Magnolol,a Lipid-lowering Active Ingredient Of Magnolia Bark,Regulates Cardiac Lipid Metabolism Through Germ Cell Nuclear Factor

Lipotoxicity in the cardiomyocyte occurs in the setting of increased substrate availability or decreased substrate utilization.Intracardiac lipid accumulation can finally cause heart failure.ANGPTL4 is one of angiopoietin-like proteins which inhibits LPL activity.Traditional Chinese medicine theory believes that cardiac lipotoxicity belongs to the category of “phlegm” or “cloudy poison”.The principal treatment of phlegm and stasis is dispelling sputum and removing toxity.Magnolol is a natural bisphenol compound extracted from the traditional Chinese medicine Magnolia,which has a variety of cardiovascular protection effects.But the regulation effect and the underlying mechanism of magnolol on ANGPTL4 expression in cardiomyocytes is still unknown.Our research had shown that GCNF,an orphone nuclear factor,regulates vascular smooth muscle cell migration.Microarray analysis indicated that ANGPTL4 is potentially regulated by GCNF.This study is to investigate whether Magnolol regulates ANGPTL4 gene through GCNF and regulates cardiac lipid metabolism.Purpose:(1)whether magnolol regulates expression of ANGPTL4 in cardiomyocytes;(2)whether ANGPTL4 is a target gene of GCNF;(3)whether GCNF-ANGPTL4 axis mediates the regulation effect of magnolol on cardiac lipid metabolism and angiogenesis.Methods:MTT-cell based toxicity sssay was performed to check the toxicity of magnolol on H9C2 cells;The m RNA expression of ANGPTL4 in rat heart,primary cardiomyocytes and H9C2 cells were analyzed by using RT-qPCR method;The subcellular localization of GCNF in H9C2 cells was analyzed by using immunofluorescent staining;The m RNA and protein expression of GCNF in magnolol treated H9C2 cells was checked by using RT-qPCR and western Blot assays;GCNF-specific siRNA was used to selectively reduce GCNF in cultured H9C2 cells;The expression level of ANGPTL4 in GCNF silenced cells was further checked by using RT-qPCR and Western Blot methods;The regulation effect of magnolol on ANGPTL4 expreesion in GCNF silenced-or overexpressed cells was analyzed by using RT-qPCR and Western Blot methods;Using luciferase reporter gene detection technology to analyze the effects of on The regulation effect of magnolol on promoter activities of ANGPTL4 and GCNF was checked by using the luciferase reporter assay;The expreesion fo GCNF in heart tissues of fasted or db/db diabetic mice was analyzed by using RT-qPCR method;The effect of H9C2 cell culture supernatant overexpressing GCNF on endothelial cell(HUVEC)migration was examined using a cell scratch-healing model.Results:(1)ANGPTL4 is expressed in heart tissue,primary cardiomyocytes and H9C2 cells;(2)Magnolol up-regulates m RNA expressions of ANGPTL4 and GCNF in H9C2 cells;(3)GCNF silencing can reduce the m RNA and protein expressionss of ANGPTL4 and inhibit the ANGPTL4 promoter activity;(4)GCNF exerts transcriptional regulation of ANGPTL4 by ANGPTL4 promoter region(-500 to-250)in H9C2 cells;(5)GCNF and PPAR? gene silencing both partially reversed the expreesion of ANGPTL4 induced by magnolol;(6)The expression of GCNF and ANGPTL4 was increased in myocardial tissue of starved rats.Conclusion: These results indicate that magnolol can positively regulate the independent GCNF-ANGPTL4 signal axis and PPAR?-ANGPTL4 signal axis to up-regulate the expression of ANGPTL4 in cardiomyocytes,and is a natural agonist of PPAR?.Magnolol rapidly regulates ANGPTL4 expression by activating PPAR?,and up-regulating GCNF to maintain ANGPTL4 expression,which is expected to be a potential drug for the treatment of cardiac lipotoxicity.