目的：

研究在外阴阴道念珠菌病（VVC）中阴道上皮细胞表皮生长因子受体（Epidermal Growth Factor Receptor，EGFR）/丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路在白念珠菌和非白念珠菌诱导的固有免疫反应中的作用。

方法：

（1）将阴道上皮细胞（VK2/E6E7细胞）和白念珠菌标准菌株SC5314共培养，首先利用二代测序、蛋白免疫印记（Western blot，WB）和细胞免疫荧光检测EGFR和MAPK信号通路蛋白在RNA和蛋白质水平被白念珠菌激活的情况。然后，利用EGFR抑制剂和构建EGFR过表达的细胞（EGFR-Ad-VK2细胞）验证EGFR和MAPK信号通路蛋白的关系。利用酶联免疫吸附试验（ELISA）和实时无标记动态细胞分析技术（RTCA）检测EGFR-MAPK信号通路在调控白念珠菌诱导的炎症反应和细胞损伤中的作用。构建白念珠菌SC5314感染的小鼠VVC模型，通过EGFR抑制剂、组织病理切片和免疫荧光检测EGFR信号通路对小鼠阴道局部炎症反应和上皮损伤的影响。

（2）将阴道上皮细胞分别与白念珠菌敏感菌株SC5314和临床耐药菌1052共培养，利用WB、EILSA、RTCA等实验技术分析两株菌在诱导EGFR-MAPK信号通路蛋白激活、炎症反应和细胞损伤三方面的差异。构建SC5314和1052感染的小鼠VVC模型，进一步分析两株菌在体内实验中诱导EGFR信号通路蛋白激活、炎症反应和阴道上皮损伤方面的差异。利用显微镜成像对比SC5314和1052在形态上的差异。

（3）将阴道上皮细胞分别与4株白念珠菌和5株非白念珠菌（光滑念珠菌、近平滑念珠菌和热带念珠菌和2株耳念珠菌）共培养，利用Western blot、ELISA和RTCA技术分析白念珠菌和各非白念珠菌在诱导EGFR-MAPK信号通路蛋白激活、炎症反应和细胞损伤三个方面的差异。利用显微镜成像对比白念珠菌和各非白念珠菌在形态上的差异。

结果：

（1）在白念珠菌的刺激下，二代测序结果显示，VK2/E6E7细胞的EGFR和MAPK信号通路显著上调；在白念珠菌的刺激下，阴道上皮细胞的EGFR和MAPK信号蛋白在不同时间点被不同程度地激活，总体来说，白念珠菌激活的EGFR、ERK1/2、p38、c-Fos和p65蛋白的磷酸化水平明显高于未加菌的对照组(P <0.05)。而且这种固有免疫反应的激活模式不同于口腔上皮细胞。EGFR抑制剂可显著降低白念珠菌激活的EGFR、ERK1/2和p38蛋白的磷酸化，但EGFR的过表达只能导致EGFR和p38磷酸化水平的升高，且都有统计学意义(P <0.05)。ELISA结果显示EGFR、ERK1/2和p38抑制剂都可以降低白念珠菌诱导的细胞因子分泌（CCL20、G-CSF、GM-CSF、IL-1β、IL-6和IL-17A），其中p38抑制剂的抑制效果最显著；而EGFR的过表达可以导致G-CSF、GM-CSF、IL-1β、IL-6和IL-17A水平的升高(P <0.05)，除了CCL20。RTCA结果显示只有EGFR和p38抑制剂可以改善白念珠菌诱导的阴道上皮细胞损伤。小鼠VVC模型的实验结果表明EGFR抑制剂可以有效减轻SC5314诱导的阴道中性粒细胞浸润和上皮损伤。

（2）在EGFR-MAPK信号通路蛋白的激活方面，相比SC5314，临床耐药菌1052可以激活更高水平的EGFR和p38蛋白的磷酸化，以及更低水平的ERK1/2和c-Fos蛋白的磷酸化。在诱导细胞因子的释放方面，1052可以激活VK2/E6E7细胞分泌更高水平的CCL20、G-CSF、GM-CSF、IL-1β、IL-6和IL-17A，且EGFR、ERK1/2和p38抑制剂都可以降低这些细胞因子的水平 (P <0.05)。RTCA结果显示只有EGFR和p38抑制剂可以降低这两株白念珠菌诱导的阴道上皮细胞损伤 (P <0.05)。在SC5314和1052感染的小鼠VVC模型中，1052诱导的EGFR磷酸化水平显著高于SC5314诱导的水平，而且EGFR抑制剂可以显著降低两株菌诱导的EGFR磷酸化，且差异都具有统计学意义(P <0.05)。而且1052诱导的阴道中性粒细胞浸润程度和上皮损伤程度都大于SC5314，EGFR抑制剂可以降低两种菌诱导的炎症反应和上皮损伤，差异具有统计学意义(P <0.05)。通过直接观察SC5314和1052在镜下的差异，发现1052更容易形成菌丝。

（3）在EGFR-MAPK信号通路蛋白的激活方面，相比白念珠菌，所有非白念珠菌可以激活更高水平的ERK1/2、c-Fos和p65蛋白的磷酸化，以及极低水平的EGFR和p38蛋白的磷酸化。在细胞因子的分泌方面，白念珠菌SC5314诱导的CCL20、G-CSF、GM-CSF、IL-1β、IL-6和IL-17A水平高于所有非白念珠菌（光滑念珠菌、近平滑念珠菌和热带念珠菌和耳念珠菌）。EGFR、ERK1/2和p38抑制剂可以降低这些非白念珠菌诱导的细胞因子分泌。RTCA结果显示每种念珠菌诱导的细胞损伤曲线各不相同，表现为种属特异性，而且耳念珠菌CBS14918和近平滑念珠菌诱导的细胞损伤程度高于白念珠菌、光滑念珠菌和热带念珠菌，差异具有统计学意义。只有p38抑制剂可以改善所有非白念珠菌诱导的阴道上皮细胞损伤。通过直接观察这些非白念珠菌在镜下形态，发现只有热带念珠菌可以形成假菌丝，其它非白念珠菌（光滑念珠菌、近平滑和耳念珠菌）都不能形成菌丝。

结论：EGFR-MAPK信号通路在念珠菌激活的阴道上皮细胞固有免疫反应中被显著激活，抑制EGFR-MAPK信号通路可有效改善VVC的炎症反应和上皮损伤。

Objective:

To investigate the role of epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling pathway in the activation of innate epithelial immune responses during vulvovaginal candidiasis (VVC).

Methods:

(1) The vaginal epithelial cells (VK2/E6E7 cells) were co-cultured with the standard strain of Candida albicans (C. albicans). To determine the activation of EGFR and MAPK signaling pathways in the interaction between VK2/E6E7 cells and Candida albicans, RNA sequencing, Western blot (WB) and cellular immunofluorescence were used. Then, the relationship between EGFR and MAPK signaling pathway proteins was verified by using EGFR inhibitors and constructing EGFR-overexpressing cells (EGFR-Ad-VK2 cells). Enzyme-linked immunosorbent assay (ELISA) and Real Time Cellular Analysis (RTCA) techniques were used to detect the effect of EGFR-MAPK signaling pathway in regulating the secretion of inflammatory cytokines and cell damage induced by C. albicans. The mouse model of VVC infected by C. albicans was constructed, and the function of EGFR signaling pathway on local inflammation in the mice vagina was determined with EGFR inhibitors, histopathological sections and immunofluorescence.

(2) Vaginal epithelial cells were co-cultured with sensitive C. albicans strain SC5314 and clinical drug-resistant isolate 1052 respectively, and the two strains were compared in terms of the activation of EGFR-MAPK signaling pathway, inflammatory response and cell damage via Western blotting, ELISA and RTCA. Through the mouse model of VVC, the differences in EGFR signaling pathway activation, inflammatory response and cell damage induced by the two strains were further compared in vivo. The morphological differences between the sensitive C. albicans strain SC5314 and the clinical drug-resistant isolate 1052 were directly compared under microscope.

(3) Vaginal epithelial cells were co-cultured with 4 strains of C. albicans and 5 strains of non-albicans Candida (NAC) species, including Candida glabrata, Candida parapsilosis and Candida tropicalis and 2 strains of Candida auris. WB, ELISA, RTCA technology were used to compare the differences between C. albicans and NAC species in inducing EGFR-MAPK signaling pathway activation, inflammatory response and cell damage. The morphological differences between C. albicans and 4 strains of NAC species were compared under microscope.

Result:  

(1) The results of RNA-Seq showed that the EGFR and MAPK signaling pathways were significantly upregulated in VK2/E6E7 cells under the stimulation of C. albicans; WB and immunofluorescence results showed that the phosphorylation of EGFR, ERK1/2, p38, c-Fos and p65 was significantly and differentially activated by SC5314 (P < 0.05); EGFR inhibitors significantly reduced the levels of EGFR, ERK1/2 and p38 phosphorylation, but overexpression of EGFR only led to a significant increase of EGFR and p38 phosphorylation (P < 0.05). ELISA results showed that EGFR, ERK1/2 and p38 inhibitors significantly reduced C. albicans-induced cytokine secretion (CCL20, G-CSF, GM-CSF, IL-1β, IL-6 and IL-17A), among which p38 inhibitor was the most effective; yet the overexpression of EGFR led to a significant increase of G-CSF, GM-CSF, IL-1β, IL-6 and IL-17A levels, except CCL20 (P <0.05). RTCA results showed that only EGFR and p38 inhibitor suppressed C. albicans-induced vaginal epithelial cell damage. In the mouse model of VVC, EGFR inhibitors effectively improved vaginal inflammation and epithelial damage induced by SC5314.

(2) With respect to the activation of EGFR-MAPK signaling pathway, clinical drug-resistant isolate 1052 induced higher levels of EGFR and p38 phosphorylation and lower levels of ERK1/2 and c-Fos phosphorylation compared to SC5314. In terms of cytokine release, 1052 induced higher levels of CCL20, G-CSF, GM-CSF, IL-1β, IL-6 and IL-17A. In addition, EGFR, ERK1/ 2 and p38 inhibitors significantly reduced the secretion of these cytokines (P < 0.05). RTCA results showed that only EGFR and p38 inhibitors significantly reduced vaginal epithelial cell damage induced by the two strains (P < 0.05). In the mouse model of VVC, the levels of EGFR phosphorylation induced by 1052 in the vagina was significantly higher than that induced by SC5314, and EGFR inhibitors significantly reduced the phosphorylation of EGFR (P < 0.05). Moreover, the number of neutrophil infiltration and apoptotic cells induced by 1052 were significantly greater than those of SC5314, and EGFR inhibitor also significantly reduced the inflammatory response and apoptosis induced by the two strain (P < 0.05). By directly observing the morphological difference between SC5314 and 1052 under the microscope, we found that 1052 was more filamentous.

(3) In terms of the activation of EGFR-MAPK signaling pathway, all NAC species activated higher levels of ERK1/2, c-Fos and p65 phosphorylation, and much lower levels of EGFR and p38 phosphorylation compared to C. albicans. The levels of cytokines (CCL20, G-CSF, GM-CSF, IL-1β, IL-6 and IL-17A) induced by SC5314 were higher than that by all NAC species (C. glabrata, C. parapsilosis, C. tropicalis and C. auris). Inhibition of EGFR, ERK1/2 and p38 resulted in significant reduction of CCL20, G-CSF, GM-CSF, IL-1β, IL-6 and IL-17A induced by these NAC species. RTCA results showed that the cell damage curve induced by each Candida strain was species-specific, and the cell damage induced by CBS14918 and C. parapsilosis was significantly stronger than that of C. albicans, C. glabrata and C. tropicalis. Only p38 inhibitor significantly reduced vaginal epithelial cell damage induced by all the NAC species. By directly observing the differences of these NAC species under the microscope, it was found that only C. tropicalis occasionally form pseudohyphae, while other NAC species grew only in the form of yeast.

Conclusion

The EGFR-MAPK signaling pathway plays a key role in the activation of innate epithelial immune responses and inhibition of EGFR can significantly ameliorate the inflammatory response and epithelial damage in experimental VVC.