| Objective:Citreoviridin(CIT)is a toxic secondary metabolite originally produced by Penicillum citreonigrum,Aspergillus terreus and so on CIT exerts toxicity on heart and liver.In our previous work,we found that CIT could activate the autophagy process in liver cells,but the upstream pathway of autophagy caused by CIT was still unclear.Recent studies have shown that peroxisome proliferator-activated receptor gamma(PPARγ)plays an important role in the regulation of autophagy through the mammalian target of rapamycin complex 2(mTORC2).However,the distribution and function of PPARγ are tissue-specific.In this study,we aimed to investigate the upstream pathway of autophagy induced by CIT and the role of PPARγ in CIT-induced autophagy in both liver and heart.Methods:In vivo,ICR mice were exposed to CIT at a concentration of 0.1 mg/kg body weight(bw)and 0.3 mg/kg bw through intrapertoneal injection for six weeks.We observed the morphology of mice heart and liver by hematoxylin-eosin(HE)staining.In order to measure the expressions of cardiac troponin T(cTnT)and creatine kinase-MB(CK-MB)in the serum of mice,we used the enzyme linked immunosorbent assay(ELISA).We used reverse transcription quantitative PCR(RT-qPCR)to measure the expressions of PPARγ target genes in the heart and liver of mice.The expressions of phospho-protein kinase B(p-AKT)(Ser473),microtubule-associated protein 1 light chain 3(LC3)and sequestosome 1(P62)protein in the heart and liver of mice were detected by Western blot.In vitro,we treated H9C2 cells with CIT at concentration of 2 mM to 8 mM andHepG2 cells with CIT at concentration of 1.25 mM to 5 mM for 24 h.We pretreated H9C2 and HepG2 cells with rosiglitazone,a PPARγ activator,to study the role of PPARγ.We used RT-qPCR to measure the expressions of PPARγ target genes in H9C2 and HepG2 cells.To measure the transcriptional activity of PPARγ in H9C2 cells,we used PPARγ Transcription Factor Assay Kit.The expressions of p-AKT(Ser473),LC3-II and P62 protein in H9C2 and HepG2 cells were detected by Western blot.We used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)method to measure the cells viability of H9C2 and HepG2 cells.In order to measure the expressions of cTnT and CK-MB in H9C2 cell culture medium,we used the ELISA method.Results:In vivo,the body weight,cardiac index and hepatic index of CIT-treated mice were not significantly different from those of control mice.In HE staining,the heart of mice showed disordered myocardial fibers with blurry boundaries in CIT-treated mice.The liver of mice was showed lipid vacuoles and lipid accumulation in CIT-treated mice.ELISA showed that the concentration of cTnT and CK-MB in mice serums was increased in CIT-treated mice.RT-qPCR showed that the expression of PPARγ target genes was decreased in CIT-treated hearts and livers.The level of LC3-II protein was increased dramatically and the P62 and p-AKT(Ser473)protein were decreased in CIT-treated hearts and livers.In vitro,RT-qPCR showed that the expression of PPARγ target genes was decreased in CIT-treated H9C2 and HepG2 cells.The level of LC3-II protein was increased dramatically and the P62 and p-AKT(Ser473)protein were decreased in CIT-treated H9C2 and HepG2 cells.After pretreatment with RSG,the expression of LC3-II protein was decreased and the expression of P62 and p-AKT(Ser473)protein was increased dramatically in CIT-treated H9C2 and HepG2 cells compared with cells treated with CIT alone.MTT showed that the cells viability was decreased in CIT-treated H9C2 and HepG2 cells.After pretreatment with 3-MA and RSG,the cells viability was increased in CIT-treated H9C2 and HepG2 cells.The concentration of cTnT and CK-MB in H9C2 cell culture medium was increased after CIT treatment.After pretreatment with 3-MA and RSG,the concentration of cTnT and CK-MB in H9C2 cell culture medium was decreased,compared with cells treated with CIT alone.Conclusion:CIT can activate autophagy through inhibiting PPARγ and mTORC2 in cardiomyocytes and hepatocytes in vivo and in vitro. |
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