| Chapter 1 transcriptome analysis of peripheral blood mononuclear cells in patients with cognitive impairment after ischemic strokeObjective:With the help of single-cell transcriptome sequencing technology,we screened the transcriptome characteristics of peripheral blood mononuclear cells in patients with cognitive dysfunction after ischemic stroke,and provided research clues for clarifying the pathogenesis of cognitive dysfunction in patients with ischemic stroke.Methods:the patients were randomly divided into two groups.12 patients with ischemic stroke were collected for single-cell sequencing of peripheral blood mononuclear cells,including 6 in the experimental group(ischemic stroke with cognitive dysfunction)and 6 in the control group(ischemic stroke without cognitive dysfunction).Peripheral blood was collected,mononuclear cells were extracted,single-cell transcriptome library was built,and single-cell RNA sequencing was performed:Single cell bioinformatics analysis of each cell subpopulation,identification and naming;Differential gene analysis was performed on the cell subsets of the two groups to explore the transcriptome characteristics of peripheral blood mononuclear cells in patients with cognitive impairment after ischemic stroke.Results:In this study,there is no difference in gender,age,and key clinical laboratory tests between the selected patients,indicating comparability between the two groups.We performed single-cell RNA sequencing on PBMCs from enrolled patients,and a total of 18691 effective cells were obtained after quality control.Cluster analysis of effective cells identified nine different cell clusters,each consisting of as few as 59 cells to as many as 9423 cells;Through differential screening,we obtained the marker genes of each cell cluster,and selected the top 20 up-regulated marker genes to draw a heat map.The results showed that each cell cluster had its specific high expression genes,which could be annotated according to these cell specific marker genes;Combined with the cell specific marker genes reported in the literature,nine cell clusters were annotated into eight categories of cells after comprehensive evaluation,including cd4+T cells,cd8+ T cells,monocytes,B cells,Treg,DCS,pDCs and NK.Taking p-value<0.05 and logfc absolute value>0.25 as the screening conditions,we screened the differential genes of Treg cells,and recorded the demerit and screened 103 differential genes,including CTLA-4,rp19,rp132,snhg5,cotll,traf3ip3,RPL29,rps3ap47,FOS,gbp5,junB.Conclusion:1)Compared with the control group,there were transcriptome differences in multiple cell subsets in peripheral blood of patients with cognitive dysfunction,which may be related to cognitive dysfunction;2)The abnormal expression of CTLA-4 in Treg cells in the experimental group may be involved in the pathogenesis of cognitive dysfunction in patients with ischemic stroke.Chapter Ⅱ clinical study of Naosuikang in reducing inflammatory reaction and improving cognitive impairment in patients with ischemic strokeObjective:To observe the clinical efficacy of Naosuikang and explore the pharmacological mechanism of naosui Kang in improving cognitive dysfunction;Objective to clarify the correlation between inflammation and cognitive dysfunction,and explore the clinical value of Treg cell CTLA-4.Methods:the patients were randomly divided into two groups.In this study,patients with cognitive dysfunction after ischemic stroke were divided into experimental group(naosuikang treatment group)and control group(conventional treatment group).Treatment plan:the experimental group was given naosuikang capsules orally on the basis of conventional treatment,taking 3 capsules each time,three times a day,for>6 months.The control group received routine treatment according to clinical guidelines.Patients in both groups were required not to take any other anti-inflammatory Chinese and Western medicine and other treatment measures.After the treatment,the clinical efficacy of the two groups was compared,including MMSE score,ss-qol score,MOCA score and TCM syndrome score;The inflammatory related factors of the two groups were detected:IL一10,TGF-β 1 and hsCRP,and the expression of CTLA-4 in peripheral blood Treg cells was analyzed.Results:In the initial experimental group of this study,65 cases were included,7 cases were lost to follow-up,including 1 case complicated with pneumonia,1 case changed the treatment plan due to other diseases,and finally 58 cases were included;In the control group,65 cases were lost to follow-up,of which 1 case was treated with antibiotics due to pneumonia,4 cases changed the treatment plan,and 55 cases were finally included.No adverse drug reactions occurred in the two groups except for definite disease-related complications.113 patients were included in this study,including 58 patients in the experimental group,including 32 males and 26 females;55 patients in the control group,including 33 males and 22 females;The average age of the experimental group was 68.16±7.77 years,and that of the control group was 65.27±15.33 years.There were no significant differences in gender,age,history of diabetes,drinking history and routine laboratory tests between the two groups,P>0.05,and the two groups were comparable.The clinical research results showed that:1、The NIHSS scores of the experimental group and control group before this treatment were 24.76 ± 2.04 and 24.33 ±2.27,respectively.After treatment,the NIHSS scores of the experimental group and control group were 15.38±1.71 and 18.25±2.38,respectively;The t-test results of the two groups of samples before treatment showed no statistical difference,P>0.05;After treatment,the t-test results of the two groups of samples showed statistical differences,P<0.05;The paired t-test results before and after treatment in the two groups showed that the NIHSS score in the experimental group decreased significantly before and after treatment,with a statistical difference of P<0.05.2、The MMSE scores of the experimental group before and after treatment were 19.43 ± 2.87 and 22.74 ± 2.07,respectively,while the MMSE scores of the control group before and after treatment were 19.24 ± 2.52 and 21.13 ± 2.59,respectively.After two independent samples t-test,the MMSE scores before and after treatment between the two groups were significantly different,P<0.05;The P values of MMSE scores before and after treatment in the experimental group and the control group by paired t-test were 0.703 and 0.035,respectively,suggesting that the MMSE score in the experimental group was significantly reduced after treatment,with statistical difference between the two groups,P<0.05.3、The ss-qol scores of the experimental group before and after treatment were 111.38 ± 13.77 and 118.24 ± 12.37,respectively,while the ss-qol scores of the control group before and after treatment were 110.44 ± 11.50 and 114.65 ± 9.34,respectively.By paired t test,the ss-qol scores of the experimental group and the control group before and after treatment were significantly different,P<0.05;The P values of ss-qol scores before and after treatment in the experimental group and the control group were 0.693 and 0.084.respectively,with no statistical difference between the two groups.P>0.05.4、The TCM syndrome scores of the experimental group before and after treatment were 12.62 ± 1.51 and 9.29 ± 2.06,respectively,and those of the control group were 12.24 ± 1.55 and 10.07± 1.82,respectively.There were significant differences between the TCM syndrome scores of the two groups before and after treatment,P<0.05;The P values of TCM syndrome scores before and after treatment in the experimental group and the control group were 0.185 and 0.036 after paired t-test,respectively,suggesting that there was no difference in TCM syndrome scores between the two groups before treatment,and the TCM syndrome scores in the experimental group decreased more significantly after treatment,P<0.05.5、The levels of IL-10 in the experimental group before and after treatment were 882.74 ± 78.67pg/ml and 991.29± 72.36pg/ml,respectively-β The hsCRP was 3.79 ±0.61ug/ml and 3.61± 0.38ug/ml,respectively;Paired t-test was performed before and after treatment in the experimental group,and the results showed that IL-10,TGF-β The difference was statistically significant,P<0.05;The difference of hsCRP was statistically significant,P<0.05.6、The levels of IL-10 in the control group before and after treatment were 860.58 ±96.42pg/ml and 957.24±96.08pg/ml,respectively-β 58± 49.69pg/ml,and hsCRP was 3.76 ± 0.45ug/ml and 3;Paired t-test was performed in the control group before and after treatment,and the results showed that IL-10 and TGF-β The difference was statistically significant,P<0.05,and there was no statistical difference in hsCRP,P>0.05.7、Before and after treatment,the experimental group and the control group underwent independent sample t-test for inflammatory factors,and the results showed that IL-10,TGF-β There was no difference in IL-10 and TGF between the two groups before treatment-β 1 after treatment,the level of the experimental group was higher than that of the control group,with statistical difference,P<0.05,and there was no statistical difference between the two groups after hsCRP treatment,P>0.05.8、The paired t-test results in the experimental group and the control group showed that the expression of CTLA-4 in the two groups increased after treatment,and there was a significant statistical difference compared with that before treatment,P<0.05;There was no statistical difference between the experimental group and the control group before treatment,and the expression of CTLA-4 in the experimental group was higher than that in the control group after treatment,P<0.05.1)Naosuikang can improve cognitive function after ischemic stroke and reduce TCM syndrome score;2)Naosuikang has certain anti-inflammatory ability,and can improve the expression level of CTLA-4 on Treg cells in the blood of stroke patients,which can reduce the cognitive dysfunction of ischemic stroke and is related to its anti-inflammatory function. |
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