| ?Background and Objective?Sarcoidosis is a systemic disease with non caseous epithelioid granuloma as the main pathological feature and most often involves in the lung and chest lymph nodes[1].The etiology and pathogenesis of sarcoidosis are not yet clear.It is considered that sarcoidosis is an abnormal reaction of cellular immunity,which may be associated with mycobacterium tuberculosis,dust and propionibacterium acnes[2-3].Previous studies have shown that sarcoidosis is based on the immune response of type1 helper T cell?Th1?[4].The unknown antigen is recognized by alveolar macrophages,presented to the Th1,which is stimulated to proliferate and produce a large amount of inflammatory mediators such as IL-2,IFN-gamma and tumor necrosis factor alpha,and then epithelioid macrophages,macrophages and lymphocytes?mainly CD4+T cells?aggregate together,finally the non caseous necrosis granuloma is formed[5].With further studies on CD4+T cells,two CD4+T cell subsets?helper T cells 17?Th17?and regulatory T cells?Treg??have been found to be involved in the pathogenesis of sarcoidosis.Th17 cell is a subtype of CD4+T cells.Its characteristic transcription factor is retinoic acid-related solitary nuclear receptors?t?ROR?t?,while IL-17 is the most important cell secretory factor.Treg is a CD4+T cell subgroup that regulates the immune response,which mainly secretes IL-10,TGF-?.Foxp3 is the specific transcription factor of Treg cells.Under the induction of only TGF-?,CD4+T cells can differentiate into Treg cells.In common with IL-6 and TGF-?CD4+T cells differentiate into Th17 cells.Th17 cells promote inflammation mainly through IL-17 and other cytokines,while Treg cells lead to immune suppression,the two subtypes of CD4+T cells mutually restrict balance.Study showed in the peripheral blood and bronchoalveolar lavage fluid?BALF?of active patients with sarcoidosis,the number of Th17 cells increased,and in the central part of the sarcoidosis granulomatous Th17 cells showed the formation,which prompted that Th17 cells may be involved in sarcoidosis granuloma[5-6].Most studies[5,7,8]suggest that the differentiation of Th17 in sarcoidosis enhances,while the role of Treg in sarcoidosis has a relatively large difference[9-12].Grunewald[13]found that the number of Treg cells in sarcoidosis patients decreased.The result of the other study[10]was the opposite,and the number of Treg cells expressed in the patient's BALF and peripheral blood were increased.Study[14]found that Th17 and Th17/Treg were significantly decreased in sarcoidosis patients after glucocorticoid treatment.People have a deeper understanding of the pathogenesis of sarcoidosis with the application of cytology and molecular biology technology,while no breakthrough has been made in the field of its effective and safed treatment.At present there is no radical treatment for sarcoidosis,glucocorticoid is the first choice for the treatment[15],and the second-line drugs commonly used include methotrexate,azathioprine,cyclophosphamide and other cytotoxic drugs.But the long-term use of corticosteroids or second-line drugs may bring serious adverse reactions[16].Therefore,finding succedaneous drugs with fewer side effects is the research direction in treatment of sarcoidosis.In combination with the immune mechanism of sarcoidosis,we think that finding a drug that can intervent the immunoregulation of sarcoidosis may become a breakthrough for drug therapy of sarcoidosis.In the previous clinical practice,we found that subcutaneous injection of thymosin can produce obvious curative effect in the treatment of sarcoidosis.2 patients with sarcoidosis who could not tolerate glucocorticoid accepted subcutaneous injection of thymosin alpha 1?T?1?,and then the swollen mediastinal and hilar lymph nodes were significantly reduced or disappeared,and clinical symptoms were completely relieved.So that we are interested to study the mechanism of T?1 in the treatment of sarcoidosis.T?1 is an active peptide isolated from calf thymus in 1966[17],which is mainly derived from thymic epithelial cells and thymic endocrine cells.In addition,T?1 is also found in spleen,lymph nodes,lungs and brain[18].T?1 can improve the ability of reacting to the antigen,maintaining the immune balance of T cells,and thus improve the body's resistance.As immune modulators[19],T?1 can promote the mature of T cells[20],improve ability of producing specific cytokines,and can also increase the proportion of Treg cells[21],through a variety of mechanisms acting on the immune system.T?1 has been widely used in the treatment of many diseases,such as cancer,severe infection,virus hepatitis[22-24],but study of T?1 used in the treatment of sarcoidosis has never been reported.In order to explore the role of T?1 in the treatment of sarcoidosis,we intend to construct a mouse sarcoidosis model on the basis of cell level study,and use T?1 to treat the model mice.Through the detection of pathological and various immunological indexes,the therapeutic effect of T?1 on sarcoidosis mice is verified by initial step.The results provide a reference for the exploration and innovation of drug therapy for sarcoidosis.?Methods?Part 1 Intervention of T?1 on peripheral blood mononuclear cells1.Intervention of different concentrations of T?1 on peripheral blood mononuclear cells?PBMC?in miceThe peripheral blood mononuclear cells were intervened for 48 hours with different concentrations of T?1?0,5ug/ml,10ug/ml?.The proportion of Th17 and Treg cells was detected by flow cytometry.2.Intervention of different concentrations of T?1 on human peripheral blood mononuclear cellsHuman peripheral blood mononuclear cells were intervened for 48 hours with different concentrations of T?1?0,5ug/ml,25ug/ml,50ug/ml?.The ratio of Th17 and Treg cells was detected by flow cytometry.Part2 Construction of C57BL/6 mouse sarcoidosis granuloma model10 C57BL/6 mice were randomly divided into 2 groups:5 in the blank control group and 5 in the model group.On the first day:the model group was sensitized by subcutaneous injection of 50ug Sod A incorporated into incomplete Freund's adjuvant?IFA?0.25ml,and the blank control group was given a subcutaneous injection of 0.5ml PBS.On the 14th day:the model group was given a tail vein injection of 50ug Sod A and 6000agarose beads?dissolved in PBS0.5ml?covalently coupled,while the blank control group was given a tail vein injection of PBS0.5ml.On the 22th day:5 mice in blank control group and 5 mice in model group randomly selected were sacrificed,observe the lung,lymph node and the histologic changes;immunohistochemistry was used to detect CD4+T cells and macrophages in granuloma;the levels of Th1,Th2,Th17,Treg and CD4/CD8 in peripheral blood and BALF were detected by flow cytometry;the cytokines IL-17A,IL-10in lung homogenate and plasma.PCR was used to detect the expression level of mRNA of ROR?t and Foxp3 in lung tissue of mice.Part3 Preliminary study on the mechanism of T?1 in the treatment of sarcoidosisDay 1-22:10 C57BL/6 mice were modeled according to the first partDay 22-35:the 10 mice in the model group were randomly divided into two groups,5in the model group and 5 in the drug group.The drug group was given an intraperitoneal injection of 10ug T alpha 1 every day,and the model group was given an intraperitoneal injection of 0.5ml PBS every day.On the 36th day:kill the mice.Dissect the mice and observe the lung,lymph node and the histologic changes;the levels of Th1,Th2,Th17,Treg and CD4/CD8 in peripheral blood and BALF were detected by flow cytometry;the cytokines IL-17A,IL-10 in lung homogenate and plasma.PCR was used to detect the expression level of mRNA of ROR?t and Foxp3 in lung tissue of mice.In vitro experiment:the peripheral blood mononuclear cell?PBMC?isolated from peripheral blood of mice was intervened for 48 hours with different concentrations of T?1,and the proportion of Th17and Treg cells was detected by flow cytometry.Chest CT and peripheral blood Th17/Treg monitoring were performed in 1 patient with sarcoidosis treated with T?1.?Results?Part 1 Intervention of T alpha 1 on peripheral blood mononuclear cells1.Intervention of different concentrations of T?1 on peripheral blood mononuclear cells in miceThe prognosis of T?1?0,5ug/ml,10ug/ml?of different concentrations on peripheral blood mononuclear cells in mice,the proportion of Th17 cells to CD4+T cells decreased with the increase of T?1 concentration,and the proportion of Treg cells to CD4+T cells increased with the increase of T?1 concentration,and there were statistical differences among the groups.2.Intervention of different concentrations of T?1 on human peripheral blood mononuclear cellsThe T?1?0,5ug/ml,25ug/ml,50ug/ml?of different concentrations of human peripheral blood mononuclear cells,the proportion of Th17 cells to CD4+T cells decreased with the increase of T?1 concentration,and there were statistical differences.There was no statistical difference between Treg cells in the proportion of CD4+T cells.Part 2 Construction of C57BL/6 mouse sarcoidosis like granuloma model1.The lung tissue sections of the mice in the model group were exposed to HE staining and we found a large number of lymphocytes were infiltrated,form many multiple sizes non caseous granulomas with lymphoid cells surrounding the epithelial cells and other cells.The blank control group showed normal structure of alveolar and interstitial lung and no lymphatic infiltration and granuloma.2.Immunohistochemistry showed in the model group the macrophage specific antigen Mac-2 was expressed in the granuloma center,the CD4+T cells are expressed around.3.Percentage of Th1 cells in CD4+T cells in model mice peripheral blood was higher than the control group?P<0.05?;Th17cell in CD4+T cells was significantly higher than control group?P<0.01?;there was no significant difference between the model group and the control group in the percentage of Treg and proportion of CD4/CD8;there was no significant difference in the ratio of Th1/Th2,but the ratio of Th1/Th2 in model group increased compared with blank control group;the ratio of Th17/Treg in model group was significantly higher than the control group?P<0.05?.4.There was no significant difference in the proportion of Th1,Th2,Th17,Treg,CD4/CD8 and ratio of Th17/Treg and Th1/Th2 in mice BALF of the two groups.5.The level of plasma factor IL-17A in model group was significantly higher than that in blank control group?P<0.05?,and there was no significant difference in the level of IL-6 between the two groups.The level of plasma factor TGF-?in model group was lower than that in control group?P<0.05?,IL-10 in model group was significantly lower than that in control group?P<0.01?.6.The level of IL-17A in lung tissue of model group was significantly higher than that of blank control group?P<0.01?,IL-6 of model group was higher than that of control group?P<0.05?,and there was no significant difference of TGF-?and IL-10 group in the two groups.7.The level of ROR?t mRNA expression in lung tissue of model group was higher than that of control group?p<0.05?,and there was no statistical difference in Foxp3 mRNA expression in the two groups,but there was a decreasing trend in model group.Part3 Preliminary study on the mechanism of T?1 in the treatment of sarcoidosis1.Bronchial basement membrane of model mice was thickening,different size of alveolar were destroyed,a large number of lymphocytes infiltrated,form many multiple sizes non caseous granulomas with lymphoid cells surrounding the epithelial cells and other cells.Different size of alveolar were found in the treated mouse,granuloma occasionally appeared,lymphocytic infiltration was lower than that of model group.2.There was no significant difference in the proportion of Th1,Th2,Th17,Treg,CD4/CD8 and ratio of Th17/Treg,Th1/Th2 in the peripheral blood between model group and drug group.3.There was no significant difference in the proportion of Th1,Th2,Th17,Treg,CD4/CD8 and Th17/Treg ratio and Th1/Th2 ratio in the BALF between model group and drug group.4.The concentration of IL-17A in plasma of drug group was significantly lower than that of model group?p<0.05?,and there was no significant difference in IL-6,TGF-?and IL-10 between model group and drug group.5.There was no significant difference in the concentration of IL-17A,IL-6,TGF-?and IL-10 in the lung tissue of model group and drug group.6.The level of mRNA expression of ROR?t in the lung tissue of drug group was lower than that of model group?p<0.05?,and there was no significant difference in the mRNA expression of Foxp3 between model group and drug group.7.The chest CT monitoring of the patient with sarcoidosis treated by T?1,showed that the lymph nodes were gradually reduced,and the proportion of Th17 of CD4+T cells and the ratio of Th17/Treg decreased.?Conclusion?1.C57BL/6 sarcoidosis granuloma model can be successfully constructed by Sod A as immunogen,and its pathological and immunological characteristics are consistent with sarcoidosis.2.T?1 had a certain curative effect on sarcoidosis model mouse.3.T?1 regulates immune imbalance in mice with sarcoidosis. |
PDF Full Text Request