Paeoniflorin alleviates LPS-induced aerobic glycolysis in HK-2 cells by modulating the PI3K/AKT/HIF-1α pathway

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.

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.

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).

PF reduces aerobic glycolysis in HK-2 cells and alleviates the inflammatory response by inhibiting the PI3K/AKT/HIF-1α signaling pathway.