| Objective:Anti-inflammatory effects of isoorientin has some researchs,but the specific mechanism of the effect is unclear.Recent studies have shown that isoorientin is the substrate competitive inhibitors of GSK3 beta.First,we observed the anti-inflamma-tory effect of isoorientin on RAW264.7 stimulated by LPS and its effect on the activity of GSK3β and its downstream signaling molecules.In vivo experiments,we further observed the anti-inflammatory effect and protection of the blood-brain barrier of isoorientin in the model of endotoxemia;In addition,the changes of GSK3β in the cerebral cortex were observed.This study provided an experimental basis for elucidating the anti-inflammatory mechanism and protective effect on inflammation-related brain injury of isoorientin.Methods:1.The anti-inflammatory effect of isoorientin on macrophage and its effect on GSK3β and downstream related signal molecules1.1 Construction of inflammation model on macrophageThe RAW264.7 cells were resuscitated and passed 3 passages before it were used in experiments.LPS was used to stimulate RAW264.7 to establish inflammation model on macrophage.Macrophage were stimulated with LPS 10 ng/ml,LPS 50 ng/ml or LPS 100 ng/ml,and the secretion of TNF-α in the cell supernatant was detected by ELISA to determine the dose of LPS.GSK3βinhibitor lithium chloride was used as a control.Different concentrations of isoorientin and lithium chloride were given,and the secretion of TNFα in the cell supernatant was detected by ELISA to determine the anti-inflammatory doses of isoorientin and lithium chloride.1.2 Analysis of the effect of the combined use of ISO and LiClAfter finding the anti-inflammatory doses,lithium chloride and isoorientin were given in combination for 1 hour,followed by LPS stimulation for 8 hours.ELISA was used to detect the changes of the secretion of TNFα in the supernatant.The interaction between isoorientin and lithium chloride were analyzed by Jin Zhengjun Q value.1.3 Regulation of GSK3β(phosphorylated GSK3β at Ser9)by ISOAfter finding the anti-inflammatory doses,the experiment was divided into control group,LPS group,LPS+ISO-L(6.25 μmol/L)group,LPS+ISO-H(25μmol/L)group,and LPS+LiCl(100 μmol/L)group.Cell were pre-treated with ISO and LiCl for 1 hour,and then stimulated with LPS for 30 minutes.After that,cells were collected and WB was used to detect the expression of p-GSK3&(ser9).The phosphorylated GSK3β at Ser9 is an activity-inhibiting state.This experiment was to explore the relationship between isoorientin and GSK3β activity.1.4 Effect of inhibit AKT on ISO-regulated GSK3β activityAKT is an important protein kinase of the upstream of GSK3β.MK-2206 is a highly selective inhibitor of AKT.In the experiment,MK-2206 was used to inhibit the activation of AKT,and the effect on GSK3β(phosphorylated GSK3β at Ser9)was observed to further anlyze the effect of isoorientin.The cells were pre-treated with AKT inhibitor MK2206 for 30 minutes,and then treated with ISO and LiCl for 1 hour.After that,stimulated with LPS for 30 minutes and 8 hours,respectively.The expression of p-GSK3β(ser9)was detected by WB,and the change of TNF-α was detected by ELISA.To investigate the effect of inhibiting the phosphorylation of GSK3β at Ser9 on the regulation of GSK3β activity and anti-inflammatory effect of isoorientin.1.5 Regulation of ISO on GSK3β downstream moleculesIn the same group as 1.3,the cells were collected after LPS stimulation for 15 minutes,1 hour,8 hours and 18 hours.The protein expression of ERK1/2,p-ERK1/2,NF-κ Bp65,IκB-α,COX-2,Nrf2 and HO-1 were detected by WB.In order to analyze the regulation of isoorientin on GSK3β downstream signaling protein.2.Analyze the anti-inflammatory effect and protective effect of isoorientin on brain injury in mice suffering endotoxemia2.1 The establishment of endotoxemiaIn the experiment,we established the endotoxemia model through the intraperitoneal injection of lipopolysaccharide in BALB/c mice.First,the stimulation dose and stimulation time of LPS were determined.Mice stimulated with LPS(1.25 mg/kg,2.5 mg/kg,5 mg/kg)at different times(1 hour,6 hours,18 hours).Changes of the Secretion of IL-6 and IL-1β were detected by ELISA,and the expression of COX-2 were detected by WB.Finally,we observed the changes of inflammation level in the central and peripheral nervous system.2.2 The anti-inflammatory effect and protective effect of isoorientin on brain injury in endotoxemia miceBALB/c mice were selected in the experiment.ISO(25,50 mg/kg·day,p.o.)and LiCl(100 mg/kg·day,p.o.)were given for 5 consecutive days.After thirty minutes,LPS 5 mg/kg was injected intraperitoneally to established the endotoxemia model.After 6 hours of LPS injection,blood were collected from eyeballs of mice.And then mice were sacrificed by cervical vertebra removed.and the brains(divided into hippocampus and cortex)were removed.The experiments were divided into control group,LPS group,LPS+ISO 25 mg/kg,LPS+ISO 50 mg/kg,LPS+LiCl 100 mg/kg.The Tissues were stored at-70°C.The inflammatory factors TNF-α,IL-6 and IL-1β were detected by ELISA.Wesrern Blot was used to detect the expression of ZO-1,Occludin,GSK3β and p-GSK3β in the cerebral cortex.Results:1.Isoorientin exerts anti-inflammatory effect by regulating GSK3β in RAW264.7 cell line1.1 Establishment of macrophage inflammation modelELISA results showed that LPS 50 ng/ml can significantly increase the secretion of TNF-α.Compared with the blank group,the secretion of TNFα increased significantly after stimulated with LPS 50 ng/ml;Compared with the model group,6.25 μmol/L,25 μmol/L ISO significantly reduced the secretion of TNF-α and IL-6.Lithium chloride is a selective inhibitor of GSK3β.Studies showed that 25,100 μmol/L lithium chloride can effectively reduced the secretion of TNF-α in the cell induced by LPS.1.2 Analysis of the effect of the combined use of ISO and LiClIn the experiment,isoorientin(25 μmol/L,6.25 μmol/L)was used in combination with lithium chloride(25 μmol/L,100 μmol/L).Compared with the model group,the secretion of the inflammatory factor TNF-α was significantly reduced in drugs group;it showed an antagonistic effect after combined administration.The results suggested that isoorientin and lithium chloride may have the same target.1.3 Regulation of GSK3β(phosphorylated GSK3β at Ser9)by ISOIn the experiment,AKT inhibitor MK-2206 was used to further analyze the relationship between the anti-inflammatory effect of isoorientin and GSK3β activation.WB results showed that compare with the blank group,LPS stimulation significantly reduced the expression of p-GSK3β;whereas,isoorientin can significantly increased the expression of p-GSK3β.It is suggested that isoorientin significantly inhibited the activity of GSK3βinduced by LPS.1.4 Relationship between the anti-inflammatory effect of ISO and GSK3β activationIn the experiment,MK-2206 was used to inhibit the activation of AKT,affecting the activation of GSK3β,and then we observed whether the effect of isoorientin was inhibited.ELISA was used to detect the changes of TNFα in the supernatant of cells stimulated by LPS.The results showed that MK-2206 lighte the inhibitory effect of isoorientin on TNF-α secretion.The expression of p-GSK3β(ser9)was detected by WB,we found that MK-2206 inhibited the effect of isoorientin on elevating p-GSK3β.It is suggested that MK-2206 can significantly inhibit the anti-inflammatory effect of isoorientin,and further verified that the anti-inflammatory effect of isoorientin is related to the activation of GSK3β.1.5 Regulation of ISO on GSK3β downstream signaling proteinWB detected the expression of ERK1/2,p-ERK1/2,NF-κ Bp65,IκB-α,COX-2,Nrf2 and HO-1.We found that isoorientin can reverse the expression of GSK3β downstream signaling proteins.It is suggested that isoorientin can affect the expression of GSK3β downstream signaling proteins.2.Analyze the anti-inflammatory effect and protective effect of isoorientin on brain injury in a mouse model of endotoxemia2.1 The establishe of endotoxemia modelFirst,we determined the stimulation dose and stimulation time of LPS.Mice were stimulated by LPS(1.25 mg/kg,2.5 mg/kg,5 mg/kg)at different times(1 hour,6 hours,18 hours),and ELISA was used to detect IL-6 and IL-1β in mouse serum.Compared with the blank group,LPS 5 mg/kg stimulation for 6 hours can significantly increase IL-6 and IL-1β in mouse serum.And WB results showed that LPS 5 mg/kg stimulation for 6 hours can increase the expression of COX-2 protein.2.2 The anti-inflammatory effect of isoorientin and its protective effect on brain injury on endotoxemia model miceIn vivo experiments,ELISA results showed that high concentrations of isoorientin and lithium chloride can significantly reduce the levels of peripheral inflammatory factors.WB results showed that compared with the blank control group,the expressions of tight-junction proteins ZO-1 and Occludin in the cerebral cortex of endotoxemia mice decreased;while ISO 25mg/kg,ISO 50 mg/kg and LiCl 100 mg/kg significantly increased the expression of tight-junction protein.Compared with the blank control group,the expression of p-GSK3β in the model group decreased;Compared with the model group,the expression of p-GSK3β increased in the ISO 25mg/kg,ISO 50mg/kg and LiCl 100mg/kg groups,and the expression of total GSK3β in each group wasn’t significant changed.Compared with the blank group,the expression of COX-2 in the model group increased,and the expression of COX-2 in the ISO 25 mg/kg,ISO 50 mg/kg and LiCl 100 mg/kg groups decreased.The results suggested that isoorientin can reduce the levels of peripheral inflammation and protect the integrity of the blood-brain barrier in endotoxemia.Conclusion:1.In the inflammatory response induced LPS in macrophage,isoorientin regulated the activation of GSK3β and its downstream signaling molecules,suggesting that GSK β is an important target for isoorientin to exert anti-inflammatory effects on macrophages.2.Isoorientin can reduce the peripheral inflammation levels,protect the integrity of the blood-brain barrier and affect the activation of GSK3β. |
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