| BackgroundIntracerebral hemorrhage(ICH)is an important public health problem that has caused worldwide concern due to its high mortality and morbidity rates.The mechanisms of brain injury in ICH include primary brain injury and secondary brain injury.The secondary injury is mainly the response to hematoma and elevated intracranial pressure,including inflammation,oxidative stress,excitotoxicity,and cytotoxicity.Compared with ICH-induced primary brain injury,secondary brain injury is recognized to participate in the development of neurobehavioral impairment,blood-brain barrier disruption,neuronal death,and cerebral edema.Luteolin is a naturally occurring flavonoid,with multiple potential pharmacological effects,including antioxidant,anti-inflammatory,apoptosis-inducing,and chemopreventive activities.It has been reported in many disease models such as Alzheimer’s disease,malignant tumor,and cardiovascular disease.However,the protective effect and related mechanisms of luteolin on intracerebral hemorrhage(ICH)are still lacking in research.Considering the release of severe inflammatory factors and oxidative stress injury after ICH,whether luteolin could play a neuroprotective role in secondary brain injury through antiinflammatory and antioxidation is an important research content of this subject.Aiming at the clinical treatment,this study tried to explore a new therapeutic drug and some targets for intracerebral hemorrhage,which had important clinical innovation and clinical value.Part I Luteolin has a neuroprotective effect on secondary brain injury after ICHObjective:To confirm whether luteolin could pass through the blood-brain barrier and to observe the effect of luteolin on the nervous system after ICH.Methods:In this study,Sprague Dawley(SD)rats were used to construct the rat model of autologous blood ICH;SD rats were randomly divided into treatment groups and control groups.Different doses of luteolin(5,10,20mg/kg)were injected intraperitoneally at different time points after the establishment of ICH models.After treatment,highperformance liquid chromatography was performed to verify the permeability of luteolin to the blood-brain barrier.Neurobehavioral tests,rotarod test,adhesive removal test,Morris water maze test,and brain water content test were used to study the effect of luteolin on secondary brain injury after ICH.The degenerated neurons were labeled by fluoro Jade B staining;and the effect of luteolin on the growth of vascular endothelial cells was measured by cell scratch test.Results:After constructing the rat model of autologous blood ICH,the results of highperformance liquid chromatography showed that luteolin could be detected in rat brain tissue after intraperitoneal injection of luteolin,suggesting that luteolin could play a role through the blood-brain barrier.After giving luteolin to rats in ICH,the neurobehavioral scores of the treatment groups were significantly higher than that in the control groups;the rotarod time was significantly increased while the adhesive-removal time was significantly reduced;there were significant differences in representative swimming-path traces,swimming speed at the beginning of the test,and escape latencies between two groups.Luteolin exerted a partial rescuing effect on motor impairment and cognitive impairment of rats after ICH(P<0.05).In vivo,fluoro Jade B staining showed that luteolin significantly reduced neuronal degeneration.The results showed that intraperitoneal injection of luteolin had a significant neuroprotective effect on ICH rats.In the scratch test,compared with the control group,luteolin could promote the growth of endothelial cells,particularly in the 10μM group.Conclusion:Luteolin could promote the growth of endothelial cells and has a neuroprotective effect on secondary brain injury after ICH through the blood-brain barrier.Part Ⅱ Luteolin alleviates neuroinflammation via downregulating the TLR4/TRAF6/NF-κB pathwayObjective:The activation of microglia and inflammatory responses is essential for the process of ICH.In this part of the study,the effects of luteolin on ICH-induced neuroinflammation were investigated,and the potential mechanisms were explored.Methods:SD rats were used to construct the rat model of ICH.The activation of microglia in brain tissue was detected by immunofluorescence staining to observe the effect of luteolin on microglia activation;the levels of different inflammatory factors in brain tissue and cell supernatant were detected by enzyme-linked immunosorbent assay.Toll-like receptors 4(TLR4),tumor necrosis factor receptor-associated factor 6(TRAF6),nuclear factor kappa-B(NF-κB),and other signaling pathway-related proteins were detected by Western Blot;the binding protein of luteolin was found by cellular thermal shift assay and co-immunoprecipitation.Results:In vivo,enzyme-linked immunosorbent assay showed that luteolin significantly decreased interleukin-1β and tumor necrosis factor-α in serum;fluorescence staining showed that luteolin significantly inhibited the expression of Iba1 microglia in brain tissue after ICH.These results suggested that luteolin could inhibit neuroinflammation induced by ICH.In the further exploration of the potential mechanisms behind the effect of luteolin,the levels of TLR4 and TRAF6 in the treatment rats’ brain tissue decreased significantly,suggesting that luteolin inhibited the signals of related proteins.At the same time,luteolin treatment significantly inhibited p65 protein and its phosphorylation in the rat brain;it increased the level of p65 in cytoplasmic protein while decreased the level of p65 in nuclear protein.Combined with the results of BV2 cell immunofluorescence,luteolin treatment could inhibit the translocation of p65 to the nucleus.These results showed that luteolin significantly inhibited the NF-κB signaling pathway by reducing p65 activation in the nucleus,thereby reducing the expression of inflammatory factors.Cell thermal shift assay showed that luteolin bonded to TRAF6,indicating that it may be an important target protein of luteolin.The co-immunoprecipitation experiment further showed that luteolin significantly inhibited the ubiquitination level of TRAF6 in the rat brains.The above results showed that luteolin inhibited TLR4/TRAF6/NF-κB signal pathway.Conclusion:Taken together,the results demonstrated that luteolin could attenuate neuroinflammation via inhibiting the TLR4/TRAF6/NF-κB signaling pathway and targeting TRAF6 to interfere with its ubiquitination,so as to reduce the level of inflammatory cytokines and play a neuroprotective role.Part Ⅲ Luteolin Exerts Antioxidation via Modulation of the p62/Keap1/Nrf2 PathwayObjective:Upregulation of neuronal oxidative stress is involved in the progression of secondary brain injury following ICH.In this part,the potential antioxidation and underlying mechanisms of luteolin were investigated.Methods:The effects of luteolin on mitochondrial function and free radical formation were evaluated by mitochondrial membrane potential and superoxide production.Nuclear factor erythroid 2-related factor 2(Nrf2),p62,kelch-like ECH-associated protein-1(Keap1),and other signaling pathway-related proteins were detected by Western Blot;the effect of luteolin on ubiquitination of Nrf2 was analyzed by co-immunoprecipitation.Results:The rat model of autologous blood ICH SD rats was constructed and different doses of luteolin were injected intraperitoneally.Results showed luteolin reduced the level of mitochondrial superoxide and inhibited the loss of mitochondrial membrane potential(P<0.05),suggesting luteolin can inhibit oxidative stress induced by ICH.After luteolin treatment,Nrf2 and its downstream pathway antioxidant stress proteins in rat brain increased significantly,suggesting luteolin significantly activated related protein signals.The levels of Keap1 and p62 in the treatment group decreased significantly,and the ubiquitination level of Nrf2 in brain tissue decreased significantly,indicating that luteolin enhanced the autophagic degradation process of p62-Keap1.The nuclear Nrf2 protein level increased while the corresponding cytoplasmic Nrf2 protein level decreased,indicating that luteolin increased Nrf2 nuclear translocation after ICH,thus activating the subsequent pathway.These results suggested that luteolin up-regulated the expression of antioxidant stress protein and reduced neuronal injury by activating the p62-Keap1-Nrf2 signal pathway.Conclusion:The results demonstrated that luteolin could modulate the p62/Keap1/Nrf2 pathway in ICH and up-regulate the expression levels of its downstream antioxidant proteins.Luteolin may represent a promising candidate for the treatment of ICHinduced secondary brain injury. |
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