Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China; Key Laboratory of Anesthesiology of Zhejiang Province, Wenzhou Medical University, Wenzhou, Zhejiang, China; Key Laboratory of Pediatric Anesthesiology, Ministry of Education, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Department of Anesthesiology, Rutgers New Jersey Medical School, Newark, NJ, USA.
Biochim Biophys Acta Mol Basis Dis. 2024 Jun;1870(5):167137. doi: 10.1016/j.bbadis.2024.167137. Epub 2024 Mar 23.
Postoperative Cognitive Dysfunction (POCD) has attracted increased attention, but its precise mechanism remains to be explored. This study aimed to figure out whether HDAC6 could regulate NLRP3-induced pyroptosis by modulating the functions of HSP70 and HSP90 in microglia to participate in postoperative cognitive dysfunction in aged mice.
Animal models of postoperative cognitive dysfunction in aged mice were established by splenectomy under sevoflurane anesthesia. Morris water maze was used to examine the cognitive function and motor ability. Sixteen-months-old C57BL/6 male mice were randomly divided into six groups: control group (C group), sham surgery group (SA group), splenectomy group (S group), splenectomy + HDAC6 inhibitor ACY-1215 group (ACY group), splenectomy + HDAC6 inhibitor ACY-1215 + HSP70 inhibitor Apoptozole group (AP group), splenectomy + solvent control group (SC group). The serum and hippocampus of mice were taken after mice were executed. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, cleaved-Caspase-1 (P20), IL-1β were detected by western blotting. Serum IL-1β, IL-6 and S100β were measured using ELISA assay, and cell localization of HDAC6 was detected by immunofluorescence. In vitro experiments, BV2 cells were used to validate whether this mechanism worked in microglia. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, P20, IL-1β were detected by western blotting and the content of IL-1β in the supernatant was measured using ELISA assay. The degree of acetylation of HSP90, the interaction of HSP70, HSP90 and NLRP3 were analyzed by coimmunoprecipitation assay.
Splenectomy under sevoflurane anesthesia in aged mice could prolong the escape latency, reduce the number of crossing platforms, increase the expression of HDAC6 and activate the NLRP3 inflammasome to induce pyroptosis in hippocampus microglia. Using ACY-1215 could reduce the activation of NLRP3 inflammasome, the pyroptosis of microglia and the degree of spatial memory impairment. Apoptozole could inhibit the binding of HSP70 to NLRP3, reduce the degradation of NLRP3 and reverse the protective effect of HDAC6 inhibitors. The results acquired in vitro experiments closely resembled those in vivo, LPS stimulation led to the pyroptosis of BV2 microglia cells and the release of IL-1β due to the activation of the NLRP3 inflammasome, ACY-1215 showed the anti-inflammatory effect and Apoptozole exerted the opposite effect.
Our findings suggest that hippocampal HDAC6 promotes POCD by regulating NLRP3-induced microglia pyroptosis via HSP90/HSP70 in aged mice.
术后认知功能障碍（POCD）受到越来越多的关注，但其确切机制仍有待探讨。本研究旨在探讨 HDAC6 是否可以通过调节小胶质细胞中 HSP70 和 HSP90 的功能来调节 NLRP3 诱导的焦亡，从而参与老年小鼠的术后认知功能障碍。
通过七氟醚麻醉下的脾切除术建立老年小鼠术后认知功能障碍动物模型。采用 Morris 水迷宫检测认知功能和运动能力。将 16 个月大的 C57BL/6 雄性小鼠随机分为六组：对照组（C 组）、假手术组（SA 组）、脾切除术组（S 组）、脾切除术+HDAC6 抑制剂 ACY-1215 组（ACY 组）、脾切除术+HDAC6 抑制剂 ACY-1215+HSP70 抑制剂 Apoptozole 组（AP 组）、脾切除术+溶剂对照组（SC 组）。处死小鼠后取小鼠血清和海马。采用 Western blot 检测 HDAC6、HSP90、HSP70、NLRP3、GSDMD-N、cleaved-Caspase-1（P20）、IL-1β的蛋白水平。采用 ELISA 法检测血清 IL-1β、IL-6 和 S100β，采用免疫荧光法检测 HDAC6 的细胞定位。体外实验采用 BV2 细胞验证该机制是否在小胶质细胞中起作用。采用 Western blot 检测 HDAC6、HSP90、HSP70、NLRP3、GSDMD-N、P20、IL-1β的蛋白水平，采用 ELISA 法检测上清液中 IL-1β的含量。采用免疫共沉淀法分析 HSP90 的乙酰化程度、HSP70、HSP90 和 NLRP3 的相互作用。
七氟醚麻醉下的脾切除术可延长老年小鼠的逃避潜伏期，减少穿越平台的次数，增加 HDAC6 的表达并激活 NLRP3 炎性小体诱导海马小胶质细胞发生焦亡。使用 ACY-1215 可降低 NLRP3 炎性小体的激活、小胶质细胞的焦亡程度和空间记忆损伤程度。Apoptozole 可抑制 HSP70 与 NLRP3 的结合，减少 NLRP3 的降解，并逆转 HDAC6 抑制剂的保护作用。体内实验结果与体外实验结果非常相似，LPS 刺激导致 BV2 小胶质细胞的焦亡和 IL-1β的释放，ACY-1215 表现出抗炎作用，而 Apoptozole 则产生相反的作用。
我们的研究结果表明，在老年小鼠中，海马体中的 HDAC6 通过调节 HSP90/HSP70 来促进 NLRP3 诱导的小胶质细胞焦亡，从而导致术后认知功能障碍。
