1,25-Dihydroxyvitamin D₃ Alleviates Ox-LDL-Induced Liver Sinusoidal Endothelial Cell Dysfunction By Inhibiting Pyroptosis Via MTOR/HIF-1α Pathway

Background:Non-alcoholic fatty liver disease（NAFLD）is the most common chronic liver disease worldwide,and the continued increase in its prevalence has posed a serious threat to human health.Studies have found that vitamin D deficiency is associated with the progression of NAFLD,and vitamin D supplementation is not only anti-inflammatory and anti-atherosclerosis,but also delay liver steatosis and fibrosis.Inhibition of pyroptosis in Kupffer cells（KCs）and hepatocytes was found to attenuate inflammation in NAFLD liver tissue,but the mechanism of pyroptosis in liver sinusoidal endothelial cells（LSECs）has not been studiedObjective and Significance:In this study,ox-LDL induced HLSECs was used to simulate NAFLD in vitro,aiming to investigate whether 1,25（OH）₂D₃ alleviates ox-LDL induced HLSECs dysfunction by regulating pyroptosis and clarify the mechanism of mammalian target of rapamycin（m TOR）/hypoxia inducible factor-1α（HIF-1α）signaling in this process.This study helps to illustrate the mechanism of vitamin D and pyroptosis in hepatic sinusoidal dysfunction and provide a scientific basis for vitamin D supplement for the prevention and treatment of non-alcoholic fatty liver disease.Methods:ox-LDL induced HLSECs to establish NAFLD model in vitro.1,25（OH）₂D₃,selective NLRP3 inhibitor MCC950,m TOR inhibitor rapamycin,HIF-1αinhibitor BAY 87-2243 were used to intervene cells,respectively.The expression of laminin（LN）,fibronectin（FN）,Cleaved caspase-1,GSDMD-N were detected by Western blotting.The frequency and diameter of the fenestrations were measured by scanning electron microscopy,intracellular lipid droplets were detected by a Cell Navigator?Fluorimetric Lipid Droplet Assay Kit,and the contents of IL-1βand IL-18 in cell supernatant were detected by enzyme linked immunosorbent assay（ELISA）.Statistical analysis of experimental data was performed using Graph Pad Prism 8.0 software.Results:1.Under the induction of ox-LDL,the protein expression of LN and FN in HLSECs increased in a time-and concentration-dependent manner,and was most different at a concentration of 100μg/ml for 24h.1,25（OH）₂D₃ pretreatment inhibited the expression of LN and FN induced by ox-LDL in a concentration-dependent manner,and the concentration of 1,25（OH）₂D₃ at 100 n M was the most significant（P<0.05）.2.Compared with normal control group,the fluorescence intensity of lipid droplets in HLSECs in ox-LDL group was increased,and the number and diameter of fenestrations were significantly decreased,while no significant difference between the1,25（OH）₂D₃+ox-LDL group and the normal control group（P<0.05）.3.Ox-LDL stimulated increased protein expression of NLRP3,cleaved caspase-1（p20）,and GSDMD-N in HLSECs in a concentration-dependent manner,while pretreatment with 1,25（OH）₂D₃ inhibited this change in a concentration-dependent manner（P<0.05）.4.The selective NLRP3 inhibitor MCC950 not only inhibited the activation of NLRP3/caspase-1/GSDMD and the contents of IL-1βand IL-18,but also decreased the expression of LN and FN protein and the fluorescence intensity of intracellular lipids induced by ox-LDL（P<0.05）.5.1,25（OH）₂D₃ inhibits the activation of ox-LDL-induced m TOR/HIF-1αpathway（P<0.05）.6.Following blockade of the m TOR/HIF-1αpathway with inhibitors,protein expression of the NLRP3/caspase-1/GSDMD pyroptosis pathway and the levels of IL-1βand IL-18 in the cell supernatant decreased,along with decreased LN and FN protein expression and intracellular lipid droplets（P<0.05）.Conclusion:1,25-dihydroxyvitamin D₃ alleviates ox-LDL-induced LSECs dysfunction by inhibiting pyroptosis via m TOR/HIF-1αpathway and vitamin D supplementation may be a potential treatment for non-alcoholic fatty liver disease.