Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

Structure-based virtual screening for discovery of paederosidic acid from Paederia scandens as novel P2Y14R antagonist.

BACKGROUND: The activation of P2Y14 receptor (P2Y14R) promotes osteoclast formation and causes neuropathic pain, exhibiting possible link to osteoarthritis (OA). Given lack of P2Y14R antagonist, the present study aims to search a novel P2Y14R antagonist with low toxicity and high activity from natural products as a possible drug candidate in treatment of OA. METHODS: The role of P2Y14R on OA was verified using P2Y14R knockout (KO) rats. Molecular docking virtual screening strategy and activity test in P2Y14R stably-expressed HEK293 cells were used to screen target compound from natural product library. The MM/GBSA free energy calculation/decomposition technique was used to determine the principal interaction mechanism. Next, the binding of target compound to P2Y14R was examined using cellular thermal shift assay and drug affinity responsive target stability test. Finally, the therapeutic effect of target compound was performed in monosodium iodoacetate (MIA)-induced OA mouse model. To verify whether the effect of target compound was attributed to P2Y14R, we establish the osteoarthritis model in P2Y14R KO mice to perform pharmacodynamic evaluation. Importantly, to investigate the potential mechanism by which target compound attenuate OA, expressions of the major transcription factors involved in osteoclast differentiation were detected by western blot, while markers of nerve damage in dorsal root ganglion (DRG) were evaluated by RT-qPCR and immunofluorescence techniques. RESULTS: Deficiency of P2Y14R alleviated pain behavior and cartilage destruction in MIA-induced OA rats. 14 natural compounds were screened by Glide docking-based virtual screening, among which paederosidic acid exhibited the highest antagonistic activity to P2Y14R with IC50 of 8.287 µM. As a bioactive component extracted from Paederia scandens, paederosidic acid directly interacted with P2Y14R to enhance the thermostability and decrease the protease sensitivity of target protein, which significantly inhibited receptor activator for nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis. More importantly, paederosidic acid suppressed osteoclast formation by downregulating expressions of NFAT2 and ATP6V0D2, as well as relieved neuropathic pain by decreasing expressions of CGRP, CSF1 and galanin in DRG. CONCLUSIONS: Paederosidic acid targeted P2Y14R to improve OA through alleviating osteoclast formation and neuropathic pain, which provided an available strategy for developing novel drug leads for treatment of OA.

Hydroxygenkwanin suppresses proliferation, invasion and migration of osteosarcoma cells via the miR­320a/SOX9 axis.

Hydroxygenkwanin (HGK) has an anticancer effect in a variety of tumors, but its role in osteosarcoma has not been explored. The purpose of the present study was to investigate the therapeutic effect of HGK on osteosarcoma and its specific molecular mechanism. Osteosarcoma cells (MG­63 and U2OS) treated with various concentrations of HGK were assigned to the treatment group. MTT, clone formation, wound healing and Transwell assays were performed to assess the viability, proliferation, migration, and invasion of MG­63 and U2OS cells. RT­qPCR was conducted to quantify the expression levels of of microRNA (miR)­320a and SRY­box transcription factor 9 (SOX9) in MG­63 and U2OS cells. The binding sites of miR­320a and SOX9 were predicted by starBase database, and verified using the dual­luciferase reporter assay. The expression levels of SOX9 and EMT­related proteins (N­cadherin, E­cadherin and vimentin) were detected by western blot analysis. HGK inhibited cell proliferation, migration, invasion, but promoted the expression of miR­320a in MG­63 and U2OS cells. Downregulation of miR­320a reversed the effects of HGK on proliferation, migration and invasion of MG­63 and U2OS cells, while upregulation of miR­320a had the opposite effect. HGK inhibited the expression of SOX9 by promoting the expression of miR­320a. Upregulation of SOX9 could partially reverse miR­320a­induced migration and invasion of MG­63 and U2OS cells. In addition, upregulation of miR­320a promoted E­cadherin expression and inhibited the expression of N­cadherin and vimentin, and the effect of miR­320a was also reversed by SOX9. In conclusion, HGK inhibited proliferation, migration and invasion of MG­63 and U2OS cells through the miR­320a/SOX9 axis.

Cardiovascular Protective Effects of Plant Polysaccharides: A Review.

Cardiovascular disease is a kind of heart, brain, and blood vessel injury disease by the interaction of various pathological factors. The pathogenesis of cardiovascular disease is complex with various risk factors, including abnormally elevated blood pressure, glucose, and lipid metabolism disorders, atherosclerosis, thrombosis, etc. Plant polysaccharides are a special class of natural products derived from plant resources, which have the characteristics of wide sources, diverse biological activities, and low toxicity or side effects. Many studies have shown that plant polysaccharides improve cardiovascular diseases through various mechanisms such as anti-oxidative stress, restoring the metabolism of biological macromolecules, regulating the apoptosis cascade to reduce cell apoptosis, and inhibiting inflammatory signal pathways to alleviate inflammation. This article reviews the pharmacological effects and protective mechanisms of some plant polysaccharides in modulating the cardiovascular system, which is beneficial for developing more effective drugs with low side effects for management of cardiovascular diseases.