Sevoflurane protects against cerebral ischemia/reperfusion injury via microrna-30c-5p modulating homeodomain-interacting protein kinase 1

Abstract

Sevoflurane (SEV) has been reported to be an effective neuroprotective agent for cerebral ischemia/reperfusion injury (CIRI). However, the precise molecular mechanisms of Sev preconditioning in CIRI remain largely unknown. Therefore, CIRI model was established via middle cerebral artery occlusion method. SEV was applied before modeling. after successful modeling, lentivirus was injected into the lateral ventricle of the brain. Neurological impairment score was performed in each group, and histopathologic condition, infarct volume, apoptosis, inflammation, oxidative stress, microRNA (miR)-30 c-5p and homeodomain-interacting protein kinase 1 (HIPK1) were detected. Mouse hippocampal neuronal cell line HT22 cells were pretreated with SEV, and the in vitro model was stimulated via oxygen-glucose deprivation and reoxygenation. The corresponding plasmids were transfected, and the cell growth was detected, including inflammation and oxidative stress, etc. The targeting of miR-30 c-5p with HIPK1 was examined. The results clarified that reduced miR-30 c-5p and elevated HIPK1 were manifested in CIRI. SEV could improve CIRI and modulate the miR-30 c-5p-HIPK1 axis in vitro and in vivo, and miR-30 c-5p could target HIPK1. Depressed miR-30 c-5p could eliminate the protection of SEV in vitro and in vivo. Repression of HIPK1 reversed the effect of reduced miR-30 c-5p on CIRI. Therefore, it is concluded that SEV is available to depress CIRI via targeting HIPK1 through upregulated miR-30 c-5p.

Keywords: Cerebral ischemia-reperfusion injury; Homeodomain-interacting protein kinase 1; MicroRNA-30c-5p; Sevoflurane.

Figure 1.

Amelioration of CIRI via Sev in vivo. (a) Neurological impairment score; (b-d) Inflammation factors TNF-α, IL-1β and IL-6 in rat brain tissue detected via ELISA; (e-g) the content of oxidative stress indexes MDA, SOD, GSH-Px in rat brain tissue detected via ELISA; (h) He staining to observe the pathological damage of brain tissue; (i) TTC staining images to evaluate the cerebral infarction area in each group, and the quantified relative infarct rate to evaluate the effect of SEV on MCAO-induced brain injury; (j) TUNEL staining to detect the apoptosis of brain tissue. (a) n = 12; b-j, n = 6; The data were expressed as mean ± SD; * vs. the Sham, P < 0.05; # vs. the MCAO, P < 0.05

Figure 2.

SEV has a protective influence on Ogd/ R-induced HT22 cell damage. (a-c) TNF-α, IL-1β and IL-6 in HT22 cells detected by ELISA; (d-f) MDA, SOD, GSH-Px in HT22 cells detected via ELISA; (g) ROS detection in HT22 cells; (h) Cell viability detection via CCK-8; (i) Cell proliferation detected by plate cloning; (j) Transwell to detect cell migration and invasion; (k) Flow cytometry detection of apoptosis. n = 3; The data were expressed as mean ± SD; * vs. the Ctrl, P < 0.05; # vs. the Ogd/R, P < 0.05

Figure 3.

SEV controls miR-30 c-5p and HIPK1 in vitro and vivo models of CIRI. (a) Apparent decline of miR-30 c-5p in vivo model of CIRI, while restoration of miR-30 c-5p via SEV; (b-c) Clear elevation of HIPK1 in vivo model of CIRI, while silence of HIPK1 via SEV; (d) Obvious depression of miR-30 c-5p in vitro Ogd/R model, while elevation via Sev; (e-f) Up-regulation of HIPK1 in vitro Ogd/R model, while silence via SEV; (a-c) n = 6, (d-f) n = 3; The data were expressed as mean ± SD; * vs. the Sham, P < 0.05; # vs. the MCAO, P < 0.05; + vs. the Ctrl, P < 0.05; $ vs. the Ogd/R, P < 0.05

Figure 4.

Knockdown miR-30 c-5p can remove the protection of SEV in vivo. (a) After injection of miR-30 c-5p antagonist, the qPCR detection of miR-30 c-5p in rat brain tissue; (b) Neurological impairment score; (c-e) TNF-α, IL-1β and IL-6 in rat brain tissue detected by ELISA; (f-h) MDA, SOD and Gsh-Px in rat brain tissue detected by ELISA; (i) He staining to observe the pathological damage of brain tissue; (j) TTC staining images to evaluate the cerebral infarction area in each group of rats, and the quantified relative infarct rate to evaluate the effect of repressive miR-30 c-5p on MCAO-induced brain injury; (k) TUNEL staining to detect the apoptosis of brain tissue. (b) n = 12; (a) & (c-k), n = 6; The data were expressed as mean ± SD; * vs. the SEV + antagomir nc, P < 0.05

Figure 5.

Knockdown miR-30 c-5p can remove the protection of SEV in vitro. (a) After transfection with miR-30 c-5p inhibitor, the qPCR detection of miR-30 c-5p in HT22 cells; (b-d) TNF-α, IL-1β and IL-6 in HT22 cells detected by ELISA; (e-g) MDA, SOD and Gsh-Px in HT22 cells detected by ELISA; (h) ROS detection in HT22 cells; (i) Cell viability detection via CCK-8; (j) Cell proliferation detected by plate cloning; (k) Transwell to detect cell migration and invasion; (l) Flow cytometry detection of apoptosis. n = 3; The data were expressed as mean ± SD; * vs. the S + in-NC, P < 0.05

Figure 6.

miR-30 c-5p in vitro targets HIPK1 and negatively controls its expression. (a) Through bioinformatics analysis forecasted the binding site of miR-30 c-5p with HIPK1 (http://starbase.sysu.edu.cn/); (b) In HT22 cells transfected with miR-30 c-5p mimic, the combination of miR-30 c-5p and HIPK1 verified by luciferase reporter gene assay; (c) RT-qPCR detection of HIPK1 in cells introduced with in-miR-30 c-5p; (d) Western blot detection of HIPK1 in cells introduced with in-miR-30 c-5p; n = 3; The data were expressed as mean ± SD; * vs. the miR-Nc, P < 0.05; + vs. the S + in-NC, P < 0.05

Figure 7.

Repressive HIPK1 reverses the effect of reduced miR-30 c-5p on CIRI in vitro. (a) After transfection of si-HIPK1, the qPCR detection of HIPK1 in HT22 cells; (b-d) TNf-α, Il-1β and IL-6 in HT22 cells detected by ELISA; (e-g) MDA, SOD and GSH-Px in HT22 cells detected by ELISA; (h) ROS detection in HT22 cells; (i) Cell viability detection via CCK-8; (j) Cell proliferation detected by plate cloning; (k) Cell migration and invasion detection via Transwell; (l) Flow cytometry detection of apoptosis. n = 3; The data were expressed as mean ± SD; * vs. the S + in-miR-30 c-5p + si-NC, P < 0.05