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. 2022 Jan 12:12:759027.
doi: 10.3389/fphar.2021.759027. eCollection 2021.

QiShenYiQi Inhibits Tissue Plasminogen Activator-Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice

Yang Ye  1   2   3   4   5   6 , Quan Li  2   3   4   5   6 , Chun-Shui Pan  2   3   4   5   6 , Li Yan  2   3   4   5   6 , Kai Sun  2   3   4   5   6 , Xiao-Yi Wang  1   2   3   4   5   6 , Shu-Qi Yao  1   2   3   4   5   6 , Jing-Yu Fan  2   3   4   5   6 , Jing-Yan Han  1   2   3   4   5   6
Affiliations

QiShenYiQi Inhibits Tissue Plasminogen Activator-Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice

Yang Ye et al. Front Pharmacol. .

Abstract

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model. Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration. Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment. Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

Keywords: QiShenYiQi; blood-brain barrier; hemorrhage; ischemic stroke; tissue plasminogen activator.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of QSYQ on cerebral blood flow (CBF) and infarction in tPA-treated stroke mice. (A) Schematic representation of experimental design. (B) Representative images and (C) quantitative analysis of CBF at baseline, 2.5, 4.5, 5.5, 6.5, and 28.5 h after stroke onset (n = six to seven per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (D) Representative images and (E) quantitative analysis of infarct volume (n = 8 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. CBF indicates cerebral blood flow; MCAO, middle cerebral artery occlusion; BBB, blood-brain barrier; IHC, immunohistochemistry; WB, western blot. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 2
FIGURE 2
Effect of QSYQ on animal survival and neurological dysfunction in tPA-treated stroke mice. (A) Survival rate (n = 12 per group). Data were compared by a Log-rank (Mantel-Cox) test. Neurological deficits were assessed by (B) modified neurological severity score (mNSS) and (C) neurological evaluation scale (NES) (n = 8 per group). Data were compared by a Kruskal–Wallis test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA. ns, not statistically significant compared to thrombus + tPA.
FIGURE 3
FIGURE 3
Effect of QSYQ on blood-brain barrier (BBB) permeability in tPA-treated stroke mice. (A) Representative images and (B) quantitative analysis of Evans blue extravasation (n = 7 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (C) Representative images and (D) quantitative analysis of albumin leakage (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (E) Quantitative analysis of cerabral water content (n = seven to eight per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 4
FIGURE 4
Effect of QSYQ on brain hemorrhage and basement membrane protein expression in tPA-treated stroke mice. (A) Representative slices and (B) quantitative analysis of brain hemorrhage in each group (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (C) Representative images and (D,E) quantitative analysis of immunofluorescence staining for collagen IV and laminin in indicated groups (n = 3 per group). (F) Representative images and (G,H) quantitative analysis of immunoblotting of collagen IV and laminin in mouse brain tissues (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 5
FIGURE 5
Effect of QSYQ on tight junctions and adherens junctions protein expression in tPA-treated stroke mice. (A,B) Representative images and (C,D) quantitative analysis of immunofluorescence staining for occludin and VE-cadherin in indicated groups (n = 3 per group). (E) Representative images of immunoblotting of zonula occludens-1 (ZO-1), junctional adhesion molecule-1 (JAM-1), occludin, claudin-5, VE-cadherin, α-catenin, and β-catenin in mouse brain tissues. Quantitative analysis of immunoblotting of ZO-1 (F), JAM-1 (G), occludin (H), claudin-5 (I), VE-cadherin (J), α-catenin (K), and β-catenin (L) (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 6
FIGURE 6
Effect of QSYQ on caveolae-related protein expression, energy-related protein expression, and mitochondrial complex enzyme activity in tPA-treated stroke mice. (A) Representative images and (B,C) quantitative analysis of immunoblotting of Caveolin-1, Src, and phospho-Src in mouse brain tissues (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (D) Representative images and (E,F) quantitative analysis of immunoblotting of ATP 5D and ATP synthase α in mouse brain tissues (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. (G-I) Enzyme activities of mitochondrial Complex I, II, and IV (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 7
FIGURE 7
Effect of QSYQ on matrix metalloproteinases (MMPs) expression in tPA-treated stroke mice. (A) Representative images and (B,C) quantitative analysis of immunostaining results of MMP-2 and MMP-9 in indicated groups (n = 3 per group). (D) Representative slices and (E,F) quantitative analysis of immunoblotting of MMP-2 and MMP-9 in indicated groups (n = 6 per group). (G) Representative images and (H) quantitative analysis of immunofluorescence staining for aquaporin-4 (AQP4) (n = 3 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
FIGURE 8
FIGURE 8
Effect of QSYQ on leukocyte adhesion and leukocyte infiltration in tPA-treated stroke mice. (A) Representative images and (B) quantitative analysis of leukocyte adhesion (n = 6 per group). Data were compared by a two-way ANOVA followed by Tukey’s post hoc test. Red arrows indicate adherent leukocytes. (C) Representative images and (D) quantitative analysis of immunostaining for myeloperoxidase (MPO) (n = 3 per group). (E) Double immunofluorescence staining showing the colocalization of MMP-9 with leukocyte markers (MPO, CD18) and the specific macrophage marker (CD68), but not with endothelial cell marker (von Willebrand factor, vWF) in the thrombus + tPA group (n = 3 per group). *p < 0.05 vs sham; # p < 0.05 vs thrombus; & p < 0.05 vs thrombus + tPA.
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