Effects of artesunate on cigarette smoke-induced lung oxidative damage in mice and the expression of Nrf2 and the possible mechanism

Abstract

To explore the effects of artesunate on cigarette smoke-induced lung oxidative damage in mice and the expression of Nuclear factor-E2-related factor 2 (Nrf2). In vivo: A 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 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 Westernblotting. In vitro: 16HBE cells were cultured and transfected with Nrf2 siRNA. Cigarette smoke extract (CSE) were used to stimulate the secretion of IL-8 in cells. Cells were divided into five groups: blank group, non-transfected non-artesunate group, non-transfected artesunate group, transfected non-artesunate group and transfected artesunate group. The latter four groups were incubated with CSE, and non-transfected artesunate and transfected artesunate groups were intervened with artesunate (30 μmol/L) before CSE incubation. The IL-8 levels of each group were measured using ELISA kit. In vivo: The total cell counts of BALF in artesunate group were significantly lower than the vehicle group [21.00(2.50)×10(4)/ml vs 35.50(2.50)×10(4)/ml, P<0.001], especially neutrophil counts [6.00(5.12)×10(4)/ml vs 13.60(5.25)×10(4)/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) ng/L, (38.2±8.8) vs (48.7±10.6) μmol/L, (28.5±5.8) vs (50.0±9.7) μg/L 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). In vitro: The IL-8 level of the non-transfected artesunate group was significantly lower than the non-transfected non-artesunate group [(352±26) vs (765±22) ng/L, P<0.001], while the IL-8 levels between the transfected artesunate and transfected non-artesunate groups had no significant difference. 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.