Paeoniflorin Mitigates Diabetic Liver Injury Via Regulating Ferroptosis

Background and purpose:Diabetic liver injury（DLI）is a common complication of diabetes mellitus.It has been suggested that the ferroptosis pathway plays an important role in diabetes mellitus combined with nonalcoholic fatty liver disease（NAFLD）.Paeoniflorin（PF）is a monoterpene glycoside compound extracted from the Chinese herbal medicines Peony,which is widely studied due to its specific pharmacological effects as well as its multi-targeting and low toxicity.In this study,we investigated the effects of paeoniflorin on liver lipid peroxidation and ferroptosis in vivo and in vitro,and observed the therapeutic effects of paeoniflorin on diabetic liver injury.Methods:In vivo experiments: 12 male db/m mice and 24 male db/db mice at 8 weeks of age were randomly divided into 6 groups: db/m group;db/m+PF group（100 mg/kg）;db/db group;db/db+PF low,intermediate and high dose groups（25 mg/kg,50 mg/kg,100mg/kg）,6 mice in each group.Each group was given equal amounts of saline and paeoniflorin daily according to the body weight of the mice.After 12 weeks of feeding,liver tissues were taken intact and liver weight/body weight was calculated.The levels of tail tip blood glucose,serum alanine transaminase（ALT）and aspartate transaminase（AST）,serum and liver triglycerides（TG）,total cholesterol（TC）and free fatty acids（FFA）were measured.The levels of liver 4-hydroxynonenal（4-HNE）and malondialdehyde（MDA）,the end products of lipid peroxidation were measured.The levels of liver GSH and superoxide dismutase（SOD）activity,GSH/GSSG and iron were measured.HE staining was used to observe the structural changes of mouse liver,DHE staining was used to detect ROS levels of mouse liver,and immunohistochemistry was used to detect the expression of ACSl4 and GPX4 in mouse liver.The expression of ACSL4,LPCAT3,ALOX15,GPX4,FTH1,TFR1,AMPK,P-AMPK,SREBP1 and NRF2 in mouse liver was measured by Western blot.The m RNA levels of ACSL4,LPCAT3,ALOX15,GPX4,SREBP1,ACC,FASN and SCD1 in mouse liver were measured by quantitative real-time PCR（q RT-PCR）.In vitro experiments: AML12 cells were taken and high glucose intervention was applied to establish a diabetic liver injury model.The drug toxicity of paeoniflorin was measured by MTT to screen the optimal concentration of the drug,the contents of ACSl4 and GPX4 were detected by Western blot to screen the experiment time.On these basis,the groups were divided into: normal glucose（NG）group;normal glucose +paeoniflorin（NG+PF）group（16 μmol/L）;mannitol（M）group;high glucose（HG）group;high glucose + paeoniflorin（HG+PF）low,intermediate and high dose groups（4μmol/L,8 μmol/L,16 μmol/L）,the above seven groups.In addition,high glucose +Ferrostatin-1（HG + Fer-1）group was set to observe the effect of Fer-1 on liver injury induced by high glucose.The accumulation of ROS in AML12 cells was measured by DCFH-DA;the expression of ACSL4,LPCAT3,ALOX15,GPX4,FTH1,TFR1,AMPK,P-AMPK,SREBP1 and NRF2 was measured by Western blot;the m RNA levels of ACSL4,LPCAT3,ALOX15,GPX4,SREBP1,ACC,FASN and SCD1 were measured by quantitative real-time PCR（q RT-PCR）.The binding of paeoniflorin to AMPK was verified by CETSA.Western blot was performed to detect the effect of paeoniflorin on NRF2,ACSL4,LPCAT3,ALOX15 and GPX4 after the use of AMPK inhibitor Dorsomorphin.Results:In vivo experiments: Compared with the db/db group,there were no significant changes in blood glucose and liver weight/body weight in paeoniflorin-administered mice.Paeoniflorin treatment significantly improved liver function and liver lipid deposition in mice,reduced liver lipid peroxidation products 4-HNE and MDA content,increased GSH content and SOD activity.In addition,liver GSH/GSSG increased and iron content decreased in paeoniflorin-administered mice.The treatment with paeoniflorin significantly reduced the histopathological damage to the liver of mice.Immunohistochemical results showed that the expression of ACSL4 in db/db mice was increased and the expression of GPX4 was inhibited.Western blot showed that SREBP1,ACSL4,LPCAT3,ALOX15 and TFR1 protein levels were decreased,P-AMPK,NRF2,GPX4 and FTH1 protein levels were increased in the liver of db/db mice after paeoniflorin treatment.In addition,paeoniflorin treatment significantly modulated the expression of genes related to the ferroptosis pathway,including increased GPX4 m RNA levels and decreased ACSL4,LPCAT3,ALOX15,SREBP1 and its downstream gene m RNA levels.In vitro experiments: 4 μmol/L,8 μmol/L and 16 μmol/L were selected as the experimental drug concentrations by MTT.Immunofluorescence results showed that high glucose intervention significantly promotes intracellular ROS accumulation in AML12 cells.Western blot showed that the expression of SREBP1,ACSL4,LPCAT3,ALOX15 and TFR1 increased,the expression of P-AMPK,NRF2,GPX4 and FTH1 decreased after high glucose intervention.In q RT-PCR,GPX4 m RNA level decreased and ACSL4,LPCAT3,ALOX15,SREBP1 and its downstream gene m RNA levels increased in the high glucose group.Paeoniflorin had a good protective effect on AML12 cells,and the related indexes of ferroptosis were significantly improved compared with high glucose group.Meanwhile,we found that Fer-1 could inhibit high glucose induced ferroptosis in AML12 cells and reduce ROS accumulation.Ferroptosis plays an important role in high glucose induced liver injury.AMPK was confirmed as a potential target of paeoniflorin by CESTA.Further validation using the AMPK inhibitor Dorsomorphin confirmed the protective effect of paeoniflorin via the AMPK/SREBP1 pathway.Conclusion:Paeoniflorin can inhibit hepatic lipid peroxidation and ferroptosis to reduce diabetes induced liver injury.