Research Paper Volume 11, Issue 22 pp 10167—10182

Tissue plasminogen activator disrupts the blood-brain barrier through increasing the inflammatory response mediated by pericytes after cerebral ischemia

Figure 5. The TGF-β/p-Smad2/3 pathway mediated the disruption of the BBB after rt-PA treatment. (AE) Representative western blot showing the expression of total TGF-β (44 kDa), active TGF-β (13 kDa) and p-Smad2/3 at 12 h and 24 h after treatment with or without 50 μg/ml rt-PA after OGD for 4 h. Densitometric analysis of the levels of total TGF-β (44 kDa), active TGF-β (13 kDa) and p-Smad2/3 protein at 12 h and 24 h after treatment with or without rt-PA after OGD for 4 h; n = 3 for each group. Data represent the mean ± sd, *p < 0.05, **p < 0.01. (F, G) Immunofluorescence was used to detect the expression of TGF-β on pericytes in the sham-treated mice and mice 1 d after I/R treatment with or without 9 mg/kg rt-PA; scale bar: 50 μm; n = 3 for each group. Data represent the mean ± sd, *p < 0.05, **p < 0.01. (H, I) Representative western blot showing the expression of active TGF-β (13 kDa) after treatment with TGF-β in combination with rt-PA or rt-PA alone after OGD for 4 h. Densitometric analysis of the level of active TGF-β (13 kDa) after treatment with TGF-β in combination with rt-PA or rt-PA alone after OGD for 4 h; n = 3 for each group. Data represent the mean ± sd, *p < 0.05, **p < 0.01. (J, K) The TEER and permeability of the coculture model were determined after OGD/R alone or OGD/R in combination with 50 μg/ml rt-PA, 3 ng/ml TGF-β or 200 nM TPO427736 HCL treatment; n = 3–5 for each group. Data represent the mean ± sd, *p < 0.05, **p < 0.01. (L, M) The concentrations of TNF-α and MCP-1 secreted from pericytes alone or treated with 50 μg/ml rt-PA, 3 ng/ml TGF-β or 200 nM TPO427736 HCL were determined 24 h after OGD/R; n = 3–4 for each group. Data represent the mean ± sd, *p < 0.05, **p < 0.01. (N, O) Immunofluorescence was used to detect the expression of TNF-α and MCP-1 after 1 d of treatment with 10 μg/kg TGF-β in combination with 9 mg/kg rt-PA after I/R. The results also showed that TGF-β decreased the colocalization of TNF-α and MCP-1 (green) with NG2 (red) compared with rt-PA alone.