Gualou-xiebai Herb Pair Alleviates VSMCs-derived Foam Cell Formation Via Regulating P2RY12-PI3K-mediated Lipophagy

Atherosclerosis(AS)is a chronic vascular disease characterized by the deposition of excess lipids within the wall of arteries.Foam cell is an important component of AS lesions and its persistence in the artery wall fuels the development of AS.It was previously thought that foam cell we primarily macrophage-derived cells.However,recent studies have suggested that vascular smooth muscle cells(VSMCs)-derived foam cells also account for a large proportion in the middle and late stages of AS.Therefore,inhibition of VSMCs-derived foam cell formation is an attractive mean for the prevention and treatment of AS.Lipophagy is an autophagy process with lipid droplets(LD)as autophagy objects,which can promote the degradation of intracellular LD and inhibit the formation of foam cell.It is generally considered to be an important mechanism for regulating excess exogenous lipids.Currently,little is known about the role of the lipophagy on VSMCs-derived foam cell formation is unknown.A study of the mechanism by which lipophagy regulating LD degradation and reducing intracellular lipid accumulation may provide new insights into the formation of VSMCs-derived foam cell.Nowadays,traditional Chinese medicine(TCM)has been widely used in the prevention and treatment of AS because of its good curative effects.Many TCM prescriptions are available for the AS treatment,including Gualou Xiebai Baijiu decoction,Gualou Xiebai Banxia decoction,Zhishi Xiebai Guizhi decoction,and Danlou prescription.All of these TCM classical prescriptions contain Gualou-Xiebai(GLXB)herb pair,Recent studies have shown that GLXB herb pair has vital and positive roles in the therapy of cardiovascular disease.In view of the fact that VSMCs-derived foam cell are the key target cells of AS and our previous research basis,we used the model of VSMCs-derived foam cell induced by ox-LDL to explore the regulatory mechanism of lipophagy in the formation of VSMCsderived foam cell and the intervention effect of GLXB herb pair.Taking this small compound AS the starting point,the mechanism of GLXB herb pair inhibiting the formation of VSMCs-derived foam cell is further revealed from the perspective of lipophagy,which provides a new scientific basis for the clinical application of GLXB herb pair prescription against AS.In view of the fact that VSMCs-derived foam cell are the key target cells of AS and the basis of our previous research group,this study used the model of VSMCs-derived foam cell induced by ox-LDL to investigate the effect of lipophagy on the formation of VSMCs-derived foam cell,so AS to provide a new scientific basis for studying the effect of lipophagy on the regulation of the formation of VSMCs-derived foam cell.Objective:To investigate the effect of lipophagy on the formation of VSMCs-derived foam cell,the intervention effect of GLXB herb pair on VSMCs-derived foam cell and the effect and mechanism of lipophagy regulation,and to reveal the role and regulatory mechanism of GLXB herb pair in improving the lipophagy function of VSMCs-derived foam cell,so AS to provide a new scientific basis for GLXB herb pair to “reat AS disease from sputum”.Method:Primary mouse aortic VSMCs were cultured and identified by predigestion and adherence method and immunocytochemistry.CCK8 assay was used to detect cell viability to screen the safe concentration of GLXB herb pair.The content of total cholesterol and free cholesterol in the cells was detected by full-wavelength microplate reader.Oil red O staining was used to detect intracellular lipid accumulation as the standard for the establishment of VSMCs-derived foam cell model.MDC staining and transmission electron microscopy were used to detect the number of autophagosomes.The formation of lipophagosomes was observed by transmission electron microscopy.The protein expressions of LC3Ⅱ,p62 and plin2 were detected by western blot.The colocalization of LC3 and BODIPY was detected by immunofluorescence.Chloroquine,an inhibitor of lipophagy,was used to investigate the effect of lipophagy on the inhibitory effect of GLXB herb pair on the formation of VSMCs-derived foam cell.The effect of P2RY12-PI3 K signaling pathway on GLXB herb pair-induced lipophagy was investigated using P2RY12 stimulator ADPβs.The active component of GLXB herb pair acting on the P2RY12 target was predicted by molecular docking.1.GLXB herb pair inhibited the formation of VSMCs-derived foam cells Compared with ox-LDL group,GLXB herb pair could reduce intracellular red lipid droplets,cholesterol esterification rate,reduce intracellular lipid accumulation,and inhibit the formation of VSMCs-derived foam cell in a concentration-dependent manner.2.GLXB herb pair inhibits the formation of VSMCs-derived foam cell by inducing lipophagyTo determine the effect of GLXB herb pair on the level of lipophagy in VSMCs-derived foam cell,MDC staining and transmission electron microscopy were used to observe the effect of GLXB herb pair on the number of intracellular autophagosomes.Compared with ox-LDL group,GLXB herb pair significantly increased the number of intracellular autophagosomes,reduced the number of intracellular lipid droplets,and promoted the formation of intracellular lipid vacuoles.GLXB herb pair could upregulate the expression of LC3 II,inhibit the expression of p62 and plin2,and increase the colocalization coefficient of LC3 and lipid droplets.To determine the effect of lipophagy on GLXB inhibiting the formation of VSMCsderived foam cell,lipophagy inhibitor chloroquine was used for intervention.Chloroquine aggravates lipid accumulation in VSMCs-derived foam cell,up-regulates the expression of LC3 II,p62 and plin2,decreases the colocalization coefficient of LC3 and lipid droplets,and inhibits lipophagy.The addition of chloroquine and GLXB herb pair abolished the inhibitory effect of GLXB herb pair on lipid accumulation and lipophagy.3.GLXB herb pair inhibits the formation of VSMCs-derived foam cell by inducing lipophagy through P2RY12-PI3KTo determine the effect of GLXB herb pair on P2RY12-PI3 K pathway in VSMCsderived foam cell,different concentrations of GLXB herb pair were used for intervention.Compared with ox-LDL group,GLXB herb pair inhibited the protein expression of P2RY12,PI3 K and p-PI3 K in a concentration-dependent manner.To further investigate whether GLXB herb pair mediates P2RY12-PI3 K pathway to induce lipophagy and inhibit the formation of VSMCs-derived foam cell,cells were treated with P2RY12 stimulator ADPβs.ADPβs abolished GLXB herb pair’s upregulation of LC3 II,p62,and plin2 expression,down-regulation of P2RY12 and PI3K/p-PI3 K expression,and increased the colocalization coefficient of LC3 and lipid droplets.4.The active components such as saponins and flavonoids may be the material basis of GLXB herb pair acting on P2RY12 targets.In order to predict the active components of GLXB herb pair acting on the P2RY12 target to exert anti-AS effect,virtual molecular docking was performed between the 48 major active components of GLXB herb pair and the P2RY12 target using Auto Duck software.The docking results showed that P2RY12 target could bind to all 48 major active components of GLXB herb pair.Conclusions:1.GLXB herb pair can reduce intracellular lipid accumulation and inhibit the formation of VSMCs-derived foam cells.2.GLXB herb pair inhibits the formation of VSMCs derived foam cells by inducing lipophagy and reducing intracellular lipid accumulation.3.GLXB herb pair activate P2RY12-PI3K-mediated lipophagy to inhibit VSMCsderived foam cell formation;The active components such as saponins and flavonoids may be the material basis of GLXB herb pair acting on P2RY12 targets.