| Part Ⅰ: The effects of omega-3 fatty acids on SAH induced-EBI in ratsObjectiveTo evaluate the effects of omega-3 fatty acids on SAH induced-EBI.MethodsExperiment 1: To determine the concentration of EPA and DHA in rat brain induced by omega-3 fatty acids pretreatment.1.Animal groups: 16 rats were randomly divided into 4 groups(4 rats per group): control group,placebo group,2-week fish oil pretreatment group,and 3-week fish oil pretreatment group.Fish oil pretreatment groups received oral gavage of 30% omega-3 fatty acids at 1 g/kg body weight once every 24 hours for indicated time.Placebo group received oral gavage of an equal volume of corn oil using the same procedure for 3 weeks.2.After the indicated treatments,lipid extraction from brains was performed.Fatty acid compositions were detected by capillary gas chromatography using a Clarus 500 Gas Chomatograph.Components were determined by comparing the peak retention times to a 30-component methyl ester standard.Experiment 2: To evaluate the effects of omega-3 fatty acids on SAH induced-EBI.1.Animal groups: 60 rats were randomly divided into 4 groups(n=12): sham group,SAH group,SAH + placebo group,and SAH + omega-3 fatty acids group.2.Animal model making : a rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern in 20 s.24 hours after the SAH model established,all the rats in the experiment were killed and cerebral tissue samples were taken for analysis.3.Detection methods and indicators: The index of brain edema was evaluated using the wet/dry method.TUNEL staining and FJB staining was performed to examine brain cell apoptosis and neuronal degeneration.Western blot analysis was used to determine the protein levels of COX-2,chemokine MCP-1,and cytokine iNOS in brain tissues.Results1.Both 2-week and 3-week fish oil pretreatments resulted in significant increases in the concentrations of EPA and DHA in rat brains(p < 0.05).However,there was no significant differences between two groups(p < 0.05).2.Brain water content was found to be significantly higher in brain samples of SAH group than that in rats subjected to the sham group,while the mean brain water content was lower in rats with SAH + omega-3 fatty acids pretreatment than in the SAH + placebo group(p < 0.05).3.24 hours after SAH,the number of TUNEL and FJB positive cells in the brain tissues increased significantly compared with the sham group(P < 0.01).However,compared with SAH + placebo group,the number of TUNEL-and FJB-positive cells was significantly reduced in the omega-3 fatty acids pretreatment group(P < 0.05).4.Compared with the sham group,the levels of these proteins were all significantly elevated in the SAH group(P < 0.01).However,compared with SAH + placebo group,omega-3 fatty acids pretreatment significantly downregulated the levels of these proteins(P < 0.05).Conclutions1.The concentrations of EPA and DHA in rat brains can be increased significantly with 2-week fish oil pretreatment.But one more week fish oil pretreatment could not make further improvement on the concentrations of EPA and DHA.2.Omega-3 fatty acids pretreatment could inhibit SAH-induced brain cell apoptosis and neuronal degeneration,and suppress inflammatory responses significantly in the brain tissue in rats.Part Ⅱ: The possible mechanisms of the effects of omega-3 fatty acids on SAH induced-EBIObjectiveTo investigate the possible mechanisms of the effects of omega-3 fatty acids on SAH induced-EBI.Methods1.Animal groups: 60 rats were randomly divided into 4 groups(n=12): sham group,SAH group,SAH + placebo group,and SAH + omega-3 fatty acids group.2.Animal model making : a rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern in 20 s.24 hours after the SAH model established,all the rats in the experiment were killed and cerebral tissue samples were taken for analysis.3.Detection methods and indicators: To examine behavioral activity in all groups.And then all the rats were killed and samples were collected.Western blot analysis was performed to determine the protein level of GPR120 and the phosphorylation levels of TAK1,MEK4,JNK,and IKK in brain tissues.Immunoprecipitation analysis was performed to determine the binding between GPR120 and β-arrestin2,between β-arrestin2 and TAK1,and between TAK1 and TAB1.EMSA was performed to determine the activation of NF-κB.Results1.Compared with the sham group,the GPR120 protein level was significantly reduced in the SAH group(P < 0.01).Furthermore,omega-3 fatty acids significantly inhibited the reduction in SAH-induced GPR120 protein levels(P < 0.01).2.Compared to the SAH + placebo group,omega-3 fatty acids pretreatment significantly enhanced the binding between GPR120 and β-arrestin2,and between β-arrestin2 and TAK1,and significantly inhibited binding between TAK1 and TAB1(P < 0.05).3.After SAH,phosphorylation levels of TAK1,MEK4,JNK,and IKK in brain tissues were significantly enhanced,and omega-3 fatty acids pretreatment significantly inhibited phosphorylation of these proteins(P < 0.05).4.EMSA results showed that Omega-3 fatty acids pretreatment significantly suppressed activation of NF-κB(P < 0.05).ConclutionsOmega-3 fatty acids may exert anti-apoptosis and anti-inflammatory effects via GPR120/β-arrestin2/TAB1/TAK1 signaling pathways in the brains of SAH rats.Part Ⅲ: Omega-3 fatty acids acts in a GPR120-dependent mannerObjectiveTo investigate the protective effects of Omega-3 fatty acids was mediated by GPR120 via β-arrestin2/TAB1/TAK1 signaling pathway.Methods1.Animal groups: 24 rats were randomly divided into 3 groups(n=6): SAH + omega-3 fatty acids group(6/7 survived),SAH + omega-3 fatty acids + control si RNA group(6/8 survived),and SAH + omega-3 fatty acids + si GPR120 group(6/9 survived).2.Animal model making : a rat SAH model was induced by injection of 0.3ml fresh arterial,nonheparinized blood into the prechiasmatic cistern in 20 s.24 hours after the SAH model established,all the rats in the experiment were killed and cerebral tissue samples were taken for analysis.All rats received omega-3 fatty acids by oral gavage beginning at 2 weeks before SAH.Transfection of si RNA in rat brain was performed 48 hours before SAH.3.Detection methods and indicators: To examine behavioral activity in all groups.And then all the rats were killed and samples were collected.Western blot analysis was performed to determine the protein level of GPR120 and the phosphorylation levels of TAK1,MEK4,JNK,and IKK in brain tissues.Immunoprecipitation analysis was performed to determine the binding between GPR120 and β-arrestin2,between β-arrestin2 and TAK1,and between TAK1 and TAB1.EMSA was performed to determine the activation of NF-κB.Western blot analysis was performed to determine the protein levels of COX-2,chemokine MCP-1,and cytokine iNOS in brain tissues.Results1.After SAH + omega-3 fatty acids pretreatment,GPR120 siRNA significantly reduced GPR120 protein levels(p < 0.001).2.GPR120 si RNA blocked omega-3 fatty acids regulation of the binding between β-arrestin2/TAB1,and TAB1/TAK1 interactions in brain tissues(p = 0.033).3.Because of GPR120 siRNA,Omega-3 fatty acids could no longer suppress the SAH-induced phosphorylation of TAK1,MEK4,JNK,and IKK and activation of NF-κB(p = 0.015).4.GPR120 silencing inhibited the ability of omega-3 fatty acids to suppress SAH-induced brain cell apoptosis and neuronal degeneration(p = 0.041).5.GPR120 siRNA significantly suppressed the anti-inflammatory effects of omega-3 fatty acids pretreatment(p < 0.05).Conclutions1.Omega-3 fatty acids could suppress the SAH-induced phosphorylation of TAK1,MEK4,JNK,and IKK and activation of NF-κB.Omega-3 fatty acids may exert anti-apoptosis and anti-inflammatory effects via GPR120/β-arrestin2/TAB1/TAK1 signaling pathways in the brains of SAH rats.GPR120 siRNA could block omega-3 fatty acids regulation of the β-arrestin2/TAB1/TAK1 pathway.2.Following SAH,the protective effects of Omega-3 fatty acids was mediated by GPR120 via β-arrestin2/TAB1/TAK1 signaling pathway.3.GPR120 may be a potential therapeutic target for subarachnoid hemorrhage treatment because of its key role in β-arrestin2/TAB1/TAK1 signaling pathway. |
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