[Effect of electroacupuncture in regulating miRNA-21-5p/A20 to induce macrophages polarization in primary dysmenorrhea rats].

OBJECTIVE

To explore the mechanism of electroacupuncture on macrophages polarization by regulating microRNA-21-5p (miR-21-5p)/ubiquitin-editing enzyme A20 (A20) in primary dysmenorrhea (PDM) rats.

METHODS

Twenty-four female SD rats of SPF grade in diestrus were randomly divided into a blank group, a model group, an electroacupuncture group and an ibuprofen group, 6 rats in each group. Except for the blank group, in the other 3 groups, PDM model was established using estradiol benzoate combined with oxytocin. In the electroacupuncture group, electroacupuncture was applied at "Guanyuan" (CV4) and bilateral "Sanyinjiao" (SP6), with continuous wave, in frequency of 50 Hz, 20 min each time, once a day for 10 days. In the ibuprofen group, ibuprofen solution of 125 mg/100 mL was given by gavage administration, 0.8 mL each time, once a day for 10 days. The writhing behavior was observed, the ultrastructure of M1 macrophages in the endometrium was observed by transmission electron, the numbers and distribution of M1/M2 macrophages in the uterus were analyzed by immunofluorescence double-labeling, the serum levels of prostaglandin F2α (PGF2α), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) were measured by ELISA, the targeting relationship between miR-21-5p and A20 was validated by dual-luciferase reporter assay, the mRNA expression of miR-21-5p, A20, nuclear transcription factor-κB p65 subunit (NF-κB p65), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1β (IL-1β), cluster of differentiation 86 (CD86), iNOS, cluster of differentiation 206 (CD206) and Arg-1 in the uterus was detected by real-time PCR, and the protein expression of A20, CD86, iNOS, CD206, Arg-1 in the uterus was detected by Western blot.

RESULTS

The bioinformatics prediction result indicated that A20 was a high-affinity target gene of miR-21-5p. Compared with the blank group, in the model group, the writhing behavior could be observed obviously; the M1 macrophages in the endometrium had severe edema, matrix dissolution and organelle damage; the density of CD86 positive cells was increased in the uterus (P<0.01); the PGF2α, TNF-α and iNOS levels were increased (P<0.05, P<0.01), the Arg-1 level was decreased (P<0.01) in the serum; the mRNA expression of miR-21-5p, NF-κB p65, NLRP3, IL-1β and CD86, as well as the protein expression of CD86 and iNOS were increased (P<0.01), the mRNA and protein expression of A20 and CD206, as well as the protein expression of Arg-1 were decreased (P<0.05, P<0.01) in the uterus. Compared with the model group, in the electroacupuncture group and the ibuprofen group, the writhing numbers and writhing scores were decreased (P<0.05), the writhing latency was prolonged (P<0.01); the edema degree of the M1 macrophages was reduced and the swelling condition of organelles was improved of in the endometrium; the density of CD206 positive cells was increased in the uterus (P<0.05, P<0.01); the PGF2α, TNF-α and iNOS levels were decreased (P<0.01, P<0.05), the Arg-1 levels were increased (P<0.01) in the serum; the mRNA expression of miR-21-5p, NF-κB p65, NLRP3, IL-1β and CD86, as well as the protein expression of CD86 and iNOS were decreased (P<0.01), the mRNA expression of A20, CD206 and Arg-1, as well as the protein expression of A20 and Arg-1 were increased (P<0.01, P<0.05) in the uterus. Compared with the ibuprofen group, in the electroacupuncture group, the mRNA expression of IL-1β was decreased (P<0.01), while that of CD86 was increased (P<0.05) in the uterus.

CONCLUSION

Electroacupuncture induces the polarization of M1 macrophages to M2 macrophages by down-regulating miR-21-5p and up-regulating A20, thus reducing the inflammatory reaction and relieving pain in PDM rats.