Recombinant PEP-1-SOD1 improves functional recovery after neural stem cell transplantation in rats with traumatic brain injury

Abstract

The transplantation of neural stem cells (NSCs) has been demonstrated as a potential treatment strategy for traumatic brain injury (TBI). Cu, Zn-superoxide dismutase (SOD1) is an important antioxidant enzyme that detoxifies intracellular reactive oxygen species, thereby protecting cells from oxidative damage. PEP-1, a peptide carrier, is able to deliver full-length native peptides or proteins into cells. Therefore, the current study investigated the effect of the transplantation of NSCs in combination with PEP-1-SOD1 for the treatment of experimental TBI in rats. Initially, the effect of PEP-1-SOD1 on the proliferation of NSCs was evaluated by MTT assay. PEP-1-SOD1 (0.5, 2.5 and 4.5 µM) significantly increased the proliferation rates of NSCs at 24, 48 and 72 h in a dose-dependent manner. PEP-1-SOD1 also promoted the differentiation of NSCs in vitro. The in vivo experiment showed that PEP-1-SOD1 in combination with NSC transplantation significantly improved the functional recovery of rats following TBI compared with NSC transplantation alone. A significant increase in brain aquaporin-4 (AQP4) mRNA and protein expression levels was observed 4 days post-TBI in PEP-1-SOD1, NSCs and PEP-1-SOD1 + NSCs groups compared with the saline group. The PEP-1-SOD1 + NSCs group showed a further increase of AQP4 mRNA and protein expression levels compared with the NSCs and PEP-1-SOD1 groups. In conclusion, the current data suggests that PEP-1-SOD1 may promote the proliferation and differentiation of NSCs, and thereby improve the functional recovery of TBI model rats following NSCs transplantation through upregulating the expression of AQP4.

Keywords: aquaporin-4; neural stem cells; superoxide dismutase; traumatic brain injury.

Figure 1.

Isolation and identification of NSCs. (A) Isolated NSCs (magnification, ×40) observed by light microscopy, and (B) the expression of nestin in NSCs as revealed by immunocytochemistry assay (magnification, ×400). NSCs, neural stem cells.

Figure 2.

PEP-1-SOD1 promoted the proliferation of NSCs in vitro. NSCs incubated with different concentrations (0, 0.5, 2.5 and 4.5 µM) of PEP-1-SOD1 for the indicated times and the proliferation of the NSCs was examined using the MTT method. Data are expressed as mean ± standard deviation (n=3). **P<0.01 vs. control group. NSCs, neural stem cells; SOD1, Cu, Zn-superoxide dismutase.

Figure 3.

PEP-1-SOD1 promoted the differentiation of NSCs. NSCs were incubated with different concentrations (0, 0.5, 2.5 and 4.5 µM) of PEP-1-SOD1 for 6 days and then immunocytochemical staining was performed to detect the expression of RIP, NeuN and GFAP (magnification, ×400). NSCs, neural stem cells; SOD1, Cu, Zn-superoxide dismutase; RIP, receptor interacting protein; NeuN, neural nuclear antigen; GFAP, glial fibrillary acidic protein.

Figure 4.

Bederson scores of rats at different time points. PEP-1-SOD1 promoted the neurological recovery after NSCs transplantation in TBI rats. Data are expressed as mean ± standard deviation (n=20 for days 1 and 3; n=14 for days 7, 14, 21 and 28). *P<0.05 and **P<0.01 vs. control (saline) group; ^##P<0.01 vs. NSCs group. NSCs, neural stem cells; SOD1, Cu, Zn-superoxide dismutase.

Figure 5.

PEP-1-SOD1 enhanced the effect of NSC transplantation on brain AQP4 expression. (A) Reverse transcription-quantitative polymerase chain reaction analysis of AQP4 mRNA expression; (B) western blot analysis of AQP4 expression; and (C) quantitative data from the densitometric analysis of AQP4 expression. Data are expressed as mean ± standard deviation (n=6) **P<0.01 vs. control (saline) group; ^#P<0.05 and ^##P<0.01 vs the PEP-1-SOD1 + NSCs group. NSCs, neural stem cells; SOD1, Cu, Zn-superoxide dismutase; AQP4, aquaporin-4.