The Effect And Mechanism Of Arsenic Trioxide On The CD4~+T Cell Apoptosis In The Acute Asthma Mouse

Chapter1The effect of Arsenic trioxide on airway hyperreactivity and airway inflamation in the acute asthma mouseObjective(1Establishing a mouse model of acute asthma, and identify the model;(2)Observe the effect of ATO (Arsenic Trioxide, ATO) on airway hyperreactivity and airway inflamation in the acute asthma mouse.MethodThirty SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains10mice. Establishing a mouse model of acute asthma after all the mice are raise adaptively for one week according to the following methods. The mice of PBS group were intraperitoneal injected with0.2ml PBS buffer(PH7.2-7.4) at the1st and13th day, and were atomized with6ml PBS buffer(PH7.2-7.4) for30min from the19th to the24th day successively (sum6days). The mice of the PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/PBS group were intraperitoneal injected with0.2ml mixed solution of OVA(ovalbumin, OVA) and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/PBS group received the intraperitoneal injection of0.2ml PBS buffer(PH7.2-7.4)30min before each atomization. The mice of OVA/ATO group were intraperitoneal injected with0.2ml mixed solution of OVA and aluminium hydroxide gel(10μg OVA and2mg aluminium hydroxide gel were dissolved in PBS buffer thoroughly) at the1st and13th day, and were atomized with6ml 5%OVA(Grade V) for30min from the19th to the24th day successively (sum6days). The mice of the OVA/ATO group received the intraperitoneal injection of0.2ml ATO solution (2.5mg/kgxmouse weights)30min before each atomization. The mice were sacrificed at25th day, the following characters were detected,(1)the ordinary behavior of the mice,(2)airway hyperreactivity was determined after methacholine challenge by invasive pulmonary impedance method,(3) collecting the BALF and carrying out the cell count and cell classification,(4) making lung pathological slices and observing inflammatory cell infiltration and mucus secretion. All the data was presented with Mean±SD, at first, the data was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett’s T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.ResultPrior to the experiment, there is no difference between each group mice in appearance, behavior, the response to stimulation, activity and weight. During the sensitization phase, there is no red, swollen and ulcerate in the injection site of each mice. During the challenge phase, the mice of PBS group feed normally and possess sensitive reflection with clean and smooth fur. The mice of OVA/PBS group displayed exciting, sneezing and scratching in early stage, which displayed crawling and somnolence in the later stage. The mice of OVA/ATO group showed mild symptoms of exciting and sneezing than the OVA/PBS group, and didn’t show obvious somnolence. Meanwhile, there was no activity decrease, anorexia and weight decrease occurred in the mice of OVA/ATO group.Compared with the PBS group, airway responsiveness statistically increased in the OVA/PBS group (P<0.05); airway hyperreactivity (AHR) was eased in the OVA/ATO group compared with that in the OVA/PBS group (P<0.05). In the BALF of OVA/PBS group, the amount of different cell is list as following, the total of leukocytes is21.11±3.28x104/ml, the Eos is3.10±0.50×104/ml, the Lym is6.60±0.97×104/ml, the Neu is4.92±0.45×104/ml, which are significantly increased (all P<0.01) compared with PBS group. In the OVA/ATO group, the amount of different cell is list as following, the total of leukocytes is13.04±2.58×104/ml, the Eos is1.06±0.19×104/ml, the Lym is2.43±0.28×104/ml, the Neu is2.36±0.29×104/ml, which are significantly decreased (all P<0.01) compared with the OVA/PBS group.Compared with the PBS group, inflammatory cell infiltration, goblet cell hyperplasia and mucus secretion in lung were far more obvious in OVA/PBS group (P<0.01); airway inflammation and mucus hypersecretion of OVA/ATO group were significantly alleviated than OVA/PBS group (P<0.05).Conclusion(1)The mouse model of acute asthma established in this experiment was successful.(2)Treatment of ATO can alleviate the AHR and airway inflammation of the acute asthma mouse. Chapter2Arsenic Trioxide promotes the CD4+T cells apoptosis in the acute asthma mouseObjectiveDetecting of the effect of arsenic trioxide on the CD4+T cells apoptosis in the acute asthma mouse.MethodIn vivo:Eighteen SPF BALB/c female mice were randomly divided into three groups:the normal group(PBS group), the asthma group(OVA/PBS group) and the ATO group(OVA/ATO group), each group contains6mice. Establishing a mouse model of acute asthma according the method in the chapter1. The mice were sacrificed at25th day, Splenic CD4+T cells of each group were separated and purified by magnetic cell sorting device (MACS). The number of CD4+T cells of each group was counted. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) were added. The CD4+T cells were cultured in the incubator(5%C02,37℃)for24hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells.In vitro:Splenic CD4+T cells of mice in the OVA/PBS group were separated and purified by MACS. Then CD4+T cells were cultured with RPMI-1640according to the concentration of2×106cell/ml, meanwhile, the ConA(5ug/ml) and ATO were added. The CD4+T cells were divided into four groups according to the concentration of the added ATO which including OμM group, luM group,3μM group, and5μM group. All the CD4+T cells were cultured in the incubator(5%CO2,37℃) for20hours. When finishing the culture, flow cytometry(FC) was conducted to detect the apoptosis rate of CD4+T cells. All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett’s T3method was conducted. Analysis of Variance(ANOVA) was conducted for Comparison the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.ResultIn vivo, in the OVA/PBS group, the number of spleen CD4+T cells is (17.88±2.13)×108/L, which is significantly increased when compared with PBS group(P<0.01). The number of the OVA/ATO group is (12.92±2.31)×108/L, which is significantly decreased when compared with OVA/PBS group(P<0.05). In OVA/PBS group, the apoptotic rate of CD4+T cells is20.69±2.68%, which is significantly decreased when compared with PBS group(P<0.05). In OVA/ATO group, the apoptotic rate of CD4+T cells is34.71±0.98%, which is significantly increased when compared with OVA/PBS group(P<0.05).In vitro, in the3μM group, the apoptotic rate of CD4+T cells is31.85±3.73%, which is significantly increased when compared with OμM group(P<0.05). In the5μM group, the apoptotic rate of CD4+T cells is40.54±1.99%, which is significantly increased when compared with OμM group or3μM group (P<0.01or P<0.05).ConclusionArsenic trioxide promotes the CD4+T cell apoptosis in the acute asthma mouse. Chapter3Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxideObjective(1)Probing the effect of arsenic trioxide on the protein expression of GRP78and CHOP in CD4+T cells.(2)Expounding the role of CHOP protein in the CD4+T cell apoptosis which induced by arsenic trioxide.MethodPart one, Splenic CD4+T cells of OVA/PBS group were separated and purified by MACS. The CD4+T cells were cultured with5μM ATO for different time which including Oh,2.5h,5h and7.5h. And the protein expression of GRP78and CHOP were detected.Part two, Splenic CD4+T cells of OVA/PBS group were divided into CHOP siRNA group and control siRNA group. Transfection of CHOP siRNA can silent the protein expression of CHOP. But the control siRNA doesn’t have any silent effection which was used as a negative control. First we transfected the CD4+T cells in CHOP siRNA group with CHOP siRNA and transfected the CD4+T cells in control siRNA group with control siRNA. Then we detected the expression of CHOP mRNA by real-time PCR and the protein expression by western blot to estimate the silent effect of CHOP siRNA. After the successful transfection, the CD4+T cells of both group were cultured with5μM ATO for20hours. Flow cytometry(FC) were conducted to detect the apoptotic rate of CD4+T cells.All the data was presented with Mean±SD. At first, the data of each group was taken test of normality and homogeneity test of variance. If the data does not conform to normal distribution, the natural logarithmic transformation was conducted. In the case of unequal variances, the Dunnett’s T3method was conducted. Independent-samples T test were conducted to compare the difference between two groups. Analysis of Variance(ANOVA) was conducted for comparing the difference between multiple sets of mean, and the LSD test was conducted for the pairwise comparison of multiple group data. P<0.05brings the statistics significant.ResultThe protein expression of GRP78in2.5h group,5h group and7.5h group was significantly increased when compared with Oh group (P<0.05, P<0.01, P<0.05respectively). The protein expression of GRP78in5h group also is more than the2.5h group and7.5h group(both P<0.05). There is no obvious difference between2.5h group and7.5h group(P>0.05).The protein expression of CHOP in5h group is more than Oh group,2.5h group and7.5h group (all P<0.05).The mRNA and protein expression of CHOP in CHOP siRNA group both significantly decreased when compared with control siRNA group (both P<0.01).After cultured with ATO, the CD4+T cell apoptotic rate in CHOP siRNA group is32.39±2.30%, which is significantly decreased when compared with control siRNA group (P<0.05).Conclusion(1)Arsenic trioxide effects the protein expression of GRP78and CHOP in CD4+T cells. (2)Transfection of CHOP siRNA silents the protein expression of CHOP and partially blocks the CD4+T cells apoptosis, which induced by arsenic trioxide.(3)Endoplasmic reticulum stress-CHOP pathway involved in the CD4+T cell apoptosis which induced by arsenic trioxide.