Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways

Abstract

Renal ischemia-reperfusion (I/R) injury leads to irreversible brain damage with serious consequences. Activation of oxidative stress and release of inflammatory mediators are considered potential pathological mechanisms. Butylphthalide (NBP) has anti-inflammatory and antioxidant effects on I/R injuries. However, it is unclear whether NBP can effectively mitigate renal I/R secondary to brain injury as well as its mechanism, which are the aims of this study. Both renal I/R injury rats and oxygen and glucose deprivation cell models were established and pre-intervened NBP. The Morris water maze assay was used to detect behavior. Hippocampal histopathology and function were examined after renal I/R. Apoptosis and tube-forming capacity of brain microvascular endothelial cells (BMVECs) were tested. Immunohistochemistry and Western blot were used to measure protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathway and NOD-like receptor C2 (NOD2)/Mitogen-activated protein kinases (MAPK)/Nuclear factor kappa-B (NF-κB) pathway. NBP treatment attenuated renal I/R-induced brain tissue damage and learning and memory dysfunction. NBP treatment inhibited apoptosis and promoted blood-brain barrier restoration and microangiogenesis. Also, it decreased oxidative stress levels and pro-inflammatory factor expression in renal I/R rats. Furthermore, NBP enhanced BMVECs' viability and tube-forming capacity while inhibiting apoptosis and oxidative stress. Notably, the alleviating effects of NBP were attributed to Nrf2/HO-1 pathway activation and NOD2/MAPK/NF-κB inhibition. This study demonstrates that NBP maintains BBB function by activating the Nrf2/HO-1 pathway and inhibiting the NOD2/MAPK/NF-κB pathway to suppress inflammation and oxidative stress, thereby alleviating renal I/R-induced brain injury.

Keywords: NOD2/MAPK/NF-κB pathway; Nrf2/HO-1 pathway; Renal I/R; blood-brain barrier; brain injury; butylphthalide.

Graphical abstract

Figure 1.

NBP promotes recovery of learning memory capacity and suppresses levels of inflammation in rats with renal I/R. (A-D) the Morris water maze Behavioral assay was used to examine the learning memory capacity of rats (n = 6). rat swimming path (a), rat escape latency (B), activity time in platform quadrant (C), and times of crossing platform (D) were shown (n = 6). (E) ELISA was used to measure the levels of IL-18, IL-1β and TNF-α in serum (n = 6). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. NBP: Butylphthalide; I/R: Ischemia/reperfusion; IL-18: Interleukin 18; IL-1β: Interleukin 1β; TNF-α: Tumor necrosis factor-α.

Figure 2.

NBP alleviates kidney injury and reduces oxidative stress in rats with renal I/R. (A) HE staining was used to observe the histopathological changes in the rat kidney (magnification, ×400) (50 μm). (B) Serum levels of BUN and Scr were examined by kit. The expression levels of MDA and SOD in hippocampal tissue (C), renal tissue (D) and serum (E) (n = 6). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. HE: Hematoxylin-Eosin; BUN: Blood urea nitrogen; Scr: Serum creatinine; MDA: Malondialdehyde; SOD: Superoxide dismutase.

Figure 3.

NBP reduces hippocampal injury in rats with renal I/R. (A, B) The histopathological Images and scores of the rat brain were observed by HE staining (magnification, ×400) (50 μm). (C, D) Nissl staining was used to detect changes in the number of neurons in the CA1, CA3 and DG regions of the hippocampus (magnification, ×400) (50 μm) (n = 3). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. DG: Dentate gyrus.

Figure 4.

NBP reduces the level of apoptosis and microvessel density in the cerebral cortex of rats with renal I/R. (A-B) Tunel staining was performed to detect the effect of NBP pretreatment on the level of apoptosis in the cerebral cortex of rats with IR (magnification, ×200) (n = 3). (C-E) the expression levels of Caspase-3, Bcl-2, Bax and vWF in the brain tissues were determined by Immunohistochemistry (magnification, ×400) (50 μm) (n = 3). (F) Meanwhile, Western blot was used to detect the expression levels of Caspase-3, Bcl-2 and Bax in the brain tissues (n = 3). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. Bcl-2: B-cell lymphoma-2; Bax: Bcl-2 assaciated X protein; vWF: Von Willebrand Factor.

Figure 5.

NBP promotes blood-brain barrier repair and neovascularization in renal IR rats. (A) Immunofluorescence staining was used to examine the effect of NBP pretreatment on the expression levels of claudin-5 and ZO-1 of the brain of I/R rats (magnification, ×200). the expression levels of Claudin-5, ZO-1 (B, C) and VEGF, VEGFR2 (D, E) in the rat brain were measured by Western blot (n = 3). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. ZO-1: Zonula occluden-1; VEGF: Vascular endothelial growth factor; VEGFR2: VEGF receptor-2.

Figure 6.

NBP activates Nrf2/HO-1 and inhibits NOD2/MAPK/NF-κB signaling pathway in the brain tissue of renal IR rats. (A-D) Western blot was used to detect the protein expression levels of Nrf2, HO-1, NLRP3, Caspase-1, NOD2, p-ERK1/2, p-JNK, p-p38, p-P65 and p-IkBa in the rat brain (n = 3). Data were presented as the mean ± standard deviation, ^▲p < 0.05, ^▲▲p < 0.01.vs. sham group; ^↔p < 0.05, ^↔↔p < 0.01.vs. I/R group. Nrf2: NF-E2-related factor 2; HO-1: Heme oxygenase-1.

Figure 7.

NBP enhances OGD-BMVECs viability and inhibits apoptosis. (A) MTT assay was carried out to detect the effect of NBP on the cell viability of BMVECs (n = 5). (B, C) Flow cytometry was used to examine the effect of different concentrations of NBP on OGD-BMVECs apoptosis (n = 3). (D, E) the expression levels of Caspase-3, Bcl-2, and Bax in the BMVECs were determined by Western blot (n = 3). ^▲p < 0.05, ^▲▲p < 0.01.vs. OGD group; ^↔p < 0.05, ^↔↔p < 0.01.vs. 10 μM NBP + OGD group; ^#p < 0.05, ^##p < 0.01.vs. 10 μM NBP + MDP + OGD group. OGD: Oxygen and glucose deprivation; BMVECs: Brain microvascular endothelial cells; MDP: Muramyl dipeptide.

Figure 8.

NBP Protects BMVECs by activating the Nrf2/HO-1 and inhibiting NOD2/MAPK/NF-κB signaling pathway. (A-D) Western blot was used to detect the protein expression levels of Nrf2, HO-1, NLRP3, Caspase-1, NOD2, p-ERK1/2, p-JNK, p-p38, p-P65 and p-IkBα in the total BMVEC protein (n = 3). ^▲p < 0.05, ^▲▲p < 0.01.vs. OGD group; ^↔p < 0.05, ^↔↔p < 0.01.vs. 10 μM NBP + OGD group; ^#p < 0.05, ^##p < 0.01.vs. 10 μM NBP + MDP + OGD group.

Figure 9.

NBP promotes tube-forming capacity and suppresses oxidative stress and inflammation levels in the BMVECs. (A, B) Effect of NBP on BMVECs tube-forming capacity was assessed and the number of tubes was counted (n = 3). (C) Western blot was used to examine the effect of NBP pretreatment on the expression levels of VEGF, claudin-5 and ZO-1 in OGD-BMVECs (n = 3). (D) In addition, the effect of NBP on the MDA, SOD, IL-18 and IL-1β in the supernatant of BMVECs was examined (n = 6). ^▲p < 0.05, ^▲▲p < 0.01.vs. OGD group; ^↔p < 0.05, ^↔↔p < 0.01.vs. 10 μM NBP + OGD group; ^#p < 0.05, ^##p < 0.01.vs. 10 μM NBP + MDP + OGD group.