The Role Of Alk-SMase In Regulating Cholesterol Metabolism In Intestinal Epithelial Cells

Background and PurposeCholesterol is a kind of multi-effector molecule,and it is necessary to maintain normal physiological functions by maintaining cholesterol homeostasis.Abnormal blood cholesterol levels have been linked to many diseases,such as atherosclerosis,cancer,neurodegenerative diseases,and diabetes.Alkaline sphingomyelinase is an extracellular enzyme that is expressed in intestinal mucosal cells and plays a key role in the hydrolysis of alkaline sphingomyelinase（SM）into ceramides.In the study of alk-SMase knockout mice,our research group found that exogenous cholesterol in the feces of mice increased significantly,which preliminarily suggested that alk-SMase might be involved in the regulation of intestinal cholesterol homeostasis.At the same time,studies have found that alk-SMase activity is significantly decreased when diseases such as high-fat diet,intestinal inflammation,and tumor occur,which may be the risk of hypercholesterolemia.In order to confirm this point of view,this topic will conduct a study on the regulatory role of alk-SMase in intestinal cholesterol metabolism and its molecular mechanism through animal experiments and cell experiments,so as to lay a theoretical foundation for clarifying the physiological regulatory role of intestinal alk-SMase in cholesterol homeostasis.MethodsThe mice were divided into WT mice(alk-SMase^+/+)and KO mice(alk-SMase^-/-)by gene identification,with 4-6 mice in each group.The mice were killed at the age of12 to 16 weeks.Their blood,intestinal contents,and intestinal mucosal tissues were collected.The concentrations of cholesterol in intestinal contents,intestinal mucosal tissues,and serum of mice were detected.Western blotting assay was used to detect the protein levels of cholesterol transport-related proteins NPC1L1,NPC2,ABCG8,ABCA2,and Caveolin-1 in intestinal mucosal tissues.The m RNA levels of ABCA1,ABCG8,NPC2,ABCA2,NPC1L1,and transcription factor LXRαin intestinal mucosa were detected by RT-q PCR.The expression and localization of NPC2,ABCG8,ABCA1,and ABCA2 were detected by the immunofluorescence.After polarization culture,the Caco-2 cells were transfected with si RNA and plasmid to detect the concentration of cholesterol in the cells and in the upper and lower media.Filipin staining was used to detect the changes in cholesterol in cells.The protein levels of NPC1L1,NPC2,and ABCA1 cells were detected by Western blotting assay,and the m RNA levels of LXRαcells were detected by RT-q PCR.ResultsCompared with WT mice,the total cholesterol content in the intestinal mucosa of KO mice was significantly decreased（P<0.05）,the total cholesterol content in intestinal contents was significantly increased（P<0.05）,and the cholesterol content in plasma was significantly increased（P<0.01）.Intestinal mucosal transporters were detected and the expression of NPC1L1,a key transporter responsible for intestinal apical membrane absorption,was significantly decreased in KO mice（P<0.01）.The expression of the NPC2 transporter,responsible for intracellular cholesterol transport,was significantly increased（P<0.05）.ABCG8 also significantly increased the expression of a cholesterol efflux transporter in KO mice（P<0.05）;Lysosomal cholesterol transport ABCA2 was significantly decreased in KO mice（P<0.01）.Caveolin-1 was involved in vesicle transport and the expression of caveolin-1 was significantly increased in KO mice（P<0.01）.Compared with WT mice,the transcription levels of NPC1L1 and ABCA2 in KO mice were significantly decreased（P<0.01）.The m RNA expressions of NPC2,ABCG8,ABCA1 and Caveolin-1 were significantly increased（P<0.05）.Immunofluorescence results showed that the expression of NPC2,ABCG8,and ABCA1 increased in KO mice,while the expression of ABCA2 decreased.The expression of transcription factor LXRαwas further detected,which could regulate the expression of ABCA1,NPC2,ABCG5,and ABCG8 downstream.It was found that the m RNA level of LXRαin KO mice was significantly increased（P<0.05）.In cell experiments,cholesterol concentration detection and filipin results showed that the cholesterol content of the Caco-2 cell si RNA group decreased,but the difference was not significant.The cholesterol concentration in the upper medium was significantly increased（P<0.05）,and the cholesterol concentration in the lower medium was slightly increased,but the difference was not significant.The cholesterol content in the OE group was significantly increased（P<0.05）,the cholesterol concentration in the upper medium was slightly decreased,the difference was not significant,and the cholesterol concentration in the lower medium was significantly decreased（P<0.05）.The expression of transcription factor LXRαin the si RNA group was significantly increased（P<0.05）.The expression in the OE group was slightly decreased with no significant difference.Cholesterol transporter analysis showed that the expression of NPC1L1 was decreased in the si RNA group,while the expression of NPC2 and ABCA1 was slightly increased,but the difference was not significant.The expression of NPC1L1 and NPC2increased in the OE group,but the difference was not significant,while the expression of ABCA1 decreased significantly（P<0.01）.ConclusionsAlk-SMase is involved in the metabolic process of cholesterol absorption,transport,and efflux in mouse intestinal mucosal cells.The molecular mechanism may be that alk-SMase regulates LXRα,and then regulates downstream cholesterol transporter to participate in cholesterol metabolism.This provides a theoretical basis for the physiological function of intestinal alk-SMase in maintaining intestinal cholesterol homeostasis,but further verification is needed.