Deng Q-F, Liu Y, Chu H, Peng B, Li X, Cao Y-S
The Second Department of Pediatric Urology Surgery, Anhui Provincial Children's Hospital, Children's Hospital of Fudan University-Anhui Campus, Hefei, China.
Physiol Res. 2024 Mar 11;73(1):117-125. doi: 10.33549/physiolres.935163.
To explore the mechanism whereby cGAS-STING pathway regulates the pyroptosis of cryptorchidism cells, with a view to finding a new strategy for clinically treating cryptorchidism-induced infertility. Spermatogonial GC-1 cells were heat stimulated to simulate the heat hurt microenvironment of cryptorchidism. The cell viability was assayed by CCK-8, and cellular DNA damage was detected by gamma-H2AX immunofluo-rescence assay. Flow cytometry was employed to assess pyroptosis index, while western blot, ELISA and PCR were used to examine the expressions of pyroptosis-related proteins (Caspase-1, IL-1beta, NLRP3) and cGAS-STING pathway proteins (cGAS, STING). After STING silencing by siRNA, the expressions of pyroptosis-related proteins were determined. Pyroptosis occurred after heat stimulation of cells. Morphological detection found cell swelling and karyopyknosis. According to the gamma-H2AX immunofluorescence (IFA) assay, the endonuclear green fluorescence was significantly enhanced, the gamma-H2AX content markedly increased, and the endonuclear DNA was damaged. Flow cytometry revealed a significant increase in pyroptosis index. Western blot and PCR assays showed that the expressions of intracellular pyrogenic proteins like Caspase-1, NLRP3 and GSDMD were elevated. The increased STING protein and gene expressions in cGAS-STING pathway suggested that the pathway was intracellularly activated. Silencing STING protein in cGAS-STING pathway led to significantly inhibited pyroptosis. These results indicate that cGAS-STING pathway plays an important role in heat stress-induced pyroptosis of spermatogonial cells. After heat stimulation of spermatogonial GC-1 cells, pyroptosis was induced and cGAS-STING pathway was activated. This study can further enrich and improve the molecular mechanism of cryptorchidism.
为探究cGAS-STING通路调控隐睾症细胞焦亡的机制，以期找到临床治疗隐睾症所致不育症的新策略。对精原细胞GC-1进行热刺激，以模拟隐睾症的热损伤微环境。采用CCK-8法检测细胞活力，通过γ-H2AX免疫荧光法检测细胞DNA损伤。运用流式细胞术评估焦亡指数，同时采用蛋白质免疫印迹法、酶联免疫吸附测定法和聚合酶链反应检测焦亡相关蛋白（半胱天冬酶-1、白细胞介素-1β、NLRP3）和cGAS-STING通路蛋白（cGAS、STING）的表达。在用小干扰RNA沉默STING后，测定焦亡相关蛋白的表达。细胞热刺激后发生焦亡。形态学检测发现细胞肿胀、核固缩。根据γ-H2AX免疫荧光（IFA）检测，细胞核内绿色荧光显著增强，γ-H2AX含量明显增加，细胞核内DNA受损。流式细胞术显示焦亡指数显著增加。蛋白质免疫印迹法和聚合酶链反应检测显示，细胞内半胱天冬酶-1、NLRP3和GSDMD等致热蛋白的表达升高。cGAS-STING通路中STING蛋白和基因表达增加表明该通路在细胞内被激活。沉默cGAS-STING通路中的STING蛋白可显著抑制焦亡。这些结果表明，cGAS-STING通路在热应激诱导的精原细胞焦亡中起重要作用。精原细胞GC-1热刺激后，诱导了焦亡并激活了cGAS-STING通路。本研究可进一步丰富和完善隐睾症的分子机制。
