Diethylstilbestrol Inhibit ConA-induced Acute Autoimmune Hepatitis In Mouse

Background and purposeThe incidence of autoimmune diseases is different between female and male. Females are always seemed like more sensitive than males. These different are causally related to the gender differences in their serum level of sex hormones. The sex hormone is very important to keep the gender specific for mammals. Estrogen and androgens is main sex hormone in female and male. Autoimmune hepatitis is one of the autoimmune diseases, it’s incidences is different between female and male, female/male (4:1), this means the estrogen maybe paly a critical role in the autoimmune diseases.As we know that the immune system can recognize the antigens that not belong to our body, what may cause diseases. After that the system can induced multiple kinds of types of cells, that can cause increased the cytokines and attack cells(killing cells) can reduce the pathogen to protect ourselves. But, if this reaction is too strong, the cytokines, antibodies and complement may cause the host disease. These diseases are named immune diseases. For example, autoimmune diseases, graft-versus-host diseases (GVHD). The host gets hurt because the over-immune-reaction.So, in these decades, some researchers found that, there were some anti-inflammatory mechanism in immune system. It conclude cytokines, like IL-10, TGF-βand some immune suppressor cells, for example, regulatory T cells(Tregs) and myeloid-derived-suppressor cells(MDSCs). They can inhibit the cells active and proliferation when the antigen present, and they also can secret some suppressor cytokines to reduced the immune reaction. Keeping the balance between pro-inflammation and anti-inflammation is very important. The immune response can decrease the infection from pathogen and also can keep the immune reaction appropriate to avoid tissue immune injury.Estrogen can infect the immune system. Now, there are3types of estrogen in environment, the nature one (17β-estrogen), the environmental estrogen (the herbal-steroidal-estrogen), and the synthetic non-steroidal estrogen. They can recognize the estrogen receptor(ER), regulate some genes transcription. There are three reasons, people to chase the estrogen and their role in immune system. First, there was the estrogen receptor in the thymus and bone marrow where the immune cells grow up and get maturity, this means the immune system maybe the estrogen’s target organ. Second, as we just demonstrated, the incidence of autoimmune diseases is different between female and male, it means that the estrogen can play a critical role in the process of autoimmune diseases. Third, the level of estrogen in parental can infect the mature of immune organ in fetus.The role of estrogen in autoimmune diseases is complicated. As we know the incidence of autoimmune diseases prefer females to males. But, there is a interesting things. The women with rheumatoid arthritis and multiple sclerosis(MS), the syndrome resolved when they carried babies. It seems that the estrogen can release the autoimmune diseases. But, the patients with SLE get worse replace the remission. From these we speculate that the role of estrogen in patient with autoimmune disease is complicated. What actor it play in the autoimmune hepatitis, we do not know yet.Diethylstilbestrol (DES) is a synthetic non-steroidal estrogens. Its structure is just like the estrogen, it can recognize the estrogen receptor and mimic the function of estradiol. Since it is funded, it was used to relieve the pregnant reaction and avoid miscarry in women carried babies. Cause about its function of mimic the estrogen, it can supply the absence or shortage of estrogen in some special physiological stage. What’s more, people found that DES can significantly reduce the incidence of osteoporosis in postmenopausal women.Our purpose is what role of estrogen in the autoimmune hepatitis. We choose DES, a synthetic estrogen, to mimic the function of estrogen, to explore the mechanism of autoimmune hepatitis and what’s estrogen role in the autoimmune disease? Is estrogen a new treatment choice for patient with AIH?Methods and resultsThere are there steps for this project.At first, to explore the function of DES for the proliferation of T cells in vitro. We choose adult female C57B/L6mouse, detect the spleen, isolate the splenocytes. We use the96-well-plate to culture the cells we got. We add0.5million cells/well, stimuli with2.5ug/mL conA, and with different dosage of DES, co-cultured for24hrs or48hrs, at last, add3H-thymidine to test the proliferation of T cells. We found that DES can significantly reduce the proliferation of T cell, when the dosage is more than20uM. It means that the DES can reduce the proliferation of T cells in vitro.Next, we choose conA induced liver damage as our in vivo experimental model. We choose adult female C57B/L6mouse. There are4groups in our model, Naive (no-treatment), Control (conA+veh), Treatment (conA+DES), DES only (DES). Pre-administration of DES (twice, sc.1/day for2days.). Polyclonal activation of T cells, following injection of concanavalin A (ConA), in mice caused acute hepatitis, characterized by significant increase in aspartate transaminase (AST), induction of inflammatory cytokines, and infiltration of mononuclear cells in the liver, leading to severe liver injury. After ConA challenge, the DES administration groups mouse significant inhibited hepatitis associated inflammation markers. And interesting, the immune suppression cells Tregs(regulate T cells) and MDSCs(myeloid-derived suppressor cells) are significant increased from the liver infiltrated cells in treatment and DES only groups compared with the control group. What’s more, we find that the apoptosis in hepatocytes is decreased in treatment group.What’s more, to confirm the DES can increase the Tregs. This experiment, we use Foxp3-EGFP knock-in mouse, isolated the Foxp3+,CD4+T cells (Tregs) from spleen, co-cultured with different DES for48hrs, and then to test the proliferation of Tregs. We found that when dosages are5uM and20uM, DES can significant increase the proliferation of Tregs.ConclusionsThis study demonstrates that the DES as a synthetic compound of the estrogen can inhibit the conA induced autoimmune hepatitis. Maybe the Tregs and MDSCs play a critical role in the very earlier phase of the disease. And the estrogen and its synthetic or mimic compounds may constitute a therapeutic selection to treat inflammatory diseases.