Analysis Of The Active Components Of Erdun-Uzil And Its Study On The Regulation Of Gene Expression In Brain Neurons

Objective:1.To analyze the neural-related active components of Mongolian Medicine Eerdun Wurile.2.To investigate the mechanism of the neuroprotective effects and neuroregenerative activity of EW by focusing on the regulation of gene expression in the brain of rat model of stroke.Method:1.Analysis of neuroactive components of Eerdun Wurile:Eerdun Wurile is pulverized and then extracted by(Ⅰ)distilled water,(Ⅱ)anhydrous ethanol,(Ⅲ)petroleum ether,(Ⅳ)ethyl acetate and(Ⅴ)n-butanol,respectively.According to the report of the chemical composition of the EW medicinal materials which were associated with nerves system,the information on the chemical constituents were collected and that chemical compositions structure were drawn through the "Chemdraw" software,building the molecular formula and theoretical relative molecular mass database.UPLC-QTof-MS was used to analyze the neural-related effective components of Eerdun Wurile.2.Study of gene expression regulation by Eerdun Wurile:A total of 110 Wistar rats(body weight 220±20 g)were randomly divided into 7 groups:blank group(NT),model group(IS),nimodipine group(NI),Eerdun Wurile low-dose group(EWD),Eerdun Wurile high dose group(EWG),N8 low dose group(N8D),N8 high dose group(N8G).The rat model of middle cerebral artery occlusion/reperfusion(MCAO/R)wasestablished by Zea-Longa suture method.After 90 minutes of cerebral ischemia,the torsion was removed from the artery and started reperfusion.Treatment:After 24 hours of cerebral ischemia,the rats were continuously injected with distilled water,Nimodipine(positive control)and EW by oral administration once a day for 2 weeks.The doses for each group were as following:NT and IS(1 ml/100 g,distilled water);NI group(4 mg/100 g,Nimodipine);EWD group(61.7 mg/100 g,EW);EWG group(123.4 mg/100 g,EW);N8D group(61.7 mg/100 g,N8)and N8G group(123.4 mg/100 g).After 2 weeks(day 15),the experimental observations were performed.(1)The neurologic status of each rat was evaluated carefully 24 h after surgery or after treatment;(2)TTC staining was observed to investigate the ischemic condition;(3)Brain tissue of rats were stained with HE to observe morphological changes of the neurons and glia cells;(4)Total RNAs from the cerebral cortex of rat MCAO models treated with either EW or distilled water(NT)were extracted and analyzed by transcriptome sequencing.Differentially expressed genes were analyzed for their functions during the recovery of ischemic stroke.(5)The expression level of significantly differentially expressed genes such as insulin-like growth factor 2(Igf2),microglia marker iba-1 and neuron marker NeuN growth factors in the lesion was further validated by RT-qPCR and Immunofluorescence technique.(6)Liver toxicity was investigated through hepatic tissue HE staining,and measuring serum AST and ALT levels.Results:1.UPLC-QTOF-MS analysis:A total of 16 active compounds were detected from the Eerdun Wurile component.2.Neurological function score(Bederson scale):Before the treatment,the neurological deficits in the model group were significantly(P<0.05);after 2 weeks of treatment,Bederson scores in each group were significantly lower than those in the model group(P<0.05);There was no significant difference between the administration groups(P>0.05).3.The results of TTC staining showed that there was no pale tissue in the brain tissue of the blank group,and the pale white tissue of the model group was obvious,which was more significant than other administration groups.4.The results of HE staining showed that in the blank group,the cerebral cortex was normal,and the cell morphology,structure,arrangement,and cell density were all normal,and no obvious softening lesions and slight reduction of nerve cells were observed.Model group:The cerebral cortex lesions were pale in color and distinctly bordered with the surrounding boundaries.The cytoplasm was concentrated and red stained,the nucleus was pyknotized as a triangle,the interstitial softening was evident,the cell density was decreased,the distribution was more sparse than normal tissue,and the arrangement was irregular and edema.Obviously loose.Compared with the model group,cytotoxicity,cytoplasmic nucleus pyknosis,interstitial softening and sparseness of the cytoplasm of each administration group were not obvious.In the EWG and N8G groups,the nerve nerve cell injury density decreased significantly.The results of HE staining of liver tissue in each group showed that there was no obvious abnormality in the liver tissue of each group.The results of serum AST and ALT levels showed that serum AST levels were normal in all groups.The ALT content in the nimodipine group was significantly higher than in the other groups(P>0.05).5.RNA-seq analysis showed that compared with the model group,the Eerdun Wurile group rats up-regulated 186 genes in the cerebral cortex,such as Igf2,Igfbp,Tgf-bl Grn,Vim,microglia markers CD68,Aifl,Csflr and complement Components C3,Clqa,etc.6.RT-qPCR results showed that comp,ared with the model group,mRNA expression of Igfl,Igf2,Grn Vim,and Igfbp2 in the cerebral cortex of the Eerdun Wurile group was significant.The expression of Igf2 is extremely significant.7.Immunofluorescence staining showed that Eerdun Wurile significantly increased the expression of Ig2 in neurons and microglia of cerebral ischemic region of rats compared with the model group.Conclusion:1.A total of 16 active compounds were detected in the Eerdun Wurile component,confirming that Eerdun Wurile’s formulation process maintains the integrity of most active compounds.2.Eerdun Wurile and N8 have neurological recovery,neuroprotection and nerve regenerationf u n cti o n..3.Neuroprotective mechanisms of Eerdun Wurile relay on its active components,which up-regulated a group of gene expression,including certain growth factors in microglia cells in the lesion,potentially prompting a M1 to M2 polarization of microglia.