Trifluoperazine regulates blood-brain barrier permeability via the MLCK/p-MLC pathway to promote ischemic stroke recovery

doi:10.1016/j.isci.2024.109156

. 2024 Feb 13;27(3):109156.

doi: 10.1016/j.isci.2024.109156. eCollection 2024 Mar 15.

Trifluoperazine regulates blood-brain barrier permeability via the MLCK/p-MLC pathway to promote ischemic stroke recovery

Wentao Zhang¹, Sisi Chen¹, Bin Ma¹, Yingmei Ding¹, Xiaofen Liu¹, Caijun He¹, Biao Wang¹, Mei Yuan^{1

2}

Affiliations

¹ The Second Affiliated Hospital, Department of Neurology, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China.
² Affiliated Nanhua Hospital, Department of Neurology, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China.

PMID: 38439960
PMCID: PMC10910233
DOI: 10.1016/j.isci.2024.109156

Trifluoperazine regulates blood-brain barrier permeability via the MLCK/p-MLC pathway to promote ischemic stroke recovery

Wentao Zhang et al. iScience. 2024.

. 2024 Feb 13;27(3):109156.

doi: 10.1016/j.isci.2024.109156. eCollection 2024 Mar 15.

Authors

Wentao Zhang¹, Sisi Chen¹, Bin Ma¹, Yingmei Ding¹, Xiaofen Liu¹, Caijun He¹, Biao Wang¹, Mei Yuan^{1

2}

Affiliations

¹ The Second Affiliated Hospital, Department of Neurology, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China.
² Affiliated Nanhua Hospital, Department of Neurology, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China.

PMID: 38439960
PMCID: PMC10910233
DOI: 10.1016/j.isci.2024.109156

Abstract

Blood-brain barrier (BBB) disruption following ischemic stroke (IS) can induce significant aftereffects. Elevated calmodulin (CaM) expression following stroke causes calcium overload-a key contributor to BBB collapse. Trifluoperazine (TFP), a CaM inhibitor, reduces CaM overexpression following IS. However, it remains unclear whether TFP participates in BBB repair after IS. We administered TFP to mice subjected to middle cerebral artery occlusion (MCAO) and bEnd.3 cells subjected to oxygen-glucose deprivation (OGD). TFP treatment in MCAO mice reduced cerebral CaM expression and infarct size and decreased BBB permeability. OGD-treated bEnd.3 cells showed significantly increased CaM protein levels and reduced tight junction (TJ) protein levels; these changes were reversed by TFP treatment. Our results found that TFP administration in mice inhibited actin contraction following cerebral ischemia-reperfusion by suppressing the MLCK/p-MLC pathway, thereby attenuating cell retraction, improving TJ protein integrity, and reducing BBB permeability. Consequently, this treatment may promote neurological function recovery after IS.

Keywords: Molecular biology; Physiology.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

**Figure 1**
BBB permeability and CaM expression are both increased after MCAO in mice (A) Representative mean fluorescent intensity of brain sections stained with TTC one day after MCAO. (B) Infarct volume (n = 6). (C) mNSS score (n = 6). (D) Representative immunofluorescence pattern of ischemic brain samples from EB and FITC-dextran leakage tests taken before and after I/R in WT mice (scale bar, 50 μm). (E and F) Quantitative analysis of the radiant efficiency of EB and FITC-dextran in (D) (n = 3 per group). (G) Representative immunofluorescence images of ZO-1 or claudin-5 (red), CD31 (green), and DAPI (blue) staining of ischemic and normal brain areas in WT mice after I/R (scale bars, 50 μm). (H and I) Quantitative analysis of CD31⁺ZO-1⁺ and CD31⁺claudin-5⁺ cells as a percentage of CD31⁺ cells (n = 6 per group). (J) Western blotting analysis of the levels of the TJ-related proteins ZO-1, occludin, and claudin-5 in WT mice before and after I/R. (K) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (J) (n = 3 per group). (L) Western blotting analysis of CaM protein expression levels in WT mice before and after I/R. (M) Quantitative analysis of the expression levels of CaM in (L) (n = 3 per group). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

**Figure 2**
EC permeability and CaM expression are both increased after OGD in bEnd.3 cells (A and B) FITC-dextran transport assay showing the permeability of bEnd.3 cells before and after OGD (n = 3 per group). (C) Representative immunofluorescence images of TJ proteins (claudin-5, occludin, and ZO-1) in bEnd.3 cells after OGD (scale bar, 20 μm). (D–F) Quantitative evaluation of the fluorescence intensity of claudin-5, occludin, and ZO-1 in (C) (n = 6 per group). (G) Western blotting analysis of the levels of the TJ proteins ZO-1, occludin, and claudin-5 in WT mice after OGD. (H) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (F) (n = 3 per group). (I) Western blotting analysis of the CaM protein expression levels in bEnd.3 cells before and after OGD. (J) Quantitative analysis of the expression levels of CaM in (I). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

**Figure 3**
TFP reduces BBB disruption and improves stroke outcomes after IS (A) Western blotting analysis of CaM protein expression levels in WT mice after I/R followed by treatment with TFP at different concentrations (2, 5, 10, and 20 mg/kg). (B) Quantitative analysis of the expression levels of CaM in (A) (n = 3 per group). (C) Representative brain sections stained with TTC. (D) Infarct volume (n = 6). (E) mNSS score (n = 6). (F) Representative immunofluorescence images from EB-leakage tests taken before and after I/R followed by treatment with TFP in WT mice (scale bar, 50 mm). (G and H) Quantitative analysis of the radiant efficiency of EB in (F) (n = 3 per group). (I) Representative immunofluorescence images of ZO-1 or claudin-5 (red), CD31 (green), and DAPI (blue) staining of ischemic and normal brain areas in WT mice after I/R (scale bars, 50 μm). (J and K) Quantitative analysis of CD31⁺ZO-1⁺ and CD31⁺claudin-5⁺ cells as a percentage of CD31⁺ cells in (I) (n = 6 per group). (L–N) RT-qPCR verification of the mRNA levels of claudin-5, occludin, and ZO-1 in WT mice after I/R followed by treatment with TFP (n = 3 per group). (O) Western blotting analysis of ZO-1, occludin, and claudin-5 in WT mice after I/R followed by TFP treatment. (P) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (O) (n = 3 per group). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

**Figure 4**
TFP represses CaM expression levels and decreases BBB breakdown after OGD (A and B) FITC-dextran transport assay showing the permeability of bEnd.3 cells treated with TFP before and after OGD (n = 3 per group). (C) Western blotting analysis of CaM protein expression levels in bEnd.3 cells treated with TFP after OGD. (D) Quantitative analysis of the expression levels of CaM in (C) (n = 3 per group). (E) Representative immunofluorescence images of TJ proteins (claudin-5, occludin, and ZO-1) in bEnd.3 cells subjected to OGD (scale bar, 20 μm). (F–H) Quantitative evaluation of the fluorescence intensity of claudin-5, occludin, and ZO-1 in (E) (n = 6 per group). (I–K) RT-qPCR verification of the mRNA levels of claudin-5, occludin, and ZO-1 in bEnd.3 cells after OGD followed by treatment with TFP (n = 3 per group). (L) Western blotting analysis of ZO-1, occludin, and claudin-5 in bEnd.3 cells treated with TFP after OGD. (M) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (L) (n = 3 per group). Data are represented as mean ± SEM. ns, p > 0.05, ∗∗p < 0.01.

**Figure 5**
MLCK plays a key role in the regulation of BBB permeability after MCAO (A) Representative immunofluorescence images of MLCK (red), CD31 (green), and DAPI (blue) staining of the mouse brain after MCAO followed by treatment with TFP (scale bars, 50 μm). (B) Quantitative analysis of CD31⁺MLCK⁺ cells as a percentage of CD31⁺ cells in (A) (n = 6 per group). (C) Representative immunofluorescence images of MLCK (green), CD31 (red), and DAPI (blue) staining of bEnd.3 cells after OGD (scale bars, 20 μm). (D) Quantitative analysis of the fluorescence intensity of MLCK in (C) (n = 6 per group). (E) Brain tissue lysates from the indicated MCAO mice were immunoprecipitated with CaM antibody. The pellets were subjected to western blotting with antibodies against MLCK. Data are represented as mean ± SEM. ns, p > 0.05, ∗p < 0.05, ∗∗p < 0.01.

**Figure 6**
MLCK overexpression reverses the TFP-mediated reduction in the permeability of bEnd.3 cells (A and B) FITC-dextran transport assay showing the permeability of bEnd.3 cells in the OGD, OGD+TFP, OGD+TFP+LV CON, and OGD+TFP+LV MLCK-OE groups before and after OGD (n = 3 per group). (C) Representative immunofluorescence images of TJ proteins (claudin-5, occludin, and ZO-1) in bEnd.3 cells in the OGD, OGD+TFP, OGD+TFP+LV CON, and OGD+TFP+LV MLCK-OE groups after exposure to OGD (scale bar, 20 μm). (D–F) Quantitative evaluation of the fluorescence intensity of claudin-5, occludin, and ZO-1 in (C) (n = 6 per group). (G) Western blotting analysis of ZO-1, occludin, and claudin-5 in bEnd.3 cells in the OGD, OGD+TFP, OGD+TFP+LV CON, and OGD+TFP+LV MLCK-OE groups. (H) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (G) (n = 3 per group). Data are represented as mean ± SEM. ns, p > 0.05, ∗∗p < 0.01.

**Figure 7**
Overexpression of MLCK reversed the TFP-mediated reduction in BBB permeability in MCAO mice (A) Representative immunofluorescence images of ischemic brain samples from the EB and FITC-dextran tests of mice in the MCAO, MCAO+TFP, MCAO+TFP+LV CON, and MCAO+TFP+LV MLCK-OE groups (scale bar, 50 μm). (B and C) Quantitative analysis of the fluorescence intensity of EB and FITC-dextran in (A) (n = 3 per group). (D) Representative immunofluorescence images of ZO-1 or claudin-5 (red), CD31 (green), and DAPI (blue) staining of the ischemic brain tissues of mice after I/R in the MCAO, MCAO+TFP, MCAO+TFP+LV CON, and MCAO+TFP+LV MLCK-OE groups (scale bars, 50 μm). (E and F) Quantitative analysis of CD31⁺ZO-1⁺ and CD31⁺claudin-5⁺ cells as a percentage of CD31⁺ cells in (D) (n = 6 per group). (G) Western blotting analysis of the levels of the TJ-related proteins ZO-1, occludin, and claudin-5 in mice after I/R in the MCAO, MCAO+TFP, MCAO+TFP+LV CON, and MCAO+TFP+LV MLCK-OE groups. (H) Quantitative analysis of the expression levels of ZO-1, occludin, and claudin-5 in (G) (n = 3 per group). (I) mNSS score (n = 6 per group). Data are represented as mean ± SEM. ns, p > 0.05, ∗p < 0.05, ∗∗p < 0.01.

**Figure 8**
TFP regulates BBB permeability via the MLCK/p-MLC signaling pathway and contributes to neurological recovery (A) Western blotting analysis of MLCK, p-MLC, and MLC in mice after I/R in the MCAO, MCAO+TFP, MCAO+TFP+LV CON, and MCAO+TFP+LV MLCK-OE groups. (B) Quantitative analysis of the expression levels of MLCK and p-MLC in (A) (n = 3 per group). (C) Representative immunofluorescence images of p-MLC (red), CD31 (green), and DAPI (blue) staining of mouse brain tissues after I/R in the sham, MCAO, MCAO+TFP, MCAO+TFP+LV CON, and MCAO+TFP+LV MLCK-OE groups (scale bars, 50 μm). (D) Quantitative analysis of CD31⁺p-MLC⁺ cells as a percentage of CD31⁺ cells in (D) (n = 6 per group). Data are represented as mean ± SEM. ns, p > 0.05, ∗p < 0.05, ∗∗p < 0.01.

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References

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1. van den Berg L.A., Berkhemer O.A., Fransen P.S.S., Beumer D., Lingsma H., Majoie C.B.M., Dippel D.W.J., van der Lugt A., van Oostenbrugge R.J., van Zwam W.H., et al. Economic Evaluation of Endovascular Treatment for Acute Ischemic Stroke. Stroke. 2022;53:968–975. doi: 10.1161/strokeaha.121.034599. - DOI - PubMed
1. Suzuki K., Matsumaru Y., Takeuchi M., Morimoto M., Kanazawa R., Takayama Y., Kamiya Y., Shigeta K., Okubo S., Hayakawa M., et al. Effect of Mechanical Thrombectomy Without vs With Intravenous Thrombolysis on Functional Outcome Among Patients With Acute Ischemic Stroke: The SKIP Randomized Clinical Trial. JAMA. 2021;325:244–253. doi: 10.1001/jama.2020.23522. - DOI - PMC - PubMed

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[1] GBD 2019 Stroke Collaborators Global, regional, and national burden of stroke and its risk factors, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol. 2021;20:795–820. doi: 10.1016/s1474-4422(21)00252-0. - DOI - PMC - PubMed

[2] GBD 2019 Stroke Collaborators Global, regional, and national burden of stroke and its risk factors, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Neurol. 2021;20:795–820. doi: 10.1016/s1474-4422(21)00252-0. - DOI - PMC - PubMed

[3] Roger V.L., Go A.S., Lloyd-Jones D.M., Adams R.J., Berry J.D., Brown T.M., Carnethon M.R., Dai S., de Simone G., Ford E.S., et al. Heart disease and stroke statistics--2011 update: a report from the American Heart Association. Circulation. 2011;123:e18–e209. doi: 10.1161/CIR.0b013e3182009701. - DOI - PMC - PubMed

[4] Roger V.L., Go A.S., Lloyd-Jones D.M., Adams R.J., Berry J.D., Brown T.M., Carnethon M.R., Dai S., de Simone G., Ford E.S., et al. Heart disease and stroke statistics--2011 update: a report from the American Heart Association. Circulation. 2011;123:e18–e209. doi: 10.1161/CIR.0b013e3182009701. - DOI - PMC - PubMed

[5] Cerasuolo J.O., Mandzia J., Cipriano L.E., Kapral M.K., Fang J., Hachinski V., Sposato L.A. Intravenous Thrombolysis After First-Ever Ischemic Stroke and Reduced Incident Dementia Rate. Stroke. 2022;53:1170–1177. doi: 10.1161/strokeaha.121.034969. - DOI - PubMed

[6] Cerasuolo J.O., Mandzia J., Cipriano L.E., Kapral M.K., Fang J., Hachinski V., Sposato L.A. Intravenous Thrombolysis After First-Ever Ischemic Stroke and Reduced Incident Dementia Rate. Stroke. 2022;53:1170–1177. doi: 10.1161/strokeaha.121.034969. - DOI - PubMed

[7] van den Berg L.A., Berkhemer O.A., Fransen P.S.S., Beumer D., Lingsma H., Majoie C.B.M., Dippel D.W.J., van der Lugt A., van Oostenbrugge R.J., van Zwam W.H., et al. Economic Evaluation of Endovascular Treatment for Acute Ischemic Stroke. Stroke. 2022;53:968–975. doi: 10.1161/strokeaha.121.034599. - DOI - PubMed

[8] van den Berg L.A., Berkhemer O.A., Fransen P.S.S., Beumer D., Lingsma H., Majoie C.B.M., Dippel D.W.J., van der Lugt A., van Oostenbrugge R.J., van Zwam W.H., et al. Economic Evaluation of Endovascular Treatment for Acute Ischemic Stroke. Stroke. 2022;53:968–975. doi: 10.1161/strokeaha.121.034599. - DOI - PubMed

[9] Suzuki K., Matsumaru Y., Takeuchi M., Morimoto M., Kanazawa R., Takayama Y., Kamiya Y., Shigeta K., Okubo S., Hayakawa M., et al. Effect of Mechanical Thrombectomy Without vs With Intravenous Thrombolysis on Functional Outcome Among Patients With Acute Ischemic Stroke: The SKIP Randomized Clinical Trial. JAMA. 2021;325:244–253. doi: 10.1001/jama.2020.23522. - DOI - PMC - PubMed

[10] Suzuki K., Matsumaru Y., Takeuchi M., Morimoto M., Kanazawa R., Takayama Y., Kamiya Y., Shigeta K., Okubo S., Hayakawa M., et al. Effect of Mechanical Thrombectomy Without vs With Intravenous Thrombolysis on Functional Outcome Among Patients With Acute Ischemic Stroke: The SKIP Randomized Clinical Trial. JAMA. 2021;325:244–253. doi: 10.1001/jama.2020.23522. - DOI - PMC - PubMed

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Trifluoperazine regulates blood-brain barrier permeability via the MLCK/p-MLC pathway to promote ischemic stroke recovery

Affiliations

Trifluoperazine regulates blood-brain barrier permeability via the MLCK/p-MLC pathway to promote ischemic stroke recovery

Authors

Affiliations

Abstract

Conflict of interest statement

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Abstract

Conflict of interest statement

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