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[Study on Huanglian Jiedu Decoction improving ox-LDL-induced LC3-related phagocytosis dysfunction in RAW264.7
Song-Fan Wang1, Ying-Yi Jiang1, Xing-Yuan Li1
1Heart Center,the First Affiliated Hospital of Henan University of Chinese Medicine Zhengzhou 450000, China Collaborative Innovation Center of Prevention and Treatment of Major Diseases by Chinese and Western Medicine Zhengzhou 450000, China the First Clinical Medical College of Henan University of Traditional Chinese Medicine Zhengzhou 450000, China.
Abstract:
To investigate whether the anti-atherosclerotic(AS) effect of Huanglian Jiedu Decoction(HLJDD) is associated with activation of extracellular signal-regulated kinase 5(ERK5) and the consequent improvement of microtubule-associated protein 1 light chain 3(LC3)-associated phagocytosis(LAP) dysfunction in macrophages. RAW264.7 cells were randomly divided into the normal control group, oxidized low-density lipoprotein(ox-LDL) group, HLJDD group, simvastatin(simva) group, and RAW264.7-ERK5 gene knockout(ERK5-KO) group. According to the experimental protocol, cells in each group were treated for 24 h with normal control rat serum, ox-LDL, HLJDD-containing serum, or simva-containing serum. Apoptotic Jurkat cells were added to each group and co-cultured for 90 min. After removal of the supernatant, LC3-Ⅱ labeling was performed to observe LAPosomes in macrophages, and the clearance of apoptotic Jurkat cells was assessed. The expression levels of phosphorylated(p)-ERK5, ERK5, LAP-related signaling molecules [T-cell immunoglobulin and mucin-domain-containing molecule-4(TIM-4), vacuolar protein sorting 34(VPS34), Beclin-1, RUN domain Beclin-1-interacting and cysteine-rich domain-containing protein(Rubicon), autophagy-related protein 5(ATG5), and autophagy-related protein 7(ATG7)], pro-inflammatory cytokines [interleukin-1β(IL-1β), interleukin-6(IL-6)], and anti-inflammatory cytokines [interleukin-10(IL-10), transforming growth factor-β(TGF-β)] were measured. RESULTS:: showed that, compared with the control group, the ox-LDL group exhibited decreased LAPosome percentage and reduced clearance of apoptotic cells, downregulated expression of p-ERK5, TIM-4, VPS34, Beclin-1, Rubicon, ATG5, and ATG7, increased IL-1β and IL-6 levels, and no significant changes in IL-10 or TGF-β expression. Compared with the ox-LDL group, the HLJDD and simva groups showed increased LAPosome percentage and enhanced apoptotic cell clearance, upregulated expression of p-ERK5, TIM-4, VPS34, Beclin-1, Rubicon, ATG5, and ATG7, decreased IL-1β and IL-6 levels, and increased IL-10 and TGF-β expression. Compared with the HLJDD group, the ERK5-KO group exhibited significantly reduced expression of ERK5 and p-ERK5, and all other indicators were reversed. In conclusion, HLJDD may ameliorate macrophage LAP dysfunction and exert anti-AS effects by activating ERK5.