| Objective: Enhanced angiogenesis facilitates neurovascular remodeling processes and promotes brain functional recovery after stroke. Salvianolic acids(Sals) is hydrophilic components isolated from Salvia miltiorrhiza, of which more than 50% is Salvianolic acid B. Salvianolic acids has been reported to enhance angiogenesis after myocardial infarction. The JAK2/STAT3 signaling pathway has been shown to regulate the expression of genes involved in cell survival, cell proliferation, cell-cycle progression and angiogenesis after cerebral insults. In this study, we investigated whether Sals promoted angiogenesis after ischemic stroke through JAK2/STAT3 signaling pathway. Adult male mice were subjected to permanent distal middle cerebral artery occlusion(d MCAO) and were treated with or without different doses(10 and 30mg/kg) of Sals, starting from 24 hours after d MCAO and daily for 14 days. Neurological functional tests were measured. Angiogenesis and the interaction of endothelial cells, pericytes, and astrocytic endfeet were examined by confocal microscope. Angiogenic factors and p JAK2, p STAT3 expression were measured by Western blot.Methods: Male C57BL/6 mice(age 2 to 3 months, weight 25 to 30 g) were subjected to d MCAO, as described previously. Sals(Low dose: 10mg/kg; High dose: 30mg/kg, i.p, Tianshili) was administrated from 24 hours after d MCAO and then once daily for up to 14 days.In the first set of studies, functional tests and infarct volume were examined. Stroke mice were randomly assigned to 3 groups:(1) d MCAO group: animals received d MCAO and equal volume of 0.9% Na Cl.(2) Sals-L group: animals underwent d MCAO and treated with a low dose of Sals(10 mg/kg, Sals-L).(3) Sals-H group: animals underwent d MCAO and were treated with a high dose of Sals(30 mg/kg, Sals-H).In the second set of studies, stroke mice used for histochemical analysis of angiogenesis and Evans blue studies. They were randomly assigned to 3 groups:(1) Sham group: animals received Sham operation and equal volume of 0.9% Na Cl.(2) d MCAO group.(3) Sals-H group(30 mg/kg). These animals were received injections of bromodeoxyuridine(Brd U, Sigma Chemical, 50 mg/kg in 0.9% Na Cl), intraperitoneally(i.p.) starting from 24 hours after d MCAO and daily for up to 14 days. Animals were sacrificed at three time points after d MCAO(day 7, day 14, day 28).In the third set of studies, mice were used for Western blot to analyze angiogenic factors and p JAK2, p STAT3 expression, randomly assigned to 3 groups:(1) Sham group.(2) d MCAO group.(3) Sals-H group(30 mg/kg).Results:1 Sals promoted functional outcome and had no effect on lesion volumeRota-Rod and m NSS tests were performed on day 1, 7, 14, 28 after d MCAO, respectively. The results showed balanced neurologic deficits on day 1 between d MCAO and Sals-treated groups. Still, no significant difference in functional outcome was detected in the experimental groups on day 7. However, during 14 to 28 days after treatment, mice treated with 30 mg/kg Sals significantly improved functional recovery on Rota-Rod and m NSS tests, compared with the 10 mg/kg Sals-treated group and d MCAO group. Meanwhile, mice treated with 10 mg/kg Sals showed no remarkble functional improvement when compared to d MCAO during the whole experiment. The lesion volume was measured on day 28 after treatment, and there were no diffrences among the three experimental groups(Fig. 1).2 Sals enhanced post-ischemic angiogenesisMicrovessel density was significantly enhanced on day 14 and 28 after d MCAO,(day 14 Sals-H group vs. d MCAO group and Sham group: 13.14%±0.60% vs. 11.12%±0.38% and 7.06%±0.27%, day 28 Sals-H group vs. d MCAO group and Sham group: 14.70%±0.34% vs. 10.46%±0.24% and 7.74%±0.29%, P < 0.01). However, there was no difference between the two groups on day 7(Sals-H group vs. d MCAO group and Sham group: 11.90%±0.16% vs. 11.25% ± 0.33% 和 8.25% ± 0.13%, P > 0.1). To determine endothelial cell proliferation, the number of endothelial cells positive for both Brd U and CD31 was also quantified on day 14. And it was significantly increased compared to the d MCAO group(Sals-H group vs. d MCAO group: 16.08%±0.50% vs. 8.47%±0.39%, P < 0.01)(Fig.2A, 2B, 2E).Vascular permeability was quantified by Evans blue extravasation. The Figure showed that there was no extravasation of Evans blue dye in the brain on day 1 after d MCAO and Evans blue extravasation was highly detected on day 7 and decreased significantly from day 14 to 28 suggesting new vessels return to integrity. Furthermore, Sals treated mice showed more extravasation than d MCAO group on day 7, whereas no significant difference was observed on day 14.(P < 0.05)(Fig. 2C, 2D).3 Sals promoted pericyte proliferation, recruitment and coverage around microvesselsWe assessed coronal brain sections double labeled with NG2(pericyte marker) and CD31 antibodies. The Figure showed that NG2-positive pericytes were rarely detached from the capillary endothelium on day 7 after d MCAO. However, from day 14, the morphology of the pericytes changed to cells within the basement membrane that elongate along the long axis of capillaries, and wrap around endothelial cells with shorter processes. Meanwhile, the ratio of covered capillary surface by pericyte endfeet over the capillary endothelium in Sals-H group was significantly increased compared with the d MCAO group(Sals-H group vs. d MCAO group and Sham group:69.88%±1.05% vs. 56.42%±1.09% and 43.08%±1.18%, P<0.01), and this effect persisted to 28 days after d MCAO compared with d MCAO(Sals-H group vs. d MCAO group and Sham group:82.35%±1.42% vs. 64.66%±2.01% and 41.29%±1.12%, P<0.01). In addition, a double-immunofluorescence analysis for NG2 and Brd U was shown in our study. These vascular associated pericytes were NG2 and Brd U positive, suggesting that they were newly formed and contributed to new vessels proliferation in the peri-infarct regions(Fig. 3).4 Sals promoted astrocytic foot processes wrapping the blood vessels and astrocytes proliferation in the peri-Infarct cortexTo evaluate the detachment of GFAP-positive astrocytic endfeet from the capillary endothelium, double immunofluorescence staining was employed by CD31 and GFAP on day 7, 14 and 28 after d MCAO. The results showed that the activated astrocytes wrapped their foot processes around the blood vessels starting from day 7. The coverage ratio was increased on day 14(Sals-H group vs. d MCAO group and Sham group: 84.20%±0.85% vs. 70.20%±0.80% and 38.80%±0.64%, P < 0.01), and reached the peak on day 28(Sals-H group vs. d MCAO group and Sham group: 91.92%±1.31% vs. 67.09%±1.49% and 34.72%±1.22%, P < 0.01). The Sals-H group showed a great increase compared with the d MCAO( P < 0.05) on day 14 and 28 after d MCAO. The number of astrocytes positive for both Brd U and GFAP was also significantly increased after Sals treatment(data not shown)(Fig.4).5 Sals mediated angiogenic factors and p JAK2, p STAT3 expressionMice were sacrificed on day 3, 7, 14 after d MCAO, and both ipsilateral and contralateral cortex were obtained to be analyzed. Compared with the Sham group, protein levels of p JAK2 and p STAT3 in the peri-ischemic cortex tended to be higher in d MCAO and Sals-H groups in the ipsilateral cortex(P < 0.05). Sals significantly upregulated the phosphorylation of JAK2 and STAT3 in the ipsilateral cortex at the three time points, compared with the d MCAO group(P < 0.05). The Figure showed that the level of VEGF was all significantly upregulated after systemic administration of Sals on day 3, 7 and 14 in the ipsilateral cortexl cortex(P < 0.05). For Ang-1, significantly difference between Sals-H and d MCAO groups started from day 7, persisting up to day 14(P < 0.05). Ang-2 was significantly reduced in the Sals-H group on day 7 and 14(P < 0.05), but no significantly difference was seen on day 3. The protein level of contralateral cortex showed no differences(Fig.5).Conclusions: Sals enhanced angiogenesis which may contribute to improvement of functional outcome after stroke and angiogenetic effect was likely mediated by activating JAK2/STAT3 signaling pathway. |
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