Eye Center, RenMin Hospital of Wuhan University, Wuhan, Hubei Province, People's Republic of China.
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Eye Institute of Shandong First Medical University, Qingdao, Shandong Province, People's Republic of China.
Invest Ophthalmol Vis Sci. 2022 Jun 1;63(6):28. doi: 10.1167/iovs.63.6.28.
Heat shock protein B8 (HspB8) can be upregulated rapidly in many pathologic processes, but its role in traumatic optic neuropathy remains unclear. In this study, we investigated the involvement of autophagy in the effects of HspB8 by using the optic nerve crush (ONC) model.
Male C57BL/6J mice were intravitreally injected with recombinant adeno-associated virus type 2 (AAV2-shHspB8 or AAV2-GFP) and subsequently received ONC by a self-closing tweezers. Western blot and immunohistochemistry staining were used to evaluate the expression of HspB8. We conducted retinal flat-mount immunofluorescence to measure the quantities of retinal ganglion cells (RGCs), and full-field flash electroretinogram (ff-ERG) and optomotor response (OMR) were used to evaluate retinal function. The autophagy level was reflected by western blot, immunohistochemistry staining, and transmission electron microscope (TEM) images. We also applied 3-methyladenine (3MA) and rapamycin (Rapa) to regulate autophagy level in optic nerve injury.
ONC stimulated the expression of HspB8. Declines of RGCs and ff-ERG b-wave amplitudes resulting from ONC can be alleviated by HspB8 downregulation. Increased autophagy activity after ONC was observed; however, this change can be reversed by intravitreal injection of AAV2-shHspB8. Furthermore, application of autophagy inhibitor 3MA had the same neuroprotective effects as AAV2-shHspB8, as illustrated by ff-ERG and quantities of RGCs. Also, protection of AAV2-shHspB8 was compromised by the autophagy activator Rapa.
Inhibition of HspB8 in mice optic nerve injury had neuroprotective effects, which may be derived from its downregulation of autophagy.
热休克蛋白 B8（HspB8）在许多病理过程中可以迅速上调，但它在创伤性视神经病变中的作用尚不清楚。在这项研究中，我们通过视神经钳夹伤（ONC）模型研究了自噬在 HspB8 作用中的参与情况。
雄性 C57BL/6J 小鼠通过玻璃体内注射重组腺相关病毒 2（AAV2-shHspB8 或 AAV2-GFP），随后用自封闭镊子进行 ONC。Western blot 和免疫组织化学染色用于评估 HspB8 的表达。我们进行视网膜铺片免疫荧光测量视网膜神经节细胞（RGCs）的数量，并使用全视野闪光视网膜电图（ff-ERG）和光运动反应（OMR）评估视网膜功能。Western blot、免疫组织化学染色和透射电镜（TEM）图像用于反映自噬水平。我们还应用 3-甲基腺嘌呤（3MA）和雷帕霉素（Rapa）调节视神经损伤中的自噬水平。
ONC 刺激 HspB8 的表达。下调 HspB8 可减轻 ONC 引起的 RGCs 减少和 ff-ERG b 波幅度下降。ONC 后观察到自噬活性增加；然而，玻璃体内注射 AAV2-shHspB8 可逆转这种变化。此外，自噬抑制剂 3MA 的应用与 AAV2-shHspB8 具有相同的神经保护作用，如 ff-ERG 和 RGCs 的数量所示。同样，AAV2-shHspB8 的保护作用被自噬激活剂 Rapa 削弱。
在小鼠视神经损伤中抑制 HspB8 具有神经保护作用，这可能源自其对自噬的下调。
