Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans

Abstract

Ethnopharmacological relevance: Pulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC.

Aim of the study: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated.

Materials and methods: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1β and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot.

Results: After administration of BEPD-containing serum, the levels of IL-1β, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins.

Conclusions: BEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

Keywords: Candida albicans; Dectin-1/Sky; N-butanol alcohol extract of Pulsatilla decoction; NLRP3 inflammasome; TLRs/MyD88; Vulvovaginal candidiasis.