Abstract
BACKGROUND: Spinal cord injury (SCI) is a devastating central nervous system disorder that remains a global health challenge. SCI-induced oxidative stress in the postinjury microenvironment limits tissue repair by provoking the excessive production of reactive oxygen species (ROS). Tea polyphenols (TP), as a natural plant polyphenol, could effectively reduce ROS. In recent years, stem cell-based therapy combined with cell sheet technology has been widely used in the treatment of SCI. Therefore, we constructed human umbilical cord mesenchymal stem cell sheet loaded with TP (CS-TP) and evaluated their therapeutic effects and mechanisms both in vitro and in vivo in SCI rats. METHODS: Human umbilical cord mesenchymal stem cell sheet (CS) were prepared by temperature-responsive cell culture method and successfully loaded with TP. The protective effect of CS and CS-TP on cells against oxidative stress was tested by Live/Dead cell staining and CCK-8 assay. CS and CS-TP were co-cultured with PC12 cells and human umbilical vein endothelial cells (HUVECs), respectively, and their effects on reducing ROS production were evaluated using flow cytometry and ROS fluorescence assays. Immune fluorescence (IF) and Western blot analysis of the mechanism by which CS-TP affects PC12 cells and HUVECs in vitro. Wound healing assay, transwell Chamber invasion experiment and tube formation assay were performed to evaluate the effects of CS and CS-TP on the biological behaviors of HUVECs. (Basso-Beattie-Bresnahan) BBB scores and gait analysis were performed to assess the recovery of motor function in rats. Molecular modeling is used to study the affinity between the main active ingredient epigallocatechin gallate (EGCG) in TP and target proteins. Western blot analyzes the mechanism of action of CS and CS-TP in SCI animals and the expression levels of antioxidant proteins. Tissue IF staining was used to evaluate angiogenesis, neuron regeneration and axonal extension. RESULTS: Compared with CS, CS-TP could effectively reduce cellular ROS production and increase cell viability under high oxidative stress conditions and significantly enhance its biological activity. In vitro, CS-TP can significantly activate the Keap-1/Nrf2/HO-1 pathway, thereby affecting PC12 cells and HUVECs. After transplantation in SCI rats, CS-TP also activates the Keap-1/Nrf2/HO-1 pathway, influencing the repair of SCI and upregulating the expression of SOD1 and SOD2. CS-TP can more effectively promote angiogenesis, neuronal regeneration, and axonal extension in injured spinal cords, greatly improving the motor function of the rats. CONCLUSION: CS-TP not only significantly enhances the resistance of CS to ROS, activates the Keap-1/Nrf2/HO-1 pathway, and regulates the level of antioxidant proteins in the body. Compared to CS, it can also more effectively increase the number of new blood vessels, promote neuron regeneration and axon extension, thereby more effectively repairing SCI.