| Part Ⅰ: The vitro study on in vitro inhibition of IL-21 / IL-21 R mediated inflammatory response in rheumatoid arthritisObjectvive: To fully explore the regulation of rheumatoid arthritis immune response process by targeting IL-21,determine the mediating relationship between it and IL-21 / IL-21 R and JAK1/STAT3 signaling pathway in the regulation process,determine the value of chuanchen in the occurrence and development of rheumatoid arthritis,and provide targeted research guidance for the later clinical treatment.Materilals and Methods: We use RASFs cell line,namely MH7 A cells,which were obtained from the patient with RA and considered to be the main effector cells in RA.MH7 A cell lines were cultured in 1640 supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37°C,which were divided into Control group(without treatment).IL-21 group(50 mg/ml IL-21 treated RA cells);Il-21 +Nobiletin 25 micron group(50 ng/ml IL-21 + 25 micron mol /L Nobiletin treated RA cells);IL-21 +Nobiletin 50 micron group(50 ng/ml IL-21 + 50 micron mol /L Nobiletin treated RA cells);After the intervention of RA cells,the related items were analyzed after 48 hours of continuous culture: Cytokine analysis: expression levels of il-6,tnf-alpha,HMGB1,mmp-3 and mmp-13 in each group were determined by ELISA.ROS(reactive oxygen species)of RA cells in each group were detected.RA cells in each group were detected by 4-hne(4-hydroxynonenal).RT-PCR was used to detect m RNA expression concentrations of key proteins in IL-21 R,TNF-α,IL-6,mmp-3 and mmp-13.Western-blot analysis of key protein expressions in IL-21 R and JAK1/STAT3 signaling pathways.Statistical data,give the conclusion.Results: ELISA: TNF-α,IL-6: the expression trend of RA cells in each group was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.HMGB1 protein: the expression trend of RA cells in each group was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.MMP-3 and MMP-13: the expression trend of RA cells in each group was Control< IL-21 +Nobiletin 50 microns < IL-21+Nobiletin 25 microns < IL-21.4-HNE detection: the expression trend of RA cells in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.ROS detection: the expression trend of RA cells in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.RT-PCR detection: IL-21R: the m RNA expression trend of RA cells in each group was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21;TNF-α,IL-6: the expression trend of RA cells in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21;MMP-3 and MMP-13: the expression trend of RA cells in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.Western-blot detection: IL-21R: the expression trend of RA cells in each group was Control< IL-21 +Nobiletin 50 microns<IL-21+Nobiletin 25 microns < IL-21;JAK1/STAT3 signaling pathway: there was little difference in the expression of JAK1 and STAT3 proteins in RA cells in each group(P>0.05,the difference was not statistically significant).In the process of phosphoric acid expression,the expression trend of RA cells in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.Conclusions:(1)Nobiletin can target IL-21 receptor to make immune response to RA cells,fully reduce the concentration changes of interleukin and MMP in RA cells,reduce the inflammatory process of RA cells,and promote the repair process of RA cells.With the increase of Nobiletin concentration,this trend is more obvious.(2)Nobiletin can target IL-21 receptor to regulate the response of the classical signaling pathway JAK1/STAT3,reduce the phosphorylation level of the key proteins JAK1 and STAT3 in the signaling pathway,and reduce the inflammatory process,which is consistent with the above conclusion.It can be concluded that Nobiletin may have a good research market in the occurrence and development of rheumatoid arthritis,providing a targeted research orientation for the later clinical treatment.Part Ⅱ: In vivo study on the inhibition of IL-21 / IL-21 R mediated inflammatory response in rheumatoid arthritisObjective: The basis of the research on the in vitro and in vivo studies to determine sichuan dried tangerine or orange peel,targeted IL-21 regulate immune response process of rheumatoid arthritis,determine its and IL-21 / IL-21 R and JAK1/STAT3 signaling pathway in the process of regulation mediated relationship,determine the sichuan dried tangerine or orange peel element in rheumatoid arthritis and value,in the process of development for the clinical treatment of late targeted research orientation.Materials and Methods: After 1 week of adaptive feeding,48 Wistar rats were randomly divided into 4 groups,with the exception of control rats,and the rest were established as CIA model.Specific groups were as follows: Control group(rats did not do treatment);IL-21 group(CIA model +50 ng/ml IL-21,intraperitoneal injection);IL-21 +Nobiletin 25 micron group(CIA model +50 ng/ml IL-21 + 25 micron mol /L Nobiletin,intraperitoneal injection);IL-21 +Nobiletin 50 microns(CIA model +50 ng/ml IL-21 +50 microns /L Nobiletin,intraperitoneal injection);After feeding for 6 weeks after immunization,the rats were initially dead,and relevant items were analyzed: the success rate of each group of rats was tested: general observation,body mass index,arthritis and joint index were analyzed.Morphological analysis: HE staining analysis of rats in each group;ELISA assay: IL-21 R,TNF-alpha,IL-6,HMGB1,MMP-3 and MMP-13 proteins.RT-PCR was used to analyze the m RNA expressions of IL-21 R,TNF-alpha,IL-6,MMP-3 and MMP-13.Western-blot analysis of IL-21 R,TNF-alpha,IL-6,MMP-3,MMP-13 and JAK1/STAT3 pathways was conducted to analyze the data and draw conclusions.Results: Test of the success rate of the model: clinical manifestations: the rats in the control group were in good mental state,showed a steady increase in body weight,and had shiny hair.The rats in the model group were obsessed with their will,and their food intake was gradually reduced.Their hair was shiny.After 2 weeks,they were unable to bear weight.Weight change: after two weeks,the weight change trend of the rats in each group was IL-21< IL-21 +Nobiletin 25 microns <IL-21 +Nobiletin 50 microns < Control.Arthritis incidence/joint index: Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21,the difference was statistically significant(P<0.05).Morphological analysis: HE staining: the anklebone tissue cells in the control group were densely arranged,with regular morphology and obvious rotunda.In the model group,the cell layer became thicker and the leukocyte and lymphocyte became more abundant and less continuous.After the intervention of hesperidin,the tissue cells of the ankle joint of the rats were densely arranged and strengthened,and the morphology of the cells tended to be normal.ELISA: TNF-α,IL-6: the expression trend was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.HMGB1 protein: the expression trend was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.MMP-3 and MMP-13: the expression trend was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.RT-PCR detection: IL-21R: the m RNA expression was Control< IL-21 +Nobiletin 50 microns < IL-21 + Nobiletin 25 microns < IL-21;TNF-α,IL-6: the expression trend was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21;MMP-3 and MMP-13: the expression was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.Western-blot detection: IL-21R: the expression trend in each group was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21;TNF-α,IL-6: the expression trend was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21;MMP-3 and MMP-13: the expression trend in each group was was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.JAK1/STAT3 signaling pathway: there was little difference in the expression of JAK1 and STAT3 proteins in each group(P>0.05,the difference was not statistically significant).In the process of phosphoric acid expression,the expression trend in each group was Control< IL-21 +Nobiletin 50 microns < IL-21 +Nobiletin 25 microns < IL-21.Conclusions:(1)Pericarpitin can improve the arthritis effect of rats in the model group,fully improve the mental state of rats,fully reduce arthritis and joint index.Provide help for the follow-up research.(2)There are consistent with the results in vitro,nobiletin improve rats main rheumatoid arthritis by targeting the body internal IL-21 R concentration change and regulating the JAK1 / STAT3 activity state,sufficient to reduce interleukins,tumor necrosis factor,high mobility of protein expression and gradually restore the articular cartilage synthesis and decomposition mechanism of balance,accelerate the recovery of disease,rheumatoid arthritis,reach the cure effect of arthritis,give clinical must be inspired. |
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