| Hypoxia/reoxygenation(H/R)and oxidative stress injuries occur frequently in human tissues,especially in the respiratory system,on account of the lung and bronchi are directly exposed to the external environment and exchange gas with the ambient air.A large number of studies have confirmed that H/R usually results in an increase in the accumulation of reactive oxygen species(ROS),which then initiates uncontrolled oxidative stress damage and subsequent cellular apoptosis.These changes are accompanied by organelles dysfunction and the occurrence of multiple diseases.On the other hand,the role of environmental factors in the pathological mechanism of airway inflammatory disease has become more and more concerned with the deterioration of atmospheric pollution.To a large extent,many pulmonary inflammation(such as chronic obstructive pulmonary disease(COPD),bronchial asthma,lung cancer,emphysema,etc.)are characterized with their chronic course and frequent recurrence.Due to the the limitation of ventilation,these diseases accompany and even aggravate long-term,repeated hypoxia/reoxygenation injury and oxidative stress state.Therefore,it is especially important to develop respiratory system drugs to resist inflammation of airway and oxidative stress.Bronchial epithelial cell is the first line of defense against external stimuli.Air pollutants,cigarette smoke(CS)and mechanical injury can lead to bronchial epithelial cell damage.Bronchial epithelial cells are also the main target of H/R injury.Persistence of oxidative stress causes damage to the bronchial and lung tissues,epithelial cell apoptosis,abscission,mucus secretion,and release of a variety of inflammatory factors,aggravating the inflammatory reaction.In the first part of this dissertation,a lipopolysaccharide(LPS)-induced nitric oxide(NO)model in murine peritoneal macrophages was established.The anti-inflammatory activity was screened for the TSG under 1.25 ?M to 640 ?M of concentrations(2 × diluted),and the IC50 equals 1.20 × 10-4 M of which the compound inhibited LPS-induced rat peritoneal macrophages to produce NO.Furthermore,the effects of TSG on LPS-stimulated TNF-?,IL-6 and IL-1? production in murine peritoneal macrophages under 1.25?m to 640 ?m of concentrations were observed.The results showed that the TSG had partly anti-inflammatory activity,which inhibited the production of NO in LPS-induced murine primary peritoneal macrophages,but there was no significant difference in the effect of inhibition of TNF-?,IL-6 and IL-1?production.This suggests that,although the TSG had anti-inflammatory activity,it is necessary to further evaluate the systematic activity.According to some report,TSG may have some antioxidant activity rather than anti-inflammation.Therefore,a H/R in-vitro damage model was further developed to observe the effects of TSG in human bronchial epithelial cells(BEAS-2B)on hypoxia/reoxygenation injury.The results showed that the proliferation of BEAS-2B cells was inhibited after H/R treatment,and the pretreatment of TSG could significantly improve the survival rate of BEAS-2B cells after H/R treatment with the relatively small cytotoxicity.Further experiments observed that even at the concentration of 12.5 ?M TSG could significantly inhibit the generation of cellular ROS,while also inhibiting the production of malondialdehyde(MDA).No significant difference was observed in the activity of superoxide dismutase(SOD).In the second part of this dissertation,the mechanism of the anti-oxidative stress damage of TSG was discussed.On the basis of the previous results,it was further experimentally demonstrated that the TSG can inhibit the BEAS-2B apoptosis of bronchial epithelial cells induced by H/R.With GFP-Bax-stable transfected human breast cancer cell lines MCF-7/GFP-Bax as a tool,the activity of TSG inhibiting translocation of pro-apoptotic protein Bax to mitochondrial and inhibiting nuclear damage was observed.Hoechst 33258 staining results showed that the TSG(50.0?M,100 ?M)could significantly inhibit H/R-induced nuclear crenation and fragmentation of MCF-7/GFP-Bax.Under fluorescence microscope,we observed that the TSG(25.0 ?M,50.0 ?M,100 ?M)inhibited the translocation of Bax to mitochondria.Through Western Blot,the same effect of TSG which inhibited the translocation of Bax to mitochondria in BEAS-2B cells was observed.In H/R-induced BEAS-2B cells,TSG inhibited the decrease of mitochondrial membrane potential,cytochrome C release and caspase-9,caspase-3 activation.Mitogen-activated protein kinase(MAPK)plays an important role in mediating inflammation,oxidative stress stimulation and downstream cell response.When H/R occurs,oxidative stress activates the cascade reaction of MAPK determining cell viability or apoptosis.p38 MAPK and SAPK/JNK 1/2 in this family are activated by inflammatory cytokines and environmental stress factors,and ERK 1/2 is activated by growth factors and cytokines,which plays a role in promoting cell proliferation.The regulative effect of TSG on MAPK signaling pathway through Western Blotting was observed.The results showed that the TSG could significantly reduce the phosphorylated protein/total protein ratio of p38 and JNK 1/2 in H/R-treated BEAS-2B cells.On the other hand,it was also observed that the TSG can reduce the phosphorylation activation of nuclear protein p53 by lowering the expression of hypoxia-inducible factor 1(HIF-1),which synergisticly inhibited the cellular apoptosis.In summary,this research consists of two parts:(1)The establishment of in-vitro H/R injury model and the observation of the effects of compound TSG in anti-inflammatory and anti-oxidative stress injury in murine peritoneal macrophages.(2)H/R-induced mitochondrial apoptosis pathway and the mechanism of the protective effect of TSG on oxidative stress injury induced by H/R in bronchial epithelial cells.This research provides the research ideas and experimental basis for the the protection of ischemic-reperfusion injury and the prevention and treatment of asthma,chronic obstructive pulmonary disease,emphysema and other respiratory diseases. |
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