Superoxide Dismutase Activity In Lung Tissue Research Articles

Effects of simvastatin on iNOS and caspase‑3 levels and oxidative stress following smoke inhalation injury.

The overexpression of inducible nitric oxide synthase (iNOS) induces cell apoptosis through various signal transduction pathways and aggravates lung injury. Caspase-3 is an important protein in the apoptotic pathway and its activation can exacerbate apoptosis. Simvastatin, a hydroxymethyl glutaryl-A reductase inhibitor, protects against smoke inhalation injury by inhibiting the synthesis and release of inflammatory factors and decreasing cell apoptosis. Following the establishment of an animal model of smoke inhalation injury, lung tissue and serum were collected at different time points and the protein and mRNA expression of iNOS and caspase-3 in lung tissue by immunochemistry, western blot and reverse transcription-quantitative polymerase chain reaction, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in lung tissue and serum were analyzed using thiobarbituric acid method and the WST-1 method. The results were statistically analyzed. The lung tissues of the rats in the saline group and the low-, middle- and high-dose groups exhibited clear edema and hemorrhage, and had significantly higher pathological scores at the various time points compared with the rats in the control group (P<0.05). Furthermore, lung tissue and serum samples obtained from these four groups had significantly higher mRNA and protein expression levels of iNOS and caspase-3 (P<0.05), significantly lower SOD activity and higher MDA content (P<0.05). Compared with the saline group, the low-, middle- and high-dose groups had significantly lower pathological scores (P<0.05), significantly lower mRNA and protein expression levels of iNOS, caspase-3 and MDA content in lung tissues (P<0.05) and significantly higher SOD activity in lung tissues and serum. The middle- and high-dose groups had significantly lower pathological scores (P<0.05), significantly decreased iNOS and caspase-3 mRNA and protein expression in lung tissues, significantly higher SOD activity in lung tissues and serum and a significantly lower MDA content (P<0.05) compared with the low-dose group. With the exception of SOD activity in lung tissues at 24 and 72 h and MDA content in serum at 48 h, no significant differences were observed between the middle- and high-dose groups. The present study demonstrated that there was an association between the therapeutic effect and dosage of simvastatin within a definitive range. In rats with smoke inhalation injury, simvastatin inhibited iNOS and caspase-3 expression in lung tissues and mitigated oxidative stress, thereby exerting a protective effect. In addition, the effect and dose were associated within a definitive range.

Effect of dexmedetomidine on acute lung injury induced by "double hit" in rats

Objective To evaluate the effect of dexmedetomidine on acute lung injury (ALI) induced by double hit in rats. Methods Forty SPF male Sprague-Dawley rats, aged 8 weeks, weighing 215-225 g, were divided into 4 groups(n=10 each) using a random number table method: sham operation group (Sham group), double hit -induced ALI group (ALI group), double hit -induced ALI plus dexmedetomidine group (ALI+ Dex group) and double hit -induced ALI plus dexmedetomidine plus yohimbine group (ALI+ Dex+ Y group). ALI was induced by blunt chest trauma and hemorrhagic shock-resuscitation in pentobarbital sodium-anesthetized rats in ALI, ALI+ Dex and ALI+ Dex+ Y groups.In group ALI+ Dex, dexmedetomidine was intravenously infused for 10 min in a loading dose of 1 μg·kg-1·h-1 starting from the time point immediately after blunt chest trauma, followed by continuous infusion of 5 μg·kg-1·h-1 until the end of operation.Yohimbine 0.1 mg/kg was injected after giving the loading dose of dexmedetomidine and at 3 h after blunt chest trauma in ALI+ Dex+ Y group.Rats were sacrificed at 6 h after successful establishment of the model, blood samples were obtained for determination of concentrations of TNF-α and IL-6 in serum (by enzyme-linked immunosorbent assay), and lung tissues were obtained for determination of wet/dry weight (W/D) ratio, expression of Fas, FasL and caspase-3 in lung tissues (by SABC assay), cell apoptosis (by TUNEL), content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) and for examination of the pathological changes which were scored.The apoptosis index (AI) was calculated. Results Compared with Sham group, the lung injury score, AI, levels of Fas, FasL, caspase-3 and MDA, and concentrations of TNF-α and IL-6 in serum were significantly increased, and the SOD activity in lung tissues was decreased in ALI group (P<0.05). Compared with ALI group, the lung injury score, AI, levels of Fas, FasL, caspase-3 and MDA, and concentrations of TNF-α and IL-6 in serum were significantly decreased, and the SOD activity in lung tissues was increased in ALI+ Dex and ALI+ Dex+ Y groups (P<0.05). Compared with group ALI+ Dex, the lung injury score, AI, levels of Fas, FasL, caspase-3 and MDA, and concentrations of TNF-α and IL-6 in serum were significantly increased, and the SOD activity in lung tissues was decreased in group ALI+ Dex+ Y (P<0.05). Conclusion Dexmedetomidine reduces ALI induced by double hit in rats, and the mechanism may be partially related to activating alpha-2 adrenergic receptor and inhibiting inflammatory responses, lipid peroxidation and cell apoptosis. Key words: Dexmedetomidine; Thoracic injury; Shock, hemorrhagic; Acute lung injury

Effects of budesonide on bronchopulmonary bysplasia and HMGB1/TLR4/NF-κB pathway in neonatal mice induced by hyperoxia

Objective To investigate the effects of budesonide (BUN) on high mobility group protein 1 (HMGB1)/toll-like receptor 4 (TLR4) / nuclear factor κB (NF-κB) pathway in neonatal mice with hyperoxia-induced bronchopulmonary dysplasia (BPD). Methods 80 mice were randomly divided into control group, model group, low, medium and high dose BUN groups, with 16 mice in each group. Mice in the control group were exposed to air, and mice in model group and BUN low, medium and high dose groups were exposed to 60% oxygen to establish BPD model; After 24 hours of modeling, the control group and model group were inhaled with normal saline every day. The low, middle and high dose BUN groups inhaled (1, 2, 4 ml) BUN, respectively, 12 h/times, and continued to inhale until the mice were executed. On the 7th and 14th days of the model establishment, hematoxylin-eosin (HE) staining was used to observe the morphological changes of lung tissue; the levels of interleukin (IL) -1β, IL-6, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD) in lung tissues were detected by enzyme-linked immunosorbent assay (ELISA); the levels of HMGB1, TLR4 and NF-κB in lung tissue were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and protein immunoblotting (WB). Results In the model group, the lung tissue structure was severely damaged and the alveolar volume was enlarged to form bullae on the 7th and 14th day. In low, middle and high dose groups, with the increase of BUN concentration, the lung tissue deformation gradually improved and the alveolar structure gradually became intact. Compared with the control group, the number of radial alveoli in the lung tissues of the model group decreased at 7th and 14th days; SOD activity decreased; the expression levels of IL-1β, IL-6, TNF-α, MDA, HMGB1, TLR4 and NF-κB mRNA and protein were increased (P<0.05). Compared with the model group, the number of radial alveoli and SOD activity in lung tissues of BUN groups increased gradually at 7th and 14th days , while the levels of IL-1β, IL-6, TNF-α, MDA, HMGB1, TLR4 and NF-κB decreased gradually at different doses (P<0.05). Conclusions BUN may inhibit the levels of inflammatory factors and oxidative stress by regulating HMGB1/TLR4/NF-κB pathway to achieve the protective effect on BPD neonatal mice. Key words: Budesonide; Hyperoxia; Bronchopulmonary dysplasia; HMGB1 protein; Toll-like receptor 4; NF-kappa B; Mice

To explore the preventive and therapeutic effects of Xuebijing injection on acute lung injury induced by cardiopulmonary bypass in rats by regulating the expression of microRNA-17-5p and its mechanism

To investigate the preventive effect of Xuebijing injection on acute lung injury induced by cardiopulmonary bypass (CPB) and the underlying mechanism. (1) In vivo experiment: 30 Sprague-Dawley (SD) rats were randomly divided into sham group, CPB group, Xuebijing pretreatment group (XBJ+CPB group) with 10 rats in each group. CPB model was reproduced in rats; and CPB was not performed in sham group, but only through arteriovenous puncture. In the XBJ+CPB group, 4 mL/kg Xuebijing injection was injected intraperitoneally 2 hours before CPB, sham group and CPB group were injected with equal volume of normal saline at the same time. The blood from femoral artery was analyzed 4 hours after operation, and the oxygenation index (PaO2/FiO2) was calculated. Then the rats were sacrificed to collect bronchoalveolar lavage fluid (BALF), and the lung permeability index (PPI) was calculated. The lung tissues were harvested, and the wet/dry weight ratio (W/D) of lung tissue was measured. The index of quantitative evaluation of alveolar injury (IQA) was measured. The levels of interleukins (IL-1, IL-6) and tumor necrosis factor-α (TNF-α) in lung tissue and BALF were measured by enzyme-linked immunosorbent assay (ELISA). The content of malondialdehyde (MDA) and the activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) in lung tissue were detected by biochemical method. The microRNA-17-5p (miR-17-5p) expression in lung tissue was determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). (2) In vitro experiments: type II alveolar epithelial cells (AEC II) were cultured in vitro, and they were randomly divided into control group (the cells were treated by preoperative serum of CPB in patients with ventricular septal defect), CPB group (the cells were treated by serum after CPB in patients), and XBJ+CPB group (Xuebijing injection 10 g/L+serum after CPB in patients). After 12 hours of culture in each group, the expression of miR-17-5p was detected by RT-qPCR. AEC II cells were transfected with miR-17-5p mimic, inhibitor or corresponding control oligonucleotide (negative control), respectively, to observe the effect of miR-17-5p on Xuebijing regulating CPB-induced apoptosis rate and caspase-3 activity. (1) In vivo experiment: compared with the sham group, the PPI, lung W/D ratio, IQA, and IL-1, IL-6, TNF-α in lung tissue and BALF, as well as MDA content and MPO activity in lung tissue were significantly increased, PaO2/FiO2 and SOD activity in lung tissue were significantly decreased. The parameters of the XBJ+CPB group were significantly improved, suggesting that Xuebijing pretreatment could improve CPB-induced ALI in rats. The expression of miR-17-5p in lung tissue of the CPB group was significantly down-regulated as compared with sham group (2-ΔΔCt: 0.48±0.13 vs. 1.00±0.11, P < 0.05); while the expression of miR-17-5p in the XBJ group was significantly up-regulated as compared with the CPB group (2-ΔΔCt: 1.37±0.09 vs. 0.48±0.13, P < 0.05), indicating that the improvement of Xuebijing injection on lung injury after CPB might be related to miR-17-5p. (2) In vitro experiment: the changes in miR-17-5p expression in each group of AEC II cells confirmed in vivo results. After transfection of miR-17-5p mimic, the apoptotic rate and caspase-3 activity of each group were significantly lower than those transfected with negative control, and the decrease was more significant in the XBJ+CPB group [apoptotic rate: (7.37±0.95)% vs. (12.60±1.90)%, caspase-3 (A value): 0.82±0.09 vs. 1.37±0.08, both P < 0.05]. After transfection of miR-17-5p inhibitor, the apoptotic rate and caspase-3 activity of each group were significantly more than those transfected with negative control [in the XBJ+CPB group: apoptotic rate was (16.30±1.86)% vs. (12.60±1.90)%, caspase-3 (A value) was 1.78±0.13 vs. 1.37±0.08, both P < 0.05]. This indicated that the apoptosis of AEC II cells cultured in serum after CPB was significantly reduced by miR-17-5p, and further reduced by the pretreatment with Xuebijing. Xuebiing injection can reduce the inflammatory reaction and oxidative stress of lung tissue in rats with ALI induced by CPB, and improve oxygenation. The mechanism may be related to up-regulation of miR-17-5p expression in AEC II cells and inhibition of apoptosis of AEC II cells.

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

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.

Role of superoxide anion on basal and stimulated nitric oxide activity in neonatal piglet pulmonary vessels.

The superoxide anion (O2*-) appears to be an important modulator of nitric oxide bioavailability. Enzymatic scavenging of O2*- is carried out by superoxide dismutase (SOD). The present study was designed to characterize the developmental changes on pulmonary vascular reactivity induced by 1) exogenous Cu/Zn SOD, 2) several putative SOD mimetics, and 3) endogenous SOD inhibition. We also analyzed age-related changes on pulmonary SOD activity and vascular O2*- levels. SOD (1-300 U/mL) produced endothelium-dependent relaxation of U46619-contracted intrapulmonary arteries (fourth branch) and veins from 12- to 24-h-old and 2-wk-old piglets. SOD-induced relaxation was greater in pulmonary arteries and was abolished by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. SOD induced a greater pulmonary artery relaxation in the 2-wk-old than in the 12- to 24-h-old piglet. SOD (100 U/mL) did not modify acetylcholine-induced relaxation in pulmonary arteries. In contrast, endogenous SOD inhibition by diethyldithiocarbamate (3 mM) impaired acetylcholine-induced relaxation in pulmonary arteries from newborn but not from 2-wk-old piglets. Total SOD activity in lung tissue did not change with postnatal age. With the use of dihydroethidium, an oxidant-sensitive fluorescent probe, we did not find significant age- or vessel-related differences in O2*- presence. From the putative SOD mimetics tested, only the metal salts MnCl2 and CuSO4 reproduced the vascular effects of SOD. In summary, SOD produces endothelium-dependent pulmonary vascular relaxation by protecting nitric oxide from destruction by O2*-. This effect was less marked in newborns than in 2-wk-old piglets. In contrast, pulmonary arteries from newborn piglets are more sensitive to the inhibition of endogenous SOD.