Existing data have indicated a growing prevalence and burden of ALD, particularly end-stage advanced fibrosis, cirrhosis, and carcinoma resulted from excessive alcohol consumption in the most of world's adult population[39]. Therefore, it is of particular clinical significance for early prevention and effective interventions of AFLD[40]. In the present study, we found that quercetin reversed the damage caused by chronic alcohol which decreased the expression of the membrane fusion protein (GTP-Rab7, Stx17, SNAP29, VAMP8) and normalized the lipophagy level. More importantly, we found the expression of TBC1D5 was down-regulated after alcohol administration and quercetin intervention restored the expression of TBC1D5 and Rab7 cycle. We demonstrated that quercetin exerted a protective effect on AFLD by restoring the normal circulation of Rab7 to improve lipophagy.
AFLD is associated with lipid metabolism disorder and insulin resistance which lead to alcoholic steatosis[41, 42]. Alcohol consumption leads to fatty acid synthesis increase (upregulate SREBP1c in liver[43]) and decrease of fatty acid transport and oxidation (downregulate PPARα[44] and mitochondrial β-oxidation[45]) as the ‘first hit’, and chronic alcohol inhibits lipophagy though may activate when acute alcohol administration as a compensatory to against alcoholic liver injury[46, 47]. Lipid accumulation occurs when lipophagy, a conserved protective manner of body, is impared[48]. The mechanism of lipophagy dysfunction in ALFD still not fully elucidated, but several molecules and pathways involved have been studied[12, 49, 50]. For example, TFEB and Dyn2, responsible for lysosomes recovery and biogenesis, were inactivated in alcohol-fed rats and alcohol-induced hepatitis patients[12, 51, 52]. In addition, chronic alcohol increases the intrahepatic leucine which further enhances the activity of mTORC1, showing autophagy inhibition[53–55]. Meanwhile, the fusion of autophagosomes and lysosomes is impaired by chronic alcohol and Rab7 as the key regulator of autophagosome-lysosome fusion plays an important role in this process[38]. A previous study showed that primary hepatocytes from rats fed with alcohol diet for 6 weeks were insensitive to starvation-induced lipophagy[15]. Consistently, we showed that the chronic alcohol decreased autophagic flux which leads to lipophagy dysfunction in the liver was due to a decrease in the Rab7 activation. However, Eid et al. showed that chronic alcohol increased the autophagosomes in rat livers[10]. The difference may be due to the difference in detection methods and measurement criteria of autophagic flux.
Quercetin is mostly in the form of quercetin glycosides found in fruits and vegetables[56]. Quercetin glycosides are hydrolyzed by β-glucosidases and then extensively metabolized in the enterocytes and then in liver[57]. Quercetin inhibits low-density lipoprotein oxidation by suppressing myeloperoxidase-catalyzed oxidation and plays an anti-inflammatory, antioxidation and lipid-lowing role in ALD by decreasing serum and hepatic NF-κB, p65, COX-2, IL-6 and TG levels[26, 58–60]. It is reported that quercetin mediates up-regulation of HO-1 through ERK/Nrf2 pathway to reduce the production of ROS, thereby exerting the protective effect against ALD[26, 61]. A recent study in zabrafish model showed that quercetin can down-regulation the P2X7R and through PI3K/Keap1/Nrf2 pathway to protect liver function in ALD[62]. In addition, our previous research showed that quercetin decreased the activated AMPK and perilipin 2 (PLIN2) levels and increased the co-location of LC3II and PLIN2 to promote lipophagy in ALD[27]. In present this study, we proved that quercetin plays a protective role in AFLD by restoring the normal circulation of Rab7 to restore lipophagy, and indicated that quercetin increased the expression of TBC1D5 which controls the inactivation of Rab7 under alcohol administration. However, the mechanism of how quercetin restores the Rab7 cycle needs to be further explored.
Rab7, as a regulator of vesicle transport, is important for many cellular processes. For instance, in lipophagy[18], mitophagy, apoptosis[63], protein sorting, maturation of endosome, fusion of lysosome and autophagosome[13], and lysosome biogenesis[64]. A recent study found that rab7-mediated endocytosis plays an important role in the maintenance of the Wnt signaling by removing the Wnt antagonist Dkk1[65], suggesting that Rab7 is closely related to cancer and vascular calcification[66–68]. The immunoblot analysis indicated that Rab7-like Ypt7p associated with LDs, Vma13p and V1 part of vacuolar (H+) ATPase (V-ATPase) as the partners of Ypt7p coordinate regulated the LDs dynamic and membrane trafficking[69], this indicates that Rab7 plays a central regulator role in lipophagy. The function of Rab7 is guaranteed by a cyclical mechanism to switch between activation (GTP-bound state) and inactivation (GDP-bound state). Consistent with previous studies[11, 15], we found that chronic alcohol administration did not change the expression of total Rab7 but can significantly reduce the activation of Rab7, but other studies have shown that alcohol treatment can reduce the expression of total Rab7[16, 70], this may be due to differences in model, duration and dose of intervention. However, we indicated that overexpression of the active mutation Rab7Q67L is less effective than Rab7Wt in reducing lipid deposition. Similarly, previous studies indicated that Rab7Q67L showed lower GTPase activity compared to Rab7Wt, and Rab7Q67L also dramatically reduced the turnover from lysosomes[71, 72], all of this suggests that the normal Rab7 cycling is more important for liver health than Rab7 activation.
The most important regulator of Rab7 cyclical is Guanine nucleotide Exchange Factors (GEFs) which are required for GTP capture and GDP dissociation and GTPase-Activating Proteins (GAPs) which are required for GTP hydrolysis. When lack retromer-bound TBC1D5 (one of the GAPs), Rab7 can’t sharply localize and accumulates over the lysosomes in active state, therefore active Rab7 can not release from lysosome so that is unavailable locally activated elsewhere[19]. In addition, the Rab7 cycle catalyzed by TBC1D5 was critical for HPV retrograde nuclear entry[73]. In order to explore why Rab7Wt can better recover the level of lipophagy that decreased by alcohol administration, we measured the expression of TBC1D5, and the results showed chronic alcohol significantly reduce the expression of TBC1D5, indicated alcohol not only reduces Rab7 activation but also causes cyclical mechanism disturbed. According to a recent study that ER-localized TMEM16K bound GTP-Rab7 and endolysosomal PtdIns3P to play an important role in endosomal retrograde[74] and other studies indicated that PI4K2A kinase conversion PI4P to PI (4,5) P2 cause Rab7 inactivation and release of PLEKHM1 and deletion of PI4K2A led to the problem of Rab7 inactivation and defective fusion of autophagosomes and lysosomes[13]. This shows that the inactivation of Rab7 also can be regulated by phosphoinositides and provides a new direction for the study of Rab7 cyclical mechanism. In addition, since the circulation of Rab7 plays an important role in maintaining the normal function of the membrane contact sites (MCSs), including the formation of ER-endosome MCSs mediated by GTP-Rab7 and the maintenance of normal mitochondria-lysosome MCSs dependent on its hydrolysis[75, 76], which emphasizes the important role of Rab7 circulation in maintaining cellular homeostasis. Thus, the formation defect of the MCSs caused by Rab7 will be further explored in the ALD next step. To our knowledge, there is no previous show the regulation of quercetin on Rab7 and TBC1D5, thus our finding further illustrate how quercetin improve lipophagy and exert ALD protection by restoring the Rab7 cycle.