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

XIE Jing; LI Ruonan; GAO Huihui; YANG Shunkai; MA Yuqing

doi:10.16016/j.2097-0927.202505035

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

Basic Medicine | 更新时间：2025-12-25

- Paeoniflorin alleviates LPS-induced aerobic glycolysis in HK-2 cells by modulating the PI3K/AKT/HIF-1α pathway
- Journal of Army Medical University Vol. 47, Issue 20, Pages: 2483-2494(2025)
- 作者机构：
  
  1.兰州大学：第一临床医学院，甘肃兰州
  2.兰州大学：第一医院麻醉科，甘肃兰州
- 作者简介：
- 基金信息：
  
  the General Project of Natural Science Foundation of Gansu Province(24JRRA1075);the Fund of the First Hospital of Lanzhou University(ldyyyn2022-5)
- DOI：10.16016/j.2097-0927.202505035
  CLC： R285.5;R329.26;R692.6
- Received：15 May 2025，
  
  Revised：2025-09-25，
  
  Accepted：26 September 2025，
  
  Published：30 October 2025
- 稿件说明：
移动端阅览
谢菁，李若男，高慧慧，等.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［J］.陆军军医大学学报，2025，47（20）：2483-2494.DOI：10.16016/j.2097-0927.202505035.

XIE J，LI R N，GAO H H，et al.Paeoniflorin alleviates LPS-induced aerobic glycolysis in HK-2 cells by modulating the PI3K/AKT/HIF-1α pathway［J］.J Army Med Univ，2025，47（20）：2483-2494.DOI：10.16016/j.2097-0927.202505035.
谢菁，李若男，高慧慧，等.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［J］.陆军军医大学学报，2025，47（20）：2483-2494.DOI：10.16016/j.2097-0927.202505035. DOI：

XIE J，LI R N，GAO H H，et al.Paeoniflorin alleviates LPS-induced aerobic glycolysis in HK-2 cells by modulating the PI3K/AKT/HIF-1α pathway［J］.J Army Med Univ，2025，47（20）：2483-2494.DOI：10.16016/j.2097-0927.202505035. DOI：

摘要

目的

探讨芍药苷（paeoniflorin，PF）对脂多糖（lipopolysaccharide，LPS）诱导的肾小管上皮细胞（HK-2细胞）有氧糖酵解的影响及其作用机制。

方法

本研究分为预实验和正式实验两部分。①预实验：体外培养HK-2细胞，CCK-8实验检测细胞活力，RT-qPCR检测炎症因子mRNA的水平，进行LPS最佳造模浓度筛选、芍药苷毒性实验和浓度筛选。正式实验：将HK-2细胞分成对照组（CON组）、LPS组、LPS+PF组和LPS+PF+740Y-P组。通过建立LPS诱导的SA-AKI肾小管细胞损伤的体外模型，分别给予PF和PI3K激活剂740Y-P干预，造模24 h后取材，CCK-8实验检测细胞活力；细胞外酸化率（extra cellular acidification rate，ECAR）试剂盒检测ECAR；ELISA检测IL-1β、IL-18、乳酸（lactic acid，Lac）以及乳酸脱氢酶A（lactate dehydrogenase A，LDHA）；Western blot检测p-PI3K、p-AKT、HIF-1α、丙酮酸激酶2（pyruvate kinase 2，PKM2）、NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）的表达；免疫荧光观察有氧糖酵解关键酶PKM2的表达和分布情况。

结果

①筛选出LPS最佳造模浓度为20.0 μg/mL，芍药苷安全剂量为0~100.0 μmol/L，芍药苷的最佳治疗浓度为25.0 μmol/L。②与CON组相比，LPS组细胞活力明显下降（

0.05），ECAR明显升高（

0.05），IL-1β、IL-18、Lac、LDHA水平明显增加（

0.05），p-PI3K、p-AKT、HIF-1α、p-PKM2、NLRP3蛋白相对表达量明显增加（

0.05），细胞核内PKM2荧光强度明显增强（

0.05）。与LPS组相比，LPS+PF组ECAR明显下降（

0.05），IL-1β、IL-18、Lac、LDHA水平明显下降（

0.05），p-PI3K、p-AKT、HIF-1α、p-PKM2、NLRP3蛋白相对表达量明显减少（

0.05），细胞核内PKM2荧光强度明显减弱（

0.05）。与LPS+PF组相比，LPS+PF+740Y-P组ECAR明显升高（

0.05），IL-1β、IL-18、Lac、LDHA水平明显增加（

0.05），p-PI3K、p-AKT、HIF-1α、p-PKM2、NLRP3蛋白相对表达量明显增加（

0.05），细胞核内PKM2荧光强度明显增强（

0.05）。

结论

芍药苷通过抑制PI3K/AKT/HIF-1α信号通路，降低HK-2细胞有氧糖酵解水平，减轻炎症反应。

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 (

0.05)

increased ECAR (

0.05)

elevated contents of IL-1β

IL-18

Lac and LDHA (

0.05)

up-regulated protein levels of p-PI3K

p-AKT

HIF-1α

p-PKM2 and NLRP3 (

0.05)

and enhanced fluorescence intensity of PKM2 in the nucleus of cells (

0.05). Compared with the model group

PF treatment reversed all above effects induced by LPS stimulation (all

0.05). Compared with the LPS+PF group

in the LPS+PF+740Y-P group

ECAR was elevated (

0.05)

the contents of IL-1β

IL-18

Lac and LDHA were increased (

0.05)

and the relative expression levels of p-PI3K

p-AKT

HIF-1α

p-PKM2 and NLRP3 were increased (

0.05)

and the fluorescence intensity of PKM2 was strengthened (

0.05) and enhanced in the nucleus (

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.

关键词

Keywords

references

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