Rab7 Turnover And Repair Mediate Alcoholic Lipophagy Disorder,Iron Deposition In The Liver,and The Quercetin Intervention Mechanism

Objective:Alcoholic liver disease（ALD）is a series of liver lesions caused by excessive intake of alcohol,including alcoholic fatty liver with typical pathological features of steatosis,alcoholic hepatitis,fibrosis,sclerosis and liver cancer.In this process,iron overload is often secondary to further aggravating the course of ALD.At present,with the indispensability of alcohol consumption,it is one of the important strategies to prevent the occurrence and development of ALD to thoroughly clarify the pathogenesis of ALD,correct the liver lipid metabolism disorder caused by alcohol and prevent liver iron deposition.As a key protein in the regulation of vesicle transport and lysosome quality control in the late stage of lipophagy,Rab7 plays an important role in the fusion of autophagosome and lysosome,as well as in the regulation of iron uptake and iron output of the lysosome.However,whether Rab7 can regulate iron-lipid metabolism disorders induced by alcohol remains to be explored.Quercetin is a common dietary flavonoid natural phytochemical that has a wide range of biological activities,such as regulating iron and lipid metabolism,anti-inflammatory,antioxidant,and so on.It also has a good protective effect on ALD.However,whether its effect is also related to the regulation of Rab7 function has not been reported.Therefore,in this study,we use Rab7 as the core to explore the membrane fusion,V-ATPase regulation,and mitochondria-lysosomal membrane contact sites（MCSs）in ALD lipid deposition and iron deposition,and the related mechanism of the protective effect of quercetin on ALD through regulating Rab7.Methods:Eight-week-old male C57BL/6J mice were randomly divided into four groups according to body weight:normal control,ALD model,quercetin intervention,and quercetin.Animal modeling was based on the modified Gao-Binge（Chronic Plus binge ethanol feeding）model.That is,mice were fed with Lieber De Carli liquid feed with 28%ethanol for 12 weeks after a single dose of high dose alcohol（5 g/kg.bw）.The control group was fed the normal Lieber De Carli liquid diet for 12 weeks and then given maltodextrin with equal energy,and quercetin at 100mg/kg.bw for 12 weeks.All mice were executed after 9hours of gavage administration.Serum transaminase levels and triglyceride（TG）levels in serum and liver were detected.The pathological changes in liver tissue were observed by optical and transmission electron microscopy.Liver lipophage level was detected by immunofluorescence co-location.Western blot was used to detect the protein levels of membrane fusion and autophagy related proteins and MRC proteins.The levels of total iron and LIP in liver tissue were determined by atomic absorption spectrometry.The lysosomal iron levels of animals were detected by the iron assay kit colorimetric.HepG2 cells transfected with the CYP2E1 plasmid and mouse normal liver cell line AML12 cells were treated with ethanol（100 m M）and quercetin（50μmol/L）for 48 h.Then,cells were treated with the Rab7 inhibitor CID1067700（80μmol/L）or different doses of the V-ATPase inhibitors Bafilomycin A and Concanamycin A.Or Rab7 knockdown and overexpression(wild-type Rab7^Wt,continuously activated Rab7^Q67L,dominant-negative Rab7^T22N)could be performed on cells in advance.Lyso Traker and BODIPY_493/503 were co-located to detect lipophagy levels.The autophagic flux was detected by transfection of m Cherry-GFP-LC3.Western blot was used to detect the protein levels of membrane fusion and autophagy related proteins and MRC proteins.Specific fluorescence probe determined the following:ROS,LIP,Lysosome p H,iron level and membrane permeability.Ed U detected cell proliferation ability.The fluorescence recovery after photoquenching（FRAP）assay was used to detect the membrane turnover capacity of Rab7.The mitochondrial-lysosome MCSs and mitochondrial morphology were observed by rotary confocal microscopy.STRING platform was used to search Rab7 related proteins and through Cytoscape software visualization,through Genecards（https://www.genecards.org/）for functional annotation of related proteins,and further experimental verification in vivo and vitro.Result:1.Compared with the normal control group,chronic alcohol-fed mice showed increased transaminase levels,increased liver and serum TG levels,decreased co-localization of LC3 with lipdroplet membrane coated protein PLIN2 and lysosomal membrane protein LAMP2,significantly increased liver total iron,LIP,and lysosomal iron（P<0.05）,and impaired mitochondrial-lysosomal MCSs cleavage.Quercetin partially reversed the elevated transaminases,lipid and iron deposition,lipophage disfunction,and mitochondrial-lysosome MCSs dissociation induced by alcohol exposure.2.Alcohol exposure significantly reduced GTP-Rab7 by 75%（P<0.05）,decreased co-localization of lysosome and lipid droplets,blocked autophagy flux（m Cherry-GFP-LC3showed increased yellow fluorescence）,increased lysosome LMP,p H level,and decreased level of mature cathepsin D protein.These can be reversed by quercetin intervention.3.Transfection of Rab7 wild-type plasmid(Rab7^Wt)significantly recovered the decreased co-localization of lysosome and lipid droplets,blocked autophagy flux,increased levels of TG and TC,and increased levels of total iron and lysosomal iron caused by alcohol treatment.Transfection of continuous activation form Rab7(Rab7^R67L)played a lower protective role than Rab7^Wt.Inactivated Rab7^T22N had no protective effect on cell damage induced by alcohol.The activation level of Rab7 increased by 71%（P<0.05）under quercetin intervention,and Rab7 inhibition counteracted the protective effect of quercetin on ALD.FRAP demonstrated that Rab7 turnover was reduced by alcohol exposure,and quercetin intervention restored Rab7 turnover.4.Alcohol exposure resulted in a decrease in the Rab7-regulated V-ATPase subunit V1G1 protein level（P<0.05）,which resulted in a decrease in the lysosomal acidification ability of cells,an increase in LMP,a decrease in the maturity of cathepsin D,and an increase in total iron and lysosomal iron of cells.Rab7 inhibition further reduced V1G1protein level and aggravated iron deposition caused by alcohol（P<0.05）,while quercetin could restore lysosomal damage and iron deposition induced by alcohol,but this protective effect was offset by Rab7 inhibition.Concanamycin A,a V-ATPase inhibitor,increased intracellular iron and lysosomal iron levels at low concentrations,accompanied by increased IRP2 protein level,HIF1αlevel,ROS level,decreased mitochondrial iron-sulfur cluster protein synthesis,mitochondrial structure damage,and number reduction.These results suggest that V-ATPase inhibition leads to intracellular iron deposition,while increased ROS leads to decreased intracellular available iron levels,resulting in stable HIF1αexpression and mitochondrial damage.Through Rab7,quercetin restores V-ATPase function,lowering intracellular iron accumulation caused by alcohol consumption.5.The Rab7 interacting proteins and their functions were explored by STRING retrieval and gene functional annotation.The results showed that the 10 interacting proteins most related to Rab7 were mainly divided into membrane fusion proteins and related proteins involved in vesicle retrograde transport,among which TBC1D5 was highly correlated with the turnover capacity of Rab7.Gao-binge alcohol feeding and alcohol incubation reduced TBC1D5 protein levels in mouse liver and HepG2 cells transfected with CYP2E1,respectively,while quercetin intervention restored TBC1D5 protein levels.The dynamic changes of mitochondria-lysosome MCSs mediated by TBC1D5-Rab7 turnover were detected by rotary confocal microscopy.The results showed that alcohol exposure caused the dissociation disorder of mitochondria-lysosome MCSs.Quercetin restores mitochondrial lysosomal MCSs breakdown in the alcohol group through Rab7,and then restores the protein levels of mitochondrial respiratory complex and FECH,alleviating mitochondrial damage and restoring cell proliferation.Conclusions:Gao-binge feeding inhibited the turnover of Rab7 in the liver,leading to the obstruction of lipophagy.It also down-regulated the protein level of V-ATPase subunit V1G1,and caused the mitochondrial-lysosomal MCSs cleavage disorder.Inhibition of V-ATPase leads to iron deposition and associated iron utilization disorders,resulting in mitochondrial damage.Quercetin intervention can improve decreased lipophagy,iron deposition,mitochondrial-lysosomal MCSs dissociation disorder,and reduce cell proliferation caused by alcohol exposure by restoring Rab7 turnover.Alcohol exposure reduced the level of GAP protein TBC1D5,which regulates Rab7 membrane turnover,quercetin restored the level of TBC1D5 and thus regulated Rab7 turnover.