基于PKCδ/NLRC4/IL-1Ra轴对NLRP3炎症小体的负调控研究白头翁汤正丁醇提取物治疗外阴阴道念珠菌病的作用机制

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

该研究旨在从PKCδ/NLRC4/IL-1Ra轴负调控NLRP3炎症小体的角度探讨白头翁汤正丁醇提取物(n-butanol alcohol extract of Baitouweng Decoction,BAEB)治疗外阴阴道念珠菌病(vulvovaginal candidiasis,VVC)小鼠的作用机制。实验中,将雌性C57BL/6小鼠分为6组,分别为空白对照组,VVC模型组,BAEB高、中、低剂量组(80、40、20 mg·kg^(-1))和氟康唑组(20 mg·kg^(-1)),除空白对照外,其余各组利用雌激素依赖性的方法构建VVC小鼠模型,模型构建成功后,空白对照组不作处理,BAEB高、中、低剂量组分别给予80、40、20 mg·kg^(-1)BAEB治疗,氟康唑组给予20 mg·kg^(-1)氟康唑治疗,VVC模型组则灌胃等量的生理盐水。每日观察各组小鼠的一般状态和体质量;革兰染色观察小鼠阴道灌洗液中白念珠菌形态的变化;微量稀释法检测小鼠阴道分泌物的真菌载量,处死小鼠后,巴氏染色法检测阴道灌洗液中中性粒细胞的浸润程度,enzyme-linked immunosorbent assay(ELISA)法检测阴道灌洗液中炎症因子interleukin(IL)-1β、IL-18以及lactate dehydrogenase(LDH)的含量;hematoxylin-eosin(HE)染色进行阴道组织病理学分析;免疫组化(immunohistochemistry,IHC)检测阴道组织中NLRP3、PKCδ、pNLRC4、IL-1Ra的表达和分布;免疫荧光(immunofluorescence,IF)检测阴道组织中pNLRC4、IL-1Ra的表达与分布,免疫印迹(Western blot,WB)检测阴道组织中NLRP3、PKCδ、pNLRC4、IL-1Ra的蛋白表达;qRT-PCR检测NLRP3、PKCδ、pNLRC4、IL-1Ra mRNA的表达。结果显示,与空白对照组比较,VVC模型组小鼠阴道出现红肿以及白色分泌物,与VVC模型组比较,BAEB能改善VVC小鼠的一般状态。革兰染色、巴氏染色、微量稀释法以及HE染色结果显示,与空白对照组比较,阴道灌洗液中分布有大量菌丝且有大量中性粒细胞浸润,阴道灌洗液中真菌载荷显著增加,阴道组织黏膜被破坏且有大量炎性细胞浸润,BAEB可减少白念珠菌菌丝形成,BAEB中、高剂量组可显著减少中性粒细胞的浸润以及真菌载荷,BAEB低、中剂量组可减轻阴道组织的损伤,而BAEB高剂量组可将损伤的阴道组织恢复至正常水平。ELISA结果显示,与空白对照组比较,炎症因子IL-1β、IL-18、LDH的含量显著增加,与VVC模型组比较,BAEB中、高剂量组可显著减少IL-1β、IL-18以及LDH的含量。WB和PCR结果显示,与空白对照组比较,VVC模型组小鼠阴道组织中PKCδ、pNLRC4、IL-1Ra蛋白和mRNA表达显著降低,而NLRP3的蛋白和mRNA表达显著上升;与VVC模型组比较,BAEB中、高剂量组可上调阴道组织中PKCδ、pNLRC4、IL-1Ra蛋白和mRNA表达,抑制阴道组织中NLRP3蛋白和mRNA表达。该研究表明,BAEB对于VVC小鼠的治疗作用可能与促进PKCδ/NLRC4/IL-1Ra轴负调控NLRP3炎症小体有关。

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)-1β,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-1β,IL-18,and LDH in the VVC model group significantly increased compared with that in the blank control group,and the content of IL-1β,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.