Figure 6
Alterations in TLR 4 and NF- κB protein and mRNA expressions after spinal cord ischemia reperfusion (I/R) injury. (A) Representative immunoblots were probed with antibody against TLR4 and NF-κB p65, while antibody against Histone and GAPDH served as loading control for the nuclear and cytoplasmic protein, respectively. (B-E) Quantification of the densities of TLR4(B), NF-κB p65 bands in nuclear extracts (C), cytoplasmic extracts (D) and calculated as nucleocytoplasmic ratio (E) in different protocol conditions. The protein expression is presented in relative units. (F-G) Real-time PCR analysis was performed in duplicate for TLR4 and NF-κB p65 under study and normalized to GAPDH mRNA. (H) Quantification data of IL-1β production in the spinal cord at 12 h and 36 h after I/R injury, as assessed by ELISA. Ordinate represents the mean integral density values (IDVs) ratios relative to the loading control. I/R caused significant increases in TLR4, nuclear and cytoplasmic NF-κB p65 expressions, as well as nucleocytoplasmic ratio at 12 h and 36 h after I/R after normalizing to Histone and GAPDH, consistent with results from real time-PCR. Intrathecal injection with minocycline, TAK-242 and PDTC prevented NF-κB p65 upregulation in nucleocytoplasmic ratio, as the upregulated expressions of NF-κB p65 in nucleus and comparable expressions in cytoplasma. Contrarily, intrathecal injection with LPS synergistically increased the activation. IL-1β content was changed accordingly with the mRNA and protein expressions of TLR4 and NF-κB by ELISA. All data are presented as mean ± SEM. **P < .01 compared to Sham group; ##P < .05 compared to I/R group; &&P < .05 compared to I/R + P group.