Effect Of Roxburghao On The Differentiation Of Mouse TH17 Cells And Its Mechanism

Part One Naive T cells were induced toTH17 cells in vitro and the intervention study of zerumboneObjective:To establish a induced protocol for Th17 cells in vitro,evaluate the effect of zerumbone on the differentiation and function of Th17 cells and explore the correlative mechanisms.Methods:CD4+CD62L+T cells of BALB/c mice spleen(6 to 8 weeks old) were isolated and purified with magnetic bead, and were co-cultured with Anti-CD3 antibodies, Anti-CD28 antibodies, interleukin(IL)—6, transforming growth factor(TGF)-beta, IL—23 for Th17-polarization. There were five groups:the normal group with nothing induced factors; the induced group; zerumbone (1μM) group; zerumbone (10μM) group; zerumbone (30μM) group. The level of IL-17 in the supernatant of cell were detected by ELISE. The proportion of TH17 cells every group was detected by Flow cytometry. RT-PCR was used to detect the expression of ROR-yT mRNA and IL-17mRNA.Results:1.This induction protocol can make the differentiation of TH17 significantly increased. Zerumbone can inhibit the differentiation of TH17, which are dose dependent. IL-17 in the supernatant can also be inhibited by zerumbone on dose dependent. Zerumbone restrained the expression of ROR-yT mRNA and IL-17mRNA, which were both dose dependent. The difference was statistically significant.Conclusions:In vitro, Zerumbone can inhibit the differentiation of CD4+CD62L+ T cells to TH17 and the secretion of IL-17 which mechanism may be through inhibiting the expression of ROR-γT.Part Two A preliminary study zerumbone on hormone insensitive asthma mice and its mechanismObjective:To investigate the influence and mechanism of zerumbone on hormone insensitive asthma mice.Methods:6 to 8 weeks,female,BALB/C mice(20-25Kg) were purchased from Experimental Animal Center of Nanjing Medical University. The mice were randomly divided into 5 groups:the control group (A group),the asthmatic group (B group),the dexamethasone group (C group),the Low-dose zerumbone group (D group), the High-dose zerumbone group (E group) with 6 mice in each group. In addition to group A with PBS, the other four groups were intranasal sensitized with 75μg OVA +10 μg LPS at 0,1,2 and 7 days, intranasal challenged with 50μg OVA at 14,15 and 22 days.Five groups mice were respectively intraperitoneal injected with PBS (200ul), PBS (200ul), dexamethasone (1mg/kg), one low-dose zerumbone (10 mg/ kg),high-dose zerumbone (50mg/kg) before half an hour OVA intranasal challenge We removed one eye for blood,took the lung and spleen,opened the chest for bronchoalveolar lavage after the last challenge 24 hours. The peripheral blood and BALF were centrifuged and got the supernatant which were stored at-80℃. We count the total cells, neutrophils, eosinophils, etc. in the BALF under a microscope. Take the right lower lobe tissue for biopsy, we can observed tissue morphology, airway epithelial damage, the inflammatory infiltration in trachea and perivascular in HE staining,the expression of P-stat3 protein in immunohistochemistry.The level of IL-17 in the BALF and serum were detected by ELISE. The proportion of TH17 cells in the spleen was detected by Flow cytometry. RT-PCR was used to detect the expression of ROR-yT mRNA and IL-17mRNA in lung tissue, western-blotting was used for the ROR-yt and P-STAT3 protein.Results:1.The reactions of grasping ears, scratching, sneezing and so on were obviously in the asthma group and dexamethasone treatment group than zerumbone group,the inhibitory effect in high dose group was more apparen.2. The total number of cells and neutrophils in BALF were significantly decreased in zerumbone group (high dose group decreased significantly) than the asthma group and dexamethasone treatment group. The ratio of TH17 cell in mouse spleen was the least in the high-dose group, followed by the low-dose group, the differences of compared with asthma group or dexamethasone group were statistically significant. The ratio of IL-17 in the bronchoalveolar lavage fluid displayed the same. Mouse lung IL-17mRNA and ROR-yTmRNA in mice lung, after PCR testing, the results of the asthmatic group and dexamethasone group were significantly higher than the high-dose group and low dose group. The expression of ROR-γT, P-STAT3 protein can also be inhibited by zerumbone, the differences among the groups were statistically significant.Conclusions:The treatment of zerumbone on hormone insensitive asthma mice may inhibit the activation of STAT3-ROR-yT to reduce the neutrophilic airway inflammation.