| Alopecia areata is a T cell-associated autoimmune disease characterized by localized patchy alopecia.Lighter often manifests as one or more scattered round or or irregular shapes of the scalp;In severe cases,it can affect the whole body hair.The etiology and pathogenesis of alopecia areata are not clear,and may be related to genetic susceptibility,mental stress,eating habits,micronutrients,oxidative stress,infection,microbiota and other factors.At present,most scholars believe that alopecia areata is associated with T cell immune imbalance.At present,there is no cure for alopecia areata.The main treatment methods include local or systematic use of glucocorticoids,immunosuppressive agents,topical response modifiers,and contact sensitizers and physical therapy.Although these methods are effective,there are safety risks in long-term use.Poor compliance and high medical costs.Traditional Chinese medicine has a long history,a variety of methods,clear curative effect,relatively safe and so on.In recent years,we have found that Shaoyao Gancao Decoction is safe and effective in the treatment of alopecia areata.In this study,protein suspension microarray,enzyme-linked immunosorbent assay,flow cytometry,RT-PCR and other methods were used to investigate the changes of Th1,Th2,Th17 and Treg cells in the serum of severe alopecia areata,and the mechanism of the treatment of severe alopecia areata with Shaoyao Gancao Granul.1 Objective:To explore of changes in Th1,Th2,Th17 and Treg cells in serum of severe alopecia areata,and the mechanism of the treatment of severe alopecia areata with Shaoyao Gancao Granul.2 Method:2.1 Changes of Th1,Th2,Th17 and Treg cells in serum of severe alopecia areata.The levels of Th1,Th2,Th17 and Treg cytokines in the serum of Alopecia areata patients and healthy people were detected by protein suspension chip and enzyme-linked immunosorbent assay.Flow cytometry was used to detect the percentage of Th17 cells and Treg cells in peripheral blood of patients with Alopecia areata and healthy individuals.2.2 Shaoyao Gancao Granul regulate the Th17/Treg imbalance through the Notch signaling pathway to treat severe alopecia areata.The CD4 cells of severe Alopecia areata were aseptically isolated and stimulated by IL-2 and CD3/CD28.After adding 10%different serum,they were divided into 6 groups,saline group,Shaoyao Gancao Granule group,methylprednisolone group and blank group,DAPT group and DAPT + Shaoyao Gancao Granule group.After the 7th day of CD4 cell group intervention,the levels of cytokines in different groups of Th17 and Treg were detected by protein suspension chip and enzyme-linked immunosorbent assay(ELISA).The expression of Notch-1,Hes-1,IL-17 and TGF-β1 mRNA was detected by Realtime-PCR.3 Result:3.1 Changes of Th1,Th2,Th17 and Treg cells in serum of severe alopecia areata.(1)Compared with healthy controls,Thl and Th17 related cytokine IFN-γ,IL-18,IL-6,IL17 A,IL-21 and,IL-22 of patients with severe alopecia areata in serum were significantly upregulated(P<0.05,P<0.01),while Th1 function-related cytokine and IL-2 were downregulated in patients with severe alopecia areata in serum(P<0.01).The level of Th17 function-related cytokine IL-1β was lowered(P>0.05).The levels of Th2 and Treg related cytokine IL-10,TGF-β1,IL-5 and IL-4 was significantly down-regulated with(P<0.01).IL13 was up-regulated(P>0.05).(2)The results of flow cytometry showed that the Th17 cells in the peripheral blood of severe alopecia areata group were significantly higher than the healthy control group(P<0.05),while the Treg level was lower than the healthy control(P<0.05).3.2 Shaoyao Gancao Granul regulate the Th17/Treg imbalance through the Notch signaling pathway to treat severe alopecia areata.(1)Effect of Shaoyao Gancao Granule on cytokines of CD4 cells.Compared with the saline group,the levels of inflammatory cytokine related to Th17 IL-17A,IL-22 and IL-6 in the Shaoyao Gancao Granule group were down-regulated(P<0.05,P<0.01),and methylprednisolone group IL-17A,IL.-6,IL-21,IL-22 significantly decreased(P<0.05,P<0.01).Compared with the saline group,the levels of immunosuppressive cytokine related to Treg IL-10 and TGF-β1 was up-regulated(P<0.05,P<0.01).The levels of IL-10 and TGFβ1 levels were decreased in the methylprednisolone group(P>0.05).(2)Effect of Shaoyao Gancao Granule on IL-17 mRNA and TGF-β1 mRNA in CD4 cells.Compared with the saline group,the expression of IL-17 mRNA was significantly downregulated(P<0.05),while the level of TGF-β1 mRNA was significantly increased(P<0.05).The expression of IL-17 mRNA in the methylprednisolone group was significantly downregulated(P<0.05),while the level of TGF-β1 mRNA was significantly increased(P<0.05).IL-17 mRNA is a Th17 marker molecule,and TGF-β1 mRNA is a representative molecule of Treg.The results suggest that Shaoyao Gancao Granule can regulate the balance of Treg/Th17.(3)Effects of Shaoyao Gancao Granules with DAPT and DAPT on cytokines of CD4 cells.Compared with the blank group,the cytokines of the DAPT group and the DAPT+Shaoyao Gancao Granule group changed.Specifically,the inflammatory factors related to Th17 secretion in the DAPT group including IL-17A and IL-21 were significantly decreased(P<0.05,P<0.01),while The level of IL-6 and IL-22 was also down-regulated except IL-17A and IL-21 in the DAPT+Shaoyao Gancao Granules group(P<0.05,P<0.01).At the same time,DAPT+Shaoyao Gancao Granules can also increase The level of Treg-associated immunosuppression cytokines of IL-10 and TGF-β1 was also up-regulated(P<0.05,P<0.01).From the above results,it can be seen that DAPT can synergize with t level to improve the imbalance of IL-6,IL-17A,IL-21,IL-22,TGF-β1 and IL-10secreted by CD4 cells.(4)Effects of Shaoyao Gancao Granule+DAPT and DAPT on IL-17/TGF-β1 and Notch/Hesl mRNA in CD4 cells.Compared with the blank group,the level of IL-17 mRNA in the Shaoyao Gancao Granules group decreased(p<0.01),and the level of TGF-β1 mRNA increased(p<0.01).The DAPT group was able to reduce the level of IL-17 mRNA(p<0.05)and increase the gene expression of TGF-β1 mRNA(p<0.05).The down-regulation level of IL-17 mRNA and the up-regulation of TGF-β1 mRNA in DAPT+Shaoyao Gancao Granules group were even worse than those in DAPT group+Shaoyao Gancao Granules group(p<0.01,p<0.05).It is suggested that the regulation of Teg cells and Th17 cells by Shaoyao Gancao Granules is related to the inhibition of Notch signaling pathway.Compared with the blank group,the expression of Notch1 and Hesl mRNA in CD4 cells of DAPT group was significantly down-regulated(P<0.05,P<0.01),and the expression of Notch1 and Hes1 mRNA in Shaoyao Gancao Granules group was also down-regulated(P<0.05).The mRNA levels of Notchl and Hesl were down-regulated in DAPT+Shaoyao Gancao Granules group(P<0.01),and the down-regulation was most obvious in the four groups.It is suggested that Shaoyao Gancao Granules group can down-regulate the Notch signaling pathway,and DAPT can synergize with Shaoyao Gancao Granules to enhance the inhibition of Notch.4 Conclusion:(1)There is imbalance of Th1/Th2 and Th17/Treg in the serum of patients with severe alopecia areata,which is characterized by up-regulation of Th1,down-regulation of Th2,upregulation of Th17 and down-regulation of Treg.(2)Shaoyao Gancao Granules regulate the imbalance of Th17/Treg by regulating Notch signaling pathway to treat severe alopecia areata. |
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