Effects Of Smoking On Pulmonary Complications After Major Craniocerebral Injury And Mechanisms For The Protection Cigarette Smoke-induced Lung Injury By Andrographolide

Part1:The Effect of Smoking on Acute Lung Injury after Severe Craniocerebral InjuryObjective:To investigate the effect of smoking on acute lung injury after severe craniocerebral injury.Methods:Select162severe craniocerebral injury patients admitted to our hospital from January2010to December2011, male139cases and female23cases, aged18to72years old, the average age was (44.86±13.51) years. Traffic accident injury85cases, high-altitude fall injury30cases, mucker injury28cases, violence hurt8cases, the other11cases. Patients were divided into smoking group (74cases) and non-smoking group (88cases) basing on the status of smoking.Judging whether severe craniocerebral injury patients were associated with acute lung injury on the5day after admission and comparing the acute lung injury incidence rates between smoking group and non-smoking group. Measurement of the lung function in patients with severe craniocerebral injury was performed on day0and day5after admission. Lung capacity indicators:breathing frequency (BF), vital capacity (VC); pulmonary ventilation indicators:forced expiratory volume in one second (FEV1), forced expiratory vital capacity (FVC), maximum minute ventilation (MMV); small airways indicators:peak expiratory flow rate (PEF). Collecting bronchoalveolar lavage fluid on day0and day5after admission, and detecting the TNF-a, IL-8and MDA levels and SOD activity in broncho alveolar lavage fluid supernatant in accordance with the the kit instructions.Results:1.26severe craniocerebral injury patients were associated with acute lung injury and the incidence rate reached35.14%in the smoking group, while only16cases were associated with acute lung injury and the incidence rate was18.18%, the difference between the two groups was statistically significant(P=0.019).2. On the0day after admission, the BF, VC, FEV1, FVC, MMV, PEF in smoking goup were lower than the non-smoking group, and the differences of MMV, PEF between two groups were statistically significant(P=0.019). On the5day after admission comparing with on the0day after admission, the lung functions of patients in two groups both showed a downward trend, and the downward trend in smoking group was more obvious than the non-smoking group.3. On the0day after admission, the TNF-a, IL-8, MDA levels in smoking group were higher than the non-smoking group and the difference between was statistically significant(P<0.05); the SOD activity in smoking group was lower than the non-smoking group. On the5day after admission comparing with on the0day after admission, the TNF-a, IL-8, MDA levels of patients in two groups both showed a upward trend, and the upward trend in smoking group was more obvious than the non-smoking group; the SOD activity of patients in two groups showed a downward trend, and the downward trend in smoking group was more obvious than the non-smoking group. The four indicators between two groups had significant differences (P<0.05).Conclusions:Compared with the non-smoking group, the basic lung function and SOD activity of severe craniocerebral injury were lower and TNF-a, IL-8and MDA levels were higher; the decrease of lung function and SOD activity and increase of TNF-a, IL-8and MDA levels were more significant, the incidence rate of acute lung injury was higher in the smoking group after injury, which may be related to smoking and craniocerebral injury cooperatively promoting inflammatory response and oxidative stresPart2:The effects of andrographolide on passive cigarette smoke-induced acute lung injuryObjective:To investigate the effects of andrographolide on cigarette smoke-induced acute lung inflammation.Methods:32C57/BL/6mice (18-20g) were6-8weeks old. Mice were randomly divided into4groups (8mice for each group):control group (C), cigarette smoke-exposed group (CS), DMSO and CS-treated group (CS+DMSO), andrographolide and CS-treated group (CS+A). Andrographolide was given by intraperitoneal injection at a dose of1mg/kg body weight,1h before CS exposure. Animals were placed in an inhalation chamber and exposed to9cigarettes daily for4consecutive days for CS-exposed groups.Control mice were placed in the same condition but not exposed to cigarette smoke. On the fifth day, mice were killed with an overdose of intraperitoneal sodium pentobarbital. Bronchoalveolar lavage fluid (BALF) was collected. Histologial analysis of mouse lungs was performed. Total cell count in BALF was determined by a hemocytometer. Differential cell counts were performed with Wright-Giemsa method. ELISA assay was used to analyze the production of tumor necrosis factor-alpha (TNF-α), interleukine-6(IL-6), macrophage inflammatory protein-2(MIP-2), interleukine-10(IL-10), LPS-induced CXC chemokine (LIX), and interferon-gamma (IFN-γ) in BALF. The pulmonary myeloperoxidase (MPO) activity was measured. Lung epithemial cell line A549cells were used as an in-vitro cell mdoel. DNA fragmentation assay was done to analyze the effect of andrographolide on cigarette smoke extract-induced apoptosis of A549cells. Western blot analysis was used to analyze the expression of apoptosis-associated factors in CS-exposed mice. Results:Mice in response to CS exposure had pulmonary lesions with interstitial edema and large areas of marked intravascular, interstitial, and intra-alveolar inflammatory cell infiltration Pretreatment with DMSO had no effects on CS-induced acute lung inflammation. However, the influx of inflammatory cells and edema in the lungs of CS-exposed mice was alleviated by the administration of andrographolide. Andrographolide inhibited CS-enhanced BALF cellularity, such as total cells, macrophages, neutrophils, and lymphocytes, suppressed CS-induced production of BALF TNF-a, IL-6and MIP-2, and also decreased the lung MPO activity. In vitro study showed that the treatment with cigarette smoke extract significantly enhaned the apoptosis of A549cells and increased the expresson of cleaved caspase-3and cleaved poly (ADP-ribose) polymerase, all which were markedly decreased by andrographolide.Conclusion:Andrographolide inhibits CS-induced acute lung inflammation and protects against CSE-mediated apoptosis of A549cells.Part3:The mechanisms for andrographolide-mediated protective effects against passive cigarette smoke-induced lung injuryObjective:To investigate the molecular mechanisms underlying andrographolide-mediated protective effects against cigarette smoke-induced acute lung injury, especially the involvement of heme oxygenase-1(HO-1) signaling patheay.Methods:In the present study, lung epithemial cell line A549cells were used as an in-vitro cell mdoel. RT-PCR and Western blot analysis were employed to analyze the effects of cigarette smoke and andrographolide on HO-1expression at both mRNA and protein levels.32C57/BL/6mice (18-20g) were6-8weeks old. Mice were randomly divided into4groups (8mice for each group):control group (C), cigarette smoke exposure group (CS), andrographolide treatment together with CS exposure group (A), and A+Z group, with zinc protoporphyrin IX (ZnPP) administration before andrographolide treatment and CS exposure. Andrographolide was given by intraperitoneal injection at a dose of1mg/kg body weight,1h before CS exposure. ZnPP, a potent heme oxygenase inhibitor, was administered intraperitoneally at a dose of10μmol/kg body weight,1h before andrographolide administration. Animals were placed in an inhalation chamber and exposed to9cigarettes daily for4consecutive days for CS-exposed groups.Control mice were placed in the same condition but not exposed to cigarette smoke. On the fifth day, mice were killed with an overdose of intraperitoneal sodium pentobarbital. Bronchoalveolar lavage fluid (BALF) was collected. Histologial analysis of mouse lungs was performed. RT-PCR and Western blot analysis were performed to analyze the expression of HO-1in the lung tissues. Pulmonary HO-1activity was also analyzed. Total cell count in BALF was determined by a hemocytometer. Differential cell counts were performed with Wright-Giemsa method. The total protein in BALF was measured by the BCA method. ELISA assay was used to analyze the production of tumor necrosis factor-alpha (TNF-a), interleukine-6(IL-6), macrophage inflammatory protein-2(MIP-2). The levels of MDA in the lung tissues and BALF were measured. The activity of LDH in BALF and the lung tissues and the activities of Superoxide Dismutase (SOD), Glutathione peroxidase (GSH-Px), Catalase (CAT), myeloperoxidase (MPO) were detected in mouse lung tissues.Western blot analysis was performed to analyze the effects of andrographolide on STAT3signaling pathway.Results:Both CS and andrographolide increased the expression of HO-1at both mRNA and protein levels in a dose-dependent manner, indicating that HO-1might exhibit protecitive effect against CS-induced lung injury. Mice in response to CS exposure had pulmonary lesions with interstitial edema and large areas of marked intravascular, interstitial, and intra-alveolar inflammatory cell infiltration. The influx of inflammatory cells and edema in the lungs of CS-exposed mice was alleviated by the administration of andrographolide. Interestingly, ZnPP pretreatment markedly abolished the protective effects of andrographolide on CS-induced lung injury. Pretreatment with andrographolide markedly attenuated lung inflammation in CS-exposed mice, coupled with reduced numbers of total cells, neutrophils, and macrophages, total protein level in bronchial alveolar lavage fluid (BALF), decreased production of cytokine/chemokine (TNF-a, IL-6, and MIP-2) into BALF, less MDA in the lung tissues and BALF, and lowered activities of LDH (in both BALF and the lung tissues), SOD, GSH-Px, CAT, and MPO in lung tissues. Notably, these histological and biochemical changes induced by andrographolide were blocked by prior administration of ZnPP. Moreover, andrographolide-induced phosphorylation of signal transducer and activator of transcription3was attenuated by ZnPP treatment in CS-exposed animals.Conclusion:Our data collectively demonstrate that andrographolide confers protection against CS-induced lung inflammation, partially through activation of HO-1and STAT3.