Shengui Sansheng San alleviates the worsening of blood-brain barrier integrity resulted from delayed tPA administration through VIP/VIPR1 pathway

Abstract

Background: Intravenous tissue plasminogen activator (tPA) is currently the only FDA-approved thrombolytic therapy for acute ischemic stroke (AIS), however, relative narrow therapeutic time window (within 4.5 h of AIS onset) and high risk of hemorrhagic transformation due to blood-brain barrier (BBB) disruption limit tPA therapeutic benefits for patients. In this study, we extended the time window of tPA administration (5 h after the occurrence of AIS) and investigated whether Chinese medicine classical formula Shengui Sansheng San (SSS) administration was able to alleviate BBB integrity worsening, and the mechanism was related to vasoactive intestinal peptide (VIP)/ VIP receptor 1 (VIPR1) pathway.

Methods: SSS was extracted using aqueous heating method and SFE-CO₂ technology, and quality control was performed using UHPLC/MS analysis. Male C57BL/6 mice were suffered from middle cerebral artery occlusion (MCAo), followed by the removal of a silicone filament after 5 h, then, t-PA was administered via tail vein injection at once, along with SSS administration by gavage. Hemoglobin levels and Evans blue leakage were measured to assess brain hemorrhagic transformation and BBB permeability, respectively. Transmission electron microscope (TEM) was utilized to present brain microvascular endothelial cells (BMECs) tight junction morphology. TTC staining and laser speckle contrast imaging were employed for infarct volume and cerebral blood flow measurements. The modified neurological severity score (mNSS) test was conducted to evaluate neurological function. The expressions of VIP, VIPR1, ZO-1, Occludin, Lectin, GFAP, NeuN were detected by immunofluorescence staining or western blotting. In vitro, bEnd.3 and N2a cells were insulted by oxygen-glucose deprivation (OGD), and VIPR1 siRNA, and VIP shRNA transfection were respectively performed, and the molecular docking was applied to verify the SSS in-serum active compounds interacted with VIPR1. The transwell system was utilized to detect OGD-insulted BMECs permeability.

Results: SSS treatment significantly reduced the infarct area, cerebral hemorrhage, and neurological deficits, and enhanced cerebral blood flow in AIS mice received intravenous tPA beyond 4.5 h time window. Simultaneously, the permeability of BBB declined, with increased expressions of tight junction proteins ZO-1, and Occludin and proper BMECs tight junction morphology, and it suggested that VIP was released by neurons rather than astrocytes or BMECs. It also showed high expressions of VIP and VIPR1 in the penumbra area. The inhibition of VIP in N2a cells or VIPR1 in bEnd.3 cells abolished the viability and integrity of OGD-insulted bEnd.3 cells treated by tPA after SSS-containing serum administration, and the SSS in-serum active compounds were proved have high affinity to VIPR1 by molecular docking.

Conclusion: SSS alleviates the worsening of BBB integrity resulted from delayed tPA administration, reduces hemorrhagic transformation and infarction volume, and ameliorates brain blood flow and neurological function in AIS mice. The mechanisms are associated with the activation of VIP/VIPR1 pathway to enhance BMECs viability and maintain tight junction phenotype.

Keywords: Acute ischemic stroke; Blood brain barrier; Shengui Sansheng San; Tissue plasminogen activator; VIP/VIPR1.

Fig. 1

Quality control of Shengui Sansheng San. A, B Ultra-High performance liquid chromatography (UHPLC) chromatogram of SSS extraction. A-(1) Ginsenoside Rg1, A-(2) Ginsenoside Rb1, A-(3) ferulic acid, B-(1) cinnamaldehvde, B-(2) Ligustilide. C Chemical structure of Ginsenoside Rg1, Ginsenoside Rb1, ferulic acid, cinnamaldehvde, Ligustilide

Fig. 2

SSS treatment improved therapeutic efficacies in AIS mice after delay tPA administration. A Schematic diagram of experimental design. B Representative Images of TTC Staining. C Quantitative analysis for infarct volume (n = 6). D Representative LSCI images of the ipsilateral and contralateral hemispheres. E Quantitative analysis of CBF. F Modified neurological severity score (mNSS) evaluation. *p < 0.05 vs MCAo, ^#p < 0.05 vs tPA

Fig. 3

SSS treatment maintained the integrity of blood–brain barrier and reduced the risk of hemorrhagic transformation. A Representative image of Evans blue leakage. B Quantitative analysis of Evans blue leakage. C Quantitative assessment of hemoglobin levels. D The TEM images for BBB phenotype (The tight junctions indicated by the red arrows). Upper panel scale bar = 2 μM; lower panel scale bar = 0.5 μM. ^*p < 0.05 vs MCAo, ^#p < 0.05 vs tPA. T + S: (tPA + SSS)

Fig. 4

SSS treatment increased tight junction protein expression in AIS mice after delayed tPA administration. A Western blotting of Tight junction proteins ZO-1, VIPR1, Occludin. B–D Quantification analysis of expressions of ZO-1 (B), VIPR1 (C), Occludin (D). E, F Representative images of immunofluorescent staining of ZO-1 (E) and Occludin (F). G–H Quantification analysis of expressions of ZO-1 (G) and Occludin (H). scale bar = 25 µm. ^*p < 0.05 vs MCAo, ^#p < 0.05 vs tPA

Fig. 5

SSS treatment enhanced the expressions of VIP and VIPR1 after tPA administration. A Representative images of immunofluorescent staining of VIP. B Quantification analysis of expressions of VIP. C Representative images of immunofluorescent staining of VIPR1. D Quantification analysis of expressions of VIPR1. E–G Co-localization of VIP and Lectin (E), GFAP (F), NeuN (G). H Colocalization of VIP and primary neuron. I VIP quantified by Elisa assay. 20 × images scale bar = 100 µm; 40 × images scale bar = 50 µm; 63 × images scale bar = 25 µm. *p < 0.05 vs MCAo, ^#p < 0.05 vs tPA

Fig. 6

SSS containing serum improved OGD-insulted cerebral endothelial cells viability after tPA administration. A Toxicity evaluation of SSS containing serum on bEnd.3 cells under normal conditions by CCK8 assay. B The determination for optimal dose of SSS containing serum on OGD-insulted bEnd.3 cells. *p < 0.05 vs 0%. C Toxicity assessment of tPA on bEnd.3 cells under normal conditions. D The determination of toxic dose of tPA on OGD insulted bEnd.3 cells. *p < 0.05 vs 0 μg/mL. E The SSS containing serum protected the viability of OGD insulted bEnd.3 cells suffered from tPA. *p < 0.05 vs OGD; ^#p < 0.05 vs tPA

Fig. 7

SSS treatment alleviated the worsening permeability of OGD insulted bEnd.3 cells barrier due to delayed tPA administration by VIP/VIPR1 pathway. A Schematic diagram of the co-culture of bEnd.3 cells and N2a. B tPA administration increased the permeability of OGD insulted bEnd.3 cells barrier. C SSS-containing serum treatment maintained the integrity of OGD insulted bEnd.3 cells barrier after tPA administration. D VIP knockdown in N2a cells verified by immunofluorescence. E VIPR1 knockdown in bEnd.3 cells verified by western blotting. F Quantification analysis of expressions of VIPR1 in bEnd.3 cells infected with VIPR1 siRNAs. G Knockdown of VIPR1 in bEnd.3 cells attenuated the protective effects of SSS-containing serum on OGD insulted bEnd.3 cells barrier injury resulted from tPA treatment. H Knockdown of VIP in N2a cells attenuated the protective effects of SSS-containing serum on OGD insulted bEnd.3 cells barrier injury resulted from tPA treatment. 40 × images scale bar = 50 µm *p < 0.05 vs MCAo

Fig. 8

The cytotoxicity assessment of active components from SSS for normal and OGD insulted bEnd.3and N2a cells. A–C 3D-structural complex of VIPR1 bound with 4-Hydroxycinnamic acid (A), Ginsenoside rg3 (B), ferulic acid (C). D–F 2D-binding mode of VIPR1 bound with 4-Hydroxycinnamic acid (D), Ginsenoside rg3 (E), ferulic acid (F). G–I Protective effect of 4-Hydroxycinnamic acid (G), Ginsenoside rg3 (H), ferulic acid (I) on OGD insulted bEnd.3. J–L Protective effects of 4-Hydroxycinnamic acid (J), Ginsenoside rg3 (K), ferulic acid (L) on OGD insulted N2a. *p < 0.05 vs 0 μM