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Objective To investigate the protective effects of secretomes released by three-dimensional cultured mesenchymal stem cells (MSCs) on neurons subjected to seawater immersion (SW) and stretch injury (SI), and to provide new insights into neuronal repair following SW combined with traumatic brain injury (TBI). Methods MSCs were cultured using the hanging drop method, and the conditioned medium (CM) containing MSCs secretomes was collected. A cellular model combining SW with SI was established using mouse hippocampal neuronal cells (HT22 cells). HT22 cells were randomly assigned to five groups: control, SI, SI+SW, SI+CM, and SI+SW+CM groups. Cell viability was assessed using the CCK-8 assay, apoptosis rate was measured by flow cytometry, cell migration ability was evaluated by scratch assay, and the expression levels of apoptosis-related proteins Bcl-2 and Bcl-2-associated protein (Bax), and ferroptosis-related proteins long-chain acyl-CoA synthetase 4 (ACSL4) and cyclooxygenase-2 (COX-2) were detected by Western blotting. Results Immersion in 15% seawater for 12 h significantly decreased HT22 cell viability (P<0.05). The CCK-8 assay indicated that cell viability in both the SI and SI+SW groups was significantly lower than that in control group after 12 h of treatment (P<0.05). Treatment with CM containing MSCs secretomes significantly increased cell viability in SI+CM group compared to SI group (P<0.0001), and in SI+SW+CM group compared to SI+SW group (P<0.001). Flow cytometry results revealed that the apoptosis rate in SI and SI+SW groups was significantly higher than that in control group (P<0.05 or P<0.001), while in SI+CM group was lower than that in SI group (P<0.05), and in SI+SW+CM group was lower than that in SI+SW group (P<0.05). Western blotting showed that compared to control group, SI and SI+SW groups exhibited reduced Bcl-2 expression level (P<0.01 or P<0.0001) and increased expression levels of Bax, ACSL4, and COX-2 (P<0.01 or P<0.0001). Compared to SI group, the SI+CM group displayed increased Bcl-2 expression level (P<0.05) and decreased expression levels of Bax, ACSL4, and COX-2 (P<0.05). Compared to SI+SW group, SI+SW+CM group exhibited increased Bcl-2 expression level (P<0.01) and decreased expression levels of Bax, ACSL4, and COX-2 (P<0.01 or P<0.001). Scratch assay results demonstrated that at both 12 h and 24 h, the cell migration rate in SI and SI+SW groups was significantly lower than that in control group (P<0.01 or P<0.0001), while the migration rate in SI+CM group was significantly higher than that in SI group (P<0.0001 or P<0.01), and the migration rate in SI+SW+CM group was significantly higher than that in SI+SW group (P<0.0001). Conclusion Secretomes derived from MSCs cultured using the hanging drop method can alleviate neuronal damage caused by SW and TBI, potentially offering a therapeutic approach for SW combined with TBI.
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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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