Cellular Thermal Shift Assay Research Articles

Huashi Baidu formula alleviates lipopolysaccharide-induced inflammation and acute lung injury in mice by targeting nuclear factor κB/phosphatidylinositol 3-kinase and peroxiredoxin 5

Abstract Background Acute respiratory distress syndrome induced by acute lung injury (ALI) is the main cause for the high mortality of coronavirus disease 2019 (COVID-19). Huashi Baidu formula (HSBD) with the effects of eliminating dampness, clearing heat, ventilating lung, and removing toxin has been proven to be effective in the treatment of COVID-19, especially in severe cases. However, the underlying mechanism and target proteins of HSBD remain unclear. Objective To provide evidence and decipher the mechanism of HSBD in alleviating inflammation and ALI. Materials and Methods A mouse model of ALI was induced by lipopolysaccharide (LPS), and hematoxylin-eosin staining was used to examine the protective effects of HSBD on the model mice. The cellular thermal shift assay and proteomics analysis were used to predict the target proteins. Furthermore, the A549 cells with peroxiredoxin 5 (PRDX5) knockdown were established to validate the predicted proteins. Results Huashi Baidu formula treatment mitigated ALI and inflammatory cytokine dysfunction in LPS-induced mice, thus exerting a therapeutic effect on COVID-19. Huashi Baidu formula could serve as a therapeutic agent to alleviate inflammation and lung injury via nuclear factor κB and phosphatidylinositol 3-kinase signaling and interleukin 17 inhibition as well as targeting PRDX5, which could be one of the promising targets for treating inflammation. In the A549 cell line with PRDX5 knockdown (si-Prdx5), the anti-inflammation effects of HSBD, including reversing LPS-induced increase in the nitric oxide level and reduction in the hydrogen peroxide content, were attenuated. Thus, HSBD protected A549 cells from LPS-induced inflammation mainly by targeting PRDX5. Conclusions Huashi Baidu formula alleviates ALI by targeting nuclear factor κB/phosphatidylinositol 3-kinase and PRDX5, as well as inhibiting the immune response induced by IL-17.

Protein Target Prediction and Validation of Small Molecule Compound.

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of crucial protein targets have become integral to drug development. Molecular docking is a computational tool widely utilized to investigate protein-ligand binding, especially in the context of drug and protein target interactions. For the experimental verification of the binding and to access the binding of the drug and its target directly, the cellular thermal shift assay (CETSA) method is used. This study aimed to integrate molecular docking with CETSA to predict and validate interactions between drugs and vital protein targets. Specifically, we predicted the interaction between xanthatin and Keap1 protein as well as its binding mode through molecular docking analysis, followed by verification of the interaction using the CETSA assay. Our results demonstrated that xanthatin could establish hydrogen bonds with specific amino acid residues of Keap1 protein and reduce the thermostability of Keap1 protein, indicating that xanthatin could directly interact with Keap1 protein.

Targeted inhibition of transforming growth factor-β type I receptor by AZ12601011 improves paraquat poisoning-induced multiple organ fibrosis

Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-β1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFβRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin–eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1β, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-β and TGFβRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFβRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-β/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment.

Linderane Attenuates Complete Freund's Adjuvant-Induced Pain and Anxiety in Mice by Restoring Anterior Cingulate Cortex Microglia M2 Polarization through Activating Cannabinoid 2 Receptor.

Chronic pain is a common condition that causes negative emotions as the disease progresses. The anterior cingulate cortex (ACC) is a key region in the integration of nociceptive perception and emotional response in chronic pain. Linderane (LDR) is an active ingredient from Linderae radix, a traditional Chinese medicine with anti-inflammatory, analgesic, and antibacterial properties. In this study, the analgesic and antianxiety effects of LDR were evaluated using a complete Freund's adjuvant (CFA)-induced inflammatory pain model in C57BL/6 male mice. Mechanical and thermal pain sensitivity were measured through plantar mechanical analgesia and hot plate apparatus, and anxiety-like behavior was evaluated by open field and elevated plus maze tests. The results showed that LDR-alleviated CFA-induced pain and anxiety, reduced the release of inflammatory cytokines, and inhibited ACC microglial activation. Target prediction, molecular docking, and cellular thermal shift assay demonstrated that LDR could bind to the cannabinoid 2 receptor (CB2R), a key component of the endocannabinoid system with an important role in regulating pain and related emotions. Moreover, both the analgesic effect of LDR and its regulation of microglia polarization were reversed by a CB2R antagonist (SR144528) treatment. Therefore, our results suggested that LDR exerted analgesic effects by regulating microglial polarization in ACC via CB2R activation.

N-Cinnamoylpyrrole-derived alkaloids from the genus Piper as promising agents for ischemic stroke by targeting eEF1A1

BackgroundIschemic stroke (IS) is a serious cerebrovascular disease characterized by significantly elevated mortality and disability rates, and the treatments available for this disease are limited. Neuroinflammation and oxidative stress are deemed the major causes of cerebral ischemic injury. N-Cinnamoylpyrrole alkaloids form a small group of natural products from the genus Piper and have not been extensively analyzed pharmacologically. Thus, identifying the effect and mechanism of N-cinnamoylpyrrole-derived alkaloids on IS is worthwhile. PurposeThe present research aimed to explore the antineuroinflammatory and antioxidative stress effects of N-cinnamoylpyrrole-derived alkaloids isolated from the genus Piper and to explain the effects and mechanism on IS. MethodsN-cinnamoylpyrrole-derived alkaloids were isolated from Piper boehmeriaefolium var. tonkinense and Piper sarmentosum and identified by various chromatographic methods. Lipopolysaccharide (LPS)-induced BV-2 microglia and a mouse model intracerebroventricularly injected with LPS were used to evaluate the antineuroinflammatory and antioxidative stress effects. Oxygen‒glucose deprivation/reperfusion (OGD/R) and transient middle cerebral artery occlusion (tMCAO) models were used to evaluate the effect of PB-1 on IS. To elucidate the fundamental mechanism, the functional target of PB-1 was identified by affinity-based protein profiling (ABPP) strategy and verified by cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), and circular dichroism (CD) analyses. The effect of PB-1 on the NF-κB and NRF2 signaling pathways was subsequently evaluated via western blotting and immunofluorescence staining. ResultsThe results showed that N-cinnamoylpyrrole-derived alkaloids significantly affected neuroinflammation and oxidative stress. The representative compound, PB-1 not only inhibited neuroinflammation and oxidative stress induced by LPS or OGD/R insult, but also alleviated cerebral ischemic injury induced by tMCAO. Further molecular mechanism research found that PB-1 promoted antineuroinflammatory and antioxidative stress activities via the NF-κB and NRF2 signaling pathways by targeting eEF1A1. ConclusionOur research initially unveiled that the therapeutic impact of PB-1 on cerebral ischemic injury might rely on its ability to target eEF1A1, leading to antineuroinflammatory and antioxidative stress effects. The novel discovery highlights eEF1A1 as a potential target for IS treatment and shows that PB-1, as a lead compound that targets eEF1A1, may be a promising therapeutic agent for IS.

Structural characterization and calcium absorption-promoting effect of sucrose-calcium chelate in Caco-2 monolayer cells and mice.

Sucrose emerges as a chelating agent to form a stable sucrose-metal-ion chelate that can potentially improve metal-ion absorption. This study aimed to analyze the structure of sucrose-calcium chelate and its potential to promote calcium absorption in both Caco-2 monolayer cells and mice. The characterization results showed that calcium ions mainly chelated with hydroxyl groups in sucrose to produce sucrose-calcium chelate, altering the crystal structure of sucrose (forming polymer particles) and improving its thermal stability. Sucrose-calcium chelate dose dependently increased the amount of calcium uptake, retention, and transport in the Caco-2 monolayer cell model. Compared to CaCl2 , there was a significant improvement in the proportion of absorbed calcium utilized for transport but not retention (93.13±1.75% vs. 67.67±7.55%). Further treatment of calcium channel inhibitors demonstrated the active transport of sucrose-calcium chelate through Cav1.3. Cellular thermal shift assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays indicated that the ability of sucrose-calcium chelate to promote calcium transport was attributed to its superior ability to bind with PMCA1b, a calcium transporter located on the basement membrane, and stimulate its gene expression compared to CaCl2 . Pharmacokinetic analysis of mice confirmed the calcium absorption-promoting effect of sucrose-calcium chelate, as evident by the higher serum calcium level (44.12±1.90mg/L vs. 37.42±1.88mmol/L) and intestinal PMCA1b gene expression than CaCl2 . These findings offer a new understanding of how sucrose-calcium chelate enhances intestinal calcium absorption and could be used as an ingredient in functional foods to treat calcium deficiency. PRACTICAL APPLICATION: The development of high-quality calcium supplements is crucial for addressing the various adverse symptoms associated with calcium deficiency. This study aimed to prepare a sucrose-calcium chelate and analyze its structure, as well as its potential to enhance calcium absorption in Caco-2 monolayer cells and mice. The results demonstrated that the sucrose-calcium chelate effectively promoted calcium absorption. Notably, its ability to enhance calcium transport was linked to its strong binding with PMCA1b, a calcium transporter located on the basement membrane, and its capacity to stimulate PMCA1b gene expression. These findings contribute to a deeper understanding of how the sucrose-calcium chelate enhances intestinal calcium absorption and suggest its potential use as an ingredient in functional foods for treating calcium deficiency.

Isobavachalcone, a natural sirtuin 2 inhibitor, exhibits anti-triple-negative breast cancer efficacy in vitro and in vivo.

Triple-negative breast cancer (TNBC) is the most aggressive and lethal clinical subtype and lacks effective targeted therapies at present. Isobavachalcone (IBC), the main active component of Psoralea corylifolia L., has potential anticancer effects. Herein, we identified IBC as a natural sirtuin 2 (SIRT2) inhibitor and characterized the potential mechanisms underlying the inhibition of TNBC. Molecular dynamics analysis, enzyme activity assay, and cellular thermal shift assay were performed to evaluate the combination of IBC and SIRT2. The therapeutic effects, mechanism, and safety of IBC were analyzed in vitro and in vivo using cellular and xenograft models. IBC effectively inhibited SIRT2 enzyme activity with an IC50 value of 0.84 ± 0.22 μM by forming hydrogen bonds with VAL233 and ALA135 within its catalytic domain. In the cellular environment, IBC bound to and stabilized SIRT2, consequently inhibiting cellular proliferation and migration, and inducing apoptosis and cell cycle arrest by disrupting the SIRT2/α-tubulin interaction and inhibiting the downstream Snail/MMP and STAT3/c-Myc pathways. In the in vivo model, 30 mg/kg IBC markedly inhibited tumor growth by targeting the SIRT2/α-tubulin interaction. Furthermore, IBC exerted its effects by inducing apoptosis in tumor tissues and was well-tolerated. IBC alleviated TNBC by targeting SIRT2 and triggering the reactive oxygen species ROS/β-catenin/CDK2 axis. It is a promising natural lead compound for future development of SIRT2-targeting drugs.

AAA237, an SKP2 inhibitor, suppresses glioblastoma by inducing BNIP3-dependent autophagy through the mTOR pathway

BackgroundGlioblastoma (GBM) is the most common brain tumor with the worst prognosis. Temozolomide is the only first-line drug for GBM. Unfortunately, the resistance issue is a classic problem. Therefore, it is essential to develop new drugs to treat GBM. As an oncogene, Skp2 is involved in the pathogenesis of various cancers including GBM. In this study, we investigated the anticancer effect of AAA237 on human glioblastoma cells and its underlying mechanism.MethodsCCK-8 assay was conducted to evaluate IC50 values of AAA237 at 48, and 72 h, respectively. The Cellular Thermal Shift Assay (CETSA) was employed to ascertain the status of Skp2 as an intrinsic target of AAA237 inside the cellular milieu. The EdU-DNA synthesis test, Soft-Agar assay and Matrigel assay were performed to check the suppressive effects of AAA237 on cell growth. To identify the migration and invasion ability of GBM cells, transwell assay was conducted. RT-qPCR and Western Blot were employed to verify the level of BNIP3. The mRFP-GFP-LC3 indicator system was utilized to assess alterations in autophagy flux and investigate the impact of AAA237 on the dynamic fusion process between autophagosomes and lysosomes. To investigate the effect of compound AAA237 on tumor growth in vivo, LN229 cells were injected into the brains of mice in an orthotopic model.ResultsAAA237 could inhibit the growth of GBM cells in vitro. AAA237 could bind to Skp2 and inhibit Skp2 expression and the degradation of p21 and p27. In a dose-dependent manner, AAA237 demonstrated the ability to inhibit colony formation, migration, and invasion of GBM cells. AAA237 treatment could upregulate BNIP3 as the hub gene and therefore induce BNIP3-dependent autophagy through the mTOR pathway whereas 3-MA can somewhat reverse this process. In vivo, the administration of AAA237 effectively suppressed the development of glioma tumors with no side effects.ConclusionCompound AAA237, a novel Skp2 inhibitor, inhibited colony formation, migration and invasion of GBM cells in a dose-dependent manner and time-dependent manner through upregulating BNIP3 as the hub gene and induced BNIP3-dependent autophagy through the mTOR pathway therefore it might be a viable therapeutic drug for the management of GBM.Graphical

Oridonin inhibits bladder cancer survival and immune escape by covalently targeting HK1

BackgroundHexokinase I (HK1) is highly expressed in a variety of malignancies, regulates glycolytic pathway in cancer cells, and thus considered to be one of the promising molecular targets for cancer therapy. Nonetheless, the development of a specific inhibitor against HK1 remains elusive. PurposeThis study aims to elucidate the mechanism by which oridonin inhibits the proliferation and immune evasion of bladder cancer cells, specifically through the suppression of HK1. MethodsTo examine the mechanisms by which oridonin directly binds to cysteines of HK1 and inhibits bladder cancer growth, this study utilized a variety of methods. These included the Human Proteome Microarray, Streptavidin-agarose affinity assay, Biolayer Interferometry (BLI) ainding analysis, Mass Spectrometry, Cellular Thermal Shift Assay, Extracellular Acidification Rate measurement, and Xenotransplant mouse models. ResultsAs indicated by our current findings, oridonin forms a covalent bond with Cys-813, located adjacently to glucose-binding domain of HK1. This suppresses the enzymatic activity of HK1, leading to an effective reduction of glycolysis, which triggers cell death via apoptosis in cells derived from human bladder cancer. Significantly, oridonin also inhibits lactate-induced PD-L1 expression in bladder cancer. Furthermore, pairing oridonin with a PD-L1 inhibitor amplifies the cytotoxicity of CD8+ T cells against bladder cancer. ConclusionThis research strongly suggests that oridonin serves as a covalent inhibitor of HK1. Moreover, it indicates that functional cysteine residue of HK1 could operate as viable targets for selective inhibition. Consequently, oridonin exhibits substantial potential for the evolution of anti-cancer agents targeting the potential therapeutic target HK1 via metabolism immunomodulation.

Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

A Network Pharmacology and Molecular Dynamics Simulation-Based Study of Qing Run Hua Jie Decoction in Interstitial Pneumonia Treatment.

This study is dedicated to revealing the potential mechanism of Qin Run Hua Jie (QRHJ) decoction in Interstitial pneumonia (IP) treatment. The TCMSP database predicted the chemical components and targets of QRHJ decoction, and the IP-related genes were from the Genecards database. Cytoscape software was used to establish the interaction network. R package clusterProfiler was utilized for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The molecular docking analysis of target proteins and the corresponding active pharmaceutical ingredients in the core position of the interaction network was conducted. Then, molecular dynamics (MD) simulations of a potential active substance and its key targets were performed. The binding efficiency of EGFR and luteolin, HIF1A and diosgenin was detected by cellular thermal shift assay (CETSA), and protein expression was measured by Western blot. CCK-8 was used to detect cell activity. A total of 153 active ingredients, 127 targets and 362 IP-related genes were obtained. KEGG enrichment analysis identified IP-related signaling pathways including HIF-1 signaling pathway and TNF signaling pathway. The two key components luteolin and diosgenin stably bound to the key targets EGFR and HIF1A. Cell experiments further showed that EGFR and luteolin, HIF1A and diosgenin bound to exert anti-fibrotic effects. As an active ingredient of QRHJ decoction, luteolin and diosgenin may exert therapeutic effect on IP through binding to the key target EGFR and HIF1A. This work initially revealed the key molecular mechanism of QRHJ decoction in IP treatment and offered theoretical evidence.

Identifying Marine-Derived Tanzawaic Acid Derivatives as Novel Inhibitors against Osteoclastogenesis and Osteoporosis via Downregulation of NF-κB and NFATc1 Activation.

To discover novel osteoclast-targeting antiosteoporosis leads from natural products, we identified 40 tanzawaic acid derivatives, including 22 new ones (1-8, 14-19, 27-32, 37, and 38), from the South China Sea mangrove-derived fungus Penicillium steckii SCSIO 41025. Penicisteck acid F (2), one of the new derivatives showing the most potent NF-κB inhibitory activity, remarkably inhibited osteoclast generation in vitro. Mechanistically, 2 reduced RANKL-induced IκBα degradation, NF-κB p65 nuclear translocation, the activation and nuclear translocation of NFATc1, and the relevant mRNA expression. NF-κB p65 could be a potential molecular target for 2, which has been further determined by the cellular thermal shift assay, surface plasmon resonance, and the gene knock-down assay. Moreover, 2 could also alleviate osteoporosis in ovariectomized mice by reducing the quantities of osteoclasts. Our finding offered a novel potential inhibitor of osteoclastogenesis and osteoporosis for further development of potent antiosteoporosis agents.

Dopamine modification of glycolytic enzymes impairs glycolysis: possible implications for Parkinson’s disease

BackgroundParkinson’s disease (PD), a chronic and severe neurodegenerative disease, is pathologically characterized by the selective loss of nigrostriatal dopaminergic neurons. Dopamine (DA), the neurotransmitter produced by dopaminergic neurons, and its metabolites can covalently modify proteins, and dysregulation of this process has been implicated in neuronal loss in PD. However, much remains unknown about the protein targets.MethodsIn the present work, we designed and synthesized a dopamine probe (DA-P) to screen and identify the potential protein targets of DA using activity-based protein profiling (ABPP) technology in combination with liquid chromatography-tandem mass spectrometry (LC–MS/MS). In situ pull-down assays, cellular thermal shift assays (CETSAs) and immunofluorescence were performed to confirm the DA modifications on these hits. To investigate the effects of DA modifications, we measured the enzymatic activities of these target proteins, evaluated glycolytic stress and mitochondrial respiration by Seahorse tests, and systematically analyzed the changes in metabolites with unbiased LC–MS/MS-based non-targeted metabolomics profiling.ResultsWe successfully identified three glycolytic proteins, aldolase A, α-enolase and pyruvate kinase M2 (PKM2), as the binding partners of DA. DA bound to Glu166 of α-enolase, Cys49 and Cys424 of PKM2, and Lys230 of aldolase A, inhibiting the enzymatic activities of α-enolase and PKM2 and thereby impairing ATP synthesis, resulting in mitochondrial dysfunction.ConclusionsRecent research has revealed that enhancing glycolysis can offer protection against PD. The present study identified that the glycolytic pathway is vulnerable to disruption by DA, suggesting a promising avenue for potential therapeutic interventions. Safeguarding glycolysis against DA-related disruption could be a potential therapeutic intervention for PD.

A natural small molecule aspidosperma-type alkaloid, hecubine, as a new TREM2 activator for alleviating lipopolysaccharide-induced neuroinflammation in vitro and in vivo

Neuroinflammation and oxidative stress play a crucial role in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease. The triggering receptor expressed on myeloid cells 2 (TREM2), highly expressed by microglia in the central nervous system (CNS), can modulate neuroinflammatory responses. Currently, there are no approved drugs specifically targeting TREM2 for CNS diseases. Aspidosperma alkaloids have shown potential as anti-inflammatory and neuroprotective agents. This study aimed to elucidate the potential therapeutic effect of Hecubine, a natural aspidosperma-type alkaloid, as a TREM2 activator in lipopolysaccharide (LPS)-stimulated neuroinflammation in in vitro and in vivo models. In this study, molecular docking and cellular thermal shift assay (CTSA) were employed to investigate the interaction between Hecubine and TREM2. Enzyme-linked immunosorbent assay (ELISA), quantitative PCR, immunofluorescence, Western blotting, and shRNA gene knockdown were used to assess the anti-neuroinflammatory and antioxidant effects of Hecubine in microglial cells and zebrafish. Our results revealed that Hecubine directly interacted with TREM2, leading to its activation. Knockdown of TREM2 mRNA expression significantly abolished the anti-inflammatory and antioxidant effects of Hecubine on LPS-stimulated proinflammatory mediators (NO, TNF-α, IL-6, and IL-1β) and oxidative stress in microglia cells. Furthermore, Hecubine upregulated Nrf2 expression levels while downregulating TLR4 signaling expression levels both in vivo and in vitro. Silencing TREM2 upregulated TLR4 and downregulated Nrf2 signaling pathways, mimicking the effect of Hecubine, further supporting TREM2 as the drug target by which Hecubine inhibits neuroinflammation. In conclusion, this is the first study to identify a small molecule, namely Hecubine directly targeting TREM2 to mediate anti-neuroinflammation and anti-oxidative effects, which serves as a potential therapeutic agent for the treatment of neural inflammation-associated CNS diseases.

Unveiling the Potential of Nelumbo nucifera-Derived Liensinine to Target The Myostatin Protein and to Counteract Muscle Atrophy.

Muscle atrophy refers to a decline in muscle mass and function, which has become a global concern due to the aging population. Various clinical trials have investigated the inhibitors of myostatin (MSTN). They have shown promising improvements in muscle function and quality of life. However, there are no drugs specifically targeting MSTN that have been approved for clinical use. In this study, we virtually screened liensinine (LIE), a food (Nelumbo nucifera)-derived compound, with low toxicity, from over 1.1 million compounds. We subsequently identified it as a potential candidate that targets MSTN by a cellular thermal shift assay (CETSA) and drug affinity response target stability (DARTS) assay. Further validation through cellular and in vivo studies demonstrated its promising potential in combating muscle atrophy. The mechanism of action may involve hindering the interaction between MSTN and the activin receptor type IIB (ActRIIB) and downregulating the expression of downstream proteins, including the muscle RING-finger protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx)/Atrogin-1, ultimately promoting muscle regeneration. These results provide a strong foundation for future studies to explore the therapeutic potential of LIE in clinical settings.

Deep learning based CETSA feature prediction cross multiple cell lines with latent space representation

Mass spectrometry-coupled cellular thermal shift assay (MS-CETSA), a biophysical principle-based technique that measures the thermal stability of proteins at the proteome level inside the cell, has contributed significantly to the understanding of drug mechanisms of action and the dissection of protein interaction dynamics in different cellular states. One of the barriers to the wide applications of MS-CETSA is that MS-CETSA experiments must be performed on the specific cell lines of interest, which is typically time-consuming and costly in terms of labeling reagents and mass spectrometry time. In this study, we aim to predict CETSA features in various cell lines by introducing a computational framework called CycleDNN based on deep neural network technology. For a given set of n cell lines, CycleDNN comprises n auto-encoders. Each auto-encoder includes an encoder to convert CETSA features from one cell line into latent features in a latent space Z\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathbb {Z}$$\\end{document}. It also features a decoder that transforms the latent features back into CETSA features for another cell line. In such a way, the proposed CycleDNN creates a cyclic prediction of CETSA features across different cell lines. The prediction loss, cycle-consistency loss, and latent space regularization loss are used to guide the model training. Experimental results on a public CETSA dataset demonstrate the effectiveness of our proposed approach. Furthermore, we confirm the validity of the predicted MS-CETSA data from our proposed CycleDNN through validation in protein–protein interaction prediction.

Integrating network pharmacology, experimental validation and molecular docking to reveal the alleviation of Yinhuang granule on idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the abnormal proliferation of fibroblast and excessive deposition of extracellular matrix (ECM), accompanied by inflammation and ultimately respiratory failure. Yinhuang granule (YHG), with clinical properties of clearing heat, detoxifying and anti-inflammation, is commonly used to heal upper respiratory diseases in China for decades. PurposeTo explore the improvement of YHG on bleomycin (BLM)-induced IPF in mice and its possible engaged mechanism. MethodsThe mortality rate was recorded, lung function was determined and hematoxylin-eosin (H&E) staining was carried out to explore the alleviation of YHG on BLM-caused IPF in mice. Hydroxyproline, collagen I and collagen III contents were detected, and Sirius red and Masson staining were conducted to evaluate YHG's alleviation on lung fibrosis. The underlying mechanism was predicted by network pharmacology, and confirmed by Real-time polymerase chain reaction (RT-PCR), Western-blot (WB) and enzyme linked immunosorbent assay (ELISA). The binding affinity between related key proteins and active compounds in YHG was calculated by using molecular docking, and further validated by cellular thermal shift assay (CESTA). ResultsYHG (400, 800 mg/kg) weakened lung damage and pulmonary fibrosis in mice induced by BLM. Network pharmacology and experimental validation displayed that inflammation and angiogenesis participated in the YHG-provided improvement on IPF, and key involved molecules included tumor necrosis factor-α (TNFα), vascular endothelial growth factor-A (VEGFA), interleukine-6 (IL-6), etc. The data of molecular docking presented that some main active compounds from YHG had a high binding affinity with TNFR1 or VEGFR2, and some of them were further validated by CESTA. ConclusionYHG effectively improved the BLM-induced IPF in mice via reducing inflammation and angiogenesis.

Carnosol ameliorated cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Introduction: Carnosol exhibited ameliorating effects on muscle atrophy of mice developed cancer cachexia in our previous research. Method: Here, the ameliorating effects of carnosol on the C2C12 myotube atrophy result from simulated cancer cachexia injury, the conditioned medium of the C26 tumor cells or the LLC tumor cells, were observed. To clarify the mechanisms of carnosol, the possible direct target proteins of carnosol were searched using DARTS (drug affinity responsive target stability) assay and then confirmed using CETSA (cellular thermal shift assay). Furthermore, proteomic analysis was used to search its possible indirect target proteins by comparing the protein expression profiles of C2C12 myotubes under treatment of C26 medium, with or without the presence of carnosol. The signal network between the direct and indirect target proteins of carnosol was then constructed. Results: Our results showed that, Delta-1-pyrroline-5-carboxylate synthase (P5CS) might be the direct target protein of carnosol in myotubes. The influence of carnosol on amino acid metabolism downstream of P5CS was confirmed. Carnosol could upregulate the expression of proteins related to glutathione metabolism, anti-oxidant system, and heat shock response. Knockdown of P5CS could also ameliorate myotube atrophy and further enhance the ameliorating effects of carnosol. Discussion: These results suggested that carnosol might ameliorate cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Kaempferol inhibits colorectal cancer metastasis through circ_0000345 mediated JMJD2C/β-catenin signalling pathway

BackgroundRecurrence and metastasis are the main causes of disease deterioration in colorectal cancer (CRC) patients, yet efficient therapeutic strategies are lacking. Natural compounds for efficient antitumour therapeutics are becoming increasingly prominent. Kaempferol, one of the main components of flavonoids in plants, displays a variety of pharmacological activities. Our preliminary experiments suggested that kaempferol could inhibit CRC metastasis and is significantly associated with the β-catenin signalling pathway. Moreover, we also defined the regulatory roles of JMJD2C in β-catenin signalling in our previous work. PurposeThis study aims to reveal the mechanism by which kaempferol inhibits CRC progression and regulates the JMJD2C/β-catenin signalling pathway. MethodsThe migratory capabilities of CRC cells after kaempferol intervention were measured by scratch wound healing and transwell assays. Circ_0000345 knockdown CRC stable cell lines were generated by lentivirus infection. The possible mechanism of kaempferol on circ_0000345 was verified by molecular-protein docking and verification program cellular thermal shift assay (CETSA). A dual luciferase reporter gene assay was carried out for the targeting relationship among circ_0000345, miR-205-5p and JMJD2C. Fluorescence in situ hybridization (FISH) was performed to determine the expression of circ_0000345 in tumour tissues. A pulmonary metastatic model of CRC in vitro was built to assess the antimetastatic effect and mechanism of kaempferol in vivo. ResultsIn vitro, kaempferol inhibits the ability to migrate of CRC cells by reducing the activation of the JMJD2C/β-catenin signalling pathway. MiR-205-5p is a key bridge for kaempferol to inhibit the expression of JMJD2C. The function of miR-205-5p is impeded by circ_0000345, which shows higher expression levels in human metastatic CRC tissues than nonmetastatic CRC tissues, and its formation is regulated by the RNA-binding proteins HNRNPK and HNRNPL. Mechanistically, kaempferol physically interacts with HNRNPK and HNRNPL to suppress JMJD2C by downregulating the expression of circ_0000345. In vivo, kaempferol suppresses CRC lung metastasis. Kaempferol inhibits the activation of JMJD2C/β-catenin signalling through reducing the expression of circ_0000345 in the CRC lung metastasis model. ConclusionCirc_0000345 enhances activation of the JMJD2C/β-catenin signalling pathway through miR-205-5p to promote CRC metastasis. Kaempferol inhibits CRC metastasis through the circ_0000345-mediated JMJD2C/β-catenin signalling pathway, and this effect is influenced as a direct consequence of the binding of kaempferol with HNRNPK and HNRNPL. This provides promising therapeutic and/or adjuvant agents for advanced CRC and sheds light on the multifaceted role of phytomedicine in cancer.

Lotusine ameliorates propionic acid-induced autism spectrum disorder-like behavior in mice by activating D1 dopamine receptor in medial prefrontal cortex.

Autism spectrum disorder (ASD) is a multifaceted neuropsychiatric condition for which effective drug therapy for core clinical symptoms remains elusive. Lotusine, known for its neuroprotective properties in the treatment of neurological disorders, holds potential in addressing ASD. Nevertheless, its specific efficacy in ASD remains uncertain. This study aims to investigate the therapeutic potential of lotusine in ASD and elucidate the underlying molecular mechanisms. We induced an ASD mouse model through intracerebroventricular-propionic acid (ICV-PPA) injection for 7 days, followed by lotusine administration for 5 days. The efficacy of lotusine was evaluated through a battery of behavioral tests, including the three-chamber social test. The underlying mechanisms of lotusine action in ameliorating ASD-like behavior were investigated in the medial prefrontal cortex (mPFC) using whole-cell patch-clamp recordings, western blotting, immunofluorescence staining, molecular docking, and cellular thermal shift assay. The efficacy and mechanisms of lotusine were further validated in vitro. Lotusine effectively alleviated social deficits induced by ICV-PPA injection in mice by counteracting the reduction in miniature excitatory postsynaptic current frequency within the mPFC. Moreover, lotusine enhanced neuronal activity and ameliorated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor dysfunction in ICV-PPA infusion mice by upregulating c-fos, p-GluA1 Ser 845, and p-GluA1 Ser 831 protein levels within the mPFC. Our findings also suggest that lotusine may exert its effects through modulation of the D1 dopamine receptor (DRD1). Furthermore, the rescuing effects of lotusine were nullified by a DRD1 antagonist in PC12 cells. In summary, our results revealed that lotusine ameliorates ASD-like behavior through targeted modulation of DRD1, ultimately enhancing excitatory synaptic transmission. These findings highlight the potential of lotusine as a nutritional supplement in the treatment of ASD.