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福建中医药大学 中西医结合学院,福州 350122
Received:07 September 2025,
Revised:2025-12-11,
Accepted:12 December 2025,
Online First:24 December 2025,
Published:20 May 2026
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段阳阳,纪翔伦,陈佳红等.泰山磐石散调控KEAP1/NRF2/FoxO3信号通路抑制滋养层细胞氧化应激损伤的作用机制[J].中国实验方剂学杂志,2026,32(10):12-22.
DUAN Yangyang,JI Xianglun,CHEN Jiahong,et al.Mechanism of Taishan Panshisan in Inhibiting Oxidative Stress Injury of Trophoblast Cells by Regulating KEAP1/Nrf2/FoxO3 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2026,32(10):12-22.
段阳阳,纪翔伦,陈佳红等.泰山磐石散调控KEAP1/NRF2/FoxO3信号通路抑制滋养层细胞氧化应激损伤的作用机制[J].中国实验方剂学杂志,2026,32(10):12-22. DOI: 10.13422/j.cnki.syfjx.20251808.
DUAN Yangyang,JI Xianglun,CHEN Jiahong,et al.Mechanism of Taishan Panshisan in Inhibiting Oxidative Stress Injury of Trophoblast Cells by Regulating KEAP1/Nrf2/FoxO3 Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2026,32(10):12-22. DOI: 10.13422/j.cnki.syfjx.20251808.
目的
2
探讨泰山磐石散(TSPSP)抑制人绒毛膜滋养层细胞(HTR-8/SVneo)氧化应激损伤的作用与机制,了解TSPSP治疗自然流产(SA)的机制。
方法
2
采用临床数据库(GEO)对SA进行基因差异分析,并与氧化应激相关联,利用网络药理学方法筛选TSPSP活性成分,构建“中药-成分-靶点-疾病”网络,预测TSPSP作用机制。体外验证实验将滋养层细胞HTR-8/SVneo细胞分为空白组,模型组,TSPSP含药血清2.5%、5%、10%组,核因子E
2
相关因子2(Nrf2)抑制剂组(ML385,30 μmol·L
-1
)。除空白组外,其他组加入150 μmol·L
-1
H
2
O
2
刺激3 h构建细胞氧化应激损伤模型,造模成功后,空白组和模型组给予10%的空白血清,各TSPSP含药血清组分别加入相应浓度含药血清处理,Nrf2抑制剂组在给予10% TSPSP含药血清的基础上,额外加入30 μmol·L
-1
ML385,各组细胞在上述条件下培养24 h,收集样本进行后续检测。细胞增殖活性检测(CCK-8)法检测各组细胞活力;划痕实验检测细胞迁移率;酶联免疫吸附测定法(ELISA)检测丙二醛(MDA)、Fe
2+
、谷胱甘肽(GSH)含量;活性氧荧光探针(DCF-DA)检测细胞内活性氧(ROS)水平;免疫荧光(IF)、实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞Kelch样ECH关联蛋白1(KEAP1)、Nrf2、叉头框蛋白O3(FoxO3)蛋白和mRNA表达水平;蛋白免疫印迹法(Western blot)检测细胞KEAP1、Nrf2、FoxO3、谷胱甘肽过氧化物酶4(GPX4)、超氧化物歧化酶(SOD)蛋白表达水平。
结果
2
GEO数据库获取GSE76862与GSE22490数据库,差异基因分析表明KEAP1、Nrf2、FoxO3基因均与疾病相关;同氧化应激通路匹配后,获得9条差异显著通路(
P
<
0.05),其中3条包含目的基因Nrf2、FoxO3。通过网络药理学共获得TSPSP活性成分靶点246个、SA相关靶点2 804个,取交集后得到154个潜在作用靶点。拓扑分析显示KEAP1、Nrf2等靶点度值较高,基因本体(GO)与KEGG富集分析表明,交集靶点主要涉及氧化应激反应、FoxO及丝裂原活化蛋白激酶(MAPK)信号通路等。体外实验中,与空白组比较,模型组细胞活力显著下降(
P
<
0.01);与模型组比较,各TSPSP含药血清组细胞活力显著增高(
P
<
0.01);与10% TSPSP含药血清组比较,ML385组细胞活力下降至70%左右(
P
<
0.01)。与空白组比较,模型组MDA、Fe
2+
、ROS含量均显著增高,GSH表达下降(
P
<
0.01),细胞迁移率显著降低(
P
<
0.01),模型组KEAP1、FoxO3蛋白及mRNA表达水平均升高(
P
<
0.01),Nrf2、GPX4、SOD蛋白质及mRNA表达水平降低(
P
<
0.01);与模型组比较,各TSPSP含药血清组MDA、Fe
2+
、ROS含量均显著降低,GSH表达上升(
P
<
0.01),迁移率显著升高(
P
<
0.01),KEAP1、FoxO3蛋白及mRNA表达水平均明显降低(
P
<
0.05,
P
<
0.01),Nrf2、GPX4、SOD蛋白及mRNA表达水平明显升高(
P
<
0.05,
P
<
0.01);与10% TSPSP含药血清组比较,ML385组各指标趋势逆转(
P
<
0.05,
P
<
0.01)。
结论
2
TSPSP能抑制H
2
O
2
引起的滋养层细胞氧化应激损伤,其作用机制可能与药物激活KEAP1/Nrf2/FoxO3信号通路有关。
Objective
2
To explore the effect and mechanism of Taishan Panshi powder (TSPSP) on inhibiting oxidative stress injury in human chorionic trophoblast cells (HTR-8/SVneo), and to uelucidate the underlying mechanism of TSPSP in the treatment of spontaneous abortion (SA).
Methods
2
Gene differential analysis of SA was performed using the Gene Expression Omnibus (GEO) database and correlated with oxidative stress. Network pharmacology was employed to screen the active components of TSPSP, and a "Chinese medicine-component-target-disease" network was constructed to predict the mechanism of action of TSPSP. For
in vitro
validation experiments, HTR-8/SVneo cells were divided into blank group, model group, TSPSP-containing serum 2.5%, 5%, 10% groups, and nuclear factor E
2
-related factor 2 (Nrf2) inhibitor group (ML385, 30 μmol·L
-1
). Except for the blank group, other groups were stimulated with 150 μmol·L
-1
H
2
O
2
for 3 h to establish a cell oxidative stress injury model. After successful modeling, the blank group and model group were given 10% blank serum, each TSPSP-containing serum group was treated with the corresponding concentration of drug-containing serum, and the Nrf2 inhibitor group was additionally given 30 μmol·L
-1
ML385 on the basis of 10% TSPSP-containing serum. All groups of cells were continuously cultured under the above conditions for 24 h, and then samples were collected for subseq
uent detection. Cell viability in each group was detected by CCK-8 assay. Cell migration rate was detected by scratch test. The contents of malondialdehyde (MDA), Fe
2+
, and Glutathione (GSH) were detected by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) level was detected by a fluorescent probe (DCF-DA). The protein and mRNA expression levels of Kelch-like ECH-associated protein 1 (KEAP1), Nrf2, and forkhead box protein O3 (FoxO3) in cells were detected by immunofluorescence (IF) and real-time quantitative polymerase chain reaction (Real-time PCR). The protein expression levels of KEAP1, Nrf2, FoxO3, Glutathione peroxidase 4 (GPX4), and superoxide dismutase (SOD) in cells were detected by Western blot.
Results
2
The GSE76862 and GSE22490 datasets were obtained from the GEO database. Differential gene analyses showed that the KEAP1, Nrf2, and FoxO3 genes were all associated with the disease. After matching with the oxidative stress pathway, nine significantly differential pathways were identified (
P
<
0.05), among which three contained the target genes Nrf2 and FoxO3. A total of 246 active ingredient targets of TSPSP and 2 804 SA-related targets were obtained through network pharmacology, and 154 potential action targets were obtained after taking the intersection. Topological analysis showed that targets such as KEAP1 and Nrf2 exhibited high degree values. GO and KEGG enrichment analyses indicated that the intersection targets were mainly involved in oxidative stress response, FOXO and MAPK signaling pathways, etc. In in vitro experiments, compared with the blank group, the cell viability in the model group was significantly decreased (
P
<
0.01). Compared with the model group, the cell viability in each TSPSP-containing serum group was significantly increased (
P
<
0.01). Compared with the 10% TSPSP-containing serum group, the cell viability in the ML385 group decreased to approxim
ately 70% (
P
<
0.01). Compared with the blank group, the model group showed significantly increased contents of MDA, Fe
2+
, and ROS, decreased GSH expression (
P
<
0.01), significantly reduced cell migration rate (
P
<
0.01), and increased protein and mRNA expression levels of KEAP1 and FoxO3 (
P
<
0.01), while decreased protein and mRNA expression levels of Nrf2, GPX4, and SOD (
P
<
0.01). Compared with the model group, each TSPSP-containing serum group showed significantly decreased contents of MDA, Fe²⁺, and ROS, increased GSH expression (
P
<
0.01), significantly increased migration rate (
P
<
0.01), significantly decreased protein and mRNA expression levels of KEAP1 and FoxO3 (
P
<
0.05,
P
<
0.01), and significantly increased protein and mRNA expression levels of Nrf2, GPX4, and SOD (
P
<
0.05,
P
<
0.01). Compared with the 10% TSPSP-containing serum group, the ML385 group showed reversed trends in all indicators (
P
<
0.05,
P
<
0.01).
Conclusion
2
TSPSP can inhibit H
2
O
2
-induced oxidative stress injury of trophoblast cells, and its mechanism of action may be related to the drug activating the KEAP1/Nrf2/FoxO3 signaling pathway.
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