| The metabolic syndrome caused by abnormal glucose and lipid metabolism is a kind of chronic diseases that seriously threaten human health and life safety.Among them,type 2 diabetes and nonalcoholic fatty liver disease(NAFLD)are typical representative diseases of generation syndrome.Therefore,specific and effective drugs for the prevention and treatment of NAFLD are necessary.In previous studies,we revealed the protective effects of HP against glucotoxicity and lipotoxicity by promoting pancreatic duodenal homeobox-1(PDX1)expression and activating Erk signalling in islet β-cells.On this basis,We investigated the protective effects of non-photoactivated hypericin on β-cells in vivo.Meanwhile,in this study,we also aimed to explore the preventive and therapeutic effects of HP against NAFLD and the underlying mechanisms in vitro and in vivo.The specific results of this study are mainly as follows:1.Preventive and therapeutic effects of HP against NAFLD and the underlying mechanisms in vitro and in vivoTo demonstrate whether HP has a protective effect in hepatocytes under high lipid conditions,we established a model of FFA-induced lipotoxicity using L02,Hep G2 cells or primary hepatocytes to investigate the protective effect of HP on hepatocyte viability and abnormal lipid metabolism.Firstly,the effects of HP on hepatocyte viability,oxidative damage and apoptosis under the condition of 1 m M free fatty acid(FFA)were detected by MTT,ROS,TUNEL and western blot.Lipid accumulation and content and the expression of lipid metabolism-related proteins were detected by biochemical methods and western blot.The results showed that HP improved cell viability by reducing apoptosis and attenuated lipid accumulation in hepatocytes via attenuating oxidative stress,inhibiting lipogenesis and enhancing lipid oxidization.We also demonstrated that 1 m M FFA interferes with AMPK signaling to cause hepatocyte impairment,whereas HP effective alleviates FFA-induced disorders of lipid metabolism via the recovery of AMPK signaling.To obtain systemic information regarding the effects of HP on lipid metabolism in FFA-treated hepatocytes and to gain further insight into the working mechanisms,we conducted transcriptome analysis by RNA-seq in untreated and FFA-treated L02 cells with or without HP.To identify other differentially expressed lipid metabolism-related genes in each group,we selected some related genes and validated alterations in their expression under different conditions by RT-q PCR.We concluded that HP effectively reversed abnormal lipid metabolism in hepatocytes caused by FFA.We also founded that the c AMP signaling pathway may be one of the main responsive pathways that contributes to the effects elicited by HP in hepatocytes.We hypothesize that drug targets of HP might be present in this signaling pathway.Molecular docking,capillary electrophoresis and immunofluorescence were used to identify the drug target of HP.the PKACα protein displayed high binding affinity for HP,forming two hydrogen bonds with HP.In addition,HP made hydrophobic contacts with PKACα residues(Leu61,Glu78,Gly42,Gly39,Lys59,Arg43,Val44,Gly37,Asn313,Phe174,Asp171,Gly173,Asp153).To further validate the amino acids of PKACα that are responsible for binding to HP,we mutated five amino acids randomly from the above thirteen amino acids to generate a series of mutated PKACα.HP formed two hydrogen bonds with PKACα-mut and interacted with less and altered hydrophobic residues(Lys160,Glu162,Ala43,Gly44,Asn163,Gly42,Phe179,Gly47,Gly178,Ala46,Leu66)compared with wild type PKACα.This molecular docking result indicated that PKACα-mut had decreased binding capacity with HP.To verify the predicted interaction between HP and PKACα by molecular docking,we used capillary electrophoresis and immunofluorescence experiments to demonstrate the binding of HP to PKACα.The binding of HP to PKACα is stronger than the binding of HP to PKACα-mut.Furthermore,we also detected binding between PKACα and HP in hepatocytes since HP exhibits red autofluorescence.Finally,we demonstrated that HP directly bound to PKACα and activated PKACα/AMPK signaling to elicit its effects against NAFLD,suggesting that PKACα is one of the drug targets of HP.We further explored the beneficial effects of HP in vivo.To do this,the preventive and therapeutic benefits of HP were examined in a mouse model of NAFLD,which was generated by feeding mice a HFHS diet for approximately four or five months.The preventive and therapeutic effects of HP on NAFLD were assessed by serological testing,histology,and immunohistochemical analysis.The results suggested that prophylactic or therapeutic HP treatment indeed protects mouse liver tissue from HFHS diet-induced NAFLD via attenuating oxidative stress.Additionally,the levels of lipid metabolism-related proteins,including PKACα and AMPK,in liver tissue were analyzed by Western blotting and IHC.The p-AMPK,CPT1 A and PKACαexpression levels were decreased,while the SREBP1,SCD1 and LDLR levels were increased in the liver tissue of HFHS diet-fed mice.However,prophylactic or therapeutic HP treatment normalized these protein levels in the liver tissue of HFHS diet-fed mice,which is consistent with the effects of HP on oxidative damage and apoptosis.To further observe whether HP also has beneficial effects on lipid metabolism in adipocytes,we conducted experiments in white adipocytes(WAs)differentiated from h MSCs.Oil red O staining indicated the HP decreased the number of lipid droplets in WAs in a dose-dependent manner compared to that in the control.HP also significantly increased the lipolysis of WAs,as evidenced by glycerol content analysis.Then,we conclude that HP increases lipolysis in adipocytes as it does in hepatocytes by activating PKACα.2.Preventive and therapeutic effects of HP against type 2 diabetes in vivoIn previous studies,we revealed the protective effects of HP against glucotoxicity and lipotoxicity by PDX1 expression and activating Erk signaling in vitro.On this basis,We investigated the protective effects of non-photoactivated hypericin onβ-cells in vivo.The results showed that prophylactic and therapeutic use of HP dramatically inhibited the increase in fasting blood glucose levels in HFHS-fed mice in a dose-dependent manner.Moreover,other diabetic phenotypes such as the expression of muscle glycogen and hepatic glycogen were ameliorated in a dose-dependent manner by prophylactic or therapeutic use of HP.Then,We also founded that prophylactic and therapeutic use of HP promoted PDX1 expression,inhibited islet β-cell apoptosis and enhanced the anti-oxidative ability of pancreatic tissue in HFHS-fed mice,thus alleviating β-cell loss and maintaining β-cell mass and islet size.There were no obvious lesions in the histopathology of kidney,heart muscle and muscle through pathological analysis of different organs.Therefore,the concentration of HP used in this experiment is within a safe and reasonable range,and will not cause pathological damage to organs.The HP,a natural compound,showed protective effects on hepatocyte and prevented the dyslipidemia in mice.Then,HP showed preventive effects against the onset of type 2 diabetes in mice.HP possesses potential to be developed as a preventive or therapeutic anti-NAFLD or abnormal lipid metabolism drug in the future. |
