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Objective To develop a matrix metalloproteinase (MMP)-responsive hyaluronic acid (HA)-based controlled-release material for brain-derived neurotrophic factor (BDNF) to provide a novel therapeutic strategy for intervention and repair of traumatic brain injury (TBI). Methods HA was modified with amination, followed by condensation with Suflo-SMCC carboxyl group to form amide, and then linked with glutathione (GSH) to synthesize HA-GSH. The recombinant glutathione S-transferase(GST)-tissue inhibitor of metalloproteinase(TIMP)-BDNF (GST-TIMP-BDNF) expression plasmid was constructed using molecular cloning technique with double enzyme digestion by BamH Ⅰ and EcoR Ⅰ. The recombinant GST-TIMP-BDNF protein was expressed in the Escherichia coli prokaryotic expression system, and purified by ion exchange chromatography, confirmed by Western blotting. MMP diluents were supplemented with PBS, MMP inhibitor marimastat, and varing concentrations (0.4, 0.6, 0.8 mg/ml) of GST-TIMP-BDNF or GST-BDNF. MMP-2 activity was analyzed using an MMP activity detection kit to evaluate the inhibitory effect of the recombinant protein on MMP. Primary rat neurons were extracted and cultured to establish an iron death model induced by RSL3. The effect of recombinant protein GST-TIMP-BDNF on neuronal injury was detected by immunofluorescence staining. Results MRI hydrogen spectrum identification confirmed the successful synthesis of HA-GSH. Western blotting results showed the successful expression of the recombinant protein GST-TIMP-BDNF containing the GST tag using the E. coli prokaryotic expression system. MMP activity detection results indicated that the recombinant protein GST-TIMP-BDNF had a superior inhibitory effect on MMP-2 activity compared to GST-BDNF (P<0.05). Immunofluorescence staining results showed a significant increase in fluorescence intensity in rat neurons treated with GST-TIMP-BDNF after RSL3 induction (P<0.05). Conclusion A MMP-responsive HA-based BDNF controlled-release material has been successfully developed, exhibiting a protective effect on neuron damage.
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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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