Moxibustion promotes endometrial repair in rats with thin endometrium by inhibiting the NLRP3/pyroptosis axis via upregulating miR-223-3p

doi:10.12122/j.issn.1673-4254.2025.07.04

Abstract

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

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.

Figures/Tables 9

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

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.

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.

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.

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.

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.

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.

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)

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

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