Fisetin Protects Against Intracerebral Hemorrhage-Induced Neuroinflammation In Aged Mice

Part Ⅰ Effect of Fisetin on Nerve Injury and Functional Recovery after Cerebral HemorrhagePurpose:ICH is responsible for 10–15% of stroke cases and is associated with high morbidity and mortality.In ICH,brain is subject to disruption of adjacent tissue and secondary damages as a result of inflammation,hematoma,release of reactive oxygen species,and perihematomal edema.To date,exiting treatments for ICH are mainly supportive therapies such as reduction of intracranial pressure,brain edema,and improvement of hemodynamic stability.Unfortunately,no treatment has been demonstrated to be effective in order to improve the clinical outcomes of patients with ICH.Emerging evidences indicated that inflammation is an important factor of ICH-related brain injury.Fisetin(3,7,3’,4’ tetrahydroxyflavone)is a flavonol that has been commonly used as an antioxidant and anti-inflammatory compound and exerts neuroprotective effects in neurological diseases.To the best of our knowledge,fisetin has not been tested in the treatment of ICH-induced brain injury.In this study,we aimed to test the role of fisetin in reducing ICH-induced brain damage and recovery of neurological function in a mouse model.Methods:Sixty C57BL/6J mice were randomly divided into three groups: sham operation group,ICH control group and fisetin treatment group,in which the fisetin treatment group was subdivided into four subgroups(each subgroup was administered separately 10 mg/kg,30 mg/kg,60 mg/kg,90 mg/kg),10 animals in each group.The mouse ICH model was established by injecting collagenase into the striatum of the brain.The fisetin was administered intraperitoneally for 3 days.The mNSS was used to evaluate the severity of ICH-induced neurological damage in each group.The dry/wet weight method was used to measure the brain water content of each group of mice.TUNEL staining was used to detect the number of apoptotic cells in the brain of injured mice.Results:1.Fisetin improved the neurological damage of ICH mice in a dose-dependent manner,90 mg / kg fisetin treatment had the most significant effect on reducing mNSS in ICH mice.2.Fisetin improved the brain water content of ICH mice in a dose-dependent manner,90 mg / kg fisetin treatment had the most significant effect on reducing cerebral edema in ICH mice.3.Fisetin improved the apoptosis of brain damage tissue in ICH mice,in a dose-dependent manner,90 mg / kg fisetin treatment had the most significant effect on reducing brain tissue apoptosis in ICH mice.Conclusion:In this study,we demonstrated the effect of fisetin on nerve damage and functional recovery after cerebral hemorrhage.After administration of fisetin,the neurological deficit score,the percentage of water content in cerebral hemorrhage and the apoptosis were significantly decreased in ICH elderly mice,and the dose-dependent effect was observed.At a concentration of 90 mg/kg,the neuroprotective effect was the best.This suggests that our research may extend the clinical application of fisetin and provide new tools for drug therapy for brain damage caused by ICH.Part Ⅱ Effect of Fisetin on Inflammatory Response and Activation of Astrocytes and Microglia after intracerebral hemorrhagePurpose:Fisetin have been commonly used in antioxidant and anti-inflammatory compounds and play a neuroprotective role in neurological diseases.To the best of our knowledge,no studies have shown that fisetin has inflammatory regulation and glial activation in ICH-induced brain injury.In this study,we aimed to test the effectiveness of fisetin in inflammatory regulation,astrocyte and microglial activation in a mouse model.Methods:Sixty mice were randomly divided into three groups: sham operation group,ICH control group and fisetin treatment group,in which the fisetin treatment group was subdivided into four subgroups(each subgroup was administered separately)10mg/kg,30 mg/kg,60 mg/kg,90 mg/kg),10 animals in each group.The mouse ICH model was established by injecting collagenase type VII into the striatum of the brain.The fisetin was administered intraperitoneally for 3 days.Enzyme-linked immunosorbent assay(ELISA)was then used to detect the levels of inflammatory factors in brain tissue,including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 and IL-10 levels.The number of active astrocytes in brain tissue was quantitatively detected by S100 protein staining.The number of active microglia in brain tissue was detected by the levels of ionized calcium-binding adaptor molecule1(Iba-1)in brain tissue tested by Western blotting.Results:1.Fisetin reduced the levels of pro-inflammatory cytokines(including TNF-α,IL-1β,IL-6 and IL-10)after ICH in a dose-dependent manner,90 mg / kg of fisetin treatment showed the most significant effect on reducing the levels of pro-inflammatory cytokines in ICH mice.2.90 mg / kg fisetin significantly inhibits the activation of astrocytes after ICH.3.90 mg / kg fisetin significantly inhibits the activation of microglia after ICH.Conclusion:In this study,we demonstrate the regulation of fisetin on astrocytes and microglia activation and inflammatory responses.Fisetin dose-dependently reduced the levels of pro-inflammatory cytokines following ICH and significantly inhibited the activation of astrocytes and microglia.Part Ⅲ Fisetin Attenuates NF-k B Signaling Pathway after Intracerebral HemorrhagePurpose:Fisetin can regulate a large number of intracellular signaling pathways,including mitogen-activated protein kinase,nuclear factor kappa B(NF-k B)and c-Jun N-terminal kinase signaling pathways.To the best of our knowledge,fisetin has not been tested for regulation of signaling pathways in the treatment of ICH-induced brain injury.NF-k B is a protein responsible for cytokine production and cell survival,and is often used as a target for immunosuppressive and anti-inflammatory molecules.Fisetin has been shown to exert their anti-inflammatory properties by modulating the NF-k B pathway.In this study,we aimed to test the regulatory effects of fisetin on NF-k B signaling in brain tissue following ICH in mice.Methods:Thirty mice were randomly divided into three groups: sham operation group,ICH control group and fisetin treatment group(fisetin concentration of 90 mg/kg),with 10 animals in each group.The mouse ICH model was established by injecting collagenase into the striatum of the brain.Fisetin was administered intraperitoneally for 3 days.Western blotting was used to evaluate the levels of key proteins in the NF-k B pathway in brain tissue of mice.Results:1.Fisetin increased the level of P65 in the cytoplasm and inhibits the level of P65 in the nucleus after ICH in mice.This result is in accordance with the fact that translocation of p65 from cytoplasm to nucleus is a necessity for the successful activation of downstream genes.2.Fisetin inhibited the level of Ik Bα protein phosphorylation and increases Ik Bαprotein levels in mice after ICH.Since Ik B can inhibit NF-k B activation,and the decreased in Ik B protein phosphorylation also confirms the inhibition of NF-k B signaling.Conclusion:In this study,we demonstrate the neuroprotective function of fisetin in ICH-induced brain injury,which is attributable to down-regulation of pro-inflammatory cytokines and inhibition of the NF-k B signaling pathway.