| Objective:In this study,the rat model of middle cerebral artery occlusion/reperfusion(MCAO/R)was used to evaluate whether intragastric administration of the phenolic components C,D and H can alleviate inflammation after CIRI,and to explore the potential mechanism.Methods:1.Replication of rat MCAO/R model The MCAO/R model was established by using thread-occluding method in male healthy Sprague Dawley(SD)rats.The neurological deficits were evaluated by Longa modified 5-point method,and the degree of limb injury was evaluated by balance beam test after ischemia 2h and reperfusion 24 h,7d and 14 d.TTC staining was used to evaluate the infarct volume.HE staining was used to detect the pathological morphology of brain tissue in rat MCAO/R model.2.The effect and mechanism of component C reduces inflammation by inhibiting COX-2 expression After the rat MCAO/R model was established,the Western blot methodwas used to detect the dynamic changes of COX-2 in cerebral cortex and hippocampus at the time of the injury was induced by cerebral ischemia for 2h and reperfusion for 1 day,3 days,5 days,7 days,9 days,14 days,21 days and 28 days respectively.Rat MCAO/R model was duplicated after component C pretreated for 5d.Evaluation of neurological deficits,balance beam test,TTC staining,HE staining were performed after ischemia 2h and reperfusion 24 h.The protein expression of COX-2 was detected by western blot method.3.The effect and mechanism of component D reduces inflammation by inhibiting 5-LOX expression After the rat MCAO/R model was established,the Western blot methodwas used to detect the dynamic changes of 5-LOX in cerebral cortex and hippocampus at the time of the injury was induced by cerebral ischemia for 2h and reperfusion for 1 day,3 days,5 days,7 days,9 days,14 days,21 days and 28 days respectively.The component D wastreated after 4.5h of MCAO/R model was established,and then given once a day for 7 successive days.Evaluation of neurological deficits,balance beam test,TTC staining,HE staining were performed after 0.5h of last administration.Then the rats were anesthesia.QPCR was used to detect the changes of LTB4 r,Cys LTs r1 m RNA expression in the injured cerebral cortex and hippocampus,the protein expression of 5-LOX was detected by western-blot,and the content of cytokine LTB4,Cys LTs was detected by ELISA kit.4.The effect and mechanism of component H reduces inflammation by inhibiting excessive activation of microglia After the rat MCAO/R model was established,the Western blot methodwas used to detect the dynamic changes of CD68 in cerebral cortex and hippocampus at the time of the injury was induced by cerebral ischemia for 2h and reperfusion for 1 day,3 days,5 days,7 days,9 days,14 days,21 days and 28 days respectively.The component H wastreated after 4.5h of MCAO/R model was established,and then given once a day for 14 successive days.Evaluation of neurological deficits,balance beam test,TTC staining,HE staining were performed after 0.5h of last administration.Then the rats were anesthesia.QPCR was used to detect the changes of TGF-?,TNF-?,IL-1?,IL-6,i NOS m RNA expressionin the damaged cerebral cortex and hippocampus.Western-blot was used to detect the protein expression of CD68.The expression of iba-1,a microglial activation marker,was detected by immunofluorescence technique.The level of cytokine TGF-?,TNF-? was detected byELISA kit.Results:1.Replication of rat MCAO/R modelThe rat MCAO/R model was replicated successfu L.The model group showed the increasing of the neurological score and the balance beam score decreasing.The infarct volume was significant increasing in TTC staining.HE staining showed obvious damage of the nerve cells.2.The effect and mechanism of component C reduces inflammation by inhibiting COX-2 expression Theresultsresults showed that COX-2 peaked at 1d and continued to 3d.Combined with the preliminary research of our group,we determined the interventive time points for the components.Component C was pretreated for 5 days.Theresultsresults showed that component C(20mg/kg)could decrease the neurological score and tended to increase the balance beam scoreof MCAO/R model rats.It couldcould reduce the rate of cerebral infarction in rats.Component C(20mg/kg)couldcould significantly reduce the expression of COX-2 protein in damaged brain tissuecould.3.The effect and mechanism of component D reduces inflammation by inhibiting5-LOX expression Theresultsresults showed that 5-LOX had two peaks at 5d and 14 d.Combined with the preliminary research of our group,we determined the interventive time points for the components.Component D was therapeutic administrated for 14 days.Theresultsresults showed that component D(20mg/kg)couldcould decrease the neurological score and tend to increase the balance beam scoreof MCAO/R model rats.It couldcould reduce the rate of cerebral infarction in rats.Component D(20mg/kg)couldcould significantly reduce the 5-LOX protein expression in the damaged cerebral tissue,and significantly inhibit the m RNA expression of LTB4 r and Cys LTsr1.It could significantly decrease the cytokines LTB4 and Cys LTs in the damaged brain.4.The effect and mechanism of component H reduces inflammation by inhibiting excessive activation of microglia Theresultsresults showed that in the damaged cerebral cortex and hippocampus,the protein expression of CD68 peaked at 7d and continued to 14 d.Component H wastherapeutic administrated for 7 days.Combined with the preliminary research of our group,we determined the interventive time points for the components.Theresultsresults showed that component H(10mg/kg)could decrease the neurological score and tend to increase the balance beam scoreof MCAO/R model rats.It could reduce the rate of cerebral infarction in rats.Component H(10mg/kg)could significantly inhibit CD68 protein expression in the damaged cerebral tissue of MCAO/R rats,it could significantly inhibit the positive expression of Iba-1protein in the ischemic brain tissue.Component H could obviously inhibit the TNF-? m RNA expression and decrease it release.Component H could promote TGF-? m RNA expression and increase TGF-? content.There was a tendency to inhibit the expression of IL-1?,IL-6,i NOS m RNA of component H.Conclusion:1.Components C,D,H have significant protective effectson cerebral ischemia /reperfusion injury of rat MCAO / R model.2.Component C pretreated for 5 days could inhibit the inflammatory response of COX-2 pathway at cerebral ischemia 2h and reperfusion 24 h of rat MCAO / R model.3.Component D therapeutic administrated for 14 days could inhibit the inflammatory response of 5-LOX pathway at cerebral ischemia 2h and reperfusion14 days of rat MCAO / R model.4.Component H therapeutic administrated for 7 days could inhibit the excessive activation of microglia and regulate the phenotype transformation of M1/M2 type of microglia after cerebral ischemia for 2h and reperfusion 7 days. |
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