Ventura Ana Lúcia Marques, Silva Thayane Martins, França Guilherme Rapozeiro
Neuroscience Program, Department of Neurobiology, Federal Fluminense University, Niterói CEP 24210-201, RJ, Brazil.
Department of Physiological Sciences, Federal University of the State of Rio de Janeiro, Rio de Janeiro CEP 20211-040, RJ, Brazil.
Brain Sci. 2025 Mar 10;15(3):291. doi: 10.3390/brainsci15030291.
BACKGROUND/OBJECTIVES: Activation of cannabinoid CB1 or CB2 receptors induces the death of glial progenitors from the chick retina in culture. Here, by using an enriched retinal glial cell culture, we characterized some mechanisms underlying glial death promoted by cannabinoids.
Retinal cultures obtained from 8-day-old (E8) chick embryos and maintained for 12-15 days (C12-15) were used. MTT assays revealed that the CB1/CB2 agonist WIN 55,212-2 (WIN) decreased cell viability in the cultures in a time-dependent manner, with a concomitant increase in extracellular LDH activity, suggesting membrane integrity loss. Cell death was also dose-dependently induced by cannabidiol (CBD), Δ-tetrahydrocannabinol (THC), and CP55940, another CB1/CB2 agonist. In contrast to WIN-induced cell death that was not blocked by either antagonist, the deleterious effect of CBD was blocked by the CB2 receptor antagonist SR144528, but not by PF514273, a CB1 receptor antagonist. WIN-treated cultures showed glial cells with large vacuoles in cytoplasm that were absent in cultures incubated with WIN plus 4-phenyl-butyrate (PBA), a chemical chaperone. Since cannabinoids induced the phosphorylation of eukaryotic initiation factor 2-alfa (eIF2α), these results suggest a process of endoplasmic reticulum (ER) swelling and stress. Incubation of the cultures with WIN for 4 h induced a ~five-fold increase in the number of cells labeled with the ROS indicator CM-H2DCFDA. WIN induced the phosphorylation of JNK but not of p38 in the cultures, and also induced an increase in the number of glial cells expressing cleaved-caspase 3 (c-CASP3). The decrease in cell viability and the expression of c-CASP3 was blocked by salubrinal, an inhibitor of eIF2α dephosphorylation.
These data suggest that cannabinoids induce the apoptosis of glial cells in culture by promoting ROS production, ER stress, JNK phosphorylation, and caspase-3 processing. The graphical abstract was created at Biorender.com.
背景/目的：大麻素CB1或CB2受体的激活可诱导培养的鸡视网膜神经胶质祖细胞死亡。在此，我们利用富集的视网膜神经胶质细胞培养物，对大麻素促进神经胶质细胞死亡的一些潜在机制进行了表征。
使用从8日龄（E8）鸡胚获得并维持12 - 15天（C12 - 15）的视网膜培养物。MTT分析显示，CB1/CB2激动剂WIN 55,212 - 2（WIN）以时间依赖性方式降低培养物中的细胞活力，同时细胞外乳酸脱氢酶活性增加，提示膜完整性丧失。大麻二酚（CBD）、Δ-四氢大麻酚（THC）以及另一种CB1/CB2激动剂CP55940也以剂量依赖性方式诱导细胞死亡。与WIN诱导的细胞死亡不受任何一种拮抗剂阻断不同，CBD的有害作用可被CB2受体拮抗剂SR144528阻断，但不能被CB1受体拮抗剂PF514273阻断。用WIN处理的培养物显示神经胶质细胞的细胞质中有大液泡，而在与WIN加4-苯基丁酸盐（PBA，一种化学伴侣）一起孵育的培养物中则没有。由于大麻素诱导真核起始因子2-α（eIF2α）磷酸化，这些结果提示内质网（ER）肿胀和应激过程。用WIN处理培养物4小时可使ROS指示剂CM-H2DCFDA标记的细胞数量增加约五倍。WIN诱导培养物中JNK磷酸化但不诱导p38磷酸化，并且还诱导表达裂解型半胱天冬酶3（c-CASP3）的神经胶质细胞数量增加。细胞活力的降低和c-CASP3的表达被eIF2α去磷酸化抑制剂沙芦比诺阻断。
这些数据表明，大麻素通过促进ROS产生、内质网应激、JNK磷酸化和半胱天冬酶-3加工来诱导培养的神经胶质细胞凋亡。图形摘要由Biorender.com创建。
