OBJECTIVE:To elucidate the possible mechanism of Jiawei Erzhi pill(加味二至丸,JWEZP)in the treatment of atherosclerosis(AS).METHODS:The chemical constituents of JWEZP were identified using ultra-performance liquid chr...OBJECTIVE:To elucidate the possible mechanism of Jiawei Erzhi pill(加味二至丸,JWEZP)in the treatment of atherosclerosis(AS).METHODS:The chemical constituents of JWEZP were identified using ultra-performance liquid chromatographymass spectrometry.A high-fat diet(HFD)was used to establish AS models in Apo E-/-mice.The Apo E-/-mice were randomly divided into the Model(normal saline),Simvastatin(normal saline),Low-dose JWEZP,Mediumdose JWEZP and High-dose JWEZP groups(n=15),and C57BL/6 mice on a normal diet were used as the control group.Mice were treated with JWEZP at different doses(3.9,7.8,15.6 g·kg^(-1)·d^(-1))or with simvastatin(2.6 mg·kg^(-1)·d^(-1))for ten weeks.The inhibitory effects of JWEZP on AS were assessed by measuring serum lipid levels and changes in atherosclerotic plaques,lipid peroxidation and ferroptosis.Common targets and key regulatory pathways of JWEZP-mediated inhibition of ferroptosis were predicted using network pharmacology and verified using real-time quantitative polymerase chain reaction(RT-q PCR)and Western blotting.RESULTS:We identified 46 active compounds in JWEZP.Mice in the JWEZP group had lower body weights and serum cholesterol levels compared to HFD mice.The results of Hematoxylin-Eosin and Oil Red O staining showed that JWEZP alleviated AS.Masson staining showed that JWEZP improved the stability of atherosclerotic plaques.In addition,JWEZP-treated mice had lower levels of reactive oxygen species(ROS)in thoracic aortic tissue according to ROS fluorescence staining.The ELISA results showed that JWEZP decreased the levels of iron,lipid peroxide,malondialdehyde and nicotinamide adenine dinucleotide phosphate and increased the levels of glutathione(GSH)and GSH-PX in the thoracic aortic tissues of mice.The expression of glutathione peroxidase 4 in the thoracic aorta of mice in the JWEZP group was upregulated in the results of the immunofluorescence assay.Network pharmacology results indicated that the action mechanisms of JWEZP-mediated inhibition of ferroptosis were closely related to the p53,mitogen-activated protein kinase(MAPK),and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathways.RT-q PCR and Western blotting results demonstrated that JWEZP inhibited the p53 and MAPK pathways,and activated the PI3K/Akt pathway to regulate ferroptosis.CONCLUSION:JWEZP improved AS by inhibiting ferroptosis.The study provides a scientific basis for further research and validation of JWEZP as a potential therapeutic for AS.展开更多
基金grants from the National Natural Sciences Foundation of China:Study on the Mechanism of Modified Erzhi Pills Upregulating Adiponectin to Improve the Anti-vasoconstriction Function of Active Perivascular Fat in Menopausal Rats(No.81373799)。
文摘OBJECTIVE:To elucidate the possible mechanism of Jiawei Erzhi pill(加味二至丸,JWEZP)in the treatment of atherosclerosis(AS).METHODS:The chemical constituents of JWEZP were identified using ultra-performance liquid chromatographymass spectrometry.A high-fat diet(HFD)was used to establish AS models in Apo E-/-mice.The Apo E-/-mice were randomly divided into the Model(normal saline),Simvastatin(normal saline),Low-dose JWEZP,Mediumdose JWEZP and High-dose JWEZP groups(n=15),and C57BL/6 mice on a normal diet were used as the control group.Mice were treated with JWEZP at different doses(3.9,7.8,15.6 g·kg^(-1)·d^(-1))or with simvastatin(2.6 mg·kg^(-1)·d^(-1))for ten weeks.The inhibitory effects of JWEZP on AS were assessed by measuring serum lipid levels and changes in atherosclerotic plaques,lipid peroxidation and ferroptosis.Common targets and key regulatory pathways of JWEZP-mediated inhibition of ferroptosis were predicted using network pharmacology and verified using real-time quantitative polymerase chain reaction(RT-q PCR)and Western blotting.RESULTS:We identified 46 active compounds in JWEZP.Mice in the JWEZP group had lower body weights and serum cholesterol levels compared to HFD mice.The results of Hematoxylin-Eosin and Oil Red O staining showed that JWEZP alleviated AS.Masson staining showed that JWEZP improved the stability of atherosclerotic plaques.In addition,JWEZP-treated mice had lower levels of reactive oxygen species(ROS)in thoracic aortic tissue according to ROS fluorescence staining.The ELISA results showed that JWEZP decreased the levels of iron,lipid peroxide,malondialdehyde and nicotinamide adenine dinucleotide phosphate and increased the levels of glutathione(GSH)and GSH-PX in the thoracic aortic tissues of mice.The expression of glutathione peroxidase 4 in the thoracic aorta of mice in the JWEZP group was upregulated in the results of the immunofluorescence assay.Network pharmacology results indicated that the action mechanisms of JWEZP-mediated inhibition of ferroptosis were closely related to the p53,mitogen-activated protein kinase(MAPK),and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathways.RT-q PCR and Western blotting results demonstrated that JWEZP inhibited the p53 and MAPK pathways,and activated the PI3K/Akt pathway to regulate ferroptosis.CONCLUSION:JWEZP improved AS by inhibiting ferroptosis.The study provides a scientific basis for further research and validation of JWEZP as a potential therapeutic for AS.
