Study On The Active Components Of Eerdun Wurile And Their Effects On Microglia Gene Expression

Stroke is the largest cause of death in China and the second major cause of death worldwide after ischemic heart disease.Therefore,it is of great significance to develop new anti-stroke drugs.Eerdun Wurile(EW)is one of the classic Mongolian prescriptions for the treatment of central nervous system disorders,which has been widely used in clinics because of its good neuroprotective effect and a significant effect on hemiplegia and other symptoms caused by stroke.However,the chemical composition of EW is complex and its mechanism of action is unclear.It is necessary to clarify the bioactive components of EW and its neuroprotective mechanism.In this thesis,we analyzed some of the active components of EW,and found that EW regulates the gene expression,polarization,and anti-neuroinflammatory effects of microglia in the brain of middle cerebral artery occlusion/reperfusion injury(MCAO/R)model rats.Objectives:(1)To verify the mechanism of neuroprotective activity of EW by focusing on the regulation of gene expression in the brain lesion of rat model of stroke and analyze the neuroactive compounds contained in EW;(2)To identify the major group of compounds in EW that contribute to the inhibition of neuroinflammation during stroke recovery through regulation of microglia polarization.;(3)To determine the key antiinflammatory molecules in EW fraction and their mechanism,as well as the effect on nerve cell protection and synaptic growth;(4)To explore the synergistic effects of key active molecules in EW on microglia gene expression profile.Methods:(1)Rat MCAO/R models were treated with EW for 14 days.Then,total RNA from the infarct in cerebral cortex were extracted and analyzed by RNA sequencing.Differentially expressed genes in NT group,MCAO group and EW group were analyzed for their functions during the recovery of ischemic stroke.(2)According to literature,the neuroprotective compounds in EW were collected to establish a database,named “EW neuroactive compounds database”.The Chem Draw software was used to draw the structural formula.Then,the EW soluble components were extracted with five different solvents,and their neuroactive compounds were analyzed by UPLC-QTof-MS.(3)The extracts of EW in different solvents were evaluated for their inhibitory ability of cytokine(Cxcl10)expression in mouse M1 phenotype microglia(BV2 cells).The extract with most significant regulatory effect was further separated to 12 fractionations on a semi-preparative HPLC column,and the optimized bioactive compounds groups were obtained.The bioactive compounds were identified by UPLCQTof-MS.(4)The key anti-inflammatory molecules in isolated component of EW(denoted F4-6)were identified using UPLC-QTof-MS,and the effects of F4-6 on the levels of proteins involved in NF-?B signaling pathway in LPS-stimulated BV2 microglia were analyzed using western blot.Then,the proliferation and synaptic growth of neurons(N2a cells)induced by conditioned culture medium produced from F4-6 treated in BV2 cells were analyzed.(5)LPS-stimulated BV2 cells were treated with the key active compounds in EW,i.e.dehydrodiisoeugenol,or alantolactone or the mixture of them.Then,the gene expression pattern profile was analyzed by RNA-Seq,and verified by RT-q PCR.Results:(1)Bederson scale indicated that the treatment of rat MCAO/R models with EW showed significantly lowered neurological deficits after 14 days: the score decreased from 2.16 before treatment to 0.21 after treatment(P <0.01).Compared with NT group,there were 63 differentially expressed genes in the infarct of the MCAO group.A total of 186 genes were regulated in the lesion of EW treated group compared to MCAO group.Among them,growth factors such as Igf1,Igf2,Tgf? and Grn were significantly upregulated in brain after treatment with EW.Meanwhile,greatly enhanced expression of microglia markers,as well as complementary components and secretive proteases were also detected.(2)EW was extracted with five different solvents,and UPLC-QTof-MS analysis showed that the 16 neuroactive compounds which had previously been reported in the10 herbal components of EW(including Carthamus tinctorius L.,Myristica fragrans Houtt.,Glycyrrhiza uralensis Fisch.,Amomum tsaoko Crevost & Lemarié,Melia azedarach L.,Gardenia jasminoides J.Ellis,Inula helenium L.,Saussurea costus(Falc.)Lipsch.,Piper longum L.and Nigella sativa L.)were detected.(3)EW petroleum ether extract(named EW-5)had the strongest inhibitory effect on Cxcl10 gene expression(P <0.05).After two consecutive fractionating by preparative HPLC,EW-5 were separated to 12 fractions.F4-6 showed the most significant inhibitory effects on expression of pro-inflammatory cytokines including Cxcl10,Tnf?,Il1? and Nos2(P <0.05).The result of UPLC-QTof-MS analysis showed that F4-6 contains 20 chemicals including costunolide,alantolactone,myristicin and linolenic acid,which significantly downregulated the expression of key proinflammatory cytokines in LPS stimulated BV2 cells as well as mouse primary microglia.(4)Ala and Deh were the key active anti-inflammatory components of F4-6.F4-6,Ala and Deh downregulated the expression of several pro-inflammatory genes including Ccl2,Cox2 and Il6 in LPS-treated microglia,respectively.Meanwhile,F4-6,Ala and Deh upregulated the expression of anti-inflammatory genes including Hmox1,Tgf?,Igf1 and Creb1,respectively.Moreover,the culture media obtained from F4-6treated microglia significantly enhanced proliferation of N2 a cells,and promoted neurite outgrowth possibly through upregulation of Nefh and Dlg4.Mechanistically,F4-6 strongly downregulated the expression of NF-?B p65,while also inhibiting the nuclear translocation of p65,suppressing the transcription of proinflammatory genes initiated by NF-?B.(5)RNA-seq found that Ala,Deh and Mix mainly downregulated the proinflammatory genes and upregulated antioxidant genes,respectively,in LPSstimulated microglia.The results showed that these compounds may contribute to the neuroprotective effects through anti-inflammatory and antioxidant activities.The effect of Mix group was more significant(P <0.05)than that of the other two(i.e.Ala and Deh group),and its effect was synergistic,rather than simple sum of the effects of Ala and Deh alone.Conclusion:In summary,this thesis used RNA-seq technology for the first time to systematically analyze the effect of Mongolian medicine EW on gene expression regulation in cells in peri-infarct of MCAO/R model rats.It was found that EW significantly improved the recovery of central nervous damage after ischemic stroke and promoted the polarization of microglia from M1 phenotype to M2 phenotype.Moreover,UPLC-QTof-MS was used to analyze the bioactive compounds related to neuroinflammation and neuroprotection in EW,and the synergistic effect of various active compounds in EW might contribute to the possible microglia polarization in brain lesion during the recovery of ischemic stroke,and the efficacy of EW in treating Baimaibing(neurological diseases)was verified,which provided important evidence for further study on the therapeutic mechanism of EW as well as the other traditional medicines.