| ObjectiveThe effect of Yiqi Huoxue Principle formula on the proliferation and differentiation of nueral stem cells in rat model of MCAO-R by JAK-STAT signaling pathway, through experimental study of the treatment of Rat MCAO-R model by Yiqi Huoxue formula Naoluoxintong, focuses on the mechanism of Yiqi Huoxue Principle in cellular and molecular level through the multi levels research in many ways to further improve the biological regulatory network in Yiqi Huoxue formula Naoluoxintong.Method.1.The middle cerebral artery occlusion and reperfusion (MCAO-R) rat model is established by thread occlusion . Observe and evaluate MCAO-R model from the levels of integration,system, organ, tissue, cell and molecule . The methods include: observed and recorded the changes of physiological signs and the weight of rats at preoperation and postoperation in different groups, According to the score of the neural function defect in MCAO-R rat model at 5h after the beggining of ischemia, to evaluate neurological deficit of rats. Removed the model rats'brains at 1d and 7d , observe the ischemic condition of middle cerebral artery blood supply area in model rats'brain tissue by TTC staining,calculate the ischemic area percentage, verification the rat models. The frozen sections of rat hippocampal slices were stained by Nissl, in order to observe the formation changes of pathological morphology in hippocampal Vertebral layer of MCAO-R model rats,2.Observe and evaluate the effection of Yiqi Huoxue formula Naoluoxintong, through the levels of integration, system, organ, tissue, cell and molecule. The methods include:observed and recorded the changes of physiological signs and the weight of rats at preoperation and postoperation in different groups,According to the score of the neural function defect in MCAO-R rat model at 1d,3d and 7d after the beggining of ischemia,to observe and evaluate the recoverment of neurological deficit of rats. Observe the ischemic condition of middle cerebral artery blood supply area in model rats'brain tissue by TTC staining at 1d,3d and 7d, calculate the ischemic area percentage,verification the rat models though statistical analysis. The paraffin sections of rat hippocampal slices were stained by HE, in order to observe the formation changes of pathological morphology in hippocampus of MCAO-R model rats.3.Verified by immunofluorescence staining to detect the expression of nestin, NF-H, GFAP,Gc protein, observe neural stem cell proliferation and differentiation in rats hippocampus at 7th day afer operation. Screening differential expressed genes (DEGs) in JAK-STAT pathway by the signaling pathway of PCR gene chip on the level of Fold-Change value >2 or <-2 .Using Western-Blotting to test and verify differentially expressed gene FCGR1 alpha,PRLR,IFGNR1,SOCS1 in protein levels.Results1.MCAO-R rat models with Qi deficiency and blood stasis symptoms have been lossing weight during the first 3 days after operation, and began to increase gradually since the 4th day.Compared with Normal group and Sham group, the weight index are significant differences among the groups,there are statistic meaning on the 1,2,4,5 ,6,7 day(P?0.05 ) ,and on the 3 day (P?0.01) . The neural function defect is obvious in Model group, there are significant differences compared with Sham group. Select the 1-3 scores of rats into the following experiment.The area of brain tissue stained by TTC is largest at 5h after molding operation, and it gradually decreased with timecompared with the Sham group,there are significant differences between the Model group and the Sham group at every point in time(P<0.001) .The patholagical changes are observed significantly in rat brain hippocampus through NISSL staining.2.Compared with the model group, the symptoms of qi deficiency and blood stasis in the MCAO-R rats are improved faster in the YQHX group. The weight of YQHX group rats was increasing in the 7 days after molding operation,it began to increase from the 3rd day,compared with Model group and Sham group,there are significant differences among the 3 groups on the 1st and 3rd day (P?0.05) . The neural function defect score are is obvious in YQHX group, there is no significant differences compared with Model group. Select the 1-3 scores of rats into the following experiment.TTC staining displays that,at the 7th day after molding operation,the ischemic areas of brain in YQHX group reduce significantly,compared with Model group rats, there are significantly differences(P =0.002<0.01). The patholagical changes are observed significantly in rat brain hippocampus through HE staining. In summary,Yiqi Huoxue Principle decoction Naoluoxintong maybe improve neurons proliferate,migrate,and regulate gial differentiate .3.To detect the expression of nestin, NF-H, GFAP, Gc proteins by immunofluorescence staining, the significantly increase of positive expression of nestin, NF-H, GFAP, Gc proteins are observed at the 7th day after molding operation, and the expression level of protein was different in each region of hippcampus . The differential expressed genes(DEGs) in JAK-STAT pathway, which are screened by the signaling pathway of PCR gene chips, on the level of Fold-Change value >2 or <-2, are FCGR1?, PRLR, IFGNR1, SOCS1,IL10ra. Using Western-Blot to test and verify differential expressed genes FCGR1?, PRLR,IFGNR1, SOCS1 in protein levels.Conclusion1 .The MCAO-R rat model was successfully established by the method of thread embolism,which provided a reliable basis for the following research?2.It promotes the recovery of the whole function of MCAO-R rats, and the recovery of the neurologic impairment,promote the cerebral ischemia area decreased, by gavaging with a medium dose of Naoluoxintong decoction of Yiqi Huoxue Principle for 7 days.3.The effect of Naoluoxintong decoction of Yiqi Huoxue Principle maybe could promotes the proliferation and differentiation of neural stem cells in hippocampus of rats, and promotes the recovery of neurological function after cerebral ischemia, by inhibit the expression of PRLR protein in JAK-STAT signal pathway of MCAO-R rat model. |
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