| Objective:1.To establish a HPLC method for the determination of several chemical constituents in Erigeron breviscapus injection,thus providing reference for improving quality control standards and postmarketing evaluation of drugs;2.To study the protective effect of Erigeron breviscapus injection on blood-brain barrier(BBB)injury induced by cerebral ischemia in rats by regulating ROS/RNS-MMPs-TJs signaling pathway,and to provide basis for clinical rational drug use.Methods:1.The content of chemical components in Erigeron breviscapus injection was determined by HPLC method under the chromatographic conditions of column temperature at 30 ?,with flow rate of 0.9 mL min-1,wavelength 327 nm and mobile phase acetonitrile-0.4%phosphoric acid.2.The rat model of focal cerebral ischemia-reperfusion injury(MCAO)was prepared by wire-suppository method,and the efficacy of Erigeron breviscapus injection,scutellarin and 3,5-dicaffeoylquinic acid in protecting BBB injury caused by cerebral ischemia was evaluated by using the following indexes:?The transmission rate of Evans blue in rat brain tissue was tested and The change of BBB permeability was analyzed.?Neurons and BBB ultrastrucure in cerebral cortex were observed by transmission electron microscope(TEM).?The Cerebral infarction was observed by TTC-staining.?Neurological scores was conducted.?Pathological changes in hippocampus of brain tissue were observed by HE-staining.?Expression of Nissl corpuscle was observed by Nissl staining of toluidine blue.3.Based on MCAO model rats,the following methods were used to study the mechanism of Erigeron breviscapus injection in regulating ROS/RNS-MMPS-TJS signaling pathway:Real-time PCR(RT-PCR)and Western Blot(WB)for the determination of iNOS,MMP-9,claudin-5,Occludin,ZO-1 mRNA and protein expression in brain tissue.Results:1.The following indexes such as precision,repeatability,stability and the recovery rate of a total of 10 chemical elements in Erigeron breviscapus injection,including scutellarin,apigenin-7-O-glucronide,caffeic acid,chlorogenic acid,isochlorogenic acid B,5-caffeoylquinic acid,4-caffeoylquinic acid,1,3-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid,and 4,5-dicaffeoylquinic acid were all in accordance with the requirements with RSD value less than 3%,and the linear range of the peak area according with the sampling quantity.2.Erigeron breviscapus injection,and scutellarin,3,5-dicaffeoylquinic acid can reduce the rate of cerebral infarction in rats,relieve the abnormal neurobehavioral behavior of rats,reduce the degree of brain tissue lesion,increase the number of the Nissl corpuscle cells,and repair injured rat neuron ultrastructure.At the same time,it can obviously reduce the ultrastructure damage of the blood-brain barrier in rats.Besides,all three drugs significantly reduced the content of Evans blue in ischemic brain tissue caused by cerebral ischemia in rats with BBB injury.3.Erigeron breviscapus injection,and scutellarin,3,5-dicaffeoylquinic acid can decrease the protein expression of iNOS and MMP-9 in rat ischemic brain tissue.3,5-dicaffeoylquinic acid can also increase the protein expression of claudin-5.Conclusion:1.The HPLC method for determination of 10 chemical constituents in Erigeron breviscapus injection was established.This method,characterized by simplicity accuracy and reproduction can be used for the quality control of Erigeron breviscapus injection.2.Erigeron breviscapus injection,and scutellarin,3,5-dicaffeoylquinic acid have obvious therapeutic effects on BBB and neuron injury induced by cerebral ischemia in rats,and the main pharmacodynamic substances are scutellarin,3,5-dicaffeoylquinic acid and so on.3.Erigeron Injection and Scutellarin inhibited the activation of MMP-9 by inhibiting the synthesis of iNOS,which protected the integrity of BBB structure.3,5-dicaffeoylquinic acid inhibit the expression and activation of MMP-9 by inhibiting the activation of iNOS,and reducing the generation of free radicals,thus reducing the degradation of important cytoskeleton connexin claudin-5 in the tight junction(TJs)structure by inhibiting the expression and activation of MMP-9 to,and finally BBB structure integrity was protected. |
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