| In recent years,non-alcoholic fatty liver disease（NAFLD）caused by lipid metabolism disorder has attracted widespread attention worldwide.Studies have shown that although selenium（Se）is an essential trace element,excessive Se supplementation can induce the occurrence of NAFLD.Selenoprotein H（SelH）is a type of protein that exists in mitochondria and is highly expressed in the liver.Under high-fat diet conditions,the expression of SelH in adipose tissue decreases,and there is a potential relationship between SelH and obesity.As a new hypothesis,competing endogenous RNA（ceRNA）has revealed a variety of new therapeutic mechanisms for NAFLD through the characterization of non-coding RNA functions.However,no literature has reported the ceRNA control axis involved in SelH and the potential relationship between SelH and NAFLD.To explore the role of SelH in NAFLD,wild-type C57BL/6J mice and SelH knockout C57BL/6J mice were used as experimental animals in this study.The NAFLD mouse model was replicated by feeding a 60%kcal high-fat diet（HFD）.After feeding the mice with HFD for twelve weeks,the liver tissue and serum of the mice in each group were collected.The liver coefficient of mice,the routine biochemical indicators of the liver,the changes in the histomorphology and ultrastructure of the mouse liver were observed,and the expression of genes related to lipid metabolism and other pathways was analyzed.It is clear that the knockout of SelH can mediate lipid metabolic pathways that inhibit NAFLD caused by HFD.Subsequently,the regulatory axis of circ-0000518/miR-135a-5p/SelH was predicted and constructed in vitro through mouse liver parenchyma cells（AML-12）,an in vitro fatty acid exposure model was established（1 mmol/L sodium oleate+0.5 mmol/L sodium palmitate）combined with bioinformatics,dual luciferase reporter assay and other technologies.The knockdown/overexpression model of SelH,the knockdown/overexpression model of miR-135a-5p,and the knockdown model of circ-0000518were established,and the effects of this ceRNA regulatory axis on lipid metabolism in AML-12cells were analyzed and verified.This study aims to investigate the role of circ-0000518/miR-135a-5p/SelH axis in lipid metabolism disorders from the perspective of ceRNA,to clarify the molecular mechanism of SelH knockout inhibiting HFD-induced NAFLD,and to provide a new reference for the treatment of NAFLD.The main results are as follows:（1）Histopathological and ultrastructural observations showed that compared with the mice in the WT+normal fat diet（NFD）group,the liver tissue of the mice in the WT+HFD group had obvious lipid droplet accumulation and hepatic cord,increased cell volume,shrinking nucleus,unstable nucleoli,swelling of mitochondria and cristae fracture or disappearance;No obvious presence of lipid droplets was observed in the SelH-/-+NFD group,and the liver locks were neatly arranged.Although a small amount of fat accumulation was also seen in the SelH-/-+HFD group mice,the liver cords could be arranged radially and neatly in the hepatic lobules,the nucleus,mitochondria and endoplasmic reticulum were clear,and the cell membrane structure was complete.Combined with Oil Red O staining The results confirmed that SelH knockout inhibited HFD-induced fat accumulation in the liver of C57BL/6J mice.（2）Bioinformatics predicted a potential interaction between circ-0000518/miR-135a-5p/SelH,which was confirmed by the dual-luciferase gene reporter assay.In AML-12 cells,the expression of SelH was increased when miR-135a-5p was knocked down;the expression of SelH was decreased when miR-135a-5p was overexpressed;and the expression of miR-135a-5p was increased and the expression of SelH decreased when circ-0000518 was knocked down,these three constitute the ceRNA regulatory network.Further real-time quantitative PCR and western blotting results showed that circ-0000518 could bind to miR-135a-5p,inhibit the negative feedback regulation of miR-135a-5p on SelH,and then affect the downstream PPAR-γ/CD36 lipid metabolism pathways and structural stability of MAMs.（3）Compared with the WT+NFD group,the contents of SOD and CAT in the WT+HFD group decreased,while the contents of MDA and LPO increased;Compared with the mice in the WT+HFD group,the levels of SOD,CAT,MDA and LPO in the liver of the SelH-/-+HFD group did not change significantly,and the verification results of the Keap1/Nrf2 antioxidant pathway also showed that it did not occur significant changes.In vitro,knockdown of SelH in AML-12cells in a high-fat environment did not significantly affect the Keap1/Nrf2 pathway.This indicates that under high-fat conditions,there is oxidative stress in mouse liver tissue,but knockdown of SelH does not regulate oxidative stress through the Keap1/Nrf2 pathway.（4）Under the condition of HFD feeding,the structure of MAMs in the liver tissue of wild-type mice was destroyed.Combined with the results of transmission electron microscopy,it was observed that the mitochondrial cristae were broken,and the endoplasmic reticulum and mitochondria were loosely connected.The structure of MAMs in the liver of SelH-/-+HFD mice was intact,and the expression of PERK and other genes was down-regulated.In vitro experiments showed that the expression of PERK and other genes was affected by ceRNA network.Overexpressed SelH could inhibit the decline of PERK expression caused by circ-0000518 and other factors,SelH knockout could maintain MAMs structural stability.（5）Compared with the WT+NFD group,the PI3K/AKT pathway in the liver tissue of the mice in the WT+HFD group was inhibited to a certain extent;Compared with the SelH-/-+NFD group,the PI3K/AKT pathway was also inhibited to a certain extent in the SelH-/-+HFD group,but under the HFD feeding condition,there was no significant difference between the SelH gene knockout mice and the wild-type mice.The difference indicates that SelH knockout cannot regulate fat metabolism through lipid synthesis pathway.（6）In this study,the genes related to lipid synthesis and decomposition were tested in vivo.In the synthetic pathway,SOCS3 and SREBP1 were significantly increased in mice in WT+HFD group compared with WT+NFD group.Compared with WT+HFD,SelH-/-+HFD mice significantly decreased SOCS3 and SREBP1,and SelH inhibited the synthesis of fat in NAFLD to a certain extent.In the decomposition pathway,compared with the WT+NFD group,the EHHADH and CPT2 of the WT+HFD group were increased,and the expression of these lipolytic genes increased during the process of steatosis.However,in the SelH-/-+HFD group mice,the expression of these genes still decreased to a certain extent compared with the WT+HFD group mice.SelH knockout affected the lipid decomposition pathway of NAFLD.（7）Compared with mice in WT+NFD group,the expression of PPAR family genes in mice in WT+HFD group was generally increased.Compared with mice in WT+HFD group,the PPAR family genes in the liver of SelH-/-+HFD group mice were generally down-regulated,the expression of genes related to PPAR-γ/CD36 signaling pathway was inhibited,and the fat synthesis and transport ability of the liver of mice were suppressed.In vitro,this study used an activator（rosiglitazone）and an inhibitor（GW9662）of the PPAR-γsignaling pathway to verify the regulation of lipid metabolism by the PPAR-γ/CD36 pathway upon SelH knockdown/overexpression effect.The results showed that rosiglitazone/GW9662 had an antagonistic effect on the abnormal fatty acid metabolism caused by SelH knockdown/overexpression,indicating that SelH knockout mainly regulates fat metabolism by inhibiting the PPAR-γ/CD36 signaling pathway.In summary,circ-0000518 can indirectly regulate SelH by competitively binding to the molecular sponge on miR-135a-5p,thereby forming a ceRNA regulatory network of circ-0000518/miR-135a-5p/SelH.This ceRNA network can prevent the synthesis and transport of fat by inhibiting the PPAR-γ/CD36 pathway,maintain the normal structure of MAMs,and inhibit the occurrence of fatty liver.In this experiment,the mechanism of SelH knockout inhibiting the occurrence of NAFLD was studied,and the ceRNA regulatory mechanism of SelH and the downstream lipid metabolism regulatory pathway were clarified,which provided theoretical support for the drug research and development of NAFLD and a reference for ceRNA research. |
