Effect And Mechanism Of P2RX7 On The Improvement Of Nonalcoholic Fatty Liver Through Lipophagy

PART ONE EXPRESSION CHANGES OF P2RX7 IN ANIMAL AND CELL MODELS OF NON-ALCOHOLIC FATTY LIVER DISEASEObjective To investigate the expression of P2RX7 in nonalcoholic fatty liver disease.Methods The expression of P2RX7 protein in ob/ob mice aged 4weeks,6 weeks and 8 weeks and liver tissues of db/db mice aged 8 weeks were detected by Western blot,and verified by the liver tissues of db/db mice aged 8 weeks.HepG2 cell lines were interfered with free fatty acids with different concentration gradients and time gradients to construct a lipid overload model of hepatocytes,and the expression of P2RX7 protein was detected by Western blot.Results Western blotting showed that the expression of P2RX7 was significantly decreased in the liver of both ob/ob and db/db mice(p<0.01).In the lipid overload cell model,the protein expression of P2RX7 decreased with increasing fat concentration and prolongation of intervention time(p<0.01).Conclusion The protein expression of P2RX7 is decreased in animal models of non-alcoholic fatty liver disease.The expression level was also decreased in the lipid overload cell model.SECOND TWO P2RX7 IMPROVED LESIONS IN THE LIPID OVERLOAD CELL MODELObjective To investigate the effect of P2RX7 on lipid overload cell model.Methods P2RX7 agonist and inhibitor were used to intervene HepG2 cells treated with free fatty acid,respectively.The accumulation of lipid droplets was observed by oil red staining and electron microscopy.Image J software was used to classify and count the number and size of lipid droplets.Results The results of oil red staining and electron microscopy showed that the lipid droplets of P2RX7 agonist group decreased significantly(P<0.01),the lipid droplets in the inhibitor group were significantly increased and enlarged(P<0.01).Conclusion P2RX7 can improve the accumulation of lipid droplets in lipid overloadPART THREE P2RX7 PROMOTES THE DEGRADATION OF AUTOPHAGOSOMES THROUGH LYSOSOMES Objective To explore the mechanism of P2RX7 affecting lipid droplet accumulation.Methods The mechanism of P2RX7 was analyzed by bioinformatics of GEO database.Western blot and confocal laser were used to verify the bioinformatics prediction results.Results Bioinformatics predicted that P2RX7 affected lysosomal function under high lipid condition.Western blot analysis showed that the lysosomal marker LAMP2 was significantly increased in the agonist group(P<0.01),LAMP2 in the inhibitor group was significantly decreased(P<0.01).The number of lysosomes in agonist group was significantly increased by laser confocal analysis.Conclusion P2RX7 promotes the degradation of autophagosomes by promoting lysosomal production and thus lipid droplet degradation.PART FOUR P2RX7 PROMOTES THE FORMATION OF AUTOPHAGOSOMES THROUGH THE AMPK/ULKI PATHWAY Objective To explore the mechanism of P2RX7 affecting lipid droplet accumulation.Methods The effects of P2RX7 agonists and inhibitors on autophagy were detected by mREF-GFP-LC3 and electron microscopy.Western blotting was used to detect the expression of upstream proteins AMPK,mTOR,ULK1 and their phosphorylated proteins that promote autophagosome production.Western blotting was used to detect the expression of autophagy labeled proteins LC3 and P62.Results The number of autophagosomes and autolysosomes in agonist group was significantly increased by confocal laser and electron microscopy(P<0.01).P-AMPK,P-ULK1,LC3 were significantly increased in agonist group(P<0.01),p-mTOR and P62 were significantly decreased.P-AMPK and P-ULK1 decreased significantly in the inhibitor group(P<0.01),p-mTOR also showed a decreasing trend,but not as obvious as agonist group,and P62 significantly increased(P<0.01).Conclusion P2RX7 promotes lipid droplet degradation by promoting autophagosome formation.