Paeonol Protect The Heart Via Activating PPARδ Expression And Activation

Objective:The peroxisome proliferator-activated receptors (PPARs)are ligand-activated transcription factors ofthe nuclear receptor superfamily. ThePPAR subfamily contains three members known asPPARα, PPAR δ, and PPARA.They regulate glucose, lipid, and cholesterol metabolism in response to fattyacids and their derivatives, eicosanoids, and drugs usedin the treatment of hyperlipidemia and diabetes. PPAR δ most expression in kidney and heart tissure, it enhances fatty acid catabolism and energy uncoupling in adipose tissue and muscle, and it suppresses macrophage-derived inflammation.Paeonol is an active component of Moutan Cortex Radicis and is widely used as an analgesic, antipyretic, andanti-inflammatoryagent in traditional Chinese medicine. A number of studies have revealed thatpaeonol possesses many pharmacological activities that may be useful for treating cardiovascular disease. Doxorubicin (DOX), an effective and broad-spectrumantitumor anthracycline drug, has been used for the treatmentof cancer for many years. However, its clinical application is limited bya strong, cumulative, dose-dependent cardiotoxicity. DOX leads to cardiac dysfunction through inductionof necrosis and apoptosis in highly differentiatedcardiomyocytes. Therefore, to further explore the potential protective effects and the underlying mechanisms of Paenole on DOX-induced cardiotoxicity is nessary and beneficial for the traditional Chinese medicine development and new medicine discovery.Method:(Ⅰ)In vitro:Paeonol’s effect on PPARδ expression and activity in H9C21. Cell transfection:Transfect the pFATPx3-Luc and PPARδ promoter plasmid into H9C2cells,pRL-Luc plasmid was used as internal control. Different treatments were applied after24hours of transfection.2. Luciferase Assay of pFATPx3-Luc to determine PPAR activity:the effect of Paeonol, GW0742(PPARd ligand) on Luciferase activity.3. Luciferase Assay to deterimine PPAR δ promoter activity:GW0742as positive control, Paeonol’s effect on PPARδ promoter activity.4. RT-qPCR to determinePPAR mRNA expression(Ⅱ) In vivo:Paeonol’s effect on PPAR and related target genes expression in C57BL/6mouse heart1. C57BL/6mouse treat with paeonol (i. p.)150mg/kg/per day for6times in total.2. RT-qPCR to determine PPAR δ and related target genes expression3.RT-PCR to determinePPARδ mRNA expression4. Westen Blot to determine PPAR δ protein expression5. PPAR α,δ, λ complete transcription assay to determinePPAR δ activity(Ⅲ) In vivo:Paeonol’s effect on Dox induced cardiac dysfunction nouse1. C57BL/6mouse pretreat with paeonol(i. p.)150mg/kg/per day for3days, then induced by Dox(i. p.)5mg/kg once a week for4weeks, while continue paeonol injection every other day,3times a week and12times in total2. Echocardiacgraphy before and after Dox injection,2times in total. Record the heart function image and data analysis. Collect the body weight each week, heart weight and tibia length ratio after4weeks.3. HE-staining for heart section4. RT-qPCR to determinePPAR δ mRNA, cardiac dysfunction marker ANP mRNA and BNP mRNA expression.Result:(Ⅰ)In vitro:Paeonol’s effect on PPAR δ expression andactivity.1. Luciferase Assay result of PPAR activity:Paeonol enhanced PPAR8ligand GW0742’s effect on PPAR activity;2. Luciferase Assay result of PPAR δ promtoer activity:Paeonol activated PPARδ activity;3. RT-qPCR result shows paeonol upregulated PPARδ mRNA expression.(Ⅱ)In vivo:Paeonol’s effect on PPARδ and activity in C57BL/6mouse1. RT-qPCR shows paeonol upregulated PPAR δ and related target genes expression, such as PPARα, PPARλ, MCD, LPL, LCAD, CPTla and CPTlb;2. RT-PCR shows paeonol upregulated PPARδ mRNA expression;3. Western Blot shows paeonol upregulated PPARδ protein expression;4. PPAR complete transcription factor assay indicated that paeonol activate PPAR δ activity.(Ⅲ)In vivo:Paeonol’s effect on Dox induced cardiac dysfunction mouse1. Echocardiacgraphy shows paeonol can protect the cardiac dysfunctions induced by Dox;2. HE-staining shows paeonol can protecct the pathological changes induced by Dox;3. RT-qPCR shows down-regulated cardiac dysfunction marker ANP mRNA and BNPmRNA expression and paeonol protect the heart via PPARδ expression.Conelusion:1. Paeonol activating PPAR δ and upregulated PPAR δ expression in H9C22. Paeonol activating PPAR δ and upregulated PPAR δ in C57BL/6mouse heart3. Paeonol protect the cardiac dysfunction regarding of upregulated PPAR8expression and downregulate ANP and BNP in Dox induced mice heart...