A P2RY12 deficiency results in sex-specific cellular perturbations and sexually dimorphic behavioral anomalies.

Microglia are sexually dimorphic, yet, this critical aspect is often overlooked in neuroscientific studies. Decades of research have revealed the dynamic nature of microglial-neuronal interactions, but seldom consider how this dynamism varies with microglial sex differences, leaving a significant gap in our knowledge. This study focuses on P2RY12, a highly expressed microglial signature gene that mediates microglial-neuronal interactions, we show that adult females have a significantly higher expression of the receptor than adult male microglia. We further demonstrate that a genetic deletion of P2RY12 induces sex-specific cellular perturbations with microglia and neurons in females more significantly affected. Correspondingly, female mice lacking P2RY12 exhibit unique behavioral anomalies not observed in male counterparts. These findings underscore the critical, sex-specific roles of P2RY12 in microglial-neuronal interactions, offering new insights into basal interactions and potential implications for CNS disease mechanisms.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h.

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

Integrative Analysis of Single-Cell and Bulk Sequencing Data Depicting the Expression and Function of P2ry12 in Microglia Post Ischemia-Reperfusion Injury.

P2ry12 is a microglial marker gene. Recently, increasing evidence has demonstrated that its expression levels can vary in response to different CNS disorders and can affect microglial functions, such as polarization, plasticity, and migration. However, the expression and function of P2ry12 in microglia during ischemia-reperfusion injury (IRI) remain unclear. Here, we developed a computational method to obtain microglia-specific P2ry12 genes (MSPGs) using sequencing data associated with IRI. We evaluated the change in comprehensive expression levels of MSPGs during IRI and compared it to the expression of P2ry12 to determine similarity. Subsequently, the MSPGs were used to explore the P2ry12 functions in microglia through bioinformatics. Moreover, several animal experiments were also conducted to confirm the reliability of the results. The expression of P2ry12 was observed to decrease gradually within 24 h post injury. In response, microglia with reduced P2ry12 expression showed an increase in the expression of one receptor-encoding gene (Flt1) and three ligand-encoding genes (Nampt, Igf1, and Cxcl2). Furthermore, double-labeling immunofluorescence staining revealed that inhibition of P2ry12 blocked microglial migration towards vessels during IRI. Overall, we employ a combined computational and experimental approach to successfully explore P2ry12 expression and function in microglia during IRI.

Co-expression patterns of microglia markers Iba1, TMEM119 and P2RY12 in Alzheimer's disease.

Microglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to ß-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119+-phenotypes was observed, independent of the presence of ß-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around ß-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan- or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies.

Human iPSC-derived microglia assume a primary microglia-like state after transplantation into the neonatal mouse brain.

Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease.

A distinct microglial subset at the tumor-stroma interface of glioma.

The characterization of the tumor microenvironment (TME) in high grade gliomas (HGG) has generated significant interest in an effort to understand how neoplastic lesions in the central nervous system (CNS) are supported and to devise novel therapeutic targets. The TME of the CNS contains unique and specialized cells, including the resident myeloid cells, microglia. Myeloid involvement in HGG, such as glioblastoma, is associated with poor outcomes. Glioma-associated microglia and infiltrating monocytes/macrophages (GAM) accumulate within the neoplastic lesion where they facilitate tumor growth and drive immunosuppression. However, it has been difficult to differentiate whether microglia and macrophages have similar or distinct roles in pathology, and if the spatial organization of these cells informs outcomes. Here, we characterize the tumor-stroma border and identify peritumoral GAM (PGAM) as a unique subpopulation of GAM. Using data mining and analyses of samples derived from both murine and human sources we show that PGAM exhibit a pro-inflammatory and chemotactic phenotype that is associated with peripheral monocyte recruitment, and decreased overall survival. PGAM act as a unique subset of GAM at the tumor-stroma interface. We define a novel gene signature to identify these cells and suggest that PGAM constitute a cellular target of the TME.

Transcriptome of microglia reveals a species-specific expression profile in bovines with conserved and new signature genes.

Evidence is growing that microglia adopt different roles than monocyte-derived macrophages (MDM) during CNS injury. However, knowledge about their function in the pathogenesis of neuroinfections is only rudimentary. Cattle are frequently affected by neuroinfections that are either zoonotic or related to diseases in humans, and, hence, studies of bovine neuroinfections as a natural disease model may generate fundamental data on their pathogenesis potentially translatable to humans. We investigated the transcriptomic landscape and lineage markers of bovine microglia and MDM. Although bovine microglia expressed most microglial signature genes known from humans and mice, they exhibited a species-specific transcriptomic profile, including strikingly low expression of TMEM119 and enrichment of the two scavenger receptors MEGF10 and LY75. P2RY12 was amongst the most enriched genes in bovine microglia, and antibodies against P2RY12 labeled specifically resting microglia, but also reactive microglia within neuroinfection foci in-situ. On the other hand, F13A1 was amongst the most enriched genes in bovine monocytes and MDM and, additionally, the encoded protein was expressed in-situ in monocytes and MDM in the inflamed brain but not in microglia, making it a promising marker for infiltrating MDM in the brain. In culture, primary bovine microglia downregulated signature genes, expressed markers of activation, and converged their transcriptome to MDM. However, they retained several microglia signature genes that clearly distinguished them from bovine MDM, making them a promising in-vitro tool to study mechanisms of microglia-pathogen interactions.

Activated microglia do not increase 18 kDa translocator protein (TSPO) expression in the multiple sclerosis brain.

To monitor innate immune responses in the CNS, the 18 kDa Translocator protein (TSPO) is a frequently used target for PET imaging. The frequent assumption that increased TSPO expression in the human CNS reflects pro-inflammatory activation of microglia has been extrapolated from rodent studies. However, TSPO expression does not increase in activated human microglia in vitro. Studies of multiple sclerosis (MS) lesions reveal that TSPO is not restricted to pro-inflammatory microglia/macrophages, but also present in homeostatic or reparative microglia. Here, we investigated quantitative relationships between TSPO expression and microglia/macrophage phenotypes in white matter and lesions of brains with MS pathology. In white matter from brains with no disease pathology, normal appearing white matter (NAWM), active MS lesions and chronic active lesion rims, over 95% of TSPO+ cells are microglia/macrophages. Homeostatic microglial markers in NAWM and control tissue are lost/reduced in active lesions and chronic active lesion rims, reflecting cell activation. Nevertheless, pixel analysis of TSPO+ cells (n = 12,225) revealed that TSPO expression per cell is no higher in active lesions and chronic active lesion rims (where myeloid cells are activated) relative to NAWM and control. This data suggests that whilst almost all the TSPO signal in active lesions, chronic active lesion rims, NAWM and control is associated with microglia/macrophages, their TSPO expression predominantly reflects cell density and not activation phenotype. This finding has implications for the interpretation of TSPO PET signal in MS and other CNS diseases, and further demonstrates the limitation of extrapolating TSPO biology from rodents to humans.

Microglia and Neuroinflammation: What Place for P2RY12?

Microglia are immune brain cells involved in neuroinflammation. They express a lot of proteins on their surface such as receptors that can be activated by mediators released in the microglial environment. Among these receptors, purinergic receptor expression could be modified depending on the activation status of microglia. In this review, we focus on P2Y receptors and more specifically on P2RY12 that is involved in microglial motility and migration, the first step of neuroinflammation process. We describe the purinergic receptor families, P2RY12 structure, expression and physiological functions. The pharmacological and genetic tools for studying this receptor are detailed thereafter. Last but not least, we report the contribution of microglial P2RY12 to neuroinflammation in acute and chronic brain pathologies in order to better understand P2RY12 microglial role.

Microglia in the developing retina couple phagocytosis with the progression of apoptosis via P2RY12 signaling.

BACKGROUND: Microglia colonize the developing vertebrate central nervous system coincident with the detection of developmental apoptosis. Our understanding of apoptosis in intact tissue in relation to microglial clearance of dying cells is largely based on fixed samples, which is limiting given that microglia are highly motile and mobile phagocytes. Here, we used a system of microglial depletion and in vivo real-time imaging in zebrafish to directly address microglial phagocytosis of apoptotic cells during normal retinal development, the relative timing of phagocytosis in relation to apoptotic progression, and the contribution of P2RY12 signaling to this process. RESULTS: The depletion of microglia resulted in accumulation of numerous apoptotic cells in the retina. Real-time imaging revealed precise timing of microglial engulfment with the progression of apoptosis, and dynamic movement and displacement of engulfed apoptotic cells. Inhibition of P2RY12 signaling delayed microglial clearance of apoptotic cells. CONCLUSIONS: Microglial engulfment of dying cells is coincident with apoptotic progression and requires P2RY12 signaling, indicating that microglial P2RY12 signaling is shared between development and injury response. Our work provides important in vivo insight into the dynamics of apoptotic cell clearance in the developing vertebrate retina and provides a basis to understand microglial phagocytic behavior in health and disease.

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects.

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.

Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation.

BACKGROUND: The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12. METHODS: In this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model. RESULTS: We demonstrate that Tmem119 and P2RY12 expression is evident within both CD45int and CD45high myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells. CONCLUSION: We recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the "infiltrating myeloid" population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.

Selective inhibition of mitochondrial respiratory complexes controls the transition of microglia into a neurotoxic phenotype in situ.

Microglia are tissue resident macrophages (innate immunity) and universal sensors of alterations in CNS physiology. In response to pathogen or damage signals, microglia feature rapid activation and can acquire different phenotypes exerting neuroprotection or neurotoxicity. Although transcriptional aspects of microglial phenotypic transitions have been described, the underlying metabolic reprogramming is widely unknown. Employing postnatal organotypic hippocampal slice cultures, we describe that microglia transformed into a mild reactive phenotype by single TLR4 stimulation with lipopolysaccharide (LPS), which was boosted into a severe neurotoxic phenotype by IFN-γ (LPS + INF-γ). The two reactive phenotypes associated with reduction of microglial homeostatic "surveillance" markers, increase of cytokine release (IL-6, TNF-α) as well as enhancement of tissue energy demand and lactate production. These reactive phenotypes differed in the pattern of inhibition of the respiratory chain in mitochondria, however. TLR4 stimulation induced succinate dehydrogenase (complex II) inhibition by the metabolite itaconate. By contrast, TLR4 + IFN-γ receptor stimulation mainly resulted in complex IV inhibition by nitric oxide (NO) that also associated with severe oxidative stress, neuronal dysfunction and death. Notably, pharmacological depletion of microglia or treatment with itaconate resulted in effective neuroprotection reflected by well-preserved cytoarchitecture and electrical network activity, i.e., neuronal gamma oscillations (30-70 Hz) that underlie higher cognitive functions in vivo. Our findings provide in situ evidence that (i) proinflammatory microglia can substantially alter brain energy metabolism and (ii) fine-tuning of itaconate and NO metabolism determines microglial reactivity, impairment of neural network function and neurodegeneration. These data add mechanistic insights into microglial activation, with relevance to disorders featuring neuroinflammation and to drug discovery.

microRNA-605 rs2043556 polymorphisms affect clopidogrel therapy through modulation of CYP2B6 and P2RY12 in acute coronary syndrome patients.

Clopidogrel therapy reduces the occurrence of major vascular events in acute coronary syndrome (ACS) patients, but treatment efficacy is variable. The present study aims to determine the mechanisms that underlie associations between certain miRNA polymorphisms and clinical outcomes of clopidogrel therapy. Our study focused on 9 miRNA single nucleotide polymorphisms in addition to CYP2C19*2 and CYP2C19*3. We found that the miR-605 rs2043556 AG genotype significantly decreased the risk of acute myocardial infarction (odds ratio, OR = 0.13, 95%CI 0.02-0.96, P = .045) and that the rs2043556 GG genotype significantly decreased the risk of unstable angina (OR = 0.19, 95%CI 0.05-0.65, P = .008) in ACS patients receiving clopidogrel therapy for more than one year. Dual-luciferase analysis indicated that miR-605 significantly decreased the mRNA expression of CYP2B6 and P2RY12 (P < .01). In cells treated with miR-605-A, the protein and mRNA expression of CYP2B6 and P2RY12 were significantly lower than that of cells treated with miR-605-G (P < .05). The results demonstrate that miR-605 targets the mRNA of the CYP2B6 and P2RY12 genes, and that rs2043556 A/G polymorphisms in miR-605 modulate the mRNA and protein expression of CYP2B6 and P2RY12 differently, which may impact the effect of clopidogrel in ACS patients.

P2RY12:rs7637803 TT variant genotype increases coronary artery aneurysm risk in Kawasaki disease in a southern Chinese population.

BACKGROUND: Activated-platelet increases the risk of thrombosis in Kawasaki disease (KD) patients with a coronary artery aneurysm (CAA). The ADP pathway is one of the platelet activation and aggregation pathways. The P2RY12 gene encodes the ADP receptor that is highly concentrated on platelets. However, few studies have reported on P2RY12 in relation to KD susceptibility with or without CAA. METHODS: We recruited 1335 healthy controls and 776 KD patients, including 103 with CAA, and selected five P2RY12 polymorphisms: rs9859538, rs1491974, rs7637803, rs6809699 and rs2046934. The present study focused on the relationship between the P2RY12 polymorphisms and KD with or without CAA. RESULTS: Among all of the selected polymorphisms, single-locus analysis showed no significant association between the P2RY12 polymorphism and KD susceptibility. However, we found a significant relationship between rs7637803 and CAA risk in KD patients [CT versus CC: odds ratio (OR) = 0.41, 95% confidence interval (CI) = 0.22-0.75; p = 0.0041; TT versus CC: OR = 2.90, 95% CI = 1.12-7.46; p = 0.0276]. Stratification analysis by age in KD patients indicated that the rs7637803 TT genotype increased CAA formation risk among children aged (OR = 3.90, 95% CI = 1.42-10.69; p = 0.0081) and increased the onset risk of CAA in males (OR = 6.28, 95% CI = 2.01-19.65; p = 0.0016). The combined effect of the five selected P2RY12 risk genotypes with the KD patients compared to non-mutated P2RY12 genotypes (score: 0) showed that patients with P2RY12 genotype polymorphisms (score: 1-5) had a significantly increased CAA risk (p = 0.0086). Stratification analysis for the severity of CAA found that the rs7637803 TT genotype reduced giant CAA (GCAA) risk (OR = 4.60, 95% CI = 1.70-12.41; p = 0.0026). CONCLUSIONS: The results of the present study indicate that the P2RY12 rs7637803 genotype might be used as a biomarker to predict the occurrence of GCAA.

Neuroprotective and Anti-Inflammatory Effects of a Hydrophilic Saffron Extract in a Model of Glaucoma.

Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells (RGCs). An increase in the intraocular pressure is the principal risk factor for such loss, but controlling this pressure does not always prevent glaucomatous damage. Activation of immune cells resident in the retina (microglia) may contribute to RGC death. Thus, a substance with anti-inflammatory activity may protect against RGC degeneration. This study investigated the neuroprotective and anti-inflammatory effects of a hydrophilic saffron extract standardized to 3% crocin content in a mouse model of unilateral, laser-induced ocular hypertension (OHT). Treatment with saffron extract decreased microglion numbers and morphological signs of their activation, including soma size and process retraction, both in OHT and in contralateral eyes. Saffron extract treatment also partially reversed OHT-induced down-regulation of P2RY12. In addition, the extract prevented retinal ganglion cell death in OHT eyes. Oral administration of saffron extract was able to decrease the neuroinflammation associated with increased intraocular pressure, preventing retinal ganglion cell death. Our findings indicate that saffron extract may exert a protective effect in glaucomatous pathology.

Sustained activation of ADP/P2ry12 signaling induces SMC senescence contributing to thoracic aortic aneurysm/dissection.

Thoracic aortic aneurysm/dissection (TAAD) is characterized by excessive smooth muscle cell (SMC) loss, extracellular matrix (ECM) degradation and inflammation. However, the mechanism whereby signaling leads to SMC loss is unclear. We used senescence-associated (SA)-ß-gal staining and analysis of expression of senescence-related proteins (p53, p21, p19) to show that excessive mechanical stretch (20% elongation, 3600cycles/h, 48h) induced SMC senescence. SMC senescence was also detected in TAAD specimens from both mice and humans. High-performance liquid chromatography and luciferin-luciferase-based assay revealed that excessive mechanical stretch increased adenosine diphosphate (ADP) release from SMCs both in vivo and in vitro. Elevated ADP induced SMC senescence while genetic knockout of the ADP receptor, P2Y G protein-coupled receptor 12 (P2ry12), in mice protected against SMC senescence and inflammation. Both TAAD formation and rupture were significantly reduced in P2ry12-/- mice. SMCs from P2ry12-/- mice were resistant to senescence induced by excessive mechanical stretch or ADP treatment. Mechanistically, ADP treatment sustained Ras activation, whereas pharmacological inhibition of Ras protected against SMC senescence and reduced TAAD formation. Taken together, excessive mechanical stress may induce a sustained release of ADP and promote SMC senescence via P2ry12-dependent sustained Ras activation, thereby contributing to excessive inflammation and degeneration, which provides insights into TAAD formation and progression.

A P2RY12 Deficiency Results in Sex-specific Cellular Perturbations and Sexually Dimorphic Behavioral Anomalies.

Microglia are sexually dimorphic, yet, this critical aspect is often overlooked in neuroscientific studies. Decades of research have revealed the dynamic nature of microglial-neuronal interactions, but seldom consider how this dynamism varies with microglial sex differences, leaving a significant gap in our knowledge. This study focuses on P2RY12, a highly expressed microglial signature gene that mediates microglial-neuronal interactions, we show that adult females have a significantly higher expression of the receptor than adult male microglia. We further demonstrate that a genetic deletion of P2RY12 induces sex-specific cellular perturbations with microglia and neurons in females more significantly affected. Correspondingly, female mice lacking P2RY12 exhibit unique behavioral anomalies not observed in male counterparts. These findings underscore the critical, sex-specific roles of P2RY12 in microglial-neuronal interactions, offering new insights into basal interactions and potential implications for CNS disease mechanisms.

Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

Gualou-Xiebai herb pair and its active ingredients act against atherosclerosis by suppressing VSMC-derived foam cell formation via regulating P2RY12-mediated lipophagy.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown. PURPOSE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS. METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays. RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPß abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12. CONCLUSION: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.