Background

Atherosclerosis is characterized by ascensive buildup of plaque in the arterial wall with perturbation of lipid metabolism and vascular inflammation [1]. Endothelium dysfunction results in subendothelial accumulation of oxidized low-density lipoprotein (ox-LDL), and transmigration of monocytes into arterial wall where they differentiate into macrophages that engulf excessive ox-LDL to generate lipid-laden foam cells, which ultimately triggers the initiation and development of atherosclerosis [1,2,3]. Besides, macrophages produce pro-inflammatory mediators under ox-LDL stimulation, which aggravates the atherosclerotic lesions [4, 5].

Macrophages, an immune cell population with high heterogeneity in phenotype and function, can alter own polarization state to adapt to complex external conditions [6,7,8,9]. When exposed to pro-inflammatory substances, resting macrophages turn to develop classical M1 phenotype capable of producing inflammatory factors including IL-6, IL-1β, TNF-α, and iNOS, leading to inflammation amplification and atherosclerosis development [10,11,12]. On the contrary, the alternatively activated M2 macrophages are generated in response to IL-4 or IL-13, which release anti-inflammatory factors such as IL-10, Arginase-1 and then possess anti-atherosclerotic ability [13, 14].

Autophagy, a pro-survival intracellular process, has been demonstrated to attenuate burgeoning plaques via suppressing foam cell formation and weakening inflammatory response [15, 16]. It is reported that pro-atherosclerotic factors such as ox-LDL could block autophagic cascades, whereas up-regulated autophagy pathway effectively blunts atherosclerosis progression in vitro and in vivo [17,18,19,20]. It has been suggested that autophagy displays important roles in M2 macrophage formation and there exists an association of macrophage polarization with autophagy signal transduction. Phosphoinositide3-kniase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway serves as a major regulator inhibiting the initiation of autophagic activities [21, 22]. Moreover, inhibition of PI3K/Akt/mTOR pathway has been shown to improve cholesterol efflux capacity of macrophage-derived foam cells and weaken atherosclerotic plaque inflammation by enhancing autophagy [21, 23]. mTOR inhibitor rapamycin has been indicated to play beneficial roles in alleviating development of plaque lesions [51]. Besides, autophagy was reported to modulate the polarization conversion of macrophage phenotype [52]. The disorder of autophagy process had been indicated to be associated with the impairment of lipid clearance and pro-inflammatory phenotype formation of macrophages [52]. Based on this, we hypothesized that RVS mediated the activation of autophagy to encumber lipid accumulation and facilitate M2 phenotypic polarization of macrophages. 3-MA and Rap were used as antagonist and agonist separately and our findings revealed that the beneficial effects of RVS on favoring autophagy implementation was abrogated by 3-MA in RAW264.7 cells insulted by ox-LDL, followed by invalidation of lipid accumulation amelioration and M2-like polarization formation induced by RVS, whereas Rap administration contributed to the positive roles of RVS in autophagy enhancement and subsequent improvement of lipid d and augmentation of generation of M2 phenotype. These observations suggested that RVS treatment exerts atheroprotective effects involving in reduction of foam cell formation and inflammatory phenotype switch via strengthening upstream autophagy processes.

Then we further investigated relevant mechanisms underlying RVS affected the autophagic activities. CQ, a kind of blocker targeting autophagy flux, was found to increase levels of LC3II/I ratio and p62 content [53], accompanied by deterioration of weakened autophagy in macrophages with ox-LDL intervention, while this effect was significantly alleviated by RVS treatment. PI3K/Akt/mTOR was a classic pathway regulating autophagy initiation and there was evidence revealing that the agents capable of enhancing autophagy via suppressing PI3K/Akt/mTOR pathway reduced the endothelial cell apoptosis induced by oxLDL [54]. Moreover, the inactivation of PI3K/Akt/mTOR pathway was clarified to mediate macrophage autophagy and stabilize the rupture-prone atherosclerotic plaques [21]. Our findings showed that RVS reduced the phosphorylated levels of PI3K, Akt and mTOR and elevated the activity of downstream target ULK1 in vivo and in vitro. Then, we discovered that PI3K agonist 740 Y-P abrogated the beneficial effects of RVS on impelling autophagy-related processes, while PI3K inhibitor LY294002 reinforced RVS-triggered autophagy-promoting effects in RAW264.7 cells stimulated by ox-LDL. These data suggested that RVS increased occurrence and development of autophagy via inhibiting signal transduction of PI3K/Akt/mTOR pathway and improving autophagic flux in macrophages under the lipid-laden condition (Fig. 7).

Fig. 7
figure 7

Schematic depiction of anti-atherosclerosis effects of RVS with the key role of autophagy involved in improving lipid accumulation and polarization conversion of macrophages

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Conclusions

In conclusion, the present study indicated that RVS intervention potently inhibited the atherosclerotic plaque development in ApoE−/− mice induced by high-fat diet. Our results provided the evidence that RVS was able to enhance autophagy activities via prohibiting activation of PI3K/Akt/mTOR pathway and increasing autophagic flux, thus leading to the anti-atherosclerotic effects involving suppression of lipid droplets accumulation and facilitation of anti-inflammatory M2 phenotype polarization, which thereby provided novel aspects into the molecular mechanisms of RVS against atherosclerosis development.