红霉素对烟草烟雾刺激下沉默信息调节因子1（SIRT1）的影响
发布时间：2018-09-01 19:35
【摘要】：第一部分：红霉素对烟草烟雾暴露下人巨噬细胞释放活性氧及炎症因子的作用目的：1.观察烟草烟雾刺激对人巨噬细胞产生活性氧的影响,以及红霉素能否调控由烟草烟雾暴露介导的人巨噬细胞活性氧(Reactive oxygen species, ROS)释放。2.观察烟草烟雾刺激条件下,人巨噬细胞释放的白细胞介素6(interleukin 6, IL-6)、白细胞介素8(interleukin 8, IL-8)、肿瘤坏死因子a (tumor necrosis factor a, TNF-α)水平改变,以及红霉素对于烟草烟雾暴露下人巨噬细胞释放炎症因子水平的影响。方法：研究对象为人淋巴瘤单核细胞株U937,经10nM佛波酯(phorbol 12-myristate 13-acetate, PMA)诱导24h后,U937转换成巨噬细胞,用香烟烟雾提取物(cigarette smoke extract, CSE)刺激人巨噬细胞。用四甲基偶氮唑蓝(MTT)法检测CSE及红霉素对细胞最适宜作用浓度。细胞分组：对照组,CSE组(加入1%CSE孵育24h),CSE+红霉素24h组(1μg/ml红霉素预孵育24h后,加入1%CSE继续刺激24h),CSE+红霉素48h组(1μg/ml红霉素预孵育48h后,加入1%CSE继续刺激24h)。各细胞组在红霉素(Erythromycin, EM)、CSE干预下,以2',7'-二氯二氢荧光素二乙酯(2',7'-dichlorofluorescin-diacetate, DCFH-DA, 10mM)作为荧光探针,流式细胞术检测各组细胞中ROS水平。用酶联免疫吸附法(ELISA)检测各实验组细胞上清液中IL-6、IL-8、 TNF-α释放水平。结果：1%浓度的CSE能显著上调人巨噬细胞ROS的释放(P0.01),红霉素(lug/ml)预孵育24h、48h能抑制1%CSE对细胞ROS的上调作用(p0.01),SIRT1特异性抑制剂(NAM)显著上调人巨噬细胞ROS的释放(P0.01)；CSE 1%增加人巨噬细胞释放IL-6、IL-8、TNF-α,红霉素(lug/ml)预孵育24h减少IL-6、TNF-α释放(p0.01)。红霉素(lug/ml)预孵育48h对人巨噬细胞释放IL-6、IL-8、TNF-α的抑制作用更加明显(p0.01)。结论：1.烟草烟雾刺激引起人巨噬细胞释放活性氧增多,红霉素抑制氧化应激机制之一是通过抑制活性氧释放。2.烟草烟雾刺激促进人巨噬细胞释放IL-6、IL-8、TNF-α增加,红霉素抑制炎症机制之一通过抑制人巨噬细胞释放IL-6、IL-8、TNF-α。第二部分：红霉素对烟草烟雾刺激下人巨噬细胞内SIRT1、NF-κB蛋白表达及两种蛋白之间的相互作用目的：观察人巨噬细胞中SIRT1及NF-κB蛋白表达在烟草烟雾暴露条件下的改变,以及红霉素对烟草烟雾刺激条件下SIRT1和NF-κB蛋白表达产生何种影响。观察人巨噬细胞中SIRT1及NF-κB蛋白是否存在相互作用,以及在烟草烟雾暴露、红霉素治疗下,存在何种相互作用关系。方法：1.用10nmol/ml PMA孵育24h可将人淋巴瘤单核细胞株U937诱导分化成巨噬细胞,用CSE刺激巨噬细胞,制造细胞在烟草烟雾暴露氧化应激模型。分为：对照组,CSE组,CSE+红霉素24h组,CSE+红霉素48h组,NF-κB蛋白特异性抑制剂PDTC组(20nM PDTC预孵育24h后加入1%CSE继续孵育24h),SIRT1特异性抑制剂NAM组(20mM NAM孵育24h)。用western blot (WB)方法检测各组细胞中NF-κB、SIRT1蛋白表达。2.将U937诱导分化成巨噬细胞后,对细胞分组并进行实验。分为：对照组,CSE组,CSE+红霉素24h组,CSE+红霉素48h组。提取各组细胞总蛋白,先用免疫共沉淀(co-immunoprecipitation, Co-IP)进行蛋白沉淀,再用westernblot (WB)方法检测各组细胞中NF-κB、SIRT1蛋白表达。结果：1.CSE暴露会导致人巨噬细胞中SIRT1蛋白表达明显下降,并能增加NF-κB蛋白表达,经红霉素预孵育24h后再予CSE刺激,能促进人巨噬细胞中SIRT1蛋白表达升高,同时抑制NF-κB蛋白表达升高,经红霉素预孵育48h后,对人巨噬细胞中SIRT1蛋白表达升高以及对NF-κB蛋白的抑制作用更加明显。结果：1.CSE暴露会导致人巨噬细胞中SIRT1蛋白表达明显下降,增加NF-κB蛋白表达,经红霉素预孵育24h、48h,均促进人巨噬细胞中SIRT1蛋白表达升高,同时抑制NF-κB蛋白表达升高,其中48h组更明显。2.经Co-IP检测,SIRT1和NF-κB蛋白在人巨噬细胞内存在直接相互作用,CSE刺激可导致SIRT1蛋白表达减少,从而促进NF-κB蛋白表达增加,经红霉素预孵育24h、48h,可增加人巨噬细胞中SIRT1蛋白表达,抑制NF-κB蛋白表达。结论：1烟草烟雾导致炎症的机制可能是通过氧化应激抑制人巨噬细胞SIRT1蛋白表达,进而促进NF-κB蛋白表达,从而促进炎症介质IL-6、IL-8、 TNF-α释放。2.红霉素抑制氧化应激诱导的炎症机制可能是通过红霉素增加SIRT1蛋白表达,进而抑制NF-κB蛋白表达,从而抑制炎症介质释放。第三部分：红霉素对烟草烟雾暴露小鼠肺组织以及肺组织中SIRT1与NF-κB蛋白作用目的：观察烟草烟雾暴露对小鼠肺组织的影响,以及红霉素对烟草烟雾暴露小鼠肺组织的作用。观察受烟草烟雾暴露刺激小鼠的肺组织中SIRT1及NF-κB蛋白表达的改变,以及红霉素对烟草烟雾刺激下对小鼠肺组织内SIRT1及NF-κB蛋白表达的作用。方法：将8周大小的雄性BALB/c小鼠分为：对照组,烟草烟雾暴露组,红霉素治疗组。烟草烟雾暴露组每天烟草烟雾暴露2h,持续12w,红霉素治疗组在烟草烟雾暴露前用红霉素(100mg/kg-day)灌胃治疗,余处理同烟草烟雾暴露组。建模到期后取右肺组织切片行HE染色,剩余肺组织行westernblot检测各组小鼠肺组织中NF-κB、SIRT1蛋白表达。结果：1.烟草烟雾暴露小鼠肺组织中出现炎症,支气管周围炎症细胞聚集,肺组织出现肺气肿表现。红霉素治疗组炎症反应较烟草烟雾暴露组减轻,肺气肿表现减轻。2.烟草烟雾暴露小鼠肺组织中SIRT1蛋白较对照组明显减少,NF-κB蛋白明显增加。红霉素治疗组小鼠肺组织中SIRT1蛋白较对照组减少,但较烟草烟雾暴露组表达增加。结论：1.红霉素减轻烟草烟雾暴露引起的小鼠肺组织炎症,可能与红霉素上调肺组织SIRT1蛋白表达,抑制肺组织中NF-κB蛋白表达相关。2.红霉素减轻小鼠肺气肿,可能与红霉素促进SIRT1表达相关。
[Abstract]:Part I: Effects of erythromycin on the release of reactive oxygen species (ROS) and inflammatory factors from human macrophages exposed to tobacco smoke. Objective: 1. To observe the effects of tobacco smoke on the production of reactive oxygen species (ROS) in human macrophages and whether erythromycin can regulate the release of ROS from human macrophages mediated by tobacco smoke exposure. To observe the changes of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-A (TNF-a) released by human macrophages under tobacco smoke stimulation, and the effect of erythromycin on the release of inflammatory factors from human macrophages under tobacco smoke exposure. METHODS: Human lymphoma monocyte U937 was induced by 10 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours. U937 was transformed into macrophages and stimulated by cigarette smoke extract (CSE). The optimum concentration of CSE and erythromycin on human lymphoma cells was determined by MTT assay. Cell groups: control group, CSE group (incubated with 1% CSE for 24 hours), CSE + erythromycin 24 hours group (incubated with 1 ug / ml erythromycin for 24 hours, added 1% CSE for 24 hours), CSE + erythromycin 48 hours group (incubated with 1 ugh / ml erythromycin for 48 hours, added 1% CSE for 24 hours). Cell groups were treated with Erythromycin (EM), CSE and 2', 7'-dichlorodihydrofluorescence. Optical diethyl ester (2', 7'-dichlorofluorescin-diacetate, DCFH-DA, 10mM) was used as a fluorescent probe to detect the ROS level in the cells of each group. The levels of IL-6, IL-8 and TNF-a in the supernatant of the cells of each experimental group were detected by enzyme-linked immunosorbent assay (ELISA). Results: CSE at 1% concentration could significantly up-regulate the release of ROS from human macrophages (P 0.01). Erythromycin (lug / ml) could inhibit the up-regulation of ROS by 1% CSE (p0.01), SIRT1 specific inhibitor (NAM) could significantly increase the release of ROS by human macrophages (p0.01), and 1% CSE could increase the release of IL-6, IL-8, TNF-a, and erythromycin (lug / ml) by 48 h after pre-incubation. Conclusion: 1. Tobacco smoke stimulates the release of reactive oxygen species from human macrophages. One of the mechanisms of erythromycin inhibiting oxidative stress is through inhibiting the release of reactive oxygen species. 2. Tobacco smoke stimulates the release of IL-6, IL-8, TNF-a from human macrophages, and erythromycin. One of the mechanisms of inhibiting inflammation is by inhibiting the release of IL-6, IL-8 and TNF-alpha from human macrophages. Part II: The effect of erythromycin on the expression of SIRT1, NF-kappa B protein in human macrophages stimulated by tobacco smoke and the interaction between the two proteins. To observe the interaction between SIRT1 and NF-kappa B protein in human macrophages and the interaction between SIRT1 and NF-kappa B protein in tobacco smoke exposure and erythromycin treatment. Methods: 1. Human lymphoma monocytes were incubated with 10 nmol/ml PMA for 24 hours. U937 cells were induced to differentiate into macrophages and stimulated with CSE to produce a model of oxidative stress induced by tobacco smoke exposure. The cells were divided into control group, CSE group, CSE + erythromycin 24 h group, CSE + erythromycin 48 h group, NF-kappa B protein specific inhibitor PDTC group (20nM PDTC pre-incubated for 24 h and added 1% CSE for 24 h), SIRT1 specific inhibitor NA. After U937 was induced to differentiate into macrophages, the cells were divided into control group, CSE group, CSE + erythromycin 24 h group and CSE + erythromycin 48 h group. The expression of NF-kappa B and SIRT1 protein was detected by Western blot (WB). Results: 1. The expression of SIRT1 protein in human macrophages was significantly decreased and the expression of NF-kappa B protein was increased after exposure to CSE. The expression of SIRT1 protein in human macrophages was stimulated by CSE 24 hours after incubation with erythromycin. After incubation with erythromycin for 48 hours, the expression of SIRT1 protein and the inhibition of NF-kappa B protein in human macrophages were increased. Results: 1. The expression of SIRT1 protein in human macrophages decreased significantly after exposure to CSE, and the expression of NF-kappa B protein was increased after incubation with erythromycin for 24 hours and 48 hours. Both of them increased the expression of SIRT1 protein and inhibited the expression of NF-kappa B protein in human macrophages, especially in 48h group. 2. The interaction between SIRT1 and NF-kappa B protein in human macrophages was detected by Co-IP. CSE stimulation could decrease the expression of SIRT1 protein and promote the expression of NF-kappa B protein. After incubation with erythromycin for 24h, 48 h, SIRT1 protein was increased. Conclusion: 1. Tobacco smoke may induce inflammation by inhibiting the expression of SIRT1 protein in human macrophages through oxidative stress, thereby promoting the expression of NF-kappa B protein, thereby promoting the release of inflammatory mediators IL-6, IL-8 and TNF-alpha. 2. Erythromycin inhibits oxidative stress-induced inflammation. The mechanism of inflammation may be that erythromycin increases the expression of SIRT1 protein and then inhibits the expression of NF-kappa B protein, thereby inhibiting the release of inflammatory mediators. The effect of erythromycin on the expression of SIRT1 and NF-kappa B protein in lung tissue of mice exposed to tobacco smoke was observed. Tobacco smoke exposure group, erythromycin treatment group. The expression of NF-kappa B and SIRT1 protein in lung tissue of mice in each group. Results: 1. Inflammation, aggregation of inflammatory cells around bronchi and emphysema appeared in lung tissue of mice exposed to tobacco smoke. The expression of SIRT1 protein in lung tissue of mice treated with erythromycin was lower than that of control group, but the expression of SIRT1 protein was higher than that of tobacco smoke exposure group. CONCLUSION: 1. Erythromycin can alleviate the inflammation of lung tissue induced by tobacco smoke exposure, which may be related to erythromycin up-regulating the expression of SIRT1 protein in lung tissue and inhibiting the expression of SIRT1 protein in lung tissue. The expression of NF-kappa B protein in tissues is correlated. 2. Erythromycin can alleviate emphysema in mice, which may be related to erythromycin promoting SIRT1 expression.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.9
本文编号：2218140
[Abstract]:Part I: Effects of erythromycin on the release of reactive oxygen species (ROS) and inflammatory factors from human macrophages exposed to tobacco smoke. Objective: 1. To observe the effects of tobacco smoke on the production of reactive oxygen species (ROS) in human macrophages and whether erythromycin can regulate the release of ROS from human macrophages mediated by tobacco smoke exposure. To observe the changes of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-A (TNF-a) released by human macrophages under tobacco smoke stimulation, and the effect of erythromycin on the release of inflammatory factors from human macrophages under tobacco smoke exposure. METHODS: Human lymphoma monocyte U937 was induced by 10 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours. U937 was transformed into macrophages and stimulated by cigarette smoke extract (CSE). The optimum concentration of CSE and erythromycin on human lymphoma cells was determined by MTT assay. Cell groups: control group, CSE group (incubated with 1% CSE for 24 hours), CSE + erythromycin 24 hours group (incubated with 1 ug / ml erythromycin for 24 hours, added 1% CSE for 24 hours), CSE + erythromycin 48 hours group (incubated with 1 ugh / ml erythromycin for 48 hours, added 1% CSE for 24 hours). Cell groups were treated with Erythromycin (EM), CSE and 2', 7'-dichlorodihydrofluorescence. Optical diethyl ester (2', 7'-dichlorofluorescin-diacetate, DCFH-DA, 10mM) was used as a fluorescent probe to detect the ROS level in the cells of each group. The levels of IL-6, IL-8 and TNF-a in the supernatant of the cells of each experimental group were detected by enzyme-linked immunosorbent assay (ELISA). Results: CSE at 1% concentration could significantly up-regulate the release of ROS from human macrophages (P 0.01). Erythromycin (lug / ml) could inhibit the up-regulation of ROS by 1% CSE (p0.01), SIRT1 specific inhibitor (NAM) could significantly increase the release of ROS by human macrophages (p0.01), and 1% CSE could increase the release of IL-6, IL-8, TNF-a, and erythromycin (lug / ml) by 48 h after pre-incubation. Conclusion: 1. Tobacco smoke stimulates the release of reactive oxygen species from human macrophages. One of the mechanisms of erythromycin inhibiting oxidative stress is through inhibiting the release of reactive oxygen species. 2. Tobacco smoke stimulates the release of IL-6, IL-8, TNF-a from human macrophages, and erythromycin. One of the mechanisms of inhibiting inflammation is by inhibiting the release of IL-6, IL-8 and TNF-alpha from human macrophages. Part II: The effect of erythromycin on the expression of SIRT1, NF-kappa B protein in human macrophages stimulated by tobacco smoke and the interaction between the two proteins. To observe the interaction between SIRT1 and NF-kappa B protein in human macrophages and the interaction between SIRT1 and NF-kappa B protein in tobacco smoke exposure and erythromycin treatment. Methods: 1. Human lymphoma monocytes were incubated with 10 nmol/ml PMA for 24 hours. U937 cells were induced to differentiate into macrophages and stimulated with CSE to produce a model of oxidative stress induced by tobacco smoke exposure. The cells were divided into control group, CSE group, CSE + erythromycin 24 h group, CSE + erythromycin 48 h group, NF-kappa B protein specific inhibitor PDTC group (20nM PDTC pre-incubated for 24 h and added 1% CSE for 24 h), SIRT1 specific inhibitor NA. After U937 was induced to differentiate into macrophages, the cells were divided into control group, CSE group, CSE + erythromycin 24 h group and CSE + erythromycin 48 h group. The expression of NF-kappa B and SIRT1 protein was detected by Western blot (WB). Results: 1. The expression of SIRT1 protein in human macrophages was significantly decreased and the expression of NF-kappa B protein was increased after exposure to CSE. The expression of SIRT1 protein in human macrophages was stimulated by CSE 24 hours after incubation with erythromycin. After incubation with erythromycin for 48 hours, the expression of SIRT1 protein and the inhibition of NF-kappa B protein in human macrophages were increased. Results: 1. The expression of SIRT1 protein in human macrophages decreased significantly after exposure to CSE, and the expression of NF-kappa B protein was increased after incubation with erythromycin for 24 hours and 48 hours. Both of them increased the expression of SIRT1 protein and inhibited the expression of NF-kappa B protein in human macrophages, especially in 48h group. 2. The interaction between SIRT1 and NF-kappa B protein in human macrophages was detected by Co-IP. CSE stimulation could decrease the expression of SIRT1 protein and promote the expression of NF-kappa B protein. After incubation with erythromycin for 24h, 48 h, SIRT1 protein was increased. Conclusion: 1. Tobacco smoke may induce inflammation by inhibiting the expression of SIRT1 protein in human macrophages through oxidative stress, thereby promoting the expression of NF-kappa B protein, thereby promoting the release of inflammatory mediators IL-6, IL-8 and TNF-alpha. 2. Erythromycin inhibits oxidative stress-induced inflammation. The mechanism of inflammation may be that erythromycin increases the expression of SIRT1 protein and then inhibits the expression of NF-kappa B protein, thereby inhibiting the release of inflammatory mediators. The effect of erythromycin on the expression of SIRT1 and NF-kappa B protein in lung tissue of mice exposed to tobacco smoke was observed. Tobacco smoke exposure group, erythromycin treatment group. The expression of NF-kappa B and SIRT1 protein in lung tissue of mice in each group. Results: 1. Inflammation, aggregation of inflammatory cells around bronchi and emphysema appeared in lung tissue of mice exposed to tobacco smoke. The expression of SIRT1 protein in lung tissue of mice treated with erythromycin was lower than that of control group, but the expression of SIRT1 protein was higher than that of tobacco smoke exposure group. CONCLUSION: 1. Erythromycin can alleviate the inflammation of lung tissue induced by tobacco smoke exposure, which may be related to erythromycin up-regulating the expression of SIRT1 protein in lung tissue and inhibiting the expression of SIRT1 protein in lung tissue. The expression of NF-kappa B protein in tissues is correlated. 2. Erythromycin can alleviate emphysema in mice, which may be related to erythromycin promoting SIRT1 expression.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.9
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