Nrf2 activator peptide protects the brain from cerebral vascular dysfunction in alcohol ingestion
Bibhuti Ballav Saikia, Saleena Alikunju, Yemin A. Poovanthodi, Zayan Kassim, P.M. Abdul Muneer
Bibhuti Ballav Saikia, Saleena Alikunju, Yemin A. Poovanthodi, Zayan Kassim, P.M. Abdul Muneer
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Research Article Neuroscience Vascular biology

Nrf2 activator peptide protects the brain from cerebral vascular dysfunction in alcohol ingestion

Abstract

Oxidative signaling is a central mechanism in alcohol-induced injury and has strong implications for blood-brain barrier (BBB) dysregulation and neuroinflammation. Here, by targeting oxidative signaling, we hypothesized an innovative approach to develop a clinically relevant therapeutic strategy for alleviating alcohol-mediated neurovascular damage. To accomplish this, we enhanced the endogenous activity of nuclear factor E2–related factor 2 (Nrf2) by treatment with a Nrf2 activator III TAT peptide (Nrf2 peptide [NP]) and investigated the neuroprotective role of Nrf2 in promoting antioxidant defense properties and reducing BBB damage and transmigration of leukocytes to the brain following alcohol ingestion. We administered the NP subcutaneously to alcohol-ingested mice and evaluated its therapeutic potential in alleviating alcohol-associated neurovascular impairments. We compared the results with those seen in animals treated with control peptide (random sequence with TAT). The studies showed that the NP treatment preserved the oxidant-antioxidant balance, downregulated ICAM-1 and its receptors, and mitigated BBB damage and leukocyte infiltration into the brain. We validated the effect of the NP in Nrf2-knockout (Nrf2−/−) mice. Thus, this study demonstrates that NP exerts neurovascular protective effects by regulating the oxidant-antioxidant balance, reducing oxidative stress–induced BBB disruption, and limiting transmigration of immune cells to the brain in a mouse model of alcohol ingestion.

Authors

Bibhuti Ballav Saikia, Saleena Alikunju, Yemin A. Poovanthodi, Zayan Kassim, P.M. Abdul Muneer

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Figure 2

Nrf2 peptide improves expression of antioxidant proteins in alcohol ingestion.

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Nrf2 peptide improves expression of antioxidant proteins in alcohol inge...
(A–C) Western blot analysis of Nrf2, p-Nrf2, and β-actin from mouse cortex tissue lysate of CD- and ED-fed animals in WT and Nrf2-knockout animals (Nrf2–/–) with CP or NP treatment. Bar graphs (B and C) with dot plot show ratio densitometry of Nrf2 and p-Nrf2 to β-actin (n = 8 per group). (D) mRNA expression level of p-Nrf2 using qPCR from the cortex of CD-fed animals with CP or NP and ED-fed animals with CP or NP (n = 8 per group). (E) Western blot analysis of Keap1 and β-actin from mouse cortex tissue lysate of CD- and ED-fed animals in WT and Nrf2-knockout animals with CP or NP treatment. Bar graph with dot plot shows ratio densitometry of Keap1 to β-actin (n = 8 per group). (F and G) Immunofluorescence staining of the antioxidant protein HO-1 colocalized with GLUT1 (microvessel marker) and DAPI (nucleus) in intact brain microvessels of mice fed with CD or ED with CP or NP treatments. Yellow arrows show expression of HO-1. Scale bars: white, 50 μm; yellow, 10 μm. (G) Quantification of HO-1 staining was analyzed using ImageJ software (n = 10 per group). (H–K) Western blot analysis of antioxidant proteins HO-1, GPx1, and GSTm1 and β-actin from mouse frontal cortex tissue lysate of CD- and ED-fed animals in WT and Nrf2-knockout animals with CP or NP treatment. Bar graphs with dot plot show ratio densitometry of HO-1 (I), GPx1 (J), and GSTm1 (K) to β-actin (n = 12 per group). All values are expressed as mean ± SD. Statistical analysis was performed by ANOVA (2-way for D and G, 3-way for B, C, E, I, and J) followed by Bonferroni’s post hoc test. P < 0.05 was considered statistically significant. @P < 0.05, @@P < 0.01, @@P < 0.001 vs. representative groups in WT animals. Exact P values are shown between the compared groups.