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Mycelium Of Paecilomyces Hepiali Stimulates Lipolysis In Adipocytes And Alleviates Hepatic Steatosis In Hepatocytes

Posted on:2018-11-28Degree:MasterType:Thesis
Country:ChinaCandidate:M GeFull Text:PDF
GTID:2393330512994196Subject:Botany
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Cordyceps sinensis has been used for centuries in China as one of the most valued traditional Chinese medicine.It has a wide range of pharmaceutical benefits,such as kidney and lung benefitting,hemostatic and expectorant effects,anti-cancer,anti-inflammatory and hypolipidemic effect.Because of the limited habitat,C.sinensis has been scarce in the nature.Therefore,it is increasingly important for the development of efficacy C.sinensis alternatives.In recent years,isolation of fungal strains from natural C.sinensis to achieve a large-scale production by fermentation is a trend.Paecilomyces hepialid,a fungal strain isolated from Cordyceps sinensis.The mycelia of this fungus are cultivated by artificial deep fermentation,and have been used widely as a substitute of C.sinensis in medicine and health foods.Previous studies show that fermented mycelia of Paecilmyces hepiali exhibits hypolipidemic activity.The previous work of our Lab found that methanol extract from mycelia of Paecilomyces hepiali(PHME)suppressed differentiation of preadipocytes to mature adipocytes.However,effect of PHME on lipolysis,another important pathway to suppress fat accumulation,is relatively limited.Therefore,the aim of this study was to clarify the effect of PHME on triglyceride accumulation in adipocytes and hepatocytes and its underlying mechanism.Furthermore,we performed a preliminary study on cordycepin,a bioactive ingredient of PHME,about its effect on lipid accumulation in adipocytes and hepatocytes.First of all,we extracted PHME from fermented mycelia of Paecilomyces hepiali,and then to determine the contents of the main components in it.The results showed that the contents of adenosine,guanosine,uridine,cordycepin,total sugar,reducing sugar and mannitol were 0.33%,0.44%,0.94%,0.53%,3.96%,3.18%and 25%,respectively,in the PHME.After that we used 3T3-L1 adipocytes to investigate the effect of PHME and its active components on adipocytes lipolysis and the underlying mechanisms.Lipolysis was evaluated by measuring the amount of glycerol released to the medium.To understand the molecular mechanisms,mRNA and protein expression of several lipolysis-related genes,lipases,PerilipinA,and phosphorylation levels of HSL,AMPK,and MAPKs were detected by qRT-PCR and Western blot.Results indicated that PHME promotes the basal lipolysis,isoproterenol-stimulated and forskolin-stimulated lipolysis.Western blot analysis showed that PHME enhanced phosphorylated HSL at sites of Ser563 and Ser660 and promoted the level of phosphorylated ERK.Furthermore,results of western blot and qRT-PCR analysis showed that PHME markedly downregulated the mRNA and protein levels of PerilipinA.Moreover,PD98059,an inhibitor for ERK,attenuated the PHME-caused increase in glycerol release and down-expression of PerilipinA.Meanwhile,H89,an inhibitor for PKA,inhibited the PHME-induced increase in glycerol release and HSL phosphorylation.These results demonstrate that PHME enhances lipolysis via the activation of ERK/PerilipinA and cAMP-PKA/HSL signaling pathway in 3T3-L1 adipocytes.Among five components,only cordycepin is able to promote adipocytes lipolysis by downregulating the mRNA and protein levels of PerilipinA and PPAR?.At last,we investigated the effects of PHME and cordycepin on triglyceride accumulation in hepatocytes and the molecular mechanisms.When HL7702 hepatocytes were treated with sodium oleate,significant triglyceride accumulation was observed.Under the same experimental conditions,addition of PHME and cordycepin significantly suppressed the sodium oleate-induced lipid accumulation in HL7702 hepatocytes.Meanwhile,the expression of C/EBPa,a key molecule involved in lipogenesis,was decreased in PHME and cordycepin-treated hepatocytes.PHME was found to downregulate phosphorylated levels of JNK.Moreover,we found that cordycepin downregulated the expression of PPARy,another key molecule involved in lipogenesis,whereas upregulated the expression of PPARa,which is involved in FFA oxidation.Meanwhile,cordycepin increased phosphorylation of AMPK,which attenuates FFA synthesis and promotes FFA oxidation.In conclusion,PHME and cordycepin attenuated triglyceride accumulation in mature adipocytes through promoting lipolysis and attenuated triglyceride accumulation in hepatocytes to improve hepatic steatosis.
Keywords/Search Tags:Paecilomyces hepiali, lipolysis, cAMP-PKA signaling pathway, ERK signaling pathway, hepatocytes
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