Effect of Pulsatilla decoction on vulvovaginal candidiasis in mice. Evidences for its mechanisms of action.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common infection that affects the female reproductive tract. Pulsatilla decoction (PD), a traditional Chinese herbal medicine, is a classic and effective prescription for VVC. However, its mechanism of action remains unclear. PURPOSE: This study aimed to evaluate the efficacy and potential mechanism of action of the n-butanol extract of Pulsatilla decoction (BEPD) in VVC treatment. METHODS: High performance liquid chromatography (HPLC) was used to detect the main active ingredients in BEPD. A VVC-mouse model was constructed using an estrogen-dependent method to evaluate the efficacy of BEPD in VVC treatment. Fungal burden and morphology in the vaginal cavity were comprehensively assessed. Candida albicans-induced inflammation was examined in vivo and in vitro. The effects of BEPD on the Protein kinase Cδ (PKCδ) /NLR family CARD domain-containing protein 4 (NLRC4)/Interleukin-1 receptor antagonist (IL-1Ra) axis were analyzed using by immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and reverse transcription-quantitative polymerase chain reaction (qRT-PCR). RESULTS: BEPD inhibited fungal growth in the vagina of VVC mice, preserved the integrity of the vaginal mucosa, and suppressed inflammatory responses. Most importantly, BEPD activated the "silent" PKCδ/NLRC4/IL-1Ra axis and negatively regulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thereby exerting a therapeutic efficacy on VVC. CONCLUSIONS: BEPD effects on mice with VVC were dose-dependent. BEPD protects against VVC by inhibiting inflammatory response and NLRP3 inflammasome via the activation of the PKCδ/NLRC4/IL-1Ra axis. This study revealed the pharmacological mechanism of BEPD in VVC treatment and provided further evidence for the application of BEPD in VVC treatment.

Effect of the Pulsatilla decoction n-butanol extract on vulvovaginal candidiasis caused by Candida glabrata and on its virulence factors.

Vulvovaginal candidiasis (VVC) caused by Candida glabrata (C. glabrata) is more persistent and resistant to treatment than when caused by Candida albicans (C. albicans) and has been on the rise in recent years. The n-butanol extract of Pulsatilla Decoction (BEPD) has been shown to be effective in treating VVC caused by C. glabrata, but the underlying mechanism of action remains unclear. In this study, the experimenter conducted in vitro and in vivo experiments to explore the effects of BEPD on the virulence factors of C. glabrata, as well as its efficacy, with a focus on possible immunological mechanism in VVC caused by C. glabrata. The contents of Anemoside B4, Epiberberine, Berberine, Aesculin, Aesculetin, Phellodendrine and Jatrorrhizine in BEPD, detected by high-performance liquid chromatography, were 31,736.64, 13,529.66, 105,143.72, 19,406.20, 4952.67, 10,317.03, 2489.93 µg/g, respectively. In vitro experiments indicated that BEPD moderately inhibited the growth of C. glabrata, its adhesion, and biofilm formation, and affected the expression of efflux transporters in the biofilm state. In vivo experiments demonstrated that BEPD significantly reduced vaginal inflammatory manifestation and the release of proinflammatory cytokines and LDH in mice with VVC caused by C. glabrata. Moreover, it inhibited the Phosphorylation of EGFR, ERK, P38, P65, and C-Fos proteins. The results suggested that although BEPD moderately inhibits the growth and virulence factors of C. glabrata in vitro, it can significantly reduce vaginal inflammation by down-regulating the EGFR/MAPK signaling pathway in mice with VVC infected by C. glabrata.

[Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis].

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1ß, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1ß, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1ß, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

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

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-1ß 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-1ß, 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.