| Multiple sclerosis（MS）is an autoimmune disease of the central nervous system（CNS）characterized by neuronal inflammation and demyelination.MS is a multifactorial disease that involves genetic and environmental factors.It affects more than 2.3 million people around the world,mainly young people,especially women.The incidence rate is high,usually at the age of 18-40,and the recurrence rate and disability rate are high.It mainly damages cortex,optic nerve,spinal cord,brain stem and cerebellum.The etiology and pathogenesis of MS are still unclear,and it is believed that it is caused by an abnormal response of the immune system to one or more myelin antigens,which are formed after genetically susceptible individuals are exposed to an undeter-mined cause.Clinical changes include multifocal defects in the white matter of the brain and spinal cord,ultimately leading to myelination destruction（demyelination）of nerve fibers and damage to glial cells（oligodendrocytes）.This experiment provides experimental basis for exploring the pathogenesis of MS and searching for clinically safe and effective therapeutic drugs.As a widely used human MS model,experimental autoimmune encepha-lomyelitis（EAE）is a type of central nervous system inflammation mediated by CD4+T cells,and is associated with T helper cell 1（Th1）/T helper cell 17（Th17）cell infiltration and demyelination.Naive CD4+T cells can differen-tiate into Th1,Th2,Th17 cells,and regulatory T cell（Treg）subpopulations after being stimulated by specific antigens.The differentiation of Th17 cells is a complex process regulated by a series of transcription factors,including retinoic acid and RORγt and STAT3.Through the activation and phosphory-lation of STAT3 and RORγt.The downstream regulation of results in the differentiation of naive CD4+T cells into Th17 cells.Currently,research on the pathogenesis and clinical treatment of MS is mostly based on the explora-tion of EAE.This experiment successfully established an EAE model in C57-BL/6 female mice immunized with MOG35-55 to investigate the pathogenesis of MS/EAE.Micro RNAs（MiRNAs）are single stranded non coding small RNAs that are highly conserved,with a length of approximately 18-22 nucleotides.They can bind to 3’-UTRs of target genes and regulate gene expression at the post transcriptional level.It is considered a diagnostic marker in many diseases,including MS.Each miRNA can regulate the expression of hundreds of target genes,participating not only in cell growth and proliferation,but also in processes such as embryonic development,immune inflammation,and tumor growth.Central nervous system tissue analysis showed that compared to healthy subjects,approximately 50 miRNAs were upregulated and 30 mi-RNAs were downregulated in patients with multiple sclerosis.The imbalance of these miRNAs may lead to an imbalance between pro-inflammatory and anti-inflammatory processes,as their targets are related to the regulation of Th1/Th17 and Treg cell differentiation.Therefore,analyzing the dysregulation of miRNA and changes in its target gene expression levels may help to better understand the etiology of multiple sclerosis and provide new alternative methods for its diagnosis and treatment.MiR-485 is a regulatory factor for inflammation and fibrosis.Previous research reports have found that miR-485 expression is downregulated in CD4+T cells of MS patients.However,the regulatory effect of miR-485 on Th17 in EAE is still completely unknown.STAT3 plays an important role in the differentiation process of Th17 cells.In our study,it was found that the expression level of miR-485-3p in PBMCs of acute MS patients was downre-gulated compared to the normal control group.In PBMCs of acute MS patients,there was a negative correlation between miR-485-3p and STAT3levels,and miR-485-3p may play a role in the pathogenesis of MS.Based on the above literature,I propose a hypothesis that miR-485-3p may play a role in the pathogenesis of MS.MiR-485-3p may inhibit Th17 cell diffe-rentiation by targeting STAT3,thereby affecting the occurrence of inflam-matory demyelination in diseases such as MS and EAE.This study,starting from the extraction of human peripheral blood mononuclear cell,preliminarily explored the differential expression of miR-485-3p and Th17related cytokines and transcription factors in PBMC of MS patients in acute phase,and verified the regulatory effect of miR-485-3p on Th17 cell differentiation and EAE disease progression in vivo and in vitro through animal experiments.This experiment provides experimental basis for seeking current clinical treatment drugs for MS and exploring the pathogenesis of MS.Part One Expression analysis of miR-485、Th17 related cytokines and transcription factors in multiple sclerosisObjective:Based on the analysis of peripheral blood mononuclear cell（PBMC）extracted from MS patients,the differential expression of miR-485-3p,Th17 related cytokines and their transcription factor（IL-17A、RORγt、STAT3）and other related indicators was explored to lay a foundation for further exploring the role of miR-485-3p in the process of multiple sclerosis.Methods:1.Study subjects:1)MS patients:All patients included were diagnosed acute MS inpatients or outpatient patients who visited the Neurology Depart-ment of the Second Hospital of Hebei Medical University from October 2020to December 2021.The included patients were not treated with anti-inflam-matory or immunosuppressive drugs,and healthy controls matched in age and gender were selected.The control group was composed of normal physical examination personnel from the Health Examination Center of Hebei Medical University,and relevant clinical information was recorded and peripheral blood was extracted.2.Extraction of PBMC and RNA from healthy controls and MS patients:extract fresh PBMC from experimenters with human peripheral blood lymphocyte isolation solution,and extract total RNA with RNA isolation kit.3.Application of RT-qPCR method to detect miR-485-3p,Th17 related cytokines and their transcription factors（IL-17A、RORγt、STAT3）.The expression level of m RNA in PBMC of MS patients and healthy controls,and the results were analyzed.Results:1.Demographic and general data:A total of 32 subjects were included in this experiment.16 acute MS patients（including 14 females and 2 males）who met the standards,and 16 healthy controls matched in age and gender.The average age of the MS group was 42.29±13.50 years old,while the average age of the healthy control group was 41.39±11.57 years old.There was no statistically significant difference in age between the two groups（P>0.05）.The included MS patients have not received anti-inflammatory or immuno-suppressive treatment,and have no previous history of special medication.2.Using RT-qPCR method to detect the expression level of miR-485-3p in PBMC of acute MS patients.Statistical analysis showed that the level of miR-485-3p in the MS group was significantly lower than that in the healthy control group,and there was a statistical difference between the two groups,P<0.01.3.Application of RT-qPCR method to detect the expression level of Th17 related cytokines and their transcription factors（IL-17A、RORγt、STAT3）m RNA in PBMC of acute MS patients.Statistical analysis showed that the level of（IL-17A,RORγt,STAT3）in the MS group were significantly higher than that in the healthy control group,and there was a statistical difference between the two groups,P<0.01.4.The level of miR-485-3p in PBMC of acute MS patients is negatively correlated with the level of STAT3.Conclusions:In this part of the experiment,peripheral blood mononu-clear cell of all subjects were extracted with human peripheral blood lympho-cyte isolate.The experimental results showed that compared with healthy controls,miR-485-3p expression was reduced in PBMCs of acute MS patients,while Th17 related cytokines and their transcription factor（IL-17A、RORγt、STAT3）expression were increased in PBMCs of acute MS patients.The levels of miR-485-3p and STAT3 were negatively correlated in acute MS patients,suggesting that miR-485-3p may become a new target for MS disease markers and treatment,laying the foundation for subsequent experiments.Part Two In vitro CD4+T cell transfection with miR-485 affects Th17 related cytokines and transcription factorsObjective:In this experiment,CD4+T cells were selected from spleen cells of EAE mice and transfected with mimics NC,miR-485-3p mimics,inhibitor NC,and miR-485-3p inhibitor,respectively,to investigate the effects of miR-485-3p on Th17 related cytokines and transcription factors in vitro,provi-ding new ideas for studying the pathogenesis of EAE.Methods:1.Sorting and in vitro culture of CD4+T cells from the spleen of EAE mice:SPF grade female C57BL/6 mice aged 8-10 weeks from Beijing Weitong Lihua Experimental Animal Company were selected for EAE modeling.During the peak period of the disease,the spleen was removed and prepared into a spleen single cell suspension for spleen CD4+T magnetic bead sorting.2.CD4+T cell transfection:After 24 hours of activation with anti-CD3and anti-CD28,the selected CD4+T cells were transfected with miR-485-3p mimics,mimics NC,miR-485-3p inhibitor,and inhibitor NC,respectively.After 48 hours,the cells were collected.3.Detection of miR-485-3p expression level after transfection using RT-qPCR method.4.Detection of IL-17A、IL-17F、RORγt、STAT3 m RNA expression in mimics NC,miR-485-3p mimics,inhibitor NC,and miR-485-3p inhibitor groups using RT-qPCR method.5.Application of ELISA detection method to determine the content of IL-17A in the supernatant of transfected CD4+T cell culture.6.Application of Western blot technology to detect the protein expres-sion of RORγt、p-STAT3 in the mimics NC,miR-485-3p mimics,inhibitor NC,and miR-485-3p inhibitor groups.Results:1.Using the CD4+T cell positive selection kit,CD4+T cells were selected from the spleen cells of EAE mice.The purity of the selected CD4+T cells was determined,and CD4+T cells with a purity of over 90%were obtained,which can meet the needs of subsequent experiments.2.The RT-qPCR detection results showed that the miR-485-3p mimics group showed upregulation of miR-485-3p expression compared to the mimics NC group,and there was a statistical difference between the two groups,P<0.01;The expression of miR-485-3p was downregulated in the miR-485-3p inhibitor group compared to the inhibitor NC group,and there was a statistical difference between the two groups,P<0.01.3.The RT-qPCR detection results showed that the expression of IL-17A、IL-17F、RORγt、STAT3 were down regulated in the miR-485-3p mimics group compared to the mimics NC group,with a statistical difference between the two groups（P<0.05）;The expression of IL-17A、IL-17F、RORγt、STAT3were upregulated in the miR-485-3p inhibitor group compared to the inhibitor NC group,and there was a statistical difference between the two groups,P<0.05.4.The ELISA detection results showed that in the supernatant of trans-fected CD4+T cell culture,the content of IL-17A in the miR-485-3p mimics group decreased compared to the mimics NC group,with a statistical diffe-rence between the two groups（P<0.05）;The miR-485-3p inhibitor group showed an increase in IL-17A content compared to the inhibitor NC group,and there was a statistical difference between the two groups,P<0.05.5.Western blot analysis showed that the expression of RORγt、p-STAT3were down regulated in the miR-485-3p mimics group compared to the mimics NC group,with a statistical difference between the two groups（P<0.01）;The expression of RORγt、p-STAT3 were upregulated in the miR-485-3p inhibitor group compared to the inhibitor NC group,and there was a statistical difference between the two groups,P<0.05.Conclusions:In this experiment,CD4+T cell positive selection kit was used to select CD4+T cells from the spleen cells of EAE mice,and CD4+T cells with a purity of over 90%were obtained.The selected CD4+T cells were transfected with mimics NC,miR-485-3p mimics,inhibitor NC,and miR-485-3p inhibitor,respectively.The experimental results showed that the m RNA levels of IL-17A、IL-17F、RORγt and STAT3 were downregulated in the miR-485-3p mimics group;The m RNA levels of IL-17A、IL-17F、RORγt and STAT3 in the miR-485-3p inhibitor group were upregulated.The expression of RORγt and p-STAT3 proteins in the miR-485-3p mimics group were downregulated;The expression of RORγt and p-STAT3 proteins were upregulated in the miR-485-3p inhibitor group.The IL-17A content in the supernatant of transfected CD4+T cell culture decreased in the miR-485-3p mimics group and increased in the miR-485-3p inhibitor group.The experimental results showed that miR-485-3p inhibited the expression of Th17related cytokines and transcription factors in vitro.Part Three MiR-485 improves disease progression in experimental autoimmune encephalomyelitis（EAE）mice by regulating Th1/Th17 cells and their inflammatory factorsObjective:In this part,we constructed Lentivirus expression vectors（LV-con,LV-485,LV-anti 485）that can overexpress or inhibit miR-485-3p in EAE mice,and studied their effects on Th1,Th17 cells and inflammatory factors,and their therapeutic effects on EAE.To lay the foundation for further verifying the role of miR-485-3p in the MS/EAE disease process.Methods:1.Study subjects:The animals used in this experiment were purchased from 6-8 week old SPF grade female C57BL/6 mice from Beijing Weitong Lihua Company,weighing 20±2g.Feed the mice indoors at a temperature of24±2℃,and provide alternating light and dark for 12 hours to ensure sufficient feed and clean water source for the mice.2.Establishment of EAE model and neurological function scoring:Mix MOG35-55 peptide with complete Freund’s adjuvant（CFA）and a certain amount of tuberculin to achieve a final concentration of tuberculosis bacteria of 4mg/ml.Mix the mixture thoroughly to form an oil-in-water emulsion.Inject the emulsion（0.1ml per mouse）subcutaneously on both sides of the back spine of each mouse using the 4-point method,followed by intraperi-toneal injection of 0.5ml pertussis toxin（PTX）.After 48 hours,inject the same dose of pertussis toxin again to successfully establish an EAE model.Use the Jager method（5-point method）to score the neurological function of two groups of mice,record the incidence of each group of mice,and simul-taneously measure their body weight.3.Experimental grouping:After one week of adaptive feeding,they were randomly divided into a blank control group and an EAE group,with 10animals in each group,for testing various indicators.4.Mice for Lentivirus transfection were randomly divided into three groups after adaptive feeding for one week:empty virus control group（LV-Con）,mmu-miR-485-3p analog group（LV-485）,mmu-miR-485-3p inhibitor group（LV-anti 485）,and 2×107the recombinant Lentivirus vector of transformation unit infected 15 mice in each group.The three groups of mice established EAE models 7 days later.5.Sampling and specimen processing:1)Samples were taken around the21st day after immunization（peak onset period）,and the control group and EAE model group mice were anesthetized with chloral hydrate and euthanized.The spleen,brain,spinal cord,and lymph nodes of the mice were taken out,and 1ml of Trizol was added to the extracted samples.After lysis,the samples were stored in a refrigerator at-80℃for RT-qPCR.2)For the mice transfected with Lentivirus,on the seventh day after transfection with Lentivirus vector,three mice in each group were taken,and killed after intraperitoneal injection of chloral hydrate anesthesia.The spleen,liver,brain and spinal cord of the mice were quickly taken out,and the RT-qPCR samples were added with 1ml Trizol to fully lyse the tissues,and stored in the refrigerator at-80℃.3)Lentivirus transfected mice,at the peak of the disease,three mice in each group were taken,perfused with 10%chloral hydrate and normal saline through the left ventricle,perfused and fixed with 4%parafor-maldehyde,isolated the lumbar enlargement tissue of the spinal cord of mice,and immediately paraffin embedded for the preparation of paraffin sectioning,and subsequently prepared for HE and LFB staining.In addition,6 mice from each group were anesthetized and killed.The spleen,brain,spinal cord,and lymph nodes were quickly removed,frozen in liquid nitrogen,and stored in a-80℃refrigerator for protein and RNA extraction.The remaining mice were observed for disease progression until the 25th day.6.HE staining was used to observe the infiltration of inflammatory cells in the lumbar enlargement of the spinal cord,and LFB staining was used to observe the degree of spinal cord myelin sheath loss.According to the scoring criteria for inflammatory cell infiltration and LFB myelin loss,the mice in each group were scored and the results were statistically analyzed.7.Detection of miR-485-3p expression in the central nervous system and peripheral immune organs of EAE mice using RT-qPCR method.8.Detection of m RNA expression of inflammatory factor（RORγt、STAT3、IL-17、IFN-γ）in the central nervous system of EAE mice using RT-qPCR method.9.On the 7th day after virus injection,the expression of miR-485-3p in various organs was detected using RT-qPCR method to detect in vivo trans-fection efficiency.10.Three groups of mice transfected with Lentivirus were sampled at the peak of the disease,and the expression of Th1,Th17,Treg related cytokines and transcription factors in the brain of EAE mice infected with Lentivirus were detected by RT-qPCR.11.Western blot was used to detect the protein expression of RORγt、p-STAT3 in the brain of EAE mice infected with Lentivirus.12.Application of ELISA technology to detect the protein level of IL-17A in the brain of virus infected EAE mice.13.In the brain tissue of Lentivirus infected mice,the correlation bet-ween miR-485-3p and STAT3 was detected by RT-qPCR.14.Target Scan Mouse was used to predict the target gene of miR-485-3p,and Luciferase reporter gene analysis was used to verify the target gene of miR-485-3p.Results:1.Mouse incidence:The EAE group mice began to get sick on the 10th day after immunization,and the samples were taken at the peak on the 21st day.The average score reached 3 points,and the incidence rate was 100%.Among the three groups of mice infected with Lentivirus,LV-anti 485 group mice began to show symptoms on the 9th day after immunization,and LV-485group mice began to develop symptoms on the 12th day after immunization.The incidence rate of the three groups was 100%.2.Expression levels of miR-485-3p and inflammatory factors in EAE mice:The RT-qPCR detection results showed that the level of miR-485-3p in the EAE group significantly decreased compared to the control group in the central nervous system and spleen,with a statistical difference between the two groups（P<0.01）;During the peak period of the disease,the levels of RORγt、STAT3、IL-17A、IFN-γin the spinal cord and brain tissues of mice in the EAE group were significantly higher than those in the control group;There is a statistical difference between the two groups,P<0.05.3.MiR-485-3p improves neurological function score in EAE miceOn the 7th day after virus injection,RT-qPCR was used to detect the expression of miR-485-3p in various organs to detect in vivo transfection effi-ciency.The results showed that the content of miR-485-3p was highest in the liver.Approximately 1.5 fold expression was found in the brain and spinal cord of infected mice.Approximately 1.2 fold expression was found in the spleen.Mice infected with Lentivirus were immunized with MOG35-55on the 7th day after virus injection.We compared the neurological symptom scores bet-ween the three groups from the 3rd day to the 25th day after immunization.The results showed that the LV-anti 485 group began to develop symptoms on the 9th day after immunization,while the LV-485 group developed symptoms on the 12th day after immunization;The neurological function score of LV-485 group was lower than that of LV-Con group,and there was a statistical difference between the two groups（P<0.01）;The neurological function score of the LV-anti 485 group was higher than that of the LV-Con group,and there was a statistical difference between the two groups（P<0.01）.4.MiR-485-3p reduces spinal inflammatory cell infiltration and degree of demyelination in EAE miceDuring the peak period of the disease,HE staining and LFB staining were performed on the tissue of the spinal cord and lumbar enlargement,and the degree of inflammatory cell infiltration and myelin sheath loss were evaluated.The results showed that compared with the LV-Con group,the EAE mice in the LV-485 group had lighter levels of inflammatory cell infiltration and myelin loss,and decreased inflammation and myelin scores.There was a statistical difference between the two groups（P<0.05）;The EAE mice in the LV-anti 485 group showed more inflammatory cell infiltration,with sleeve like appearance around small blood vessels.The degree of myelin loss was severe,and the inflammation and demyelination scores increased.There was a statistical difference between the two groups（P<0.05）.5.The regulatory effect of miR-485-3p on Th1/Th17/Treg cells in EAE miceThe brain tissue of the three groups of mice transfected with Lentivirus was taken out at the peak of the disease.RT-qPCR detection results showed that compared with the mice infected with LV-Con,the RORγt、STAT3、IL-17A、IL-17F、IFN-γm RNA level of the mice infected with LV-485 were significantly reduced,and there was a statistical difference between the two groups,P<0.05,Foxp3 was significantly increased,and there was a statistical difference between the two groups,P<0.05;The RORγt、STAT3、IL-17A、IL-17F、IFN-γm RNA levels in mice infected with LV-anti 485 group were significantly increased,with a statistical difference between the two groups（P<0.05）,and Foxp3 was significantly reduced.There was a statistical difference between the two groups（P<0.05）.The brain tissue of the three groups of mice transfected with Lentivirus was taken out at the peak of the disease.Western blot test results showed that compared with the mice infected with LV-Con,the RORγt、p-STAT3 protein content of the mice infected with LV-485 were significantly reduced,with a statistical difference between the two groups（P<0.05）,while the RORγt、p-STAT3 protein content of the mice infected with LV anti 485 were increased,with a statistical difference between the two groups（P<0.05）.During the peak period of the disease,samples were taken and ELISA technology was used to detect the protein level of IL-17A in the brain of EAE mice infected with the virus.Analysis of statistical results showed that compared with the mice infected with LV-Con group,the protein content of IL-17A in mice infected with LV-485 significantly decreased,with a statistical difference between the two groups（P<0.05）,while the protein content of IL-17A in mice infected with LV-anti 485 increased,with a statistical difference between the two groups（P<0.05）.6.By applying Target Scan Mouse,it was found that the action site of miR-485-3p targeting STAT3 is located in the 644-650 region of the 3’-UTR end.7.The 3’-UTR of miR-485-3p targeting STAT3 m RNA in HEK-293T cells was detected by Luciferase reporter gene analysis.Compared with the control simulant,Luciferase activity was significantly reduced.Co trans-fection with miR-485-3p mimetics did not affect the MUT 3’-UTR carrier.In addition,miR-485-3p inhibitor and wt 3’-UTR vector were co transfected into HEK-293T cells,which significantly increased Luciferase activity.In HEK-293T cells,miR-485-3p has a targeted relationship with STAT3.8.In the brain tissue of Lentivirus infected mice,we found that the expression level of miR-485-3p was negatively correlated with the expression level of STAT3.MiR-485-3p inhibits Th17 cell differentiation by inhibiting the expression of the target gene STAT3 in vivo,slowing down the progression of EAE mice.Conclusions:In this part of the experiment,C57BL/6 female mice were immunized with MOG35-55to establish an EAE model,and Lentivirus expres-sion vectors（LV-con,LV-485,LV-anti 485）that can overexpress or inhibit miR-485-3p in EAE mice were constructed.It was found that miR-485-3p was negatively correlated with the level of STAT3,which further confirmed that STAT3 was the target gene of miR-485-3p.miR-485-3p inhibited the differentiation of Th17 cells by inhibiting the expression of target gene STAT3 in vivo,and slowed down the pathogenesis of EAE mice.The results of this study further reveal the immune regulatory role of miR-485-3p in EAE and provide new targets for disease treatment. |
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