (OVOCs) still threaten the air quality in GBA. However, there lacks a systematic summary on the emission, formation, pollution and environmental effects of OVOCs in this region for further air quality management. This review focused on the researches related to OVOCs in GBA, including their pollution characteristics, detection methods, source distributions, secondary formations, and impacts on the atmosphere. Pollution profile of OVOCs in GBA revealed that the concentration percentage among total VOCs from Guangzhou and Dongguan cities exceeded 50 %, while methanol, formaldehyde, acetone, and acetaldehyde were the top four highest concentrated OVOCs. The detection technique on regional atmospheric OVOCs (e.g., oxygenated organic molecules (OOMs)) underwent an evolution of off-line derivatization method, on-line spectroscopic method and on-line mass spectrometry method. The OVOCs in GBA were mainly from primary emissions (up to 80 %), including vehicle emissions and biomass combustion. The anthropogenic alkenes and aromatics in urban area, and natural isoprene in rural area also made a significant contribution to the secondary emission (e.g., photochemical formation) of OVOCs. About 20 % in average of ROx radicals was produced from photolysis of formaldehyde in comparison with O3, nitrous acid and rest OVOCs, while the reaction between OVOCs and free radical accelerated the NOx-O3 cycle, contributing to 15 %-60 % cumulative formation of O3 in GBA. Besides, the heterogeneous reactions of dicarbonyls generated 21 %-53 % of SOA. This review also provided suggestions for future research on OVOCs in terms of regional observation, analytical method and mechanistic study to support the development of a control and management strategy on OVOCs in GBA and China.

Anti-enterovirus 71 activity of native fucosylated chondroitin sulfates and their derivatives.

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.

Glycopolymer with Sulfated Fucose and 6'-Sialyllactose as a Dual-Targeted Inhibitor on Resistant Influenza A Virus Strains.

The frequent mutations of influenza A virus (IAV) have led to an urgent need for the development of innovative antiviral drugs. Glycopolymers offer significant advantages in biomedical applications owing to their biocompatibility and structural diversity. However, the primary challenge lies in the design and synthesis of well-defined glycopolymers to precisely control their biological functionalities. In this study, functional glycopolymers with sulfated fucose and 6'-sialyllactose were successfully synthesized through ring-opening metathesis polymerization and a postmodification strategy. The optimized heteropolymer exhibited simultaneous targeting of hemagglutinin and neuraminidase on the surface of IAV, as evidenced by MU-NANA assay and hemagglutination inhibition data. Antiviral experiments demonstrated that the glycopolymer displayed broad and efficient inhibitory activity against wild-type and mutant strains of H1N1 and H3N2 subtypes in vitro, thereby establishing its potential as a dual-targeted inhibitor for combating IAV resistance.

Inhibition of influenza a virus infection by natural stilbene piceatannol targeting virus hemagglutinin.

BACKGROUND: Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. Piceatannol (PIC) is a tetrahydroxylated stilbenoid (trans-3,4,3',5'-tetrahydroxystilbene), also known as 3'- hydroxy resveratrol, which has demonstrated many different biological activities such as anti-inflammatory and antiviral activities. PURPOSE: In this study, the anti-influenza A virus (IAV) activities and mechanisms of PIC in vitro and in vivo were investigated in order to provide reference for the development of novel plant-derived anti-IAV drugs. METHODS: The viral plaque assay, RT-PCR and western blot assay were used to evaluate the anti-IAV effects of PIC in vitro. The anti-IAV mechanism of PIC was determined by HA syncytium assay, DARTS assay and Surface Plasmon Resonance assay. The mouse pneumonia model combined with HE staining were used to study the anti-IAV effects of PIC in vivo. RESULTS: PIC shows inhibition on the multiplication of both H1N1 and H3N2 viruses, and blocks the infection of H5N1 pseudovirus with low toxicity. PIC may directly act on the envelope of IAV to induce the rupture and inactivation of IAV particles. PIC can also block membrane fusion via binding to HA2 rather than HA1 and cleavage site of HA0. PIC may interact with the two residues (HA2-T68 and HA2-I75) of HA2 to block the conformational change of HA so as to inhibit membrane fusion. Importantly, oral therapy of PIC also markedly improved survival and reduced viral titers in IAV-infected mice. CONCLUSION: PIC possesses significant anti-IAV effects both in vitro and in vivo and may block IAV infection mainly through interaction with HA to block membrane fusion. Thus, PIC has the potential to be developed into a new broad-spectrum anti-influenza drug for the prevention and treatment of influenza.

Inhibitory effects and mechanisms of proanthocyanidins against enterovirus 71 infection.

Proanthocyanidins (PC), a natural flavonoid compound, was reported to possess a variety of pharmacological activities such as anti-tumor and anti-viral effects. In this study, the anti-Enterovirus 71 (EV71) activities and mechanisms of PC were investigated both in vitro and in vivo. The results showed that PC possessed anti-EV71 activities in different cell lines with low toxicity. PC can block both the adsorption and entry processes of EV71 via directly binding to virus VP1 protein. PC may competitively interfere with the binding of VP1 to its receptor SCARB2. PC can also regulate three different MAPK signaling pathways to reduce EV71 infection and attenuate virus induced inflammatory responses. Importantly, intramuscular therapy of EV71-infected mice with PC markedly improved their survival and attenuated the severe clinical symptoms. Therefore, the natural compound PC has potential to be developed into a novel anti-EV71 agent targeting viral VP1 protein and MAPK pathways.

Ligustilide covalently binds to Cys129 of HMGCS1 to ameliorate dyslipidemia.

Dyslipidemia is characterized by elevated levels of total cholesterol and triglycerides in serum, and has become the primary human health killer because of the major risk factors for cardiovascular diseases. Although there exist plenty of drugs for dyslipidemia, the number of patients who could benefit from lipid-lowering drugs still remains a concern. Ligustilide (Lig), a natural phthalide derivative, was reported to regulate lipid metabolic disorders. However, its specific targets and underlying molecular mechanism are still unclear. In this study, we found that Lig alleviated high fat diet-induced dyslipidemia by inhibiting cholesterol biosynthesis. Furthermore, a series of chemical biological analysis methods were used to identify its target protein for regulating lipid metabolism. Collectively, 3-hydroxy-3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) of hepatic cells was identified as a target for Lig to regulate lipid metabolism. The mechanistic study confirmed that Lig irreversibly binds to Cys129 of HMGCS1 via its metabolic intermediate 6,7-epoxyligustilide, thereby reducing cholesterol synthesis and improving lipid metabolism disorders. These findings not only systematically elucidated the lipid-lowering mechanism of Lig, but also provided a new structural compound for the treatment of dyslipidemia.

Design, synthesis, and biological evaluation of novel penindolone derivatives as potential inhibitors of hemagglutinin-mediated membrane fusion.

Development and design of anti-influenza drugs with novel mechanisms is of great significance to combat the ongoing threat of influenza A virus (IAV). Hemagglutinin (HA) is regarded as a potential target for the therapy of IAV. Our previous research led to the discovery of penindolone (PND), a new diclavatol indole adduct, as an HA targeting leading compound exhibited anti-IAV activity. To enhance the bioactivity and understand the structure-activity relationships (SARs), 65 PND derivatives were designed and synthesized, and the anti-IAV activities as well as the HA targeting effects were systematically investigated in this study. Among them, compound 5g possessed high affinity to HA and was more effective than PND in terms of inhibiting HA-mediated membrane fusion. Compound 5g may act on the trypsin cleavage site of HA to exhibit a strong inhibition on membrane fusion. In addition, oral administration of 5g can significantly reduce the pulmonary virus titer, attenuate the weight loss, and improve the survival of IAV-infected mice, superior to the effects of PND. These findings suggest that the HA inhibitor 5g has potential to be developed into a novel broad-spectrum anti-IAV agent in the future.

Ursolic acid alleviates tetrandrine-induced hepatotoxicity by competitively binding to the substrate-binding site of glutathione S-transferases.

BACKGROUND: Tetrandrine (TET), a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, is the only approved medicine in China for silicosis. However, TET-induced hepatotoxicity has raised safety concerns. The underlying toxic targets and mechanism induced by TET remain unclear; there are no targeted detoxification strategies developed for TET-induced hepatotoxicity. Ursolic acid (UA), a pentacyclic triterpene with liver protective effects, may have detoxification effects on TET-induced hepatotoxicity. PURPOSE: This study aims to explore toxic targets and mechanism of TET and present UA as a potential targeted therapy for alleviating TET-induced hepatotoxicity. METHODS: A TET-induced liver-injury model was established to evaluate TET toxicity and the potential UA detoxification effect. Alkenyl-modified TET and UA probes were designed to identify potential liver targets. Pharmacological and molecular biology methods were used to explore the underlying toxicity/detoxification mechanism. RESULTS: TET induced liver injury by covalently binding to the substrate-binding pocket (H-site) of glutathione S-transferases (GSTs) and inhibiting GST activity. The covalent binding led to toxic metabolite accumulation and caused redox imbalance and liver injury. UA protected the liver from TET-induced damage by competitively binding to the GST H-site. CONCLUSION: The mechanism of TET-induced hepatotoxicity is related to irreversible binding with the GST H-site and GST-activity inhibition. UA, a natural antidote, competed with TET on H-site binding and reversed the redox imbalance. This study revealed the hepatotoxic mechanism of TET and provided a targeted detoxifying agent, UA, to alleviate hepatotoxicity caused by GST inhibition.

Mechanism Assay of Honeysuckle for Heat-Clearing Based on Metabolites and Metabolomics.

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as cyclooxygenase (Cox)-1/2 inhibitor, have emerged as potent antipyretics and analgesics. However, few herbs with Cox-1/2 inhibitory activity are commonly used for heat-clearing in China. Although these are known to have antipyretic activity, there is a lack of molecular data supporting their activity. Using the traditional Chinese medicine herb honeysuckle (Hon) as an example, we explored key antipyretic active compounds and their mechanisms of action by assessing their metabolites and metabolomics. Mitogen-activated protein kinase (MAPK) 3 and protein kinase B (AKT) 1 were suggested as key targets regulated primarily by chlorogenic acid (CA) and swertiamarin (SWE). CA and SWE synergistically inhibited the production of interleukin (IL)-1 and IL-6, alleviated generation of prostaglandin E2, and played an antipyretic role equivalent to honeysuckle extract at the same dose contents within 3 h. Collectively, these findings indicated that lipopolysaccharide-induced fever can be countered by CA with SWE synergistically, allowing the substitution of a crude extract of complex composition with active compounds. Our findings demonstrated that, unlike the traditional NSAIDs, the Hon extract showed a remote and indirect mechanism for alleviating fever that depended on the phosphatidylinositol-3-kinase-AKT and MAPK pathways by regulating the principal mediator of inflammation.

Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism.

Hyperaldosteronism is a common disease that is closely related to endocrine hypertension and other cardiovascular diseases. Cytochrome P450 11B2 (CYP11B2), an important enzyme in aldosterone (ALD) synthesis, is a promising target for the treatment of hyperaldosteronism. However, selective inhibitors targeting CYP11B2 are still lacking due to the high similarity with CYP11B1. In this study, atractylenolide-I (AT-I) was found to significantly reduce the production of ALD but had no effect on cortisol synthesis, which is catalyzed by CYP11B1. Chemical biology studies revealed that due to the presence of Ala320, AT-I is selectively bound to the catalytic pocket of CYP11B2, and the C8/C9 double bond of AT-I can be epoxidized, which then undergoes nucleophilic addition with the sulfhydryl group of Cys450 in CYP11B2. The covalent binding of AT-I disrupts the interaction between heme and CYP11B2 and inactivates CYP11B2, leading to the suppression of ALD synthesis; AT-I shows a significant therapeutic effect for improving hyperaldosteronism.

Ursolic acid inhibits the cholesterol biosynthesis and alleviates high fat diet-induced hypercholesterolemia via irreversible inhibition of HMGCS1 in vivo.

BACKGROUND: In hypercholesteremia, the concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) are enhanced in serum, which is strongly associated with an increased risk of developing atherosclerosis. Ursolic acid (UA), a pentacyclic terpenoid carboxylic acid, was found to alleviate hypercholesterolemia and hypercholesterolemia-induced cardiovascular disease. However, the specific targets and molecular mechanisms related to the effects of UA in reducing cholesterol have not been elucidated. PURPOSE: In this study, we aimed to illustrate the target of UA in the treatment of hypercholesterolemia and to reveal its underlying molecular mechanism. METHODS: Nontargeted metabolomics was conducted to analyze the metabolites and related pathways that UA affected in vivo. The main lipid metabolism targets of UA were analyzed by target fishing and fluorescence colocalization in mouse liver. Molecular docking, in-gel fluorescence scan and thermal shift were assessed to further investigate the binding site of the UA metabolite with HMGCS1. C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks to induce hypercholesteremia. Liver tissues were used to verify the cholesterol-lowering molecular mechanism of UA by targeted metabolomics, serum was used to detect biochemical indices, and the entire aorta was used to analyze the formation of atherosclerotic lesions. RESULTS: Our results showed that hydroxy­3-methylglutaryl coenzyme A synthetase 1 (HMGCS1) was the primary lipid metabolism target protein of UA. The UA metabolite epoxy-modified UA irreversibly bonds with the thiol of Cys-129 in HMGCS1, which inhibits the catalytic activity of HMGCS1 and reduces the generation of precursors in cholesterol biosynthesis in vivo. The contents of TC and LDL-C in serum and the formation of the atherosclerotic area in the entire aorta were markedly reduced with UA treatment in Diet-induced hypercholesteremia mice. CONCLUSION: UA inhibits the catalytic activity of HMGCS1, reduces the generation of downstream metabolites in the process of cholesterol biosynthesis and alleviates Diet-induced hypercholesteremia via irreversible binding with HMGCS1 in vivo. It is the first time to clarify the irreversible inhibition mechanism of UA against HMGCS1. This paper provides an increased understanding of UA, particularly regarding the molecular mechanism of the cholesterol-lowering effect, and demonstrates the potential of UA as a novel therapeutic for the treatment of hypercholesteremia.

Upregulation of RCAN1.4 in spinal dorsal horn is involved in inflammatory pain hypersensitivity.

The calcium/calmodulin-dependent protein phosphase calcineurin (CaN) regulates synaptic plasticity by controlling the phosphorylation of synaptic proteins including AMPA type glutamate receptors. The regulator of calcineurin 1 (RCAN1) is characterized as an endogenous inhibitor of CaN and its dysregulation is implicated in multiple neurological disorders. However, whether RCAN1 is engaged in nociceptive processing in the spinal dorsal horn remains unrevealed. In this study, we found that RCAN1 was predominately expressed in pain-related neurons in the superficial dorsal horn of the spinal cord. Intraplantar injection of complete Freund's adjuvant (CFA) specifically increased the total and synaptic expression of the RCAN1.4 isoform in spinal dorsal horn. The CFA-induced inflammation also caused an increased binding of RCAN1.4 to CaN. Overexpression of RCAN1.4 in spinal dorsal horn of intact mice produced both mechanical allodynia and thermal hyperalgesia, which were accompanied by increased synaptic expression and phosphorylation of GluA1 subunit. Furthermore, the siRNA-mediated knockdown of RCAN1.4 significantly attenuated the development of pain hypersensitivity, meanwhile, decreased the synaptic expression of GluA1 in mice with peripheral inflammation. These data suggested that the increased expression of RCAN1.4 contributed to the development of inflammatory pain hypersensitivity, at least in part by promoting the synaptic recruitment of GluA1-containing AMPA receptor.