A Comparative Study Of Pharmacological Mechanism Of Components And Combinations Of Qingkailing On Rats After Experimental Stroke

A comparative study of pharmacological mechanism of Components andcombinations of Qingkailing on rats after experimental strokeIn the recent years, the study of substance foundation and their action mechanism,operation theory of multipathway, multitargets and their cnformity and regulation of Chinesemedication, has been a key scientific matter.1．AimTo explore the inner mechanism of components and combination of Qingkailing,comparethe gene expression and pharmacological pathway after experimental stroke rats.2．Methods2．1 Thoretic methodsThe mainline of this study is characteristic of combination of Chinese prescription. Thedevelopment history of Chinese prescription and its complex characteristics are summarized.Then with the methodology of omics study, this paper study the cut-in point of theorderlinesss study of Components and combinations of Qingkailing.2．2 Methods of experimentationThe focal cerebral ischemia-reperfusion model of sting-shutoff were operated on 15 ratsin every treatment groups. With the uncompleted design of analystical elements,we dividedall animals into 4 single component groups: Baicalin(BA),Jasminoidin(JA), UrsodeoxycholicAcid(UA), Concha margaritifera usta(CM), 4 groups of combination of the 4 components:BJ(BA+JA), JU(JA+UA), BJU(BA+JA+UA), BJUC(BA+JA+UA+CM), and 3 controlgroups:NM,SHAM and model group. (1) 3 animals were per-refusion treated after 24hours,cut in coronal tissue of brain. Then the cerebral tissue were pigmented by TTC andcalculated the ratio value of ischemic volume; (2) Total RNA were abstracted from thecerebral tissue with the method of one-step. Then cy5/cy3 sign them and gene chip of themRNA were made in accordance to our aim of our study. The gene profile of expression wererepeated 6-9 times; (3) The Genespring software calculated all the gene data, and wereclassified by gene ontology principle.The analysis of clustering and principle componentsanalysis were operated for the information of gene expression. All main pathway were foundby pathway studio 5．0 ; (4) Real-time PCR was operated to confirm the data of genechip afterdesign of primer matter of 8 key genes. 3．Results3．1 Results of Thoretic methodsAccording to the thinking of my tutors,the complex charactristics of combination ofChinese medication were abstracted. They are the integration of components,emergement ofquantitative components,dynamic charactristics. Then we need made the disease modeladministration of the combination of components of Chinese medication. From the angle ofpharmaclogical and molecular pharmacological pathway,the internal mechanism of relationbetween components and combinations was explored.3．2 Results of operations3．2．1 The effection of superficial tissue of cerebral ischemia after treatment ofcomponents and combinations of Qingkailing.All treatment groups can reduce proportion of tissue of cerebral ischemia. In thegroups,proportion of tissue of cerebral ischemia in groups of JU and BJUC are lower thanother treatment groups(p<0．01) .3．2．2 The whole analysis of the profile of gene expression of cerebral ischemia aftertreatment of components and combinations of Qingkailing.The profile of gene expression includes 1,436 genes of different expression (8．72%)(p<0．05) , and 724(4．4%) probes at false discovery rate <5% by significance analysis ofmicroarray. They are divided for 12 functions. Compared with the group of model,all groupsof treatment changed the level of gene expression. This results indicated all genes are ralatedwith the mechansim of effection by these groups of treatment.3．2．3 Comprehensive comparasion of BA, JA and BJThe overlapped gene among the groups of BA,JA and BJ are 23, 11．The results ofclustering indicated that JA first was clustered with BJ,and PCA's result indicated theeigenvector of the 2 groups in first 3 principle components are close,which suggests that the 2groups are consistent in the effection of gene expression of cerebral ischemia-reperfusioninjury.3．2．4 Comprehensive comparasion of JA,UA and JUThe overlapped gene among the groups of JA,UA and JU are 21, 25, 30．The results ofclustering indicated that JA first was clustered with JU,and PCA's result indicated theeigenvector of the 2 groups in first 3 principle components are close,which suggests that the 2groups are consistent in the effection of gene expression of cerebral ischemia-reperfusioninjury. 3．2．5 Comprehensive comparasion of BJ(BA+JA), UA and BJU(BA+JA+UA)The overlapped gene among the groups of BJ(BA+JA),UA and BJU(BA+JA+UA) are 23,40 genes. The results of clustering indicated that BJ first was clustered with BJU,and PCA'sresult indicated the eigenvector of the 2 groups in first 3 principle components areclose,which suggests that the 2 groups are consistent in the effection of gene expression ofcerebral ischemia-reperfusion injury,which indicated UA can assistant BJ for the top score ofgroup BJU.3．2．6 Comprehensive comparasion of BJU(BA+JA+UA), CM, BJUC(BA+JA+UA+CM)The overlapped gene among the groups of BJU(BA+JA+UA), CM, BJUC(BA+JA+UA+CM) are 18, 14 genes. The results of clustering indicated that BJU first wasclustered with BJUC,and PCA's result indicated the eigenvector of the 2 groups in first 3principle components are close,higher than that of CM,which suggests that BJUC and BJUhas better effect than CM in the effection of gene expression of cerebral ischemia-reperfusioninjury,which indicated CM has less effect in the all components.3．2．7 Comparasion of distinct combination and components of QKL on signalingpathwayBJUC, JA, BJ can lower the level of C-fos, Elk-1, LPS,then activate CREB andMAPK-ERK pathway ;ascend the level of STAT1,lower STAT3,then activate JAK-STATpa?hway(P-value=0．89) .BJUC,BJU and JA can retrain the transcription activity of NF-κB,iκB in different pathway,preveting their over-expression.BJ and UA can increase the level of TIMP-1 and decrease MMP-9,which activateMMPs-ECM pa?hway(P-value=0．96) ,then have the protection function of BBB.BJUC and JU can increase the level of VEGF,(?)-catenin and their down-position proteinTCF3,which activate Wnt-(?)-catenin pathway(P-value=0．92) .4．Conclusions4．1 All treatment groups can reduce proportion of tissue of cerebral ischemia in differentdegreee,which groups of JU and BJUC are better than that of other treatment groups,indicatethe superiority of all combination of 4 components.4．2 BJU(BA+JA+UA)and BJUC have protection function for brain,with the integrationof activation several pathways such as MAPK, PI3-K/Akt, JAK-STAT, NF-kB, Wnt, TGF-(?),Ca²⁺,IGF1R-CEBPA et al. 4．3 JA, as well as BJ(BA+JA) and BJU(BA+JA+UA), have similar pharmacologicaleffect on pathway,which has leading function among the 4 components,and a combinatingneed with other 3 components.4．4 The application of genechip with the technology of high data and parallel analysis andfeasible methods of bioinformatics,which include the combination of clustering and PCA,andpathway analysis of network of gene and protein,are helpful to the study of mechansim ofcombination and components of Chinese medications.In sum,we compared the proportion of ischemic tissue,the difference of gene profile,andnetwork pathway after the treatment of 4 single components and 4 combinations of QKL,thencome to the results that the effect of BJUC is better than other combinattion group with theleading action of JA,and BJUC and BJ can protect brain after ischemia with the molecularmechanism of regulation of sveral key pathway. So this study give the methodologicalexplore for study of combination mechanism of Chinese medications.