Proteome-wide Ligand and Target Discovery by Using Strain-Enabled Cyclopropane Electrophiles.

The evolving use of covalent ligands as chemical probes and therapeutic agents could greatly benefit from an expanded array of cysteine-reactive electrophiles for efficient and versatile proteome profiling. Herein, to expand the current repertoire of cysteine-reactive electrophiles, we developed a new class of strain-enabled electrophiles based on cyclopropanes. Proteome profiling has unveiled that C163 of lactate dehydrogenase A (LDHA) and C88 of adhesion regulating molecule 1 (ADRM1) are ligandable residues to modulate the protein functions. Moreover, fragment-based ligand discovery (FBLD) has revealed that one fragment (Y-35) shows strong reactivity toward C66 of thioredoxin domain-containing protein 12 (TXD12), and its covalent binding has been demonstrated to impact its downstream signal pathways. TXD12 plays a pivotal role in enabling Y-35 to exhibit its antisurvival and antiproliferative effects. Finally, dicarbonitrile-cyclopropane has been demonstrated to be an electrophilic warhead in the development of GSTO1-involved dual covalent inhibitors, which is promising to alleviate drug resistance.

Small Molecule-Induced Post-Translational Acetylation of Catalytic Lysine of Kinases in Mammalian Cells.

Reversible lysine acetylation is an important post-translational modification (PTM). This process in cells is typically carried out enzymatically by lysine acetyltransferases and deacetylases. The catalytic lysine in the human kinome is highly conserved and ligandable. Small-molecule strategies that enable post-translational acetylation of the catalytic lysine on kinases in a target-selective manner therefore provide tremendous potential in kinase biology. Herein, we report the first small molecule-induced chemical strategy capable of global acetylation of the catalytic lysine on kinases from mammalian cells. By surveying various lysine-acetylating agents installed on a promiscuous kinase-binding scaffold, Ac4 was identified and shown to effectively acetylate the catalytic lysine of >100 different protein kinases from live Jurkat/K562 cells. In order to demonstrate that this strategy was capable of target-selective and reversible chemical acetylation of protein kinases, we further developed six acetylating compounds on the basis of VX-680 (a noncovalent inhibitor of AURKA). Among them, Ac13/Ac14, while displaying excellent in vitro potency and sustained cellular activity against AURKA, showed robust acetylation of its catalytic lysine (K162) in a target-selective manner, leading to irreversible inhibition of endogenous kinase activity. The reversibility of this chemical acetylation was confirmed on Ac14-treated recombinant AURKA protein, followed by deacetylation with SIRT3 (a lysine deacetylase). Finally, the reversible Ac13-induced acetylation of endogenous AURKA was demonstrated in SIRT3-transfected HCT116 cells. By disclosing the first cell-active acetylating compounds capable of both global and target-selective post-translational acetylation of the catalytic lysine on kinases, our strategy could provide a useful chemical tool in kinase biology and drug discovery.

Structural basis for DNMT3A-mediated de novo DNA methylation.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.

Nicotinate-curcumin improves NASH by inhibiting the AKR1B10/ACCα-mediated triglyceride synthesis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver condition. However, the potential therapeutic benefits and underlying mechanism of nicotinate-curcumin (NC) in the treatment of NASH remain uncertain. METHODS: A rat model of NASH induced by a high-fat and high-fructose diet was treated with nicotinate-curcumin (NC, 20, 40 mg·kg- 1), curcumin (Cur, 40 mg·kg- 1) and metformin (Met, 50 mg·kg- 1) for a duration of 4 weeks. The interaction between NASH, Cur and Aldo-Keto reductase family 1 member B10 (AKR1B10) was filter and analyzed using network pharmacology. The interaction of Cur, NC and AKR1B10 was analyzed using molecular docking techniques, and the binding energy of Cur and NC with AKR1B10 was compared. HepG2 cells were induced by Ox-LDL (25 µg·ml- 1, 24 h) in high glucose medium. NC (20µM, 40µM), Cur (40µM) Met (150µM) and epalrestat (Epa, 75µM) were administered individually. The activities of ALT, AST, ALP and the levels of LDL, HDL, TG, TC and FFA in serum were quantified using a chemiluminescence assay. Based on the changes in the above indicators, score according to NAS standards. The activities of Acetyl-CoA and Malonyl-CoA were measured using an ELISA assay. And the expression and cellular localization of AKR1B10 and Acetyl-CoA carboxylase (ACCα) in HepG2 cells were detected by Western blotting and immunofluorescence. RESULTS: The results of the animal experiments demonstrated that NASH rat model induced by a high-fat and high-fructose diet exhibited pronounced dysfunction in liver function and lipid metabolism. Additionally, there was a significant increase in serum levels of FFA and TG, as well as elevated expression of AKR1B10 and ACCα, and heightened activity of Acetyl-CoA and Malonyl-CoA in liver tissue. The administration of NC showed to enhance liver function in rats with NASH, leading to reductions in ALT, AST and ALP levels, and decrease in blood lipid and significant inhibition of FFA and TG synthesis in the liver. Network pharmacological analysis identified AKR1B10 and ACCα as potential targets for NASH treatment. Molecular docking studies revealed that both Cur and NC are capable of binding to AKR1B10, with NC exhibiting a stronger binding energy to AKR1B10. Western blot analysis demonstrated an upregulation in the expression of AKR1B10 and ACCα in the liver tissue of NASH rats, accompanied by elevated Acetyl-CoA and Malonyl-CoA activity, and increased levels of FFA and TG. The results of the HepG2 cell experiments induced by Ox-LDL suggest that NC significantly inhibited the expression and co-localization of AKR1B10 and ACCα, while also reduced levels of TC and LDL-C and increased level of HDL-C. These effects are accompanied by a decrease in the activities of ACCα and Malonyl-CoA, and levels of FFA and TG. Furthermore, the impact of NC appears to be more pronounced compared to Cur. CONCLUSION: NC could effectively treat NASH and improve liver function and lipid metabolism disorder. The mechanism of NC is related to the inhibition of AKR1B10/ACCα pathway and FFA/TG synthesis of liver.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) have emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme's active site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles.

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

2-Ethynylbenzaldehyde-Based, Lysine-Targeting Irreversible Covalent Inhibitors for Protein Kinases and Nonkinases.

Lysine-targeting irreversible covalent inhibitors have attracted growing interests in recent years, especially in the fields of kinase research. Despite encouraging progress, few chemistries are available to develop inhibitors that are exclusively lysine-targeting, selective, and cell-active. We report herein a 2-ethynylbenzaldehyde (EBA)-based, lysine-targeting strategy to generate potent and selective small-molecule inhibitors of ABL kinase by selectively targeting the conserved catalytic lysine in the enzyme. We showed the resulting compounds were cell-active, capable of covalently engaging endogenous ABL kinase in K562 cells with long-residence time and few off-targets. We further validated the generality of this strategy by developing EBA-based irreversible inhibitors against EGFR (a kinase) and Mcl-1 (a nonkinase) that covalently reacted with the catalytic and noncatalytic lysine within each target.

N-N-Bridged Polynuclear POM-Based Coordination Polymers Based on a V-Type Ligand: Proton Conduction and Magnetism.

The construction of polyoxometalate (POM)-based coordination polymers, in the presence of a nitrogen heterocyclic ligand, is intriguing due to the potential for obtaining diverse structures. These structures exhibit extensive application possibilities in the fields of proton conductivity and magnetism. Herein, four new POM-based polynuclear coordination polymers with the formulas of {[Fe2(btb)3(H2O)2(SiW12O40)]·3H2O}n (1), {[Cd2(btb)2(H2O)6(HPMoVI10MoV2O40)]·2H2O}n (2), {[Co3(OH)2(btb)2(H2O)5(HPMoVI10MoV2O40)]·7H2O}n (3), and {[Cu3(OH)(btb)2(H2O)(HP2Mo5O23)]·6H2O}n (4) have been prepared using the V-type 1,3-bis(4H-1,2,4-triazole-4-yl)benzene (btb) ligand. Compounds 1 and 2 feature similar two-dimensional (2D) structures, derived from the binuclear Fe2N6 and Cd2N4 subunits connected by tridentate btb ligands. Meanwhile, in compound 3, hexanuclear Co6(OH)4 units are bound by quadridentate btb ligands forming a 2D layer with the same 4-c sql topology simplification as compounds 1 and 2. In compound 1, Keggin-type polyoxoanions are monodentate-coordinated to metal ions and suspended on the 2D structure, while, in compounds 2 and 3, they act as discrete counterions residing in the interstitial spaces between two adjacent layers, thereby extending the 2D structures into 3D structures through hydrogen bonding interactions. In compound 4, trinuclear Cu3(OH) subunits are further constructed into a 3D framework through cooperation with four tridentate and quadridentate btb ligands as well as Strandberg-type anions. Furthermore, the proton conduction of the four compounds has been investigated. They display high proton conductivities at 358 K and 98% RH with powdered samples, which are 1.26 × 10-3, 1.24 × 10-3, 3.24 × 10-4, and 2.57 × 10-4 S cm-1, respectively. Interestingly, by mixing with Nafion, the composite membranes of compounds 2 and 4 exhibit enhanced proton conductivities, measuring at 4.87 × 10-2 and 1.28 × 10-2 S cm-1, respectively, at 358 K and 98% RH, which suggests excellent potential for applications. In addition, compounds 1, 3, and 4 display antiferromagnetic behaviors due to similar magnetic interactions. This work can provide research insights into the assembly of 2D POM-based coordination polymers with nitrogen heterocyclic ligands and Keggin-type POMs and further promote their research progress in proton conduction.

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

Circ_C4orf36 Promotes the Proliferation and Osteogenic Differentiation of BMSCs by Regulating VEGFA.

Fracture healing is a complicated process containing the regulation of cellular process. It has been reported that circRNAs are involved in fracture healing. Our study aims to explore the role and mechanism of circ_C4orf36 in fracture healing. Here, we found that the expressions of Circ_C4orf36 and VEGFA were increased during osteoblast differentiation in MC3T3-E1 cells. Circ_C4orf36 overexpression could accelerate the proliferation and migration, as well as osteoblast differentiation in MC3T3-E1 cells, as well as increased ALP activity and osteogenic markers (Runx2, OCN) via upregulating VEGFA expression. Mechanistically, circ_C4orf36 facilitated the expression of VEGFA by recruiting EIF4A3. Taken together, our results elucidated that circ_C4orf6 promoted the migration, proliferation and osteoblast differentiation in BMSCs by upregulating VEGFA, which indicated that circ_C4orf36 might be a potential target in fracture healing treatment.

The MFF-SIRT1/3 axis, regulated by miR-340-5p, restores mitochondrial homeostasis of hypoxia-induced pulmonary artery smooth muscle cells.

Mitochondrial dynamics and quality control play a central role in the maintenance of the proliferation-apoptosis balance, which is closely related to the progression of pulmonary arterial hypertension (PAH). However, the exact mechanism of this balance remains unknown. Pulmonary artery smooth muscle cells (PASMCs) were cultured in hypoxia condition for constructing a PAH model in vitro. The expression of genes and proteins were determined by qRT-PCR and western bolt assays. Cell proliferation-apoptosis balance were tested by MTT, EdU and TUNEL assays. The mitochondrial functions were assessed by flow cytometry, JC-1, Mito tracker red staining, and corresponding kits. Besides, the molecular interaction was validated by dual-luciferase reporter assay. MFF was overexpressed in hypoxia-treated PAMSCs. Knockdown of MFF significantly repressed the excessive proliferation but enhanced cell apoptosis in hypoxia-treated PAMSCs. Moreover, MFF silencing improved mitochondrial function of hypoxia-treated PAMSCs by increasing ATP production and decreasing ROS release and mitochondrial fission. Mechanistically, MFF was a directly target of miR-340-5p, and could negatively regulate SIRT1/3 expression. Subsequently, functional rescue assays showed that the biological effects of MFF in hypoxia-treated PAMSCs were negatively regulated by miR-340-5p and depended on the regulation on SIRT1/3 pathway. These results provided evidences that miR-340-5p regulated MFF-SIRT1/3 axis to improve mitochondrial homeostasis and proliferation-apoptosis imbalance of hypoxia-treated PAMSCs, which provided a theoretical basis for the prevention and treatment of PAH.

Preparation and Characterization of Polysaccharides from Turpiniae Folium and Its Antioxidative, Anti-Inflammatory Activities and Antiproliferative Effect on VSMCs.

Turpiniae Folium, the dried leaves of Turpinia arguta Seem., is a kind of historic traditional Chinese medicine. Here, based on our previous study, we extracted the Turpiniae Folium polysaccharides (TFP) and isolated three polysaccharide fractions from TFP. Then, TFP and one of the major polysaccharide fractions (TFP-1a) were identified through HPLC, HPGPC, and ATR-FTIR. Furthermore, the evaluations of their antioxidative, anti-inflammatory activities and inhibitory effect on angiotensin II-induced vascular smooth muscle cells (VSCMs) proliferation in vitro were conducted. Both TFP and TFP-1a showed strong hydroxyl radical scavenging, DPPH radical scavenging, and Fe2+ chelating activities, and exerted strong anti-inflammatory activity. Moreover, TFP and TFP-1a also possessed a strong inhibitory effect on Ang II-induced VSCMs proliferation. On these premises, we inferred that TFP and TFP-1a could be potential and promising natural antioxidants, anti-inflammatory agents, and implicated to treat cardiovascular disease.

Cell-Active, Reversible, and Irreversible Covalent Inhibitors That Selectively Target the Catalytic Lysine of BCR-ABL Kinase.

Despite recent interests in developing lysine-targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell-active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde-based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR-ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug-resistant ABL mutations. Among them, the salicylaldehyde-containing A5 is the first-ever reversible covalent ABL inhibitor that possessed time-dependent ABL inhibition with prolonged residence time and few cellular off-targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

Developing a Peak Extraction and Retention (PEER) Algorithm for Improving the Temporal Resolution of Raman Spectroscopy.

In spectroscopic analysis, push-to-the-limit sensitivity is one of the important topics, particularly when facing the qualitative and quantitative analyses of the trace target. Normally, the effective recognition and extraction of weak signals are the first key steps, for which there has been considerable effort in developing various denoising algorithms for decades. Nevertheless, the lower the signal-to-noise ratio (SNR), the greater the deviation of the peak height and shape during the denoising process. Therefore, we propose a denoising algorithm along with peak extraction and retention (PEER). First, both the first and second derivatives of the Raman spectrum are used to determine Raman peaks with a high SNR whose peak information is kept away from the denoising process. Second, an optimized window smoothing algorithm is applied to the left part of the Raman spectrum, which is combined with the untreated Raman peaks to obtain the denoised Raman spectrum. The PEER algorithm is demonstrated with much better signal extraction and retention and successfully improves the temporal resolution of Raman imaging of a living cell by at least 1 order of magnitude higher than those by traditional algorithms.

Cargo-laden erythrocyte ghosts target liver mediated by macrophages.

Liver-targeted cargo delivery possesses great potential for the treatment of liver disease. It is urgent to find an efficient and biocompatible liver targeted delivery system. This study focused on the liver targeting properties of erythrocyte ghosts and its possible mechanism. Herein, we optimized conditions to fabricate human and mouse erythrocyte ghosts with sufficient room capable of incorporating various model substances. Erythrocyte ghosts are biocompatible cargo carriers because it is derived from autologous red blood cells (RBCs), and the cell size, zeta potential, and biconcave-disk shape of the ghosts were consistent with those of RBCs. An in vivo imaging system and positron emission tomography/computed tomography imaging showed that the ghosts were captured mainly in the liver by intravenous injection of fluorescence or 18F-fluorodeoxyglucose (FDG)-labelled ghosts into mice. In contrast, the main concentration of naked octreotide was trapped in the lungs while naked 18F-FDG was trapped in the heart. However, the concentration of cargo-loaded ghosts decreased significantly in the liver in macrophage-depleted mice. Accordingly, in vitro experiments showed that higher phosphatidylserine exposure was observed in the ghosts (38.9 %) compared to normal erythrocytes (0.69 %), and the phagocytic activity of the macrophage RAW 264.7. on the ghosts was significantly higher than that of normal erythrocytes (p < 0.001). Together they indicate that erythrocyte ghosts show liver targeting properties, and possibly owing to macrophage phagocytosis. This promising and effective therapeutic delivery system may provide therapeutic benefits for liver disease.

Sodium tanshinone IIA sulfonate protects against Aß-induced cell toxicity through regulating Aß process.

Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aß-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aß (10 µmol/L)-induced cell toxicity in a dose-dependent manner in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH-SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL-1ß, IL-6 and TNF-α) in the SH-SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin-degrading enzyme were up-regulated in the SH-SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aß. ELISA and qPCR results indicated that STS could increase α-secretase (ADAM10) activity and decrease ß-secretase (BACE1) activity. In conclusion, STS could protect against Aß-induced cell damage by modulating Aß degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Comparison of mRNA Expression of P2X Receptor Subtypes in Different Arterial Tissues of Rats.

This study aims to compare the expression of P2X receptor subtype mRNA in different arterial tissues of rats. After the rats were sacrificed, the internal carotid, pulmonary, thoracic aorta, mesenteric and caudal arteries were dissected out. Then, the P2X receptor mRNA expression in different blood vessels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction. The P2X1, P2X4 and P2X7 receptor mRNA amplification products revealed specific bands of the same size as the amplified target fragment in their respective lanes, while the P2X2, P2X3, P2X5 and P2X6 receptor mRNA amplification products did not reveal significant specific bands in their respective lanes by RT-PCR. Based on the P2X1 receptor mRNA expression of the mesenteric artery, there were no significant differences in the internal carotid, pulmonary and thoracic aorta (0.64 ± 0.07, 0.17 ± 0.11 and 1.49 ± 0.65, respectively). However, the P2X1 receptor mRNA expression level in the caudal artery significantly increased (11.06 ± 1.99, P < 0.01). Furthermore, there was no difference in P2X4 receptor mRNA expression among these five blood vessels (P > 0.05). The P2X7 receptor mRNA expression level was significantly different: pulmonary artery < tail artery = thoracic aorta < internal carotid artery < mesenteric artery. The relative P2X1 receptor mRNA expression in the caudal artery was observed to be elevated when compared to that of the internal carotid, pulmonary and thoracic aorta as well as the mesenteric arteries. The P2X7 receptor mRNA expression level is pulmonary artery < caudal artery = thoracic aorta < internal carotid artery < mesenteric artery. P2X4 receptor mRNA expression was not significantly different among these five blood vessels.

Yangxinkang tablet protects against cardiac dysfunction and remodelling after myocardial infarction in rats through inhibition of AMPK/mTOR-mediated autophagy.

Context: Acute myocardial infarction (AMI) is defined as myocardial necrosis. Clinicians use the traditional Chinese patent medicine Yangxinkang Tablet (YXK) to treat chronic heart failure.Objective: To explore the effects of YXK on heart injury following AMI and the underlying mechanisms.Materials and methods: The AMI model was produced in Wistar rats by permanent ligation of the left anterior descending coronary artery. Rats were divided into the following five groups: Sham (n = 6), MI (Model, n = 10), AICAR (AMPK agonist, 50 mg/kg/d, i.p., n = 10), Compound C (AMPK inhibitor, 10 mg/kg/d, i.p., n = 10), and YXK (0.72 g/kg/d, gavage, n = 10) groups. Cardiac function, cardiac fibrosis, apoptosis, and expression of p-AMPK, p-mTOR, and autophagy-related proteins was measured after 4 weeks of treatment after the successful modelling of the AMI.Results: Compared to MI group, both YXK and AMPK inhibitor improved cardiac dysfunction and reduced cardiac fibrosis (15.6 ± 2.3; 22.6 ± 4.6 vs. 34.6 ± 4.3%) and myocardial cell apoptosis (12 ± 3.67; 25.6 ± 6.8 vs. 54 ± 4.8%). Futhermore, YXK and AMPK inhibitor significantly decreased p-AMPK expression by 11.05% and 14.64%, LC3II/I by 25.08% and 35.28% and Beclin-1 by 66.71% and 33.85%, increased p-mTOR by 22.14% and 47.46% and p62 by 70.83% and 18.58%.Conclusions: The underlying mechanism appears to include suppression of autophagy via inhibiting AMPK/mTOR signalling, suggesting that YXK may serve as a potentially effective Chinese herbal compound for suppressing cardiac fibrosis in heart injury.