Paeoniflorin alleviates LPS-induced aerobic glycolysis in HK-2 cells by modulating the PI3K/AKT/HIF-1α pathway
10.16016/j.2097-0927.202505035
- VernacularTitle:芍药苷通过调控PI3K/AKT/HIF-1α通路减轻脂多糖诱导的HK-2细胞有氧糖酵解
- Author:
Jing XIE
1
;
Ruonan LI
;
Huihui GAO
;
Shunkai YANG
;
Yuqing MA
Author Information
1. 兰州大学第一临床医学院
- Keywords:
sepsis associated-acute kidney injury;
aerobic glycolysis;
PI3K/AKT/HIF-1α pathway;
paeoniflorin;
renal tubular epithelial cells
- From:
Journal of Army Medical University
2025;47(20):2483-2494
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of paeoniflorin(PF)on lipopolysaccharide(LPS)-induced aerobic glycolysis in renal tubular epithelial cell line HK-2 and its underlying mechanism of action.Methods This study consists of a preliminary experiment and a formal experiment.Preliminary experiment:CCK-8 assay and RT-qPCR were used respectively to measure cell viability and mRNA expression levels of inflammatory factors in HK-2 cells after LPS stimulation to determine the optimal LPS concentration for modeling as well as to evaluate the toxicity of PF and screen for its appropriate concentration.Formal experiment:HK-2 cells were divided into control group(CON group),LPS group,LPS+PF group and LPS+PF+740Y-P group.LPS was used to establish a cell model of sepsis associated-acute kidney injury(SA-AKI)in HK-2 cells,and then the cell model was treated with PF and PI3K activator 740Y-P,correspondingly for 24 h.CCK-8 assay was employed to detect cell viability,and Extracellular Acidification Rate(ECAR)Kit was utilized to measure the rate.The contents of IL-1β,IL-18,lactic acid(Lac)and lactate dehydrogenase A(LDHA)were determined with ELISA.Western blotting was applied to detect the expression of p-PI3K,p-AKT,HIF-1α,pyruvate kinase 2(PKM2,a key enzyme in aerobic glycolysis)and NOD-like receptor thermal protein domain associated protein 3(NLRP3),and immunofluorescence assay was performed to observe the expression and distribution of PKM2.Results ① Our preliminary experiment identified that the optimal concentration of LPS for modeling was 20.0 μg/mL,a safe dosage range of PF was 0～100.0 μmol/L,and its optimal therapeutic concentration was 25.0 μmol/L.② Compared with the CON group,LPS stimulation resulted in significantly decreased cell viability(P<0.05),increased ECAR(P<0.05),elevated contents of IL-1β,IL-18,Lac and LDHA(P<0.05),up-regulated protein levels of p-PI3K,p-AKT,HIF-1α,p-PKM2 and NLRP3(P<0.05),and enhanced fluorescence intensity of PKM2 in the nucleus of cells(P<0.05).Compared with the model group,PF treatment reversed all above effects induced by LPS stimulation(all P<0.05).Compared with the LPS+PF group,in the LPS+PF+740Y-P group,ECAR was elevated(P<0.05),the contents of IL-1β,IL-18,Lac and LDHA were increased(P<0.05),and the relative expression levels of p-PI3K,p-AKT,HIF-1α,p-PKM2 and NLRP3 were increased(P<0.05),and the fluorescence intensity of PKM2 was strengthened(P<0.05)and enhanced in the nucleus(P<0.05).Conclusion PF reduces aerobic glycolysis in HK-2 cells and alleviates the inflammatory response by inhibiting the PI3K/AKT/HIF-1α signaling pathway.
