健脾滋肾方改善系统性红斑狼疮小鼠肾损害：基于p53-MDM2信号轴介导的铁死亡

doi:10.12122/j.issn.1673-4254.2026.04.07

摘要/Abstract

摘要：

目的基于鼠p53-双微体基因2（MDM2）信号轴探讨健脾滋肾方对系统性红斑狼疮（SLE）小鼠肾损害铁死亡的影响和治疗作用。方法 30只SPF级MRL/lpr狼疮鼠采用随机对照表法分为模型（Model）组、健脾滋肾方（JPZS）组、泼尼松（Pred）组。另取10只C57BL/6小鼠为正常对照（Control）组。健脾滋肾方组予以JPZS悬液7.8 g·kg^-1·d^-1、泼尼松组予以泼尼松悬液5 mg·kg^-1·d^-1，正常对照组及模型组予生理盐水17.5 mL·kg^-1·d^-1，灌胃给予相应剂量药物，连续用药8周后取材监测。酶联免疫吸附法（ELISA）检测血清抗双链DNA抗体（anti-ds DNA）；自动生化分析仪检测补体3（C3）、血肌酐（Scr）；考马斯亮蓝（CBB）法检测24 h尿蛋白（24 hPRO）含量；比色法检测肾组织铁离子（Fe²⁺）含量；硫代巴比妥酸法（TBA）检测丙二醛（MDA）、WST-1法测定超氧化物歧化酶（SOD）、微板法测定微量还原型谷胱甘肽（GSH）水平；流式检测肾组织ROS水平；组织病理学染色（HE、Masson、PAS）法观察肾组织病理学改变及定量分析；透射电子显微镜下观察肾脏足细胞线粒体形态变化；实时荧光定量PCR法（RT-qPCR）检测p53、MDM2、谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7成员11（SLC7A11）、长链酰基辅酶A合成酶4（ACSL4）、Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤2蛋白（Bcl-2）和半胱氨酸天冬氨酸特异性蛋白酶3（Caspase-3） mRNA的表达情况。蛋白质印迹法检测GPX4、SLC7A11、ACSL4、Caspase-3、Bax和Bcl-2蛋白的表达情况。结果与Control组相比，Model组dsDNA、Scr、24hPRO、Fe²⁺、MDA、ROS水平、p53、MDM2 mRNA表达水平、ACSL4、Bax和Caspase-3 mRNA及蛋白表达水平均显著升高（P<0.05），C3、SOD、GSH水平、GPX4、SLC7A11、Bcl-2 mRNA及蛋白表达水平显著下降（P<0.05）；与Model组相比，JPZS、Pred组dsDNA、Scr、24hPRO、Fe²⁺、MDA、ROS水平、p53、MDM2 mRNA表达水平、ACSL4、Bax和Caspase-3 mRNA及蛋白表达水平均显著降低（P<0.01），C3、SOD、GSH水平、GPX4、SLC7A11、Bcl-2 mRNA及蛋白表达水平显著升高（P<0.01）。结论健脾滋肾方能够明显降低狼疮小鼠肾组织铁死亡水平，改善肾损害、修复肾功能，其机制或与抑制p53-MDM2信号轴有关；结合足细胞线粒体超微结构异常呈一致性改变，这一过程可能与肾小球足细胞相关的铁死亡损伤密切相关。

关键词: 系统性红斑狼疮, 肾损害, 健脾滋肾方, 铁死亡, p53-MDM2信号轴

Abstract:

Objective To investigate the therapeutic mechanism of Jianpi Ziren Formula (JPZS) for ameliorating renal injury in mice with systemic lupus erythematosus (SLE). Methods Thirty MRL/lpr lupus mice were randomly divided into model group, JPZS treatment group, and prednisone treatment group, with 10 C57BL/6 mice as the control group. After treatment with daily gavage with normal saline, JPZS (7.8 g/kg) or prednisone (5 mg/kg) for 8 consecutive weeks, the mice were examined for changes in serum levels of anti-ds DNA, C3 and Scr, 24-h urine protein (24hPRO) and renal Fe²⁺ content, MDA level, SOD activity, GSH level, and ROS level. Renal histopathological changes and ultrastructural changes were observed with HE staining and transmission electron microscopy. The changes in renal expressions of p53, MDM2, GPX4, SLC7A11, ACSL4, Bax, Bcl-2, and caspase-3 mRNAs and proteins were detected using RT-qPCR and Western blotting. Results Compared with the normal control mice, the mouse models of SLE had significantly elevated levels of dsDNA, Scr, 24hPRO, Fe²⁺, MDA, and ROS, increased renal expressions of p53, MDM2, ACSL4, Bax, and caspase-3, lowered levels of C3, SOD, and GSH, and reduced renal expressions of GPX4, SLC7A11, and Bcl-2 at both the mRNA and protein levels. Treatment with JPZS and prednisone both significantly ameliorated these abnormalities in the mouse models. Conclusion JPZS can reduce renal ferroptosis in lupus mice, ameliorate kidney injury, and promote renal function repair possibly by inhibiting the p53-MDM2 signaling axis, which is closely associated with regulation of glomerular podocyte ferroptosis.

Key words: systemic lupus erythematosus, kidney damage, Jianpi Zishen Formula, ferroptosis, p53-MDM2 signaling axis

李云飞, 黄传兵, 庞利君, 朱子衡, 李明. 健脾滋肾方改善系统性红斑狼疮小鼠肾损害：基于p53-MDM2信号轴介导的铁死亡[J]. 南方医科大学学报, 2026, 46(4): 785-793.

Yunfei LI, Chuanbing HUANG, Lijun PANG, Ziheng ZHU, Ming LI. Jianpi Zishen Formula improves renal damage in mice with systemic lupus erythematosus by inhibiting p53-MDM2 signaling axis-mediated ferroptosis[J]. Journal of Southern Medical University, 2026, 46(4): 785-793.

图/表 9

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
β-actin	120	AGTGTGACGTTGACATCCGT	TGCTAGGAGCCAGAGCAGTA
p53	157	TGTAGCTTCAGTTCATTGGG	CTGATATCCGACTGTGACTC
MDM2	157	CTCCAGGTTAGACCAAAACC	AACAATACACAATGTGCTGC
GPX4	133	TGCTTGGGACGCACAG	GGGCGTTTCCTAGGACTTT
SLC7A11	147	AAGCTGAGCTGGTGTGTAAT	GTGAGAACTCAGAGGTGTGT
ACSL4	98	AAACGCTATGGCAAAGAGAA	CTATGACAGCTAGTGAGTCG
Bax	160	CTGATGGCAACTTCAACTGG	TCTTGGATCCAGACAAGCAG
Caspase-3	105	GCTTGGAACGGTACGCTAA	CTTGCTCCCATGTATGGTCT
Bcl-2	164	AGGATTGTGGCCTTCTTTGA	TCCACAAAGGCATCCCAG

表2 各组小鼠肾功能指标和免疫学指标水平

Tab.2 Renal function and immunological indices of the mice in each group (Mean±SD)

Group	dsDNA (ng/mL)	C3 (pg/mL)	24hPRO (mg/L)	Scr (umol/L)
Control	465.20±33.76	65.21±1.04	189.6±15.30	4.10±0.72
Model	1951.00±78.84**	14.56±1.31**	1709.00±42.21**	29.03±0.83**
JPZS	1083.00±38.98^##	39.46±0.64^##	513.2±48.94^##	14.62±0.86^##
Pred	1100.00±32.00^##	39.42±1.42^##	530.2±47.61^##	15.23±0.76^##

表3 各组小鼠肾组织Fe2+、MDA、SOD、GSH水平

Tab.3 Levels of Fe2+, MDA, SOD, and GSH in the kidney tissue of the mice in each group (Mean±SD)

Group	Fe²⁺ (μmol/L)	MDA (nmol/mL)	SOD (U/mg)	GSH (μmol/L)
Control	6.66±1.07	5.15±0.25	17.58±0.55	268.81±8.82
Model	27.27±3.19**	14.54±0.68**	6.44±0.58**	88.66±10.21**
JPZS	12.67±3.21^##	9.01±0.76^##	11.35±0.28^##	144.90±6.20^##
Pred	12.79±3.30^##	9.31±0.49^##	9.78±0.72^##	143.60±10.99^##

图1 健脾滋肾方对狼疮小鼠肾组织ROS水平的影响

Fig.1 Effects of Jianpi Zishen Formula (JPZS) on renal ROS levels in lupus mice. **P<0.01 vs Control group; ##P<0.01 vs Model group. (n=10).

图2 HE染色、Masson染色、PAS染色各组小鼠肾组织病理形态学变化及定量分析

Fig.2 Histopathological changes of the kidney tissue of the mice observed using HE staining, Masson's Trichrome staining, and PAS staining (Left: original magnification: ×20; Right: ×100). **P<0.01 vs Control group; ##P<0.01 vs Model group. (n=10).

图3 电镜下各组小鼠肾组织内足细胞形态变化

Fig.3 Ultrastructural changes in renal podocytes of the mice in different groups under transmission electron microscope.

图4 健脾滋肾方对各组小鼠p53-MDM2轴和肾组织铁死亡相关基因表达的影响

Fig.4 Effects of JPZS Formula on the p53-MDM2 axis and renal expressions of iron death-related genes in the mice in different groups. **P<0.01 vs Control group; ##P<0.01 vs Model group. (n=10).

表4 健脾滋肾方对各组小鼠肾组织铁死亡相关蛋白表达的影响

Tab.4 Effects of JPZS Formula on expressions of iron death-related proteins in mouse kidney tissue in different groups (Mean±SD, n=10)

Group	Control	Model	JPZS	Pred
GPX4	0.89±0.06	0.24±0.08**	0.59±0.07^##	0.62±0.01
SLC7A11	0.81±0.05	0.18±0.06**	0.40±0.04^##	0.49±0.05^##
ACSL4	0.42±0.07	0.92±0.04**	0.67±0.01^##	0.63±0.03^##
Caspase-3	0.26±0.05	0.98±0.02**	0.68±0.04^##	0.66±0.01^##
Bax	0.20±0.07	0.89±0.07**	0.55±0.02^##	0.48±0.04^##
Bcl-2	0.89±0.09	0.32±0.07**	0.55±0.04^##	0.60±0.04^##

图5 Western blotting 检测 GPX4、SLC7A11、ACSL4、caspase-3、Bax和Bcl-2蛋白表达水平

Fig. 5 Western blotting for detecting renal GPX4, SLC7A11, ACSL4, caspase-3, Bax and Bcl-2 protein expressions.

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