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Outline
To elucidate the specific role of Ras-related C3 botulinum toxin substrate 2 (RAC2) in neuronal apoptosis induced by cerebral ischemia-reperfusion (CIR), and investigate the molecular mechanisms underlying its regulation of apoptosis.
① Cell culture and grouping (n=5): mouse hippocampal neuronal cell line HT22 was divided into normal control (NC) and oxygen-glucose deprivation/reoxygenation (OGD/R) groups. The NC group was maintained under standard culture conditions, while the OGD/R group was subjected to 8 hours’ OGD/R. Cell proliferation was assessed using CCK-8 assay and EdU staining, and cell apoptosis was evaluated by flow cytometry. Western blotting was performed to measure the protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, cleaved poly(ADP-ribose) polymerase-1 (PARP-1), RAC2, and extracellular signal-regulated kinase (ERK)/p-ERK. Quantitative real-time PCR (qRT-PCR) was applied to detect the mRNA level of RAC2. ② Gene knockdown: HT22 cells were transfected with sh-RAC2 or non-targeting control shRNA (sh-NC) before OGD/R treatment. Subsequently, the cells were divided into OGD/R:sh-NC and OGD/R:sh-RAC2 groups (n=5). Apoptosis-related proteins were detected by Western blotting. ③ Pathway validation: The ERK inhibitor FR180204 was employed to establish 4 groups (n=5): NC, OGD/R, FR180204 alone, and FR180204+OGD/R (pretreated with FR180204 followed by OGD/R treatment). Expression of cleaved caspase-3, RAC2, and p-ERK was analyzed with Western blotting.
①Compared with the NC group, OGD/R treatment significantly inhibited HT22 cell proliferation and increased apoptotic rates (P<0.05). OGD/R upregulated pro-apoptotic proteins (Bax, cleaved PARP-1, and cleaved caspase-3), downregulated the anti-apoptotic protein Bcl-2, and markedly elevated RAC2 expression at both mRNA and protein levels (P<0.05). ② Specific knockdown of RAC2 (sh-RAC2 group) partially restored Bcl-2 expression and attenuated OGD/R-induced upregulation of Bax and cleaved PARP-1 compared with the OGD/R group (P<0.05). ③ OGD/R treatment activated the ERK signaling pathway (elevated p-ERK/ERK ratio), upregulated RAC2 expression, and promoted caspase-3 cleavage compared with the NC group (P<0.05). Conversely, pretreatment with FR180204 effectively blocked OGD/R-induced RAC2 upregulation and caspase-3 activation compared with the OGD/R group (P<0.05).
RAC2 plays a key role in CIR-induced neuronal apoptosis by modulating the Bcl-2/Bax balance and PARP-1 cleavage through the ERK signaling pathway.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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