Mechanisms Of AKR1C3 Facilitating NAFLD By Reprogramming Lipid Metabolism

Objective: Nonalcoholic fatty liver is the most common chronic liver disease in the world,affecting up to 30% of adults and 70-80% of obese and diabetes patients.In China,the incidence rate of nonalcoholic fatty liver is increasing year by year.The patients with nonalcoholic fatty liver disease account for 29.2% of the total population in China,of which 10% develop cirrhosis within three years and 12.8% develop liver cancer after three years.Although some progress has been made in clarifying the pathogenesis of nonalcoholic fatty liver and developing targeted drugs for the treatment of nonalcoholic fatty liver,the current situation of treatment is not optimistic because its pathogenesis is not fully clarified.At present,FDA has not approved any drugs for the treatment of nonalcoholic fatty liver.Aldehyde-ketone reductase family 1 member C3(AKR1C3)is an aldehyde-ketone reductase that uses NADH and NADPH to reduce ketone steroids to hydroxysteroids.NAD(P)H is used to catalyze the reduction of 3-,17-and 20-ketosteroids on the steroid skeleton and side chain,and regulate the metabolism of androgen,estrogen and progesterone.This study aims to clarify the role of AKR1C3 in promoting liver cell fat storage under high lipid pressure,reveal the molecular mechanism of AKR1C3 regulating liver cell lipid metabolism,and explore whether AKR1C3 can be a new target for NAFLD treatment.Methods:(1)The intracellular lipid content was determined by lipid fluorescence staining and flow cytometry;(2)ROS staining was used to determine the intracellular ROS level.(3)CCK8 cell count test was used to detect cell viability;(4)The changes of AKR1C3 were detected by Western Blot under high lipid pressure;(5)The autophagy pathway protein was detected by Western blot;(6)MCherry-GFP-LC3 double fluorescence labeling was used to detect the changes of autophagy flow;(7)Western blot method was used to detect the protein of fat de novo synthesis pathway;(8)Oil red O staining was used to detect the changes of lipid droplets in the liver of human AKR1C3 transgenic mice;(9)Western blot was used to detect the expression of AKR1C3 gene in human AKR1C3 transgenic mice;Results :(1)The results of lipid droplet fluorescence staining and flow cytometry showed that knockdown or pharmacological inhibition of AKR1C3 could inhibit the fat storage of hepatocytes and decrease the intracellular lipid droplets;At the same time,overexpression of AKR1C3 can lead to increased lipid droplets in hepatocytes.(2)Western blot results showed that AKR1C3 were up-regulated under high lipid pressure;(3)ROS staining and CCK8 cell count showed that AKR1C3 could protect cells and reduce ROS under high lipid pressure.(4)Western Blot experiment showed that after knockdown of AKR1C3,the autophagy pathway was activated,and at the same time,the fat de novo synthesis pathway was inhibited.After overexpression of AKR1C3,the autophagy pathway was inhibited,and the fat de novo synthesis pathway was activated.(5)Consistent with Western blot results,m Cherry-GFP-LC3 double fluorescence labeled autophagy flow detection results showed that AKR1C3 knockdown cell autophagy flow was activated,and overexpression cell autophagy flow was blocked.(6)The results of oil red O staining showed that the liver fat storage of mice expressing human AKR1C3 increased and the intracellular lipid droplets increased.Conclusion: AKR1C3 can promote hepatocyte fat storage and further develop into steatosis and promote the occurrence and development of fatty liver by promoting hepatocyte fat synthesis,inhibiting hepatocyte fat autophagy and mediating hepatocyte lipid metabolism reprogramming.