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This work investigates the effects and mechanisms of inhibiting TRPC6 (a non-selective cation channel) downregulation on rat astrocyte activation and proliferation following spinal cord injury (SCI) by suppressing AQP4 expression. We used HYP9 (TRPC6-specific agonist) and TGN-020 (AQP4-specific inhibitor) to explore the relationship between TRPC6 and AQP4 and their probable protective effects on SCI.In a rat SCI model, we randomly assigned female Sprague-Dawley rats into the following four groups: Sham, SCI, SCI+HYP9, and SCI+TGN-020. Western blotting and immunofluorescence staining were used to determine protein expression among groups following SCI. TUNEL and immunofluorescence staining were used to identify changes in the rate of apoptosis and the fraction of surviving neurons after SCI. The Basso-Beattie-Bresnahan open-field locomotor scale was used to identify changes in motor function after SCI. In vitro astrocyte scratch model, we first used the CCK8 assay to test the effects of varying doses of HYP9 or TGN-020 on astrocytes and then split the astrocytes into four groups: Con, Scratch, Scratch+HYP9, and Scratch+TGN-020. Western blotting and immunofluorescence were used to identify changes in the expression of target proteins.In vivo and in vitro models, SCI dramatically decreased TRPC6 while considerably upregulating AQP4, glial fibrillary acidic protein (GFAP), and proliferating cell nuclear antigen (PCNA) expression. However, HYP9 or TGN-020 significantly suppressed activation of astrocytes, promoted neurons survival in the anterior horn of the spinal cords, and benefited the recovery of motor function in the hind limbs of rats following SCI. Interestingly, TRPC6 agonists dramatically suppressed AQP4 overexpression, indicating that the probable mechanism of HYP9 benefiting alleviation of SCI may be connected to AQP4 inhibition and astrocyte activation and proliferation reduction.we discovered for the first time that HYP9 inhibits astrocyte activation and proliferation by inhibiting AQP4 in SCI rats in vivo and in vitro models and that it preserves neuronal survival and functional recovery after SCI. |
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