Changes Of Rac1 And The Relationship With ILC2-Related Cytokines In Allergic Asthma Mice

Objective:To detect the change of Rac1 expression levels in airway tissues from asthma mice, the mouse model of asthma was established. Then, to analyze the relationship of innate type 2 helper cells(ILC2)-related cytokines such as IL-5, IL-33 and IL-13 and the influence of serious situation of airway inflammation. We want to understand the function of Rac1 in immune response regulation and possible pathogenesis of asthma in which Rac1 participate in.Methods:1. Establishment of ovalbumin(OVA)asthma model: according to asthma modeling reference literature and a little improvement, the mice in the experimental group were injected with 50 μ g OVA and 10% aluminum hydroxide adjuvant on 1d and 11 d,control group was injected with equal amount of saline, on 22 d the experimental group was treated with 2% OVA saline solution to allergic reaction, one time a day,every time 1h for continuous 5d. The control group only received saline inhalation.2. EHT1864 treatment: In the process of model induction, three days prior to the day for atomization, the EHT1864 was intraperitoneally injected. EHT1864 powder was dissolved with saline water before using. The dosage is 40mg\kg.3. Bronchial alveolar lavage, peripheral blood mononuclear cells and lung tissue suspension were obtained, and Trizol was used to extract total RNA, then c DNA was synthesized by reverse transcription reaction. Real-time fluorescence quantitative PCR was used to detect the m RNA levels of Rac1 and ILC2 related cytokines(IL-5,IL-33 and IL-13).4. After centrifugation, taking the supernatant of bronchial alveolar lavage and serum,enzyme linked immunosorbent assay(ELISA) method was used to detect the protein expression levels of ILC2s-related cytokines(IL-5, IL-33 and IL-13).5. The right lung was fixed in formalin, paraffin embedded and cut into slices, the hematoxylin-eosin(HE) staining was performed. The pathological changes were observed under light microscope.6. Paraffin sections were made from bronchus, the expression level of Rac1 was detected by immunofluorescence assay.7. Statistical analysis was carried out using Prism5 Graph Pad software. The relevance of the expression levels of Rac1 and IL-5, IL-33 or IL-13 in the lung tissue from asthmatic mice was analyzed. The difference between groups was analyzed by the non-paired T-test, P < 0.05 indicates that the difference was statistically significant.Statistical analysis of multiple groups using ANOVA 0ne-way, P < 0.05 indicates that the difference was statistically significant.Results:1.The model mice were restless, frequent itching occurs, dyspnea, sneezing,incontinence, and other asthma acute phase, there was no obvious abnormal performance in the control group.2.The result of HE staining showed that there were a large number of inflammatory cells in the alveolar and surrounding blood vessels in the lung tissue from asthmatic mice. Total Ig E and OVA-Ig E levels in serum were higher than those in the control group. The airway structure of the control group was clear, no inflammatory cells invasion.3.Immune fluorescence showed that the expression level of Rac1 in asthmatic model was significantly lower than that in control mice. After EHT1864 stimulating, Rac1 expression was further significantly decreased.4. Rac1 expression in airway and peripheral blood: The expression level of Rac1 m RNA was significantly lower than that in control group tissue, and EHT1864 stimulating promoted the reduction of Rac1 expression levels.5.The expression of ILC2 related cytokines in lung tissue of asthmatic mice: We compared the difference of m RNA expression between the model group and the control group, the expression levels of IL-5, IL-33 å'Œ IL-13 m RNA were significantly increased in asthma mice, especially in the EHT1864 treatment group.6.The expression levels of IL-5, IL-33 and IL-13 in serum of asthmatic mice were detected by ELISA method, we can learn that the expression levels of IL-5, IL-33and IL-13 in the model group were higher than those in the control group.7.Correlation analysis showed that there was a significantly negative correlation between the m RNA levels of Rac1 and ILC2 s associated cytokines(IL-5, IL-33 and IL-13) in asthma mice.Conclusion:Our results showed that the IL-5, IL-5, IL-33 and IL-13 m RNA and protein expression levels in the asthma mice were higher than that in control group, while the expression level of Rac1 is decreased obviously. The correlation analysis showed that there was a significant negative correlation between the expression of Rac1 and ILC2 s related cytokines(IL-5, IL-33 and IL-13). In order to further prove the role of Rac1 in the occurrence of asthma, the mice were treated by EHT1864 before allergen provoking,it resulted in more enhanced expression levels of ILC2 s related cytokines along with decreased expression level of Rac1, and the inflammatory was aggravated in asthmatic mice. For these reasons, it is arguable that the Rac1 can affect the pathogenesis of asthma. If the Rac1 agonist is used in asthma patient, it may effectively improve the inflammatory state. Further studies on the role of Rac1 in asthma, is beneficial to understand the immune mechanism of asthma and can help us to find new targets for the prevention and treatment of asthma.