| Part I Correlation between fractalkine and regulatory T cells in patients with systemic lupus erythematosusObjective: To detect the expression level of serum Fractalkine(FKN)and the distribution of regulatory T cells(Tregs)in peripheral blood in patients with Systemic lupus erythematosus(SLE)and healthy controls,and analyze the correlation between FKN and Treg cell level and the activity of SLE disease.To investigate the role of FKN and killer cells in the disease preliminarily.Methods: 31 patients with SLE diagnosed for the first time in the department of Nephrology Affiliated Hospital of Youjiang Medical University for Nationalities and 11 healthy people were selected.The disease activity of SLE patients was evaluated according to the systemic lupus erythematosus disease activity index(SLEDAI-2K).All subjects were divided into 3groups: healthy control group,SLE inactive group and SLE active group.The serum level of FKN in each group was detected by enzyme-linked immunosorbent assay(ELISA),and the number of Treg cells in peripheral blood of each group was analyzed by flow cytometry(FC),and the differences between the groups were compared.The correlation between FKN and Treg cells and the activity of lupus disease was analyzed by Spss23.0 statistical software.Results: Compared with the healthy control group,the serum expression level of FKN was increased significantly in the inactive group and the active group(P < 0.01),and the distribution of Treg cells was decreased significantly(P < 0.01);the serum expression level of FKN in the SLE active group was significantly higher than the SLE inactive group(P < 0.01),and the number of Treg cell was significantly lower than the SLE inactive group(P < 0.01).The expression of FKN in SLE was positively correlated with SLEDAI-2K and negatively correlated with distribution of Treg cell.Conclusion: The serum expression level of FKN in SLE patients increased with Treg cell down-regulation,FKN participated in the pathogenesis of SLE.Part II Study on the expression level of fractalkine and p38 MAPK and the distribution of Treg cells in lupus nephritis miceObjective: The expression level of Fractalkine,p38 Mitogen-activated Protein Kinase(p38MAPK)and Foxp3 in serum and the distribution of Treg cells in peripheral blood of lupus nephritis(LN)mice were detected.Compared with normal mice,the change of the expression level of Fractalkine,p38 MAPK and Foxp3 and the distribution of Treg in peripheral blood were studied.Methods: the lupus nephritis mice model was established by intraperitoneal injection of0.5ml pristane to BALB/c mice.After 12 weeks,the content of urine protein,the levels of serum antinuclear antibody and the change of renal tissue were observed to identify the successful construction of the model.Then,the mice were divided into two groups: normal group and model group.The distribution of Treg cells in peripheral blood of mice was analyzed by flow cytometry,the levels of fractalkine and Foxp3 in serum were detected by ELISA,and the expression levels of fractalkine and Foxp3 in renal tissue were detected by immunofluorescence(IFC),Western-blot and real time PCR(q RT-PCR).Results: after 12 weeks of intraperitoneal injection of pristane,compared with the control group,the content of 24-hour urine protein and the levels of serum antinuclear antibody of the model mice were increased significantly(P < 0.01).The results of renal histopathological showed that the glomerulus of the model group mice had different degrees of damage,and the identification of LN mice model was successful.Compared to the control group,the distribution of Treg cells in LN group was decreased significantly(P < 0.01).The expression level of fractalkine in serum was increased significantly,while the expression level of Foxp3 was decreased significantly(P<0.01).The expression levels of fractalkine and phosphorylated p38MAPK(p-p38AMPK)in renal tissue were increased significantly,while the expression level of Foxp3 was decreased significantly(P < 0.01).However,there was no significant change in the expression of phosphorylated p38MAPK(P > 0.05).Conclusion: Fractalkine may be involved in the pathogenesis of lupus nephritis through p38 MAPK signal pathway regulation of Treg cells.Part III Fractalkine regulates apoptosis of Treg cells in lupus nephritis through p38 MAPK signaling pathwayObjective: the key point of this study was fractalkine(FKN),and the mechanism of FKN in Treg cells was studied from the cellular level.By observing the expression level of FKN,p38 MPAK and Foxp3 in LN mice Treg cells after knockdown of FKN gene in vitro,and the specific mechanism of U-46619 and SB203580(U-46619 is the activator of p38 MAPK,SB-203580 is the inhibitor of p38MAPK)on LN mice Treg cells,to explore the role of p38 MAPK signal pathway in the process of Treg cell apoptosis and the specific mechanism of FKN on p38 MAPK signal pathway,and to further understand the mechanism of FKN in Treg cells,for the diagnosis and treatment of lupus nephritis(LN)provides a theoretical basis.Methods: FKN knockdown LN Treg call was achieved by transfection of the adenovirus vector particle-FKN.The cells were randomly divided into 7 groups:(1)normal control group;(2)negative control group;(3)FKN-KD group;(4)U-46619 group;(5)SB203580 group;(6)FKN-KD + U-46619 group;(7)FKN-KD + SB203580 group.Western blot was used to verify the successful transfection of FKN knockdown;Flow cytometry was used to detect the apoptosis rate of cells in each group;Western blotting and q RT-PCR were used to detect the Foxp3,p38 MAPK signal pathway and the expression level of apoptosis related factors in each group.Results: The phosphorylation level of p38 MAPK was decreased significantly and the expression of Foxp3 was increased significantly after knocking down FKN.U-46619 promoted the phosphorylation of p38MAPK(P < 0.01),down regulated the expression of Foxp3 in LN Treg cells(P < 0.01);SB203580 inhibited the phosphorylation of p38MAPK(P< 0.01),up regulated the expression of Foxp3 in LN Treg cells(P < 0.01);after knockdown of FKN,the expression of Bax and Cyt-c was decreased significantly(P < 0.01),the apoptosis rate was decreased significantly(P < 0.01),and the expression of Bcl-2 was increased significantly(P < 0.01).After intervention of U-46619,compared with the control group,the expression level of Bax and Cyt-c was increased significantly(P < 0.01),the apoptosis rate was up increased significantly(P < 0.01),and the expression level of Bcl-2 was decreaed significantly(P < 0.01);after SB203580 intervention,compared with the control group,the expression levels of Bax and Cyt-c was decreaed significantly(P < 0.01),the apoptosis rate was decreaed significantly(P < 0.01),and the expression level of Bcl-2 was increased significantly(P < 0.01).Conclusion: FKN participates in the process of apoptosis of Treg cells in lupus nephritis by activating p38 MAPK signal pathway,which provides experimental basis for elucidating the pathogenesis of lupus nephritis. |
PDF Full Text Request