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Xuesaitong Combined with Dexmedetomidine Improves Cerebral Ischemia-Reperfusion Injury in Rats by Activating Keap1/Nrf2 Signaling and Mitophagy in Hippocampal Tissue - PubMed

  • ️Sat Jan 01 2022

Xuesaitong Combined with Dexmedetomidine Improves Cerebral Ischemia-Reperfusion Injury in Rats by Activating Keap1/Nrf2 Signaling and Mitophagy in Hippocampal Tissue

Guo-Jie Han et al. Oxid Med Cell Longev. 2022.

Abstract

Ischemic stroke is the most common type of cerebrovascular disease with high mortality and poor prognosis, and cerebral ischemia-reperfusion (CI/R) injury is the main murderer. Here, we attempted to explore the effects and mechanism of Xuesaitong (XST) combined with dexmedetomidine (Dex) on CI/R injury in rats. First, a rat model of CI/R injury was constructed via the middle cerebral artery occlusion (MCAO) method and treated with XST and Dex alone or in combination. Then, on the 5th and 10th days of treatment, the neurological impairment was assessed using the modified neurological severity scores (mNSS), the 8-arm radial maze test (8ARMT), novel object recognition test (NORT), and fear conditioning test (FCT). H&E staining was performed to observe the pathological changes of the hippocampus. ELISA and related kits were used to assess the monoamine neurotransmitters and antioxidant enzyme activities in the hippocampus. The ATP, mitochondrial membrane potential levels, and qRT-PCR of genes related to mitochondrial function were determined to assess mitochondrial functions in the hippocampus and western blot to determine Keap1/Nrf2 signaling pathway and mitophagy-related protein expression. The results showed that XST combined with Dex significantly reduced mNSS, improved spatial memory and learning deficits, and enhanced fear memory and cognitive memory ability in CI/R rats, which was superior to single-drug treatment. Moreover, XST combined with Dex treatment improved hippocampal histopathological damage; significantly increased the levels of monoamine neurotransmitters, neurotrophic factors, ATP, and mitochondrial membrane potential; and upregulated the activities of antioxidant enzymes and the expression of mitophagy-related proteins in the hippocampus of CI/R rats. XST combined with Dex treatment also activated the Keap1/Nrf2 signaling and upregulated the protein expression of downstream antioxidant enzymes HO-1 and NQ. Altogether, this study showed that a combination of XST and Dex could activate the Keap1/Nrf2 signaling and mitophagy to protect rats from CI/R-related neurological impairment.

Copyright © 2022 Guo-Jie Han et al.

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Conflict of interest statement

All the authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Experimental design of this study. For the sham group rats, only their blood vessels were exposed, and no embolization coils were introduced. Rats in the XST, Dex, and XST+Dex groups were treated medically every day after MCAO surgery for 10 days. The mNSS of rats in each group were measured before surgery and on days 1, 2, 6, and 10 of treatment. On the 5th and 10th days of treatment, behavioral tests were performed in each group of rats, followed by the collection of hippocampal tissues. On the 10th day of treatment, the hippocampal tissues were collected from rats in each group after completing behavioral tests; then, hematoxylin and eosin (H&E) staining was performed to observe histopathological changes in these tissues.

Figure 2
Figure 2

The combination of XST and Dex improves neurological impairment in rats with cerebral CI/R. Changes in each group before surgery and on the 1st, 2nd, 6th, and 10th days of treatment were assessed by the modified neurological severity score (mNSS) system (n = 10 per group). ∗p < 0.05 vs. the sham group at the same time points, #p < 0.05 vs. the CI/R group at the same time points, &p < 0.05 vs. the XST+Dex group at the same time point, and $p < 0.05 vs. the same group of rats treated on the first day.

Figure 3
Figure 3

XST combined with Dex improves spatial learning and memory impairment and enhances cognitive memory and fear memory ability in CI/R rats. (a, b) Changes in working memory errors (WME), reference memory errors (RME), and total number of entries (TE) on the 5th (a) and 10th (b) days of treatment in each group of rats via 8-ARMT. (c, d) Fear conditioning test (FCT) to detect changes in the percentage of freezing time in context-related and tone-related tests in each group of rats on day 5 (c) and day 10 (d) after treatment. (e, f) Novel object recognition test (NORT) to check RI changes on the 5th (e) and 10th (f) days of treatment in each group of rats (n = 10 per group). ∗∗p < 0.01 vs. the sham; #p < 0.05, ##p < 0.01 vs. the CI/R; and &p < 0.05, &&p < 0.01 vs. the XST+Dex.

Figure 4
Figure 4

Combined treatment with XST and Dex alleviates pathological damage in the hippocampus of CI/R rats. Hematoxylin and eosin (H&E) staining was conducted to observe pathological changes of hippocampal tissue; 40x: scale bar = 100 μm; 100x: scale bar = 10 μm.

Figure 5
Figure 5

XST combined with Dex treatment increased the levels of monoamine neurotransmitters and neurotrophic factors in the hippocampal tissues of CI/R rats. (a–c) ELISA to detect changes in DA (a), 5-HT (b), and NE (c) levels in the hippocampus of rats on the 5th and 10th days of treatment. (d–f) ELISA to detect the changes in BDNF (d), TrkB (e), and NT-3 (f) levels in the hippocampus of rats after 10 days of treatment (n = 10 per group). ∗∗p < 0.01 vs. the sham; #p < 0.05, ##p < 0.01 vs. the CI/R; and &p < 0.05, &&p < 0.01 vs. the XST+Dex. DA: dopamine; 5-HT: 5-hydroxytryptamine; NE: norepinephrine; BDNF: brain-derived neurotrophic factor.

Figure 6
Figure 6

Improvements from XST combined with Dex treatment on oxidative stress on the hippocampal tissues of CI/R rats. (a–c) The levels of SOD (a), CAT (b), and GPx (c) in the hippocampus of rats in each group were measured by related kits on the 5th and 10th days of treatment (n = 10 per group). ∗∗p < 0.01 vs. the sham; #p < 0.05, ##p < 0.01 vs. the CI/R; and &&p < 0.01 vs. the XST+Dex. SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase.

Figure 7
Figure 7

Improvements from XST combined with Dex treatment on mitochondrial dysfunction in the hippocampal tissues of CI/R rats. (a) Adenosine 5′-triphosphate (ATP) levels in the hippocampus of rats in each group were detected by related kits. (b) Mitochondrial membrane potential in the hippocampus of rats was detected using the JC-I kit. (c, d) qRT-PCR was performed to detect the mRNA expression levels of TFAM, ATP6, Drp1, and Mfn1 in the hippocampal tissues of rats in each group (n = 10 per group). ∗∗p < 0.01 vs. the sham; ##p < 0.01 vs. the CI/R; and &&p < 0.01 vs. the XST+Dex.

Figure 8
Figure 8

Combined treatment with XST and Dex activates the Keap1/Nrf2 signaling pathway in rats' hippocampal tissues. (a–d) Western blot to determine Keap1, Nrf2, HO-1, and NQO1 protein expression levels in the hippocampus of rats in each group on the 5th (a, b) and 10th (c, d) days of treatment (n = 3 per group). ∗∗p < 0.01 vs. the sham; ##p < 0.01 vs. the CI/R; and &&p < 0.01 vs. the XST+Dex.

Figure 9
Figure 9

Combined treatment with XST and Dex activates mitophagy in the hippocampal tissues of CI/R rats. (a–d) Western blot to assess the protein expression levels of PINK1, Parkin, LC3-I, LC3-II, and p62 in the hippocampus of rats in each group on the 5th (a, b) and 10th (c, d) days of treatment (n = 3 per group). ∗∗p < 0.01 vs. the sham; ##p < 0.01 vs. the CI/R; and &&p <0.01 vs. the XST+Dex.

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