Neuroprotective effects of nobiletin on cerebral ischemia/reperfusion injury rats by inhibiting Rho/ROCK signaling pathway

Abstract

Background: Nobiletin (NOB), an active natural flavonoid component of citrus, is used in Traditional Chinese Medicine for its anti-inflammatory activity, but its efficacy in cerebral ischemia/reperfusion (I/R) injury remains unclear. Methods: In a middle cerebral artery occlusion (MCAO) rat model, MCAO rats were administered (Sham group and MCAO model group treated with an equal volume of solvent, NOB group treated with 10 or 20 mg/kg NOB) once a day for 7 days before cerebral ischemia and again after reperfusion, 2,3,5-triphenyltetrazolium chloride (TTC) staining was applied to assess the infarct area. Neurological function was evaluated by the modified neurological severity score and Morris water maze. The levels of inflammatory factors, interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), were examined by enzyme-linked immunosorbent assay (ELISA). Histopathological staining evaluated neuron apoptosis in brain tissue. In an oxygen-glucose deprivation PC12 cell (OGD PC12) model, the proliferation, migration and apoptosis of OGD PC12 cells were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and cell migration assays and flow cytometry. The gene and protein expression levels of Ras homolog gene family, member A (Rho A), ras-related C3 botulinum toxin substrate 1 (Rac 1), Rho-associated kinase 1 (ROCK 1), ROCK 2 in the Rho/ROCK pathway were measured by Realtime PCR (RT-PCR), immunohistochemistry and western blot.Results: In rats with cerebral I/R injury, NOB significantly decreased the infarcted area, neuron apoptosis in brain tissue and expressions of IL-6, IL-1β, and TNF-α. It also improved neurological deficits in brain tissue and enhanced learning and memory ability. Further, NOB had a protective effect on OGD PC12 cells, increasing proliferation and migration and decreasing apoptosis. The expressions of Rho A, Rac 1, ROCK 1 and ROCK 2 were high in cerebral I/R injury rats, but were downregulated by NOB in I/R injury rats' brain tissue and OGD PC12 cells.Conclusions: Nobiletin had a neuroprotective effect in rats with cerebral I/R injury, and its potential mechanism is decreasing neuron apoptosis by inhibiting the Rho/ROCK signaling pathway. These results suggest NOB is a promising neuroprotective agent for patients with cerebral ischemia.

Demethylnobiletin ameliorates cerebral ischemia‐reperfusion injury in rats through Nrf2/HO‐1 signaling pathway

“…Similarly, 24 h after reperfusion, neurological deficits in rats from four aspects, including motion, sensation, balance, and reflexes were evaluated using modified neurological severity score (mNSS). The score details were as previously reported 18 . Of the total 18 scores, there is a positive correlation between the severity of neurological impairment and the scores 19 …”

“…MCAO modeling in the present study was performed according to the protocol described earlier by our team 18 . In brief, 3% pentobarbital sodium (30 mg/kg, Sigma‐Aldrich, CA, US) was injected intraperitoneally to anesthetize the animals.…”

“…The score details were as previously reported. 18 Of the total 18 scores, there is a positive correlation between the severity of neurological impairment and the scores. 19…”

“…MCAO modeling in the present study was performed according to the protocol described earlier by our team. 18 In brief, 3% pentobarbital sodium (30 mg/kg, Sigma-Aldrich, CA, US) was injected intraperitoneally to anesthetize the animals. After disinfection, the skin and subcutaneous tissue were incised, which was followed by separating right common carotid artery, external carotid artery and internal carotid artery, and ligating the far-end of the external carotid artery.…”