The Mechanism Of Calcitriol Promoting Hepatocyte-like Cells Maturation And Alleviating Alcoholic Hepatocyte Damage

Calcitriol(1,25-dihydroxyvitamin D3)is an active molecule produced by the metabolism of vitamin D(VD)in vivo,and its activation involves two hydroxylation modifications in the liver and kidney.Vitamin D is well known for its classical hormonal action to promote intestinal calcium and phosphorus absorption,maintain calcium and phosphate homeostasis,and promote bone growth and maintain bone health.Recent researches have recognized that calcitriol also regulates cell proliferation and differentiation,inflammatory immune response,fibrosis,and other physiological and pathological processes.VD deficiency is a common condition worldwide,which has gradually aroused people’s attention.VD deficiency is closely associated with a variety of skeletal and non-skeletal chronic diseases.It has been reported that VD deficiency is frequently present in nonalcoholic and alcoholic fatty liver,hepatitis,liver fibrosis,and other liver diseases.There is increasing clinical and basic evidence that low VD level is positively correlated with an increase of histologic liver damage in liver disease patients.VD supplementation can improve the sustained virological response in chronic hepatitis C and alleviate hepatic toxin-induced cirrhosis in a rat model and suppress hepatic stellate cell activation in vitro.The observations that calcitriol regulates cell differentiation and is critical for liv er function led to the question that whether calcitriol regulates differentiation of huma n pluripotent stem cells into functional hepatocytes,and whether it plays any role in pr otecting against alcohol-induced hepatocyte injury.Human hepatocyte-like cells(HLCs)derived from human pluripotent stem cells(hPSCs)represent a promising cell source for pharmaceuticals toxicity testing and regenerative medicine.However,the hepatic functionality of HLCs is still significantly inferior to primary human hepatocytes.In the first part of this study,calcitriol was added during the hepatic differentiation.Then,we analyzed the expression level of hepatocyte maturation-related genes and hepatocyte function in HLCs.The results showed that calcitriol promoted the expression of mature related genes and the function of detoxification enzyme activity of the P450 system,urea production,and albumin secretion of HLCs derived from human embryonic stem cells and human-induced pluripotent stem cells.Moreover,calcitriol also enhanced mitochondrial respiratory with increased protein expression levels of the subunit of respiratory enzyme complexes in HLCs.Further analyses showed that calcitriol administration could up-regulate mitochondrial biogenesis regulators’ expression and cause mitophagy leading to improved mitochondrial function.Those improvements in functionality and mitochondrial condition were vitamin D receptor(VDR)dependent since the improvements were abolished in VDR deficiency conditions.Excessive ethanol consumption causes cellular damage leading to alcohol liver diseases.A great deal of progress has been achieved in the mechanism studies of alcohol-induced hepatocyte damage.However,there are limited preventive intervention means to reduce or rescue hepatocyte damage caused by alcohol.Calcitriol exhibit a significant protective effect on ethanol-induced hepatotoxicity in HLCs.We have found that calcitriol has a significant protective effect on ethanol-induced hepatocyte damage in HLCs,without affecting the protein expression levels of ethanol metabolizing enzymes ADH1 and CYP2E1.Therefore,the mechanism of calcitriol in alleviating ethanol-induced hepatotoxicity is worth elucidating.In the second part of this study,hepatocytes were pretreated with calcitriol and then exposed to ethanol.The results showed that calcitriol pretreatment markedly improved the cell viability,decreased cell apoptosis and oxidative stress,and alleviated the abnormal mitochondrial morphology and membrane potential of hepatocytes induced by ethanol.Notably,autophagy was significantly enhanced by calcitriol.as evident by the increasing number of autophagosomes and autolysosomes,upregulated LC3B-II and ATG5 levels,and promotion of p62 degradation.Furthermore,calcitriol pretreatment increased the colocalization of GFP-LC3-labeled autophagosomes with mitochondria,suggesting that calcitriol effectively promoted ethanol-induced mitophagy in hepatocytes.In comparison,the inhibition of autophagy attenuated the protective and preventive effect of calcitriol.Further studies on autophagy-related regulatory pathways have found that the effect of calcitriol on autophagy was regulated by AMPK/mTOR signaling,and signaling transduction was dependent on the VDR.In conclusion,calcitriol improved autophagy by activating AMPK/mTOR signaling and ameliorated ethanol-induced hepatocyte damage.In summary,we found that calcitriol promotes the functional maturation of hepatocyte-like cells,providing a cost-effective method for obtaining maturation hepatocytes derived from stem cells in vitro.Besides,the protective effect of calcitriol on ethanol-induced hepatocyte injury provided a new strategy and intervention mean to prevent alcoholic hepatocyte injury.