Objective As a high-level nerve center that regulates visceral and endocrine activity, the hypothalamus plays an important role in regulating the body’s stress response. Previous studies have shown that stress can cause damage to hypothalamic neurons. The present study aimed to further clarify the mechanism of endoplasmic reticulum stress (ERS) involvement in hypothalamic neuronal injury. Methods A 7-day stressed rat model was established with daily restraining for 8 h and forced ice-water swimming for 5 min. The rats were randomly divided into control, stress, stress + GSK2606414 (PERK phosphorylation inhibitor), stress + KIRA6 (IRE1 phosphokinase activity inhibitor), GSK2606414, and KIRA6 groups. The pathological changes of hypothalamic neurons were observed by thionine staining. Expression of ERS proteins GRP78, ATF4, ASK1, JNK, and CHOP in the hypothalamic neurons were observed by immunohistochemical staining. The expression of JNK and CHOP mRNA in the hypothalamic neurons were observed by RNA in situ hybridization (RNA Scope) and the expression of related proteins and mRNA was semiquantitatively analyzed by microscopy-based multicolor tissue cytometry (MMTC). Results Thionine staining revealed that stress exposure resulted in edema, a lack of Nissl bodies, and pyknosis in hypothalamic neurons. Immunohistochemistry and RNA Scope showed that stress exposure significantly increased the expression of GRP78, ATF4, ASK1, CHOP, JNK, JNK mRNA, and CHOP mRNA. Treatment with PERK and IRE1 inhibitors attenuated pathological damage and downregulated the expression of ATF4, ASK1, JNK, CHOP, JNK mRNA, and CHOP mRNA. Conclusion Stress caused pathological changes in rat hypothalamic neurons. ERS PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways were involved in the injury process.
The hypothalamus, which is the initial part of the hypothalamic-pituitary-adrenal (HPA) axis, plays a critical role in regulating stress in the central nervous system. The present study aimed to determine whether endoplasmic reticulum stress in hypothalamic neurons is differentially stimulated by varying durations of stress exposure, which ultimately leads to pathological changes in neurons by affecting HPA axis function. There is a need for better morphological evidence of the mechanisms involved in stress-induced neuron injury. A stress model was established in rats by restraining for 8 h and forced ice-water swimming for 5 min each day. The stress-inducing process lasted for 1, 3, 7, 14, and 21 days. Enzyme-linked immunosorbent assay (ELISA) was used to assay serum glucocorticoid levels. Thionine staining was used to observe morphological changes in hypothalamic neurons. Immunohistochemistry and microscopy-based multicolor tissue cytometry (MMTC) was used to detect changes in expression of endoplasmic reticulum stress protein GRP78, ATF4, and CHOP. Serum glucocorticoid levels significantly increased after 3 days of stress exposure and the levels peaked by 7 days. By 21 days, however, the levels were significantly decreased. Thionine staining revealed that prolonged stress exposure resulted in hypothalamic neurons with edema, a lack of Nissl bodies, and pyknotic neurons. Immunohistochemistry and MMTC showed that increasing stress periods significantly decreased GRP78 expression, although ATF4 and CHOP protein expression significantly increased. Stress resulted in pathological changes and significant dynamic changes because of endoplasmic reticulum stress in rat hypothalamic neurons. These results suggested that the endoplasmic reticulum stress PERK-ATF4-CHOP pathway may be associated with hypothalamic neuronal injury.
Objective. The present study selected PC12 cells to construct a neuronal injury model induced by glucocorticoids (GC) in vitro, aiming to explore whether the endoplasmic reticulum stress (ERS) PKR-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP-homologous protein (CHOP) and inositol requirement 1 (IRE1)-apoptosis signal regulating kinase 1 (ASK1)-C-Jun amino-terminal kinase (JNK) signaling pathways are associated with the neuronal injury process induced by GC and provide morphological evidence. Methods. Cell models with different doses and different durations of GC exposure were established. The viability of PC12 cells was detected by the CCK-8 assay, and the apoptosis rate of PC12 cells was detected by the flow cytometry assay. The expression of microtubule-associated protein 2 (Map2); glucocorticoids receptor (GR); cellular oncogene fos (C-fos); and ERS-related proteins, glucose-regulated protein 78 (GRP78), p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP, was observed by immunofluorescence staining. Results. The results of immunofluorescence staining showed that PC12 cells abundantly expressed Map2 and GR. The CCK-8 assay revealed that high-concentration GC exposure significantly inhibited the cell viability of PC12 cells. The flow cytometry assay indicated that high-concentration GC exposure significantly increased the apoptosis rate of PC12 cells. Immunofluorescence staining showed that GC exposure significantly increased the expression of C-fos, GRP78, p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP. Treatment with ERS inhibitor 4-phenylbutyric acid (4-PBA) and GR inhibitor RU38486 attenuated related damage and downregulated the expression of the abovementioned proteins. Conclusion. High-concentration GC exposure can significantly inhibit the viability of PC12 cells and induce apoptosis. PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways are involved in the above damage process.
An increasing number of people are in a state of stress due to social and psychological pressures, which may result in mental disorders. Previous studies indicated that mesencephalic dopaminergic neurons are associated with not only reward-related behaviors but also with stress-induced mental disorders. To explore the effect of stress on dopaminergic neuron and potential mechanism, we established stressed rat models of different time durations and observed pathological changes in dopaminergic neurons of the ventral tegmental area (VTA) through HE and thionine staining. Immunohistochemistry coupled with microscopy-based multicolor tissue cytometry (MMTC) was employed to investigate the number changes of dopaminergic neurons. Double immunofluorescence labelling was used to investigate expression changes of endoplasmic reticulum stress (ERS) protein GRP78 and CHOP in dopaminergic neurons. Our results showed that prolonged stress led to pathological alteration in dopaminergic neurons of VTA, such as missing of Nissl bodies and pyknosis in dopaminergic neurons. Immunohistochemistry with MMTC indicated that chronic stress exposure resulted in a significant decrease in dopaminergic neurons. Double immunofluorescence labelling showed that the endoplasmic reticulum stress protein took part in the injury of dopaminergic neurons. Taken together, these results indicated the involvement of ERS in mesencephalic dopaminergic neuron injury induced by stress exposure.
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