Division of Urology, Department of Surgery, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 11101, Taiwan.
School of Medicine, Fu Jen Catholic University, New Taipei City 24205, Taiwan.
Int J Mol Sci. 2022 May 5;23(9):5156. doi: 10.3390/ijms23095156.
Antimicrobial peptides (AMPs), which are natural antibiotics, protect against pathogens invading the urinary tract. RNase 7 with antimicrobial properties has rapid and powerful suppressive effects against Gram-positive and Gram-negative bacterial infections. However, its detailed antibacterial mechanisms have not been fully determined. Here, we investigate whether RNase 7 had an impact on bladder cells under uropathogenic (UPEC) infection in a high-glucose environment using in vitro GFP-UPEC-infected bladder cell and PE-labeled TLR4, STAT1, and STAT3 models. We provide evidence of the suppressive effects of RNase 7 on UPEC infection and UPEC-induced inflammatory responses by regulating the JAK/STAT signaling pathway using JAK inhibitor and STAT inhibitor blocking experiments. Pretreatment with different concentrations of RNase 7 for 24 h concentration-dependently suppressed UPEC invasion in bladder cells (5 μg/mL reducing 45%; 25 μg/mL reducing 60%). The expressions of TLR4, STAT1, and STAT3 were also downregulated in a concentration-dependent manner after RNase 7 pretreatment (5 μg/mL reducing 35%, 54% and 35%; 25 μg/mL reducing 60%, 75% and 64%, respectively). RNase 7-induced decrease in UPEC infection in a high-glucose environment not only downregulated the expression of TLR4 protein and the JAK/STAT signaling pathway but also decreased UPEC-induced secretion of exogenous inflammatory IL-6 and IL-8 cytokines, although IL-8 levels increased in the 25 μg/mL RNase 7-treated group. Thus, inhibition of STAT affected pSTAT1, pSTAT3, and TLR4 expression, as well as proinflammatory IL-6 and IFN-γ expression. Notably, blocking JAK resulted in the rebound expression of related proteins, especially pSTAT1, TLR4, and IL-6. The present study showed the suppressive effects of RNase 7 on UPEC infection and induced inflammation in bladder epithelial cells in a high-glucose environment. RNase 7 may be an anti-inflammatory and anti-infective mediator in bladder cells by downregulating the JAK/STAT signaling pathway and may be beneficial in treating cystitis in DM patients. These results will help clarify the correlation between AMP production and UTI, identify the relationship between urinary tract infection and diabetes in UTI patients, and develop novel diagnostics or possible treatments targeting RNase 7.
抗菌肽（AMPs）是天然抗生素，可防止尿路病原体入侵。具有抗菌特性的 RNase 7 对革兰氏阳性和革兰氏阴性细菌感染具有快速而强大的抑制作用。然而，其详细的抗菌机制尚未完全确定。在这里，我们通过体外 GFP-UPEC 感染膀胱细胞和 PE 标记的 TLR4、STAT1 和 STAT3 模型，研究了 RNase 7 在高糖环境下 Uropathogenic（UPEC）感染对膀胱细胞的影响。我们通过 JAK 抑制剂和 STAT 抑制剂阻断实验，提供了 RNase 7 通过调节 JAK/STAT 信号通路对 UPEC 感染和 UPEC 诱导的炎症反应具有抑制作用的证据。用不同浓度的 RNase 7 预处理 24 小时，浓度依赖性地抑制膀胱细胞中 UPEC 的入侵（5μg/mL 减少 45%；25μg/mL 减少 60%）。RNase 7 预处理后，TLR4、STAT1 和 STAT3 的表达也呈浓度依赖性下调（5μg/mL 减少 35%、54%和 35%；25μg/mL 减少 60%、75%和 64%）。RNase 7 在高糖环境下诱导的 UPEC 感染减少不仅下调了 TLR4 蛋白和 JAK/STAT 信号通路的表达，还降低了 UPEC 诱导的外源性炎症性 IL-6 和 IL-8 细胞因子的分泌，尽管 25μg/mL RNase 7 处理组的 IL-8 水平升高。因此，STAT 的抑制作用影响了 pSTAT1、pSTAT3 和 TLR4 的表达，以及促炎细胞因子 IL-6 和 IFN-γ 的表达。值得注意的是，阻断 JAK 导致相关蛋白的反弹表达，特别是 pSTAT1、TLR4 和 IL-6。本研究显示，RNase 7 在高糖环境下抑制 UPEC 感染和诱导膀胱上皮细胞炎症。RNase 7 可能通过下调 JAK/STAT 信号通路成为膀胱细胞中的抗炎和抗感染介质，可能有益于治疗糖尿病患者的膀胱炎。这些结果将有助于阐明 AMP 产生与 UTI 之间的相关性，确定 UTI 患者尿路感染与糖尿病之间的关系，并开发针对 RNase 7 的新型诊断或可能的治疗方法。
