IRE1-JNK-Beclin1信号通路参与PM2.5暴露致大鼠睾丸损伤机制的研究

Fine particulate matter (PM2.5) is considered as the most significant air pollutant that contributes to health risks. At present, potential adverse effects of PM2.5 on reproductive injury have raised concerns. However, the pathogenesis of male reproductive injury underlying PM2.5 has not been well clarified. Our previous research found that PM2.5 had significantly increased the expression of endoplasmic reticulum stress-sensing protein IRE1 and autophagy-specific protein Beclin1 in rat testis. Other studies have shown that JNK was the downstream effector of IRE1 signaling, and which also was a potentially triggered autophagy regulation pathway as well. Accordingly, we hypothesized that whether PM2.5 -induced rat testicular injury by activating the IRE1-JNK-Beclin1 signaling pathway. In order to verify this hypothesis, PM2.5 rat model was used in this experiment. This study was to examine the changes of IRE1, JNK and Beclin1 in the process of testicular injury from the observation of different cycles and exposure dose. Combined with in vitro cell model by pharmacological intervention and RNA interference, the study will be performed to further investigate and discuss the above hypotheses. The study will comprehensively evaluate the effect of PM2.5 on testicular injury and its association with IRE1-JNK-Beclin1 signaling pathway, and thereby will provide clues and ideas for finding new therapeutic targets for PM2.5-induced testicular injury.

细颗粒物（PM2.5）是造成健康风险最重要的大气污染物，目前有关PM2.5对生殖损伤的影响已受到学界的关注，然而PM2.5致雄性生殖损伤机制尚不明确。我们预试验研究发现，PM2.5暴露的大鼠睾丸组织中出现内质网应激感应蛋白IRE1和自噬特异性蛋白Beclin1表达升高。另有研究报道，JNK是IRE1的下游信号分子，且可调控自噬的发生。据此我们提出假说，PM2.5可能通过激活IRE1-JNK-Beclin1信号通路介导大鼠睾丸损伤。为了验证这一假说，本课题拟以PM2.5暴露大鼠模型为研究对象，从观察不同周期及暴露剂量入手，分析IRE1、JNK及Beclin1在睾丸损伤过程中的变化。同时结合体外实验，采用药理学干预及RNA干扰技术对上述关联加以验证。以此综合评价PM2.5对睾丸损伤的影响及其与IRE1-JNK-Beclin1信号通路的关联，为寻找PM2.5致睾丸损伤新的治疗靶点提供线索和思路。

细颗粒物（PM2.5）是造成健康风险最重要的大气污染物，目前有关PM2.5对生殖损伤的影响已受到学界的关注，然而PM2.5致雄性生殖损伤机制尚不明确。本研究以PM2.5暴露大鼠模型为研究对象，分析IRE1、JNK及Beclin1在睾丸损伤中的变化。同时结合体外实验，采用药理学干预及RNA干扰技术对上述关联加以验证，以明确 PM2.5 是否激活 IRE1/JNK/Beclin1途径参与睾丸损伤机制。体内实验结果发现，与对照组相比，PM2.5组的精子质量明显降低，血清睾酮含量有所下降，细胞凋亡指数明显增加，STF083010干预后，精子质量明显提高，血清睾酮含量升高，细胞凋亡指数下降；光学显微镜下可见PM2.5组生精细胞间结构疏松，生精细胞和成熟精子数量减少，STF083010干预后，生精细胞结构损伤有所改善，管腔脱落细胞减少；Western blot检测发现，PM2.5组的IRE1、JNK、p-JNK、Beclin1﹑LC3II/LC3I表达明显上调，P62蛋白表达明显下调，而STF083010干预明显抑制了以上效应。体外实验结果发现，与对照组相比，PM2.5组的IRE1、JNK和Beclin1的蛋白表达水平明显上调；经与4μ8C联合作用后，与PM2.5组相比，4μ8C明显抑制了IRE1、JNK和Beclin1表达的上调；与对照组相比，PM2.5组的JNK、Beclin1和LC3Ⅱ/LC3Ⅰ表达明显上调，P62表达明显下调；经过与SP600125联合作用后，SP600125明显抑制了上述效应；透射电镜观察自噬，结果显示，PM2.5可诱导睾丸支持细胞产生自噬，采用4μ8C或SP600125干预后自噬减轻；mRFP-GFP-LC3质粒转染观察PM2.5诱导自噬，PM2.5组可见多个明亮的红色和黄色荧光斑点，说明PM2.5 可诱导睾丸支持细胞自噬溶酶体和自噬体的形成，自噬流顺畅；PM2.5可抑制细胞增殖或增加细胞凋亡，Rapamycin干预可进一步抑制细胞增殖，加剧细胞凋亡，而3-MA干预后细胞增殖能力有所提高，细胞凋亡水平有所降低；采用siRNA技术沉默Beclin1后，与对照组或阴性对照相比，Beclin1-siRNA2组的细胞凋亡率明显降低。综上，PM2.5可能通过IRE1/JNK/自噬信号通路参与雄性大鼠生殖损伤机制，为寻找PM2.5致生殖损伤的治疗靶点提供证据。