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. 2015 Apr 22;35(8):1260–1271. doi: 10.1038/jcbfm.2015.59

Figure 2.

Figure 2

Methamphetamine (METH) impairs barrier properties via tumor necrosis factor-alpha (TNF-α)/nuclear factor-kappa B (NF-κB) pathway. (A) METH (1 μmol/L) increased TNF-α levels after 15 minutes, which was still observed after 1 hour. The blockade of NF-κB pathway with BAY only prevented TNF-α released at 1 hour after METH. The cytokine levels are expressed as mean pg/mL+S.E.M., n=3 to 9. (B) Rat endothelial cell (RBMVEC) permeability was assessed using Na-F at different time points under the following experimental conditions: 1 μmol/L METH; METH+0.01 μg/mL Ab TNF-α; METH+5 μmol/L BAY; 0.01 μg/mL TNF-α; TNF-α+BAY, n=3 to 13. (C) Transendothelial electrical resistance (TEER) was analyzed at different time periods under the same conditions as above mentioned, n=3 to 8. (D) Horseradish peroxidase (HRP) transport across RBMVECs was evaluated in the presence of METH alone or in combination with BAY for 1 hour, n=3 to 4. The results shown are means+S.E.M. (E) Representative images showing the activation of NF-κB pathway by METH (15 minutes exposure), which was evaluated by p65 translocation into the nucleus (arrowheads). This effect was prevented by BAY, as well as by Ab TNF-α. As a positive control, TNF-α was used which effect was also prevented by BAY. p65 (green), Hoechst 33342 (blue) and scale bars=20 μm. (F) Quantification of nuclear p65-positive RBMVECs under the same experimental conditions as in (E). Cells were counted within a total of 1,000 obtained from 37 to 42 visual fields acquired from 3 different slides. **P<0.01, ***P<0.001, significantly different when compared with the control (CTR or dashed line); +P<0.05, +++P<0.001 significantly different when compared with METH; ###P<0.001 significantly different when compared with TNF-α from each time point using Bonferroni's Multiple comparison test.