| ObjectivePart 1:To explore the effects of artesunate on cigarette smoke-induced lung oxidative damage in mice.Part 2:To explore the effects of artesunate on airway responsiveness and inflammation in cigarette smoke-exposed mice and glucocorticoid sensitivity.MethodsPart1A total of 40 female specific pathogen free BALB/c mice were divided randomly into four groups:normal group, cigarette smoke group, vehicle group and artesunate group. The latter three groups were exposed on cigarette smoke for 40 days. Vehicle (5% NaHCO3 containing 5% dimethyl sulfoxide,0.1 ml of each mice) or artesunate (30 mg/kg, dissolved in the 0.1 ml vehicle) was given by intraperitoneal(i.p.) injections before each passive smoking of the vehicle or artesunate group. Saline of 0.1ml was given to the normal and cigarette smoke groups as negative controls. Cells in bronchoalveolar lavage fluid (BALF) were collected and analyzed by absolute different cell counts. Interleukin (IL)-8 levels in BALF and 3-nitrotyrosine (NT) levels in lung tissue were tested by emzyme linked immunosorbent assay (ELISA). Malondialdehyde levels in serum, total superoxide dismutase (SOD) activity and total glutathione peroxidase (GPx) activity in lung tissue were detected. The pathological changes of lung tissues were observed by HE staining. And the expression levels of Nrf2 were measured by Western-blotting.Part 2In vivo:A total of 75 female specific pathogen free BALB/c mice were divided randomly into five groups:normal group, cigarette smoke-exposed asthmatic group, vehicle group, artesunate group and dexamethasone group. The latter four groups were exposed on cigarette smoke for 40 days, normal group mice were exposed to ambient air as control. The latter four groups were sensitized by i.p. injections of 20 μg OVA and 4 mg Al(OH)3 suspended in 0.1 ml saline on days 10,17 and 24. Seven days after the last sensitization, mice were challenged with 1% OVA aerosol for 30 min per day in the afternoon until day 40. Two hours before each aerosol challenge, vehicle (5% NaHCO3 containing 5% DMSO), artesunate (30mg/kg, dissolved in 5% DMSO and diluted with 5% NaHCO3), and dexamethasone (1mg/kg, dissolved in saline) was given by i.p. injection to mice in the Vehicle, artesunate, and dexamethasone groups, respectively. Normal group and cigarette smoke-exposed asthmatic group mice were saline sensitized and challenged as negative controls. Six mice in each group were measured airway hyper-responsiveness, and inspiratory and expiratory resistance and dynamic compliance were analyzed. Cells in bronchoalveolar lavage fluid (BALF) of the remaining nine mice were collected and analyzed by absolute different cell counts. IL-4, IL-8, IL-13 and TNF-a levels in BALF were tested by ELISA. The pathological changes of lung tissues were observed by HE staining. Histone deacetylase 2 (HDAC2) activity in lung tissue were tested by HDAC2 activity kit. And the expression levels of PI3Kδ、PI3Kγ p-Akt1、 Akt1、p-HDAC2、HDAC2 were measured by Western-blotting.In vitro:16HBE cells were cultured and were divided randomly into five groups: control group, CSE incubated group, dexamethasone group, common group and artesunate group,. The latter four groups were incubated with CSE for 6 hours, then were stimulated with TNF-a overnight after culture medium were changed. The latter three groups were treated with dexamethasone (10-11 to 10-6 M), both dexamethasone and artesunate befor TNF-a stimulated and artesunate (10μM), respectively, for 1 h. IL-8 levels were tested by ELISA. The 50% inhibitory concentration (IC50) values of dexamethasone for IL-8 production were calculated using GraphPad Prism version 5.0 software. And HDAC2 activity in cells were tested by HDAC2 activity kit.ResultsPart1The total cell counts of BALF in artesunate group were significantly lower than the vehicle group [21.00(2.50)x104/ml vs 35.50(2.50)x104/ml, P<0.001], especially neutrophil counts [6.00(5.12)x104/ml vs 13.60(5.25)x104/ml, P<0.001]. The IL-8 levels in BALF, malondialdehyde levels in serum,3-NT levels and total SOD activity in lung tissue of artesunate group were all drastically lower than those in the vehicle group [(508±55) vs (912±68) pg/ml, (38.2±8.8) vs (48.7±10.6) umol/L, (28.5±5.8) vs (50.0±9.7) ng/ml and (11.8±1.8) vs (18.0±5.3) U/mg protein, respectively, all P<0.05]. No significant difference of total GPx activity existed in these four groups. And the expression level of Nrf2 in artesunate group significantly increased than that in vehicle group (P=0.008).Part 2In vivo:1. As compared to the normal group, cigarette smoke-exposed asthmatic group mice have higher Rl and RE, lower Cdyn (all P<0.001). Artesunate significantly reduced Rl and RE and restored Cydn these effects were similar to the effects of dexamethasone.2. The total and differential cell counts in BALF of the cigarette smoke-exposed asthmatic group were significantly lower than the normal group (P<0.05). Artesunate decreased the neutrophil count(P<0.05), while dexamethasone had little effect on the suppression of neutrophil counts.3. The IL-4, IL-8,ll-13 and TNF-a levels in BALF of the cigarette smoke-exposed asthmatic group were all significantly higher than the normal group (all P<0.001). The vehicle has no effect on these inflammation factors. As compared to the vehicle group, artesunate significantly reduced IL-4, IL-8, IL-13 and TNF-a levels (P<0.001, P<0.001, P<0.001, P=0.001, respectively). Dexamethasone significantly reduced IL-4, IL-8 and IL-13 levels compared to the cigarette smoke-induce asthmatic group (P<0.001, P=0.017, P<0.001, respectively), but has no effect on TNF-a level (P=0.388).4. Lung sections were examined by HE staining showed that the bronchial inflammation of the cigarette smoke-exposed asthmatic and vehicle groups were significantly worse than the normal group, while artesunate and dexamethasone could significantly improve the lung inflammation.5. The cigarette smoke-exposed asthmatic group has lower HDAC2 activity than the normal group (P=0.018), while the vehicle has no effect on HDAC2 activity. As compared to the vehicle, artesunate obviously reverse HDAC2 activity (P=0.018). The vehicle has no effect on HDAC2 activity. Artesunat drastically reverse HDAC2 activity compared to the vehicle group (P=0.030). The HDAC2 activity of dexamethasone group were similar to that of the igarette smoke-exposed asthmatic group.6. Western blotting show that there were no notable differences in p11O(?) Pl3K levels normalized to β-actin protein expression among the groups. Cigarette smoke and OVA concurrent exposure resulted in higher protein expression of p110δ than the normal group. Artesunate significantly inhibited p110δ expression (P<0.001). Dexamethasone had little effect on both Pl3K(?) and Pl3Kδ expression compared with artesunate. Artesunate significantly suppressed phosphorylation of Akt1 compared with the vehicle group (P<0.001), while dexamethasone has little effect on phosphorylation of Akt1. There was no significant difference in nuclear HDAC2 expression levels among any of the groups, the level of phosphorylated HDAC2 was greater in the cigarette smoke-exposed asthmatic group than the normal group (P<0.001). Artesunate significantly suppressed phosphorylation of HDAC2 compared with the vehicle group (P<0.001), while dexamethasone has no significant effect on phosphorylation of HDAC2.In vitro:The IC50 of dexamethasone group was 2.51x10-9M, which was significantly greater than that in the common group (3.98x1O-7M; P<0.001). And the IC50 of the normal group was 6.31 X 10-9M. The HDAC2 activity of the cigarette smoke-exposed asthmatic group was significantly lower than the normal group (P=0.002). The HDAC2 activity of dexamethasone and cigarette smoke-exposed asthmatic groups has no obvious difference (P=0.409). The HDAC2 activity of artesunate and commmon groups were both obviously higher than the cigarette smoke-exposed asthmatic group (P=0.001, P=0.009).Conclusion1. Arteunate attenuates cigarette smoke-induced lung oxidative damage in mice and increases the expression level of Nrf2, and its effects might be mediated by the actions of nuclear Nrf2.2. Artesunate ameliorated cigarette smoke and OVA concurrent exposure-induced airway inflammation, inhibited the Pl3Kδ/Akt pathway, restored HDAC2 activity.3. Artesunate reversed CSE-induced glucocorticoid insensitivity in BEAS-2B cells might via reverse HDAC2 avtivity. |
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