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In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Qa-SNARE , Estresse Salino , Aquaporinas/metabolismo , Aquaporinas/genética , Arabidopsis/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Transporte Proteico , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qa-SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genéticaRESUMO
The abnormal activation of the epidermal growth factor receptor (EGFR) is strongly associated with cancer invasion and metastasis. Tools and methods are required to study and visualize EGFR activation under (patho)physiological conditions. Here, we report the development of a two-step photoaffinity probe (HX101) by incorporation of a diazirine as a photoreactive group and an alkyne as a ligation handle to quantitively study EGFR kinase activity in native cellular contexts and human tissue slices. HX101 is a multifunctional probe based on the pharmacophore of the EGFR tyrosine kinase inhibitor (EGFR-TKI) and can covalently target the EGFR upon photoactivation. The incorporated alkyne serves as a versatile ligation handle and enables HX101 to introduce distinct reporter groups (e.g., fluorophore and biotin) via click chemistry. With variable reporter tags, HX101 enables visualization and target engagement studies of the active EGFR in a panel of cancer cells using flow cytometry, confocal microscopy, and mass spectrometry. Furthermore, as a proof of concept study, we applied HX101 in stochastic optical reconstruction microscopy super-resolution imaging to study EGFR activation in live cells. Importantly, HX101 was also applied to visualize EGFR mutant activity in tumor tissues from lung cancer patients for prediction of EGFR-TKI sensitivity. Altogether, our results demonstrate the wide application of a selective photoaffinity probe in multi-modal assessment/visualization of EGFR activity in both live cells and tissue slices. We anticipate that these diverse applications can facilitate the translation of a strategically functionalized probe into medical use.
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Neoplasias Pulmonares , Tirosina , Alcinos/química , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The aberrant changes of fussion/fission-related proteins can trigger mitochondrial dynamics imbalance, which cause mitochondrial dysfunctions and result insulin resistance (IR). However, the relationship between the inner mitochondrial membrane fusion protein optic atrophy 1 (Opa1) and hepatic IR as well as the specific molecular mechanisms of signal transduction has not been fully elucidated. In this study, we explore whether abnormalities in the Opa1 cause hepatic IR and whether berberine (BBR) can prevent hepatic IR through the SIRT1/Opa1 signalling pathway. High-fat diet (HFD)-fed mice and db/db mice are used as animal models to study hepatic IR in vivo. IR, morphological changes, and mitochondrial injury of the liver are examined to explore the effects of BBR. SIRT1/Opa1 protein expression is determined to confirm whether the signalling pathway is damaged in the model animals and is involved in BBR treatment-mediated mitigation of hepatic IR. A palmitate (PA)-induced hepatocyte IR model is established in HepG2 cells in vitro. Opa1 silencing and SIRT1 overexpression are induced to verify whether Opa1 deficiency causes hepatocyte IR and whether SIRT1 improves this dysfunction. BBR treatment and SIRT1 silencing are employed to confirm that BBR can prevent hepatic IR by activating the SIRT1/Opa1 signalling pathway. Western blot analysis and JC-1 fluorescent staining results show that Opa1 deficiency causes an imbalance in mitochondrial fusion/fission and impairs insulin signalling in HepG2 cells. SIRT1 and BBR overexpression ameliorates PA-induced IR, increases Opa1, and improves mitochondrial function. SIRT1 silencing partly reverses the effects of BBR on HepG2 cells. SIRT1 and Opa1 expressions are downregulated in the animal models. BBR attenuates hepatic IR and enhances SIRT1/Opa1 signalling in db/db mice. In summary, Opa1 silencing-mediated mitochondrial fusion/fission imbalance could lead to hepatocyte IR. BBR may improve hepatic IR by regulating the SIRT1/Opa1 signalling pathway, and thus, it may be used to treat type-2 diabetes.
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Berberina , Resistência à Insulina , Camundongos , Animais , Berberina/farmacologia , Berberina/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fígado/metabolismo , Transdução de Sinais , Mitocôndrias/metabolismoRESUMO
Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM.
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Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Transtorno Autístico/genética , Calmodulina/química , Humanos , Simulação de Acoplamento Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/química , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Ligação ProteicaRESUMO
Eating vegetables with pesticide residues over a long period of time causes serious adverse effects on the human body, such as acute poisoning, chronic poisoning, and endocrine system interference. To achieve the goal of a healthy society, it is an urgent issue to find a simple and effective method to detect organic pesticides. In this work, two fluorescent porous organic polymers, LNU-45 and LNU-47 (abbreviation for Liaoning University), were prepared using π-conjugated dibromopyrene monomer and boronic acid compounds as building units through a Suzuki coupling reaction. Due to the large π-electron delocalization effect, the resulting polymers revealed enhanced fluorescence performance. Significantly, in sharp contrast with the planar π-conjugated polymer framework (LNU-47), the distorted conjugated structure (LNU-45) shows a higher specific surface area and provides a broad interface for analyte interaction, which is helpful to achieve rapid response and detection sensitivity. LNU-45 exhibits strong fluorescence emission at 469 nm after excitation at 365 nm in THF solution, providing strong evidence for its suitability as a luminescent chemosensor for organic pesticides. The fluorescence quenching coefficients of LNU-45 for trifluralin and dicloran were 5710 and 12,000 (LNU-47 sample by ca. 1.98 and 3.38 times), respectively. Therefore, LNU-45 serves as an effective "real-time" sensor for the detection of trifluralin and dicloran with high sensitivity and selectivity.
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Many neuroactive steroids potently and allosterically modulate pentameric ligand-gated ion channels, including GABAA receptors (GABAAR) and nicotinic acetylcholine receptors (nAChRs). Allopregnanolone and its synthetic analog alphaxalone are GABAAR-positive allosteric modulators (PAMs), whereas alphaxalone and most neuroactive steroids are nAChR inhibitors. In this report, we used 11ß-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAAR PAM, to characterize steroid-binding sites in the Torpedo α2ßγδ nAChR in its native membrane environment. We found that F4N3Bzoxy-AP (IC50 = 31 µm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [3H]tenocyclidine to nAChRs in the desensitized state. At 300 µm, neither steroid inhibited binding of [3H]tetracaine, a closed-state selective channel blocker, or of [3H]acetylcholine. Photolabeling identified three distinct [3H]F4N3Bzoxy-AP-binding sites in the nAChR transmembrane domain: 1) in the ion channel, identified by photolabeling in the M2 helices of ßVal-261 and δVal-269 (position M2-13'); 2) at the interface between the αM1 and αM4 helices, identified by photolabeling in αM1 (αCys-222/αLeu-223); and 3) at the lipid-protein interface involving γTrp-453 (M4), a residue photolabeled by small lipophilic probes and promegestone, a steroid nAChR antagonist. Photolabeling in the ion channel and αM1 was higher in the nAChR-desensitized state than in the resting state and inhibitable by promegestone. These results directly indicate a steroid-binding site in the nAChR ion channel and identify additional steroid-binding sites also occupied by other lipophilic nAChR antagonists.
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Proteínas de Peixes/química , Simulação de Acoplamento Molecular , Pregnanolona , Receptores Nicotínicos/química , Esteroides/química , Animais , Sítios de Ligação , Proteínas de Peixes/metabolismo , Pregnanolona/análogos & derivados , Pregnanolona/química , Receptores Nicotínicos/metabolismo , Esteroides/metabolismo , Tetracaína/química , TorpedoRESUMO
According to the fact that gecko-inspired vertically aligned carbon nanotubes (VA-CNTs) exhibit ultrastrong adhesion, dopamine is utilized to make a modification to this traditional biomimetic material. The composite material is tested for adhesion performance under different environmental conditions by an atomic force microscope. The adhesion force of the modified VA-CNTs does not show obvious fluctuation during the gradual heating process; however, the material gains improved adhesion when increasing the ambient humidity. In addition, the modified CNTs show a stronger adhesion force than the original CNTs in their performance tests. The dopamine polymer has a good combination with CNTs, which is responsible for the aforementioned excellent performance. Overall, this modification method is simple, convenient, efficient, and environmentally friendly, which all indicates a promising future in its application. The modified CNTs are expected to be used for super-adhesion in harsh environments, as well as in the field of microelectronics.
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Dopamina/química , Nanotubos de Carbono/química , Umidade , Polímeros/químicaRESUMO
Nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid type A receptors (GABAARs) are members of the pentameric ligand-gated ion channel superfamily. Drugs acting as positive allosteric modulators of muscle-type α2ßγδ nAChRs, of use in treatment of neuromuscular disorders, have been hard to identify. However, identification of nAChR allosteric modulator binding sites has been facilitated by using drugs developed as photoreactive GABAAR modulators. Recently, R-1-methyl-5-allyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (R-mTFD-MPAB), an anesthetic and GABAAR potentiator, has been shown to inhibit Torpedo α2ßγδ nAChRs, binding in the ion channel and to a γ+-α- subunit interface site similar to its GABAAR intersubunit binding site. In contrast, S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (S-mTFD-MPPB) acts as a convulsant and GABAAR inhibitor. Photolabeling studies established that S-mTFD-MPPB binds to the same GABAAR intersubunit binding site as R-mTFD-MPAB, but with negative rather than positive energetic coupling to GABA binding. We now show that S-mTFD-MPPB binds with the same state (agonist) dependence as R-mTFD-MPAB within the nAChR ion channel, but it does not bind to the intersubunit binding site. Rather, S-mTFD-MPPB binds to intrasubunit sites within the α and δ subunits, photolabeling αVal-218 (αM1), δPhe-232 (δM1), δThr-274 (δM2), and δIle-288 (δM3). Propofol, a general anesthetic that binds to GABAAR intersubunit sites, inhibited [3H]S-mTFD-MPPB photolabeling of these nAChR intrasubunit binding sites. These results demonstrate that in an nAChR, the subtle difference in structure between S-mTFD-MPPB and R-mTFD-MPAB (chirality; 5-propyl versus 5-allyl) determines selectivity for intra- versus intersubunit sites, in contrast to GABAARs, where this difference affects state dependence of binding to a common site.
Assuntos
Barbitúricos/química , Proteínas de Peixes/química , Receptores de GABA-A/química , Receptores Nicotínicos/química , Animais , Sítios de Ligação , Propofol/química , Subunidades Proteicas , TorpedoRESUMO
WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Naphthalene-etomidate, an etomidate analog containing a bulky phenyl ring substituent group, possesses very low γ-aminobutyric acid type A (GABAA) receptor efficacy and acts as an anesthetic-selective competitive antagonist. Using etomidate analogs containing phenyl ring substituents groups that range in volume, we tested the hypothesis that this unusual pharmacology is caused by steric hindrance that reduces binding to the receptor's open state. METHODS: The positive modulatory potencies and efficacies of etomidate and phenyl ring-substituted etomidate analogs were electrophysiology defined in oocyte-expressed α1ß3γ2L GABAA receptors. Their binding affinities to the GABAA receptor's two classes of transmembrane anesthetic binding sites were assessed from their abilities to inhibit receptor labeling by the site-selective photolabels [H]azi-etomidate and tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. RESULTS: The positive modulatory activities of etomidate and phenyl ring-substituted etomidate analogs progressively decreased with substituent group volume, reflecting significant decreases in both potency (P = 0.005) and efficacy (P < 0.0001). Affinity for the GABAA receptor's two ß - α anesthetic binding sites similarly decreased with substituent group volume (P = 0.003), whereas affinity for the receptor's α - ß/γ - ß sites did not (P = 0.804). Introduction of the N265M mutation, which is located at the ß - α binding sites and renders GABAA receptors etomidate-insensitive, completely abolished positive modulation by naphthalene-etomidate. CONCLUSIONS: Steric hindrance selectively reduces phenyl ring-substituted etomidate analog binding affinity to the two ß - α anesthetic binding sites on the GABAA receptor's open state, suggesting that the binding pocket where etomidate's phenyl ring lies becomes smaller as the receptor isomerizes from closed to open.
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Anestésicos Intravenosos/farmacologia , Etomidato/farmacologia , Receptores de GABA/metabolismo , Animais , Técnicas de Cultura de Células , Humanos , Oócitos , Receptores de GABA/efeitos dos fármacos , XenopusRESUMO
Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca2+/CaM/CaMKII cascade modulates NaV1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM12, CaM34, and CaM1234), and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Cálcio/química , Calmodulina/química , Canal de Sódio Disparado por Voltagem NAV1.1/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Acoplamento Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TermodinâmicaRESUMO
Drugs that block the cardiac K(+) channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K(+) channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K(+) channel, which is discussed in the light of findings on other ion channels.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Fenetilaminas/metabolismo , Bloqueadores dos Canais de Potássio/metabolismo , Ensaio Radioligante/métodos , Sulfonamidas/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Células HEK293 , Humanos , Fenetilaminas/química , Fenetilaminas/farmacologia , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Ligação Proteica/fisiologia , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Wearable devices with continuous monitoring capabilities are critical for the daily detection of epileptic seizures, as they provide users with accurate and comprehensible analytical results. However, current AI classifiers rely on a two-stage recognition process for continuous monitoring, which only reduces operation time but remains challenged by the high cost of additional hardware. To address this problem, this article proposes a novel fusion architecture for AI processors, which enables event-triggered cross-paradigm integration and computation. Our method introduces a distributed-aggregated classification architecture (D-ACA) that facilitates the reuse of hardware resources across two-stage recognition, thereby obviating the need for standby hardware and enhancing energy efficiency. Integrating a non-encoding biomedical circuit method based on spiking neural networks (SNNs), the architecture eliminates encoded neurons at the hardware level, significantly optimizing energy consumption and hardware resource utilization. Additionally, we develop a configurable and highly flexible control method that supports various neuron modules, enabling continuous detection of epileptic seizures and activating high-precision recognition upon event detection. Finally, we implement the design on the Xilinx ZCU 102 FPGA board, where the AI processor achieves a high classification accuracy of 98.1% while consuming extremely low classification energy (3.73 µJ per classification).
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SHP2 plays a critical role in modulating tumor growth and PD-1-related signaling pathway, thereby serving as an attractive antitumor target. To date, no antitumor drugs targeting SHP2 have been approved, and hence, the search of SHP2 inhibitors with new chemical scaffolds is urgently needed. Herein, we developed a novel SHP2 allosteric inhibitor SDUY038 with a furanyl amide scaffold, demonstrating potent binding affinity (KD = 0.29 µM), enzymatic activity (IC50 = 1.2 µM) and similar binding interactions to SHP099. At the cellular level, SDUY038 exhibited pan-antitumor activity (IC50 = 7-24 µM) by suppressing pERK expression. Furthermore, SDUY038 significantly inhibited tumor growth in both xenograft and organoid models. Additionally, SDUY038 displayed acceptable bioavailability (F = 14%) and half-life time (t1/2 = 3.95 h). Conclusively, this study introduces the furanyl amide scaffold as a novel class of SHP2 allosteric inhibitors, offering promising lead compounds for further development of new antitumor therapies targeting SHP2.
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Amidas , Antineoplásicos , Desenho de Fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Animais , Regulação Alostérica/efeitos dos fármacos , Amidas/farmacologia , Amidas/química , Amidas/síntese química , Camundongos , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Furanos/farmacologia , Furanos/química , Furanos/síntese química , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proliferação de Células/efeitos dos fármacos , Camundongos NusRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by hepatocyte steatosis and adipose accumulation with the main lesion in the hepatic lobule, but without a history of excessive alcohol consumption. NAFLD ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), and may further accumulate fibrosis leading to cirrhosis. Many studies have found that ginseng can treat NAFLD. (20 R)-Panaxadiol (PD) is a panax ginseng diol type compound, has been proved that can treat the obesity. This study wants to investigate the effect of PD on non-alcoholic liver disease. We used 20 ob/ob mice and 10 C57BL/6 J mice. C57BL/6 J mice as CONTROL group, ob/ob mice were divided into model group and PD group. In PD group, ob/ob mice were treated with PD for eight weeks(10 mg/kg, the CON and OB group was given the same amount of sodium carboxymethyl cellulose), detected the weight, food intake and serum index, observed the HE staining of liver and intestine, performed the 16 S rRNA and untargeted metabolomics analysis used mice feces, and verify the results by detect the expression of TNF-α, MDA and SOD. In vivo results, PD can improve abnormal glucose and lipid metabolism and liver function. In 16 S rRNA result, we found beneficial bacteria Muribaculaceae and Lactobacillus increased; in untargeted metabolomics analysis, inflammatory metabolites prostaglandin (PG) and lipopolysaccharide (LPS) decreased, antioxidant metabolites FAD and lipoic acid increased. Then, we proceeded the association analysis of gut microbiota and metabolites, the result showed gut microbiota have strongly associated with anti-inflammatory and antioxidant metabolites. In addition, PD improves intestinal wall integrity. Meanwhile, the expression of TNF-αãMDA and SOD were detected, it was verified that PD has the effect of antioxidant and anti-inflammation. Our study showed that PD, as an active ingredient of ginseng, can play an anti-inflammatory and antioxidant role by improving intestinal metabolites, thereby preventing and treating non-alcoholic fatty liver disease to a certain extent.
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Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Antioxidantes/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Obesidade/tratamento farmacológico , Superóxido Dismutase , Metabolômica , FezesRESUMO
Introduction: Obesity is an important cause of a range of metabolic diseases. However, the complex mechanisms of obesity and its related diseases make some weight loss methods ineffective or have safety issues. Ginseng, a specialty of Jilin Province in China with both edible and medicinal value, contains mainly ginsenosides and other components. In order to study the anti-obesity effect of ginseng, network pharmacology was used to predict and screen the active ingredients, action targets and signaling pathways of ginseng. We found (20R)-panaxadiol (PD) is a more desirable active ingredient due to its high drug-like properties and high bioavailability. Moreover, it is closely related to cAMP pathway which is more important in metabolism regulation. The corresponding pharmacodynamic targets of PD include ADRB2 (the gene encoding the ß2-adrenoceptor receptor). Our study aimed to investigate whether Panaxadiol can promote white adipocyte beigeing and increase thermogenesis through modulating the ß2/cAMP pathway to exert anti-obesity effects. Methods: In vivo, we established high-fat feeding obesity model, genotypically obese mice (ob/ob) model, and administered PD (10 mg/kg). PD treatment in ob/ob mice along with ß2 receptor inhibitor ICI118551. In vitro, differentiated mature 3T3-L1 cells were given palmitate (PA) to induce hypertrophy model along with PD (20 µM). Results: The results of this study demonstrated that PD significantly reduced body weight, improved glucose tolerance and lipid levels in high-fat-induced obese mice and ob/ob mice, and also reduced lipid droplet size in PA-treated hypertrophic adipocytes in vitro. Molecular biology assays confirmed that cAMP response element binding protein (CREB) phosphorylation was increased after PD administration, and the expression of thermogenesis-related proteins UCP1, PRDM16 and mitochondrial biosynthesis-related proteins PGC-1α, TFAM and NRF1 were increased. Molecular docking results showed a low binding energy between ß2 receptors and PD, indicating an affinity between the ß2 receptor and PD. In addition, the ß2 receptor inhibition, reversed the anti-obesity effect of PD on the body weight, lipid droplets, the expression of thermogenesis-related proteins and CREB phosphorylation in ob/ob mice. Discussion: These results suggest that PD may promote the expression of thermogenic proteins through phosphorylation of CREB via ß2 receptor activation, and thus exert anti-obesity effects.
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The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly metabolized by monoacylglycerol lipase (MAGL) in the brain. Selective inhibitors of MAGL provide valuable insights into the role of 2-AG in a variety of (patho)physiological processes and are potential therapeutics for the treatment of diseases such as neurodegenerative disease and inflammation, pain, as well as cancer. Despite a number of MAGL inhibitors been reported, inhibitors with new chemotypes are still required. Here, we developed a substrate-based fluorescence assay by using a new fluorogenic probe AA-HNA and successfully screened a focused library containing 320 natural organic compounds. Furthermore, we applied activity-based protein profiling (ABPP) as an orthogonal method to confirm the inhibitory activity against MAGL in the primary substrate-based screening. Our investigations culminated in the identification of two major compound classes, including quinoid diterpene (23, cryptotanshinone) and ß-carbolines (82 and 93, cis- and trans-isomers), with significant potency towards MAGL and good selectivity over other 2-AG hydrolases (ABHD6 and ABHD12). Moreover, these compounds also showed antiproliferative activities against multiple cancer cells, including A431, H1975, B16-F10, OVCAR-3, and A549. Remarkably, 23 achieved complete inhibition towards endogenous MAGL in most cancer cells determined by ABPP. Our results demonstrate the potential utility of the substrate-based fluorescence assay in combination with ABPP for rapidly discovering MAGL inhibitors, as well as providing an effective approach to identify potential targets for compounds with significant biological activities.
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The epithelial growth factor receptor (EGFR) is abnormally overexpressed on the cell surface of cancer cells and is strongly associated with cancer cell proliferation, migration, differentiation, apoptosis, and angiogenesis. Tools enabling the visualization of EGFR in a structure-function approach are highly desirable to predict EGFR mutations and guide EGFR tyrosine kinase inhibitor (TKI) treatment making. Here, we describe the design, synthesis, and application of new, potent and selective clickable probes 13 (HX03), 20 (HX04) and 24 (HX05) by introducing an alkyne ligation handle to visualize EGFR activity in living cancer cells and tissue slices. These clickable probes are versatile chemical tools based on the key pharmacophore (4-anilinoquinazoline) of EGFR-TKIs (e.g., canertinib, dacomitinib and afatinib) and are able to irreversibly target the kinase domain of EGFR. Among them, 13 exhibits the highest reactivity towards EGFR kinase, particularly to EGFR kinase with primary mutations. Using activity-based protein profiling strategy, 13 showed high sensitivity and selectivity in labeling of endogenous EGFR in a native cellular context. Moreover, 13 was applied to visualize EGFR mutant activity in tumour tissues from non-small-cell lung cancer (NSCLC) xenograft mouse models, and patients with NSCLC for the prediction of EGFR-TKI sensitivity. These results demonstrate that strategically designed EGFR-TKI-based probes allow discriminating EGFR mutations in human tissues and hold promise as useful diagnostic tools in predicting EGFR-TKI therapy response.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Afatinib , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fatores de CrescimentoRESUMO
Rose is one of the most important ornamental flowers, accounting for approximately one-third of the world's cut flower market. Powdery mildew caused by Podosphera pannosa is a devastating fungal disease in rose, mainly infecting the young leaves and causing serious economic losses. Therefore, a study on the mechanism of the fungus infecting the rose leaves and the possibility to improve resistance hereby is interesting and meaningful. Accordingly, we conducted transcriptome sequencing of rose leaves infected by P. pannosa at different time points to reveal the molecular mechanism of resistance to powdery mildew. The high-quality reads were aligned to the reference genome of Rosa chinensis, yielding 51,230 transcripts. A total of 1,181 differentially expressed genes (DEGs) were identified in leaves during P. pannosa infection at 12, 24, and 48 hpi. The transcription factors of ERF, MYB, bHLH, WRKY, etc., family were identified among DEGs, and most of them were downregulated during P. pannosa infection. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the hormone signal transduction pathway, especially ethylene signal-related genes, was consistently showing a downregulated expression during powdery mildew infection. More importantly, exogenous 1-MCP (inhibitor of ethylene) treatment could improve the rose leaves' resistance to P. pannosa. In summary, our transcriptome of rose leaf infected by powdery mildew gives universal insights into the complex gene regulatory networks mediating the rose leaf response to P. pannosa, further demonstrating the positive role of 1-MCP in resistance to biotrophic pathogens.
RESUMO
The bactericidal effect on biofilm is the main challenge currently faced by antibacterial agents. Nanoscale drug-delivery materials can enhance biofilm penetrability and drug bioavailability, and have significant applications in the biomedical field. Dental caries is a typical biofilm-related disease, and the acidification of biofilm pH is closely related to the development of dental caries. In this study, a pH-responsive core-shell nano micelle (mPEG-b-PDPA) capable of loading hydrophobic antibacterial agents was synthesized and characterized, including its ability to deliver antibacterial agents within an acidic biofilm. The molecular structure of this diblock copolymer was determined by hydrogen-1 nuclear magnetic resonance (1H-NMR) and gel permeation chromatography (GPC). The characters of the micelles were studied by dynamic light scattering (DLS), TEM, pH titration, and drug release detection. It was found that the hydrophilic micelles could deliver bedaquiline, a hydrophobic antibacterial agent on S. mutans, in acidic environments and in mature biofilm. No cytotoxic effect on the periodontal cells was detected within 48 h. This pH-responsive micelle, being able to load hydrophobic antibacterial agent, has good clinical application potential in preventing dental caries.