Effects And Mechanism Of Arsenic Trioxide On Th17/IL-17 Axis In A Mouse Model Of Asthma

Background:Asthma is a choronic aiway inflammatory disease associated with autoimmune disorder, and " Thl/Th2 unbalance, Th2 dominant differentiation" theory have played a major role in asthma pathogenesis. Th17 cells is a newly subtype of CD4+T cells, which take effection by secretingⅠ1-17. Recent researchs show that Thl7/IL-17 participate in the pathogenesis and progress of asthma. Pehaps that is why it is difficult to cure it. ATO is an age-old but wangderful traditional Chinese medicine. Clinical datas show that ATO plays a role in relieving asthma, however the mechanism is unknown recently. And it has not been reported whether the effection of ATO is assoiated with inhibiting the expession of Th17/IL-17 and what is the detail mechanism.Objectives:To evaluate the role of arsenic trioxide on relieving asthma and the expession of Th17/IL-17, and compare with the effect of dexamethasone. And then to preliminarily explore if its role in treating bronchial asthma was associated with inducing CD4+T cell apoptosis in asthmatic mice through mitochondria-related pathway.Methods:①40 SPF female BALB/c mice were randomly divided into 4 groups:Normal group(PBS/PBS),Asthma group(OVA/PBS),ATO treatment group(OVA/ATO,DXM treatment group(OVA/DXM), The asthmatic model was reproduced by sensitizing and changing with OVA, 24h after the last changing, the mice were killed. The behaviors of mice were observed, The changes in airway responseveness were determined, Lung tissue sections were used for observing inflammatory infiltration and mucus secretion, the white cells count,neutrophils,eosinophils,lymphocytes from BALF were measured, the concentration of IL-17 in BALF were detected.②Grinding the spleen of mice, preparation of mononuclear cell suspension, CD4+T cells were isolated by MACS, the number and the vability of spleenic CD4+T cells in each group were evaluated, ratio of positive Thl7 cells were detected by FCM.③spleenic CD4+T cells from asthma group were divided into 3 groups:Asthma CD4+T cell group(OVA),asthma CD4+T cell+ATO group(OVA/ATO),asthma CD4+T cell+DXM group (OVA/DXM), cultured for 20h, and early apoptotic CD4+T cells were detected by FCM, Mitochondrial membrane potential and intracellular Ca+concentration were analyzed, IL-17 concentration in culture supernatant of OVA group and OVA/ATO group were detected by ELISA.④Correlation analysis were performed to evaluate the following items:whether the positive ratio of Th17 cells in spleenic CD4+T cells from asthma group is assoiated with the number of neutrophil, whether the IL-17 concentration in BALF of asthmatic mice is assoiated with the number of neutrophil, whether the IL-17 concentration in culture Supernatant of asthma CD4+T+ATO group is assoiated with the ratio of early apoptotic CD4+T cells.Results:(1) Typical asthma-like symptoms were showed in Asthma group, asthma-like symptoms were eased in ATO treatment group and DXM treatment group, while Normal group showed normal;(2) Compared with Normol group, airway responsiveness significantly increased in Asthma group (P<0.05); while AHR was eased in ATO treatment group and DXM treatment group compared with that in Asthma group(P<0.01, respectively), and there was no significant difference between 2 groups(P>0.05);(3) Compared with Normol group, higher levels of inflammatory cells infiltration, goblet cell hyperplasia and airway mucus hypersecretion were found in Asthma group (P<0.01, respectively); airway inflammation and mucus secretion were eased in ATO treatment group and DXM treatment group (respectively P<0.01), and there was no significant difference between 2 groups(P>0.05);(4) Compared with Normol group, the number of total white cell, Neu, Eos, and Lym in the BALF from Asthma group obviously increased (respectively P<0.01), while the number of total white cell, Neu, Eos, and Lym obviously decreased in ATO treatment group and DXM treatment group (P<0.01, respectively), however, ATO had a better inhibitory effect on Neu than DXM (P<0.01);(5) Compared with Normol group, IL-17 concentration in the BALF from Asthma group obviously increased(P<0.01), while the concentration of IL-17 significantly decreased in ATO treatment group and DXM treatment group (P＜0.01, respectively), and there was no significant difference between 2 groups(P>0.05);(6) Compared with Normol group, the number of spleenic CD4+T cells obviously increased in Asthma group (P＜0.01), while the number of CD4+T cells significantly reduced in ATO treatment group and DXM treatment group (P＜0.01, respectively), and there was no significant difference between 2 groups(P>0.05);(7) Compared with Normol group, the positive ratio of Th17 cells in spleenic CD4+T cell obviously increased in Asthma group (P<0.01), ATO treatment group and DXM treatment group had a lower level of positive ratio of Thl7 cells than that in Asthma group (respectively P＜0.01), and there was no significant difference between 2 groups (P>0.05);(8) Compared with Asthma CD4+T cell group, the ratio of early apoptotic CD4+T cells obviously increased in Asthma CD4+T cell+ATO group and Asthma CD4+T cell+DXM group (P<0.01, respectively), and there was no significant difference between 2 groups (P>0.05);(9) Compared with Asthma CD4+T cell group, Mitochondrial membrane potential obviously decreased in Asthma CD4+T cell+ATO group (P＜0.01);(10) Compared with Asthma CD4+T cell group, the intracellular Ca2+ concentration obviously increased in Asthma CD4+T cell+ATO group (P＜0.01);(11) Compared with Asthma CD4+T cell group, IL-17 concentration of culture supernatants obviously decreased in Asthma CD4+T cell+ATO group (P＜0.01);(12) The positive ratio of Thl7 cells in spleenic CD4+T cell form Asthma group positively correlated with the number of Neu in the BALF form Asthma group (r=0.998, P<0.05);(13) IL-17 concentration in the BALF form Asthma group positively correlated with the number of Neu in the BALF form Asthma group (r=0.982,p<0.05);(14) IL-17 concentration in culture Supernatants of asthma+ATO group negatively correlated with the apoptosis ratio of CD4+T cells (r=-0.956,P<0.05).Conclusions:(1) In this experiment, asthmatic mouse model was reproduced successfully, Th17/IL-17 axis was involved in the pathogenesis of asthma;(2) ATO has potential value in the treatment of asthma, especially non-eosinophilic asthma mediated by neutrophils;(3) ATO could play a good role in relieving asthma, and this effect might be related to inhibition of the expression of asthma Th17/IL-17 axis;(4) To induce CD4+T cell from asthma mice apoptosis through mitochondria-related pathway may be one of the mechanisms that ATO inhibit the expression of asthmatic Th17/IL-17 axis.