Inhibitory effect and mechanism of Danyu formula on oxidized low-density lipoprotein-induced foam cell formation in rat aortic endothelial cells

SONG Xiaolong; PAN Renyou; TU Dongyun; GU Yuexing; SONG Jun; WU Deming; NI Qimeng

doi:10.3969/j.issn.1002-266X.2023.35.009

Abstract：Objective To explore the inhibitory effect of Danyu formula on foam cell formation in rat aortic endothe-lial cells(RAECs)induced by oxidized low-density lipoprotein(Ox-LDL)and its possible mechanism.Methods RAECs were randomly divided into the control group,model group,low-dose Danyu formula group,medium-dose Danyu formula group,and high-dose Danyu formula group,and atorvastatin group.Except for the control group,RAECs in the rest of the five groups were treated with 200 μg/mL Ox-LDL for 72 h,and the low-,medium-,and high-dose Danyu formu-la groups and the atorvastatin group were treated with 50,100,and 200 μg/L Danyu formula solution and 300 μg/L atorv-astatin,respectively.After 24,48,and 72 h of treatment,the cell proliferation ability was detected by CCK-8.The foam formation was observed by oil red O staining in the cells treated for 72 h in each group.The protein levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),malondialdehyde(MDA),and superoxide dismutase(SOD)in the cell superna-tant were measured by ELISA.The reactive oxygen species(ROS)content was determined by the 2-hydroxyethylfluoresce-in oxidation fluorescence reaction.The relative mRNA expression levels of protein tyrosine kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3)were detected by real-time fluorescence quantitative PCR.The relative protein expression levels of JAK2,p-JAK2,STAT3,and p-STAT3 were determined by Western blotting,and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were calculated.Results Compared with the control group,the OD values of the model group decreased at the same time points(all P＜0.05).Compared with the model group at the same time points,the OD values of the high-dose Danyu formula group and the atorvastatin group increased,and the OD values of the medium-dose Danyu formula group increased at 48 and 72h(all P＜0.05).The control group cells did not exhibit foam formation,while the cells in the model group showed obvious foam formation.Compared with the model group,the foam formation in the atorvastatin group and the high-,medium-,and low-dose Danyu formula groups improved,with the high-dose Danyu formula group and the atorvastatin group showing the most significant improvement,almost approaching the control group.Compared with the control group,the levels of TNF-α,IL-6,ROS,JAK2 mRNA expression,STAT3 mRNA expression,p-JAK2/JAK2,p-STAT3/STAT3,and MDA in the cell supernatant of the model group increased,while the expression of SOD in the cell supernatant decreased(all P＜0.05).Compared with the model group,the levels of TNF-α,IL-6,ROS,JAK2 mRNA expression,STAT3 mRNA expression,p-JAK2/JAK2,p-STAT3/STAT3,and MDA in the cell supernatant of the atorvastatin group and the high-,medium-,and low-dose Danyu formula groups decreased,while the expression of SOD in the cell supernatant increased,with the most significant changes in the atorvastatin group and the high-dose Danyu formula group(all P＜0.05).There were no significant differences in the relative protein expression levels of JAK2 or STAT3 among these groups(all P＞0.05).Conclusion The high-dose,medium-dose,and low-dose Danyu formula can inhibit foam formation in Ox-LDL-induced RAECs,with the significant effect in the high-dose Danyu formula,and its mechanism may be related to the inhibition of inflammatory reaction,oxidative stress response,and JAK2/STAT3 signal-ing pathway.