艾灸通过调控miR-223-3p/NLRP3焦亡通路修复薄型子宫内膜

doi:10.12122/j.issn.1673-4254.2025.07.04

摘要/Abstract

摘要：

目的探讨艾灸通过miR-223-3p/NLRP3/pyroptosis 信号轴促进子宫内膜修复的机制。方法将SD大鼠随机分为对照组（CON）、模型组（MOD）、艾灸组（MOX），16只/组。使用95%无水乙醇造模，MOX组给予艾灸关元穴治疗。检索公共数据库结合高通量测序寻找薄型子宫内膜（TE）的靶基因；双荧光素酶验证miR-223-3p和NLRP3的靶向关系；HE染色法观察子宫病理形态；RT-qPCR法检测miR-223-3p、NLRP3表达；ELISA法检测IL-1β、IL-18水平；Western blotting法检测NLRP3、ASC、Caspase-1、GSDMD的表达；统计大鼠妊娠情况。结果共检索到13个TE与细胞焦亡相交的靶基因，差异基因富集提示TE中炎症反应上调。双荧光素酶实验验证了miR-223-3p靶向抑制NLRP3的表达。与CON组比，MOD大鼠子宫内膜厚度、腺体及血管数量减少（P<0.01），NLRP3的mRNA表达增强（P<0.05），血清IL-1β、IL-18水平升高（P<0.01），NLRP3、ASC、Caspase-1、GSDMD蛋白表达增加（P<0.05），患侧妊娠率降低（P<0.05），患侧及健侧妊娠数量和妊娠总数均减少（P<0.01）；与MOD比，MOX组大鼠内膜厚度、腺体和血管数增加（P<0.01），miR-223-3p表达上调（P<0.05），IL-1β、IL-18含量减少（P<0.05），NLRP3、ASC、Caspase-1、GSDMD的蛋白表达降低（P<0.05），健侧妊娠数量和妊娠总数量增多（P<0.05）。结论艾灸关元穴可以上调miR-223-3p靶向抑制NLRP3，缓解细胞焦亡，促进子宫内膜修复和妊娠功能恢复。

关键词: 薄型子宫内膜, 中医药, 艾灸, 关元穴, 细胞焦亡, miR-223-3p/NLRP3/pyroptosis 信号轴

Abstract:

Objective To explore the mechanism through which moxibustion promotes endometrial repair in rats with in thin endometrium (TE). Methods Female SD rats were randomized into control group, 95% anhydrous ethanol-induced TE model group and moxibustion (at "Guan Yuan") group. High-throughput sequencing was used to identify the target genes of TE, and the targeting relationship between miR-223-3p and NLRP3 was verified using a dual luciferase assay. Histopathological of rat uterus was observed with HE staining, and expressions of miR-223-3p and NLRP3 were detected using RT-qPCR; serum levels of IL-1β and IL-18 of the rats were detected using ELISA, and protein expressions of NLRP3, ASC, caspase-1 and GSDMD in the uterus were detected with Western blotting. The pregnancies of the rats after treatment were counted. Results Enrichment analysis of the differential genes suggested up-regulated inflammatory response in TE, and dual luciferase assay verified targeted inhibition of NLRP3 expression by miR-223-3p. The rat models of TE had significantly decreased endometrial thickness and reduced endometrial glands and blood vessels with enhanced mRNA expression of NLRP3, increased serum levels of IL-1β and IL-18, up-regulated protein expressions of NLRP3, ASC, caspase-1 and GSDMD, lowered pregnancy rates on both the affected and unaffected sides and the overall number of pregnancies. Treatment of the rat models with mo-xibustion obviously increased the endometrial thickness and the density of glands and blood vessels, up-regulated miR-223-3p expression, lowered serum IL-1β and IL-18 levels and the protein expressions of NLRP3, ASC, caspase-1 and GSDMD, and significantly increased the number of pregnancies. Conclusion Moxibustion at "Guan Yuan" acupoint up-regulates the expression of miR-223-3p, which results in targeted inhibition of NLRP3 to suppress pyroptosis and promote endometrial repair in rat models of TE.

Key words: thin endometrium, Chinese medicine, moxibustion, Guan Yuan acupoint, pyroptosis, miR-223-3p/NLRP3/pyroptosis signaling pathway

周海忆, 何斯怡, 韩瑞芳, 关永格, 董丽娟, 宋阳. 艾灸通过调控miR-223-3p/NLRP3焦亡通路修复薄型子宫内膜[J]. 南方医科大学学报, 2025, 45(7): 1380-1388.

Haiyi ZHOU, Siyi HE, Ruifang HAN, Yongge GUAN, Lijuan DONG, Yang SONG. Moxibustion promotes endometrial repair in rats with thin endometrium by inhibiting the NLRP3/pyroptosis axis via upregulating miR-223-3p[J]. Journal of Southern Medical University, 2025, 45(7): 1380-1388.

图/表 9

表 1 qRT-PCR引物序列

Tab.1 Primers equence of qRT-PCR

Gene	Primers sequence (5'→3')
miR-223-3p	F: ACACTCCAGCTGGGTGTCAGTTTGTCAAA
miR-223-3p	R: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGGTATT
U6	F: CTCGCTTCGGCAGCACA
U6	R: AACGCTTCACGAATTTGCGT
NLRP3	F: ACCAACCAGAACCTCACACA
NLRP3	R: GGCTCTGGTTATGGGTCAGA
GAPDH	F: CTGGAGAAACCTGCCAAGTATG
GAPDH	R: GGTGGAAGAATGGGAGTTGCT

图1 韦恩图和GO富集分析Top 20柱状图

Fig.1 Venn diagram and histograms of the top 20 GO terms. A: Intersection of the target genes of TE and pyroptosis. B: Histogram of the top 20 GO terms for differentially expressed mRNAs. C: Histogram of the top 20 GO terms for differentially expressed proteins.

图2 靶基因结合位点及双荧光素酶实验

Fig.2 Target gene binding sites (A) and results of dual-luciferase assay (B). **P<0.01 vs NC-NLRP3+miR-223-3p; ##P<0.01 vs wt-NLRP3+miR-223-3p; ^^P<0.01 vs TRAF6+NC-miR-223-3p group.

图 3 各组大鼠子宫病理形态比较及统计分析

Fig.3 Comparison of pathological morphology of the uterus among the 3 groups. A: HE staining of uterine tissue of the rats in each group (blue arrows indicate the endometrial thickness, black arrows indicate the glands, and green arrows indicate blood vessels). B: Statistical analysis of endometrial thickness, number of glands and blood vessels in each group. **P<0.01, ***P<0.001 vs CON group; ##P<0.01, ###P<0.001 vs MOD group.

图 4 各组大鼠子宫组织的基因表达情况

Fig.4 Expressions of miR-223-3p and NLRP3 mRNA in the uterine tissue of the rats in each group. *P<0.05 vs CON group; #P<0.05 vs MOD group.

图 5 各组大鼠血清IL-1β、IL-18含量

Fig.5 Serum levels of IL-1β and IL-18 levels of the rats in each group. **P<0.01 vs CON group; #P<0.05, ###P<0.001 vs MOD group.

图6 各组大鼠子宫组织中焦亡相关因子的蛋白表达情况

Fig.6 Protein expression of pyroptosis-related factors in the uterine tissues of rats in each group. A: Western blotting for detecting the protein expressions of pyroptosis factors in rat uterine tissues. B: Relative expression of pyroptosis-related factors. *P<0.05, **P<0.01 vs CON group; #P<0.05, ##P<0.01 vs MOD group.

表2 各组大鼠妊娠情况统计

Tab.2 Pregnancy rates and number of embryos of the rats in the 3 groups (n=8)

Group

Pregnancy rate on the left side (%)

Embryo counts on the left

(Mean±SD)

Embryo counts on the right (Mean±SD)

Bilateral embryo counts

(Mean±SD)

图7 各组大鼠妊娠情况代表图

Fig.7 Representative diagram of pregnancy in each group of rats.

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