Effect Of Sinomenine On The Treatment Of Ankylosing Spondylitis By Regulating The Function Of Group 3 Innate Lymphoid Cells

Object: In this study,we combined in vitro experimental studies to explore the number and function of Group 3 innate lymphoid(ILC3)cells in patients with Ankylosing Spondylitis(AS),and then to explore the correlation with the number of ILC3 and disease activities,and further elucidate the mechanism of Sinomenine regulating ILC3 function in the treatment of AS.Methods:1.Peripheral blood of 8 AS patients,8 healthy volunteers and 8 RA patients was collected and disease activity(ASDAS-CRP)of AS patients was recorded.Flow cytometry was used to analyze the correlation between the percentage of ILC3 in ILC in peripheral blood of AS patients and disease activity.2.Peripheral blood mononuclear cell(PBMC)was extracted from the peripheral blood of healthy volunteers and AS patients,and the functional differences of IL-17 A,IL-22 and IFN-γ secreted by ILC3 cells were analyzed by flow cytometry.3.The Celltiter-Glo ? chemiluminescence cell viability assay was used to detect the effect of Sinomenine(SIN)on the cell activity of MNK-3 and to define the intervention concentration range of experimental drugs in vitro.4.SIN was used to intervene PBMC of AS patients in vitro,and DMSO was added into blank control group.Flow cytometry was used to compare the ability of ILC3 cells to secrete cytokines IL-22,IL-17 A and IFN-γ between the two groups.5.ILC3 cell line model MNK-3 was cultured in vitro and divided into blank control group and drug treatment group.The cytokines IL-22,IL-17 A and IFN-γ in cell supernatant were detected by ELISA,and the m RNA expression levels of Rorc、Il22、Il23r、Ifng and Il17 a in cell lysate were detected by RT-q PCR,to compare the differences between the two groups.Results:1.The study found that the percentage of ILC3 in ILC in PBMC of AS patients was higher than that of healthy subjects(p< 0.001,n=8).Among them,the proportion of NKP44+ILC3 increased,the proportion of NKP44-ILC3 decreased,and the proportion of NKP44+ILC3/NKP44-ILC3 increased,which had statistical differences compared with healthy people and RA patients(p< 0.05,n=8).There was no significant difference in the percentage of ILC3 in ILC between RA patients and healthy subjects(p> 0.05,n=8).The proportion of ILC3 in PBMC of AS patients was positively correlated with disease activity(p < 0.05,n=8).Compared with healthy subjects,the proportion of CD45+ cells positive for IL-22,IL-17 A and IFN-γ in peripheral blood of AS patients was significantly higher(p<0.01,n=4);IL-22,IL-17 A and IFN-γ positive ILC1 and ILC3 cells were also significantly increased(p<0.05,p<0.05,p<0.001,n=4).2.Cell viability was evaluated by Celltiter-Glo ? photoluminescence assay.The results showed that activity of MNK-3 cells was not affected by SIN at lower concentration(0.1-1.0u M)(P > 0.05,n=3).The cell viability was significantly affected when the drug concentration exceeded 2μM(p< 0.05,n=3).3.Sin(1.0u M)was used to intervene PBMC of AS patients in vitro,and the blank control group was added with equal volume DMSO.Flow analysis results showed that the proportion of CD45+ immune cells capable of producing IL-22,IL-17 A and IFN-γ in SIN treatment group was significantly lower than that in control group(p<0.01,p<0.05,p<0.001,n=3),the proportion of ILC cells capable of secreting IL-22,IL-17 A and IFN-γ was significantly lower than that of the control group(p<0.05,p<0.05,p<0.01,n=3).4.The expression levels of IL-22,IL-17 A and IFN-γ in MNK-3 cells were analyzed by ELISA.The results showed that the cytokines IL-22(p<0.05,n=4)、IL-17A(p<0.05,n=4)and IFN-γ(p<0.01,n=4)expressed in MNK-3 cells treated with SIN were significantly higher than those treated with SIN;5.SIN(1.0 μm)was used to intervene MNK-3 cell lines in vitro,and q PCR was used to detect the expression levels of Ifng,Il17 a and Il22 as well as transcription factors Rorc and Il23 r.The results showed that:after SIN treatment,the m RNA expression levels of Ifng(p<0.001,n=3),Il17a(p<0.001,n=3)and Il22(p<0.001,n=3)were significantly decreased.The m RNA expression levels of transcription factors Rorc(p<0.001,n=3)and Il23r(p<0.01,n=3)were also significantly decreased.Conclusion:1.The level of ILC3 in PBMC of AS patients is higher than that of healthy subjects and RA patients.The ILC3 level was positively correlated with disease activity in AS patients.2.The levels of ILC3 secreting inflammatory cytokines IL-22,IL-17 A and IFN-γ in peripheral blood of AS patients were significantly higher than those of healthy controls,suggesting that ILC3 may be involved in the inflammatory response in the development of AS disease.3.SIN can inhibit the levels of IL-22,IL-17 A and IFN-γ secreted by ILC3 in the peripheral blood of AS and MNK-3,suggesting that SIN can reduce the inflammatory response in AS diseases by inhibiting the function of ILC3,thus achieving the effect of treating AS.4.SIN can inhibit the expression levels of transcription factors RORγt and IL-23 R in ILC3,suggesting that SIN may inhibit the activation and function of ILC3 by regulating the molecular mechanism of RORγt or IL-23 signaling pathway.