Danhong Injection Combined With t-PA Improves Thrombolytic Therapy in Focal Embolic Stroke

Abstract

Background: Hemorrhagic transformation, neurotoxicity, short treatment time windows, and other defects are considered as the major limitations for the thrombolytic therapy. This study is devoted to figure out whether Danhong injection (DHI) combined with tissue-plasminogen activator (t-PA) could extend the treatment time windows and ameliorate brain injury, hemorrhagic complication and BBB disruption after focal embolic stroke. Methods:In vitro, the combined concentrations of DHI and t-PA were added to wells reacted with plasminogen and D-Val-Leu-Lys-AMC. The optimum ratio of the combination of DHI plus t-PA was explored by detecting relative fluorescent. In vivo experiments, we firstly investigated the optimal dose of t-PA and Danhong injection for focal embolic stroke. The neurological deficit score, infarct volume and brain edema were assessed. Secondly, we proved that the combination group extended the thrombolytic window for treatment of focal embolic stroke. The neurological deficit score, infarct volume, brain edema and hemorrhagic complication were assessed, while levels of BAX, Bcl-2 and caspase-3 in brain tissue were analyzed by real-time polymerase chain reaction. Finally, to ask whether combination therapy with DHI plus t-PA protected the blood-brain barrier in a rat model of focal embolic stroke, neurological deficit score, ELISA, RT-PCR, western blot and fluorescence were used to detect the indicators of blood-brain barrier, such as tight junction protein, blood-brain barrier permeability and related gene expression. Results:In vitro, plasmin activity assays showed that the combination of t-PA with DHI at about 1:1.6 w/v ratio increased by almost 1.4-fold the plasmin-generating capability of t-PA. In vivo experiments, the results showed that the combination of Danhong injection (4 mL/kg) and t-PA (2.5 mg/kg) could extend the t-PA treatment time windows to 4.5 h. And the combination t-PA (2.5 mg/kg) with DHI (4 mL/kg) ameliorated neurological score, cerebral infarction, brain edema, brain hemorrhage, and BBB disruption. Conclusion: Combination therapy with Danhong injection (4 mL/kg) plus t-PA (2.5 mg/kg) could extend the t-PA treatment time windows to 4.5 h, ameliorate BBB disruption, reduce infarction, brain swelling and hemorrhage after ischemic stroke.

Keywords: Danhong injection; cerebral ischemia; combination therapy; embolic; t-PA.

FIGURE 1

Effect of DHI on tPA-dependent plasmin generation in vitro and acute brain tissue outcomes of different combination therapy in stroke rats. (A) A range of concentrations of tPA (1, 2.5, 5, and 10 μg/mL) with or without the indicated concentrations of DHI (1, 2, 3, and 4 μL/mL) were added to wells of 96-well plate. Plasmin activity was represented as fold of plasmin activity related to 1 μg/mL of tPA alone. (B) At 24 h after stroke, neurological score were quantified. (C) Ischemic infarct volume rates were quantified at 24 h after stroke. (D) Brain edema was assessed at 24 h after stroke. Data were expressed as mean ± SD, n = 6 per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group. ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI-M+tPA-M.

FIGURE 2

Representative images for TTC stained ischemic brain infarctions. (A) Effect of different combination groups on cerebral infarction volume. (B) Effect of treatment time window on cerebral infarction volume.

FIGURE 3

Effect of different treatment time on neurological score, cerebral infarction volume and cerebral edema. (A) At 24 h after stroke, neurological score were quantified. (B) Ischemic infarct volume rates were quantified at 24 h after stroke. (C) Brain edema was assessed at 24 h after stroke. Data were expressed as mean ± SD, n = 6 per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI+tPA+4.5 h.

FIGURE 4

Effect of different treatment time on the mRNA transcriptions of Bax, Bcl-2, caspase-3. (A–C) The real time PCR was performed to determine the relative levels of Bax, Bcl-2, caspase-3 mRNA transcriptions at 24 h after stroke. Data were expressed as mean ± SD, n = 6 per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group. ^&P < 0.05, ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI+tPA+4.5 h.

FIGURE 5

Effect of DHI combinated with tPA in neurological core and the levels of MMP-9, PAI-1, P-selectin at 4.5 h after initiation of ischemia. (A) At 24 h after stroke, neurological score were quantified. (B–D) The levels of MMP-9, PAI-1, P-selectin were tested at 24 h after stroke. Data were expressed as mean ± SD, n = 6 per group. ^∗∗P < 0.01 vs. vehicle group. ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI+tPA.

FIGURE 6

(A–C) The real time RT-PCR was performed to determine the relative levels of claudin-5, occludin, ZO-1 mRNA transcriptions at 24 h after stroke. (D) Evans Blue extravasation in the ischemic hemi sphere was quantified by fluorometry. Data were expressed as mean ± SD, n = 6–8 per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group. ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI+tPA.

FIGURE 7

At 24 h after stroke, the expression of MMP-2 and occludin protein treated with saline, DHI, tPA, and DHI combinated with tPA. (A) Representative blots of the MMP-2, occludin in the five groups. (B,C) The relative expression level of MMP-2 and occluding signaling proteins. Data were expressed as mean ± SD, n = 6–8 per group. ^∗P < 0.05, ^∗∗P < 0.01 vs. vehicle group. ^&&P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. DHI+tPA.