哮喘小鼠模型中内质网应激变化及谷氨酰胺的干预作用机制
发布时间：2018-09-03 12:03
【摘要】：背景与目的：全世界哮喘发病率逐渐增加,我国哮喘患者也逐年上升,因此研究哮喘发病机制并寻求有效而副作用小的药物成为研究热点。内质网应激(ERS)是真核细胞的一种保护性应激反应,病毒感染,氧化应激、缺血缺氧、营养不足和钙失衡等均可引起ERS,因此ERS参与多种疾病的病理和生理过程,包括肝功能损伤,神经退行性病变,循环系统疾病,肿瘤的发生和发展等。ERS以多种机制作用于炎症反应通路,与机体的炎症反应密切相关,所以ERS也参与了很多炎症性疾病,呼吸系统疾病中有较多关于ERS与慢阻肺的关系报道,但ERS与哮喘关系的研究报道国内甚少,而哮喘本质是气道慢性炎症,故推测ERS也参与了哮喘的发病。哮喘治疗目前临床上主要应用激素及支气管扩张药等,但长期应用可带来较多副作用,对儿童生长发育也有一定影响。谷氨酰胺是体内非必需氨基酸,主要用做胃粘膜保护剂治疗慢性胃炎,溃疡等,但也有很多学者研究发现谷氨酰胺有抗炎,调节免疫,保护粘膜屏障等作用,而哮喘发病与T细胞免疫功能异常有关,而且以慢性气道炎症为其特点,推测谷氨酰胺可应用于哮喘的治疗。本论文主要探讨哮喘小鼠模型中内质网应激变化及炎症因子的改变,并应用谷氨酰胺干预后观察了谷氨酰胺对哮喘的治疗作用及其可能的作用机制。 方法： 1.6-8周龄BALB/C小鼠,以卵蛋白及氢氧化铝致敏后雾化吸入卵蛋白激发气道制备哮喘小鼠模型。小鼠随机分成5组,每组各10只。A正常对照组；B哮喘模型组；C低剂量谷氨酰胺治疗组：0.4g/kg；D中剂量谷氨酰胺治疗组：0.75g/kg；E大剂量谷氨酰胺治疗组：1.5g/kg。 2.取各组小鼠血清用ELISA法测定IL-6,IL-4,IgE,TNF α,IL-12水平； 3.取各组小鼠BALF离心后的细胞涂片进行Diff-quik染色,检测白细胞数及嗜酸细胞数； 4.取各组小鼠BALF上清液用ELISA法测定IL-6,IL-4。 5.小鼠一侧肺组织甲醛固定后进行HE染色观察肺部炎症改变。 6.另一侧肺用Western Blot方法检测肺组织JNK,pJNK,CHOP,GRP78IRE-1,BAX,Bcl2,Caspase12表达. 7.利用衣霉素制备细胞ERS模型：体外培养人肺腺癌细胞株A549细胞,用MTT法检测不同浓度(0.1ug/ml,1ug/ml,10ug/ml)衣霉素对A549细胞作用不同时间(6,12,24,48h)的细胞增值抑制率,选定应用衣霉素合适浓度及时间。 8.MTT法检测不同浓度谷氨酰胺(2,4,8,12,16mmol/1)对产生内质网应激反应的A549细胞的作用不同时间点(12,24,48小时)的细胞增殖抑制率,选定应用谷氨酰胺的合适浓度及时间。 9.细胞培养分5组,空白对照组：在培养液中只有A549细胞及；衣霉素组：培养液中加入A549细胞及衣霉素1ug/ml:不同浓度谷氨酰胺组,分3组：GLN4组：培养液中加入A549细胞,衣霉素lug/ml以及4mMmmol/1谷氨酰胺；GLN8组：培养液中加入A549细胞,衣霉素1ug/ml及8mmol/1谷氨酰胺；GLN12组：培养液中加入A549细胞,衣霉素lug/ml及12mmol/1谷氨酰胺。Western Blot方法检测不同细胞组培养12小时后的JNNK,pJNK.CHOP,GPR78表达水平。 结果： 1.B组(模型组)血清IL-6,IL-4,IgE,TNF α水平较A组(对照组)明显升高,IL-12水平明显降低,差异有显著性(P0.05)。C,D,E,组血清IL-6,IL-4,IgE,TNF α较B组明显降低,IL-12水平明显升高,有统计学意义(P0.05)。 2.Diff-quik染色结果：B组(模型组)肺泡灌洗液中白细胞数及嗜酸细胞计数明显高于A组(正常组)差异有显著性(p0.05)；C,D,E组肺泡灌洗液中白细胞数及嗜酸细胞数较模型组明显减少,但高于正常组,差异有显著性(P0.05)； 3.B组(模型组)肺泡灌洗液中工L-6,IL-4水平较A组(对照组)明显升高,差异有显著性(P0.05)。C,D,E组肺泡灌洗液中IL-6,IL-4较B组明显降低,有统计学意义(P0.05)。 4.肺组织病理结果：HE染色结果,B组与A组比较,支气管及血管周围大量淋巴细胞和嗜酸性粒细胞等炎性细胞浸润；支气管上皮细胞多处脱落,基底膜可见轻度增厚,支气管平滑肌有增生表现,细小的支气管内可见到较多粘液栓和炎性渗出物,肺间质和肺泡腔内也可见炎性细胞浸润；C.D.E组较模型组比较,支气管及血管周围,肺间质炎性细胞浸润明显减轻,支气管上皮脱落减少,肺泡腔内炎性细胞减少。 5.B组与A组比较,小鼠肺组织pJNK.CHOP,GPR78,IRE1表达水平明显升高,而JNK表达无明显差异,说明哮喘发病时肺部发生了内质网应激反应。谷氨酰胺治疗组的pJNK.CHOP,GPR78,IRE1表达水平较模型组明显降低,差异有显著性,说明谷氨酰胺对内质网应激有保护作用：B组与A组比较,小鼠肺组织BAX/Bcl2,Caspase12表达水平明显升高,谷氨酰胺治疗组其值降低,说明谷氨酰胺通过抑制内质网应激反应抑制了细胞凋亡的发生： 6.细胞毒性MTT方法检测结果,衣霉素处理细胞A549浓度为lug/m1,作用时间为12小时时细胞增值抑制率为51.3%,最接近50%,故选择衣霉素作用浓度为lug/ml,作用时间为12h。 7.MTT法检测不同浓度谷氨酰胺(2,4,8,12,16mmol/1)对产生内质网应激反应的A549细胞作用不同时间点(12,24,48小时)的细胞增殖抑制率结果为谷氨酰胺8mmol/l作用12小时的细胞增殖抑制率为48.9%,最接近50%,故选择此浓度及时间点为合适作用浓度及时间。 8.体外细胞培养试验中,衣霉素组较空白对照组pJNK.CHOP,GPR78表达水平明显升高,而JNK表达无明显差异,说明衣霉素诱导A549细胞产生了内质网应激。谷氨酰胺+衣霉素组的pJNK.CHOP,GPR78表达水平较衣霉素组明显降低,差异有显著性,说明谷氨酰胺对内质网应激有保护作用。 结论： 1.哮喘时有IL-4, IL-6, IgE, TNFα这些促炎症因子水平增高,血清抑炎因子IL-12水平降低；谷氨酰胺治疗可降低促炎症因子的释放,增加抑炎因子水平,说明谷氨酰胺通过抑制哮喘的炎症反应而起到对哮喘气道损伤的保护作用。 2.哮喘时肺组织产生了内质网应激反应,并有细胞凋亡及炎症的发生,谷氨酰胺对内质网应激有保护作用,并进一步抑制了细胞凋亡及气道炎症,从而对哮喘有治疗作用。 3.体外细胞培养结果证实,在细胞水平,谷氨酰胺对产生内质网应激的A549细胞有保护作用。
[Abstract]:BACKGROUND & OBJECTIVE: The incidence of asthma is increasing all over the world, and the incidence of asthma in China is also increasing year by year. Therefore, the study of the pathogenesis of asthma and the search for effective and less side-effect drugs have become a hot spot. Endoplasmic reticulum stress (ERS) is a protective stress response of eukaryotic cells, including viral infection, oxidative stress, ischemia and hypoxia, undernutrition and so on. Calcium imbalance can cause ERS, so ERS is involved in many pathological and physiological processes, including liver function damage, neurodegenerative diseases, circulatory system diseases, tumor occurrence and development. There are many reports about the relationship between ERS and COPD in respiratory diseases, but there are few reports about the relationship between ERS and asthma in China, and the essence of asthma is chronic airway inflammation, so it is speculated that ERS is also involved in the pathogenesis of asthma. Glutamine is a non-essential amino acid in the body, mainly used as a gastric mucosal protective agent in the treatment of chronic gastritis, ulcers, etc., but many scholars have found that glutamine has anti-inflammatory, immune regulation, protection of mucosal barrier and other effects, and the incidence of asthma is related to abnormal T cell immune function, and to Chronic airway inflammation is its characteristic, and glutamine may be used in the treatment of asthma. This paper mainly discusses the changes of endoplasmic reticulum stress and inflammatory factors in asthmatic mice model, and observes the therapeutic effect of glutamine on asthma and its possible mechanism after intervention with glutamine.
Method:
1.6-8 weeks old BALB/C mice were sensitized with ovalbumin and aluminium hydroxide and then inhaled with aerosolized ovalbumin to induce airway asthma in mice. Amido treatment group: 1.5g/kg.
2. serum levels of IL-6, IL-4, IgE, TNF alpha and IL-12 were measured by ELISA in each group of mice.
3. Diff-quik staining was used to detect the number of leukocytes and eosinophils in BALF-centrifuged cells.
4. the supernatants of BALF from each group were determined by ELISA, IL-6, IL-4.
5. one lung tissue of mice was fixed with formaldehyde, then HE staining was used to observe the changes of lung inflammation.
6. on the other side of the lung, the expression of JNK, pJNK, CHOP, GRP78IRE-1, BAX, Bcl2 and Caspase12 in lung tissue were detected by Western Blot.
7. Establishment of cell ERS model with chlamydia: Human lung adenocarcinoma cell line A549 was cultured in vitro. The inhibition rate of proliferation of A549 cells treated with Chlamydia at different concentrations (0.1ug/ml, 1ug/ml, 10ug/ml) for different time (6, 12, 24, 48h) was detected by MTT method.
8. MTT assay was used to detect the inhibitory rate of proliferation of A549 cells at different time points (12,24,48 hours) induced by different concentrations of glutamine (2,4,8,12,16 mmol/1).
9. Cell culture was divided into 5 groups, blank control group: A549 cells and Chlamycin group: A549 cells and Chlamycin 1ug/ml were added into the culture medium, and divided into 3 groups: GLN4 group: A549 cells, Chlamycin lug/ml and 4 mMmmol/1 glutamine were added into the culture medium; GLN8 group: A549 cells were added into the culture medium. GLN12 group: A549 cells were added into the culture medium, and the expressions of JNNK, pJNK.CHOP and GPR78 were detected by Western Blot method 12 hours after culture.
Result:
1. The serum levels of IL-6, IL-4, IgE and TNF-alpha in group B were significantly higher than those in group A (control group), and IL-12 was significantly lower (P 0.05). The serum levels of IL-6, IL-4, IgE and TNF-alpha in group C, D, E were significantly lower than those in group B (P 0.05).
2. Diff-quik staining results: Group B (model group) alveolar lavage fluid white blood cell and eosinophil counts were significantly higher than group A (normal group) difference was significant (p0.05); C, D, E alveolar lavage fluid white blood cell and eosinophil counts were significantly lower than the model group, but higher than the normal group, the difference was significant (P 0.05);
3. The levels of IL-6 and IL-4 in alveolar lavage fluid of group B (model group) were significantly higher than those of group A (control group) (P 0.05). The levels of IL-6 and IL-4 in alveolar lavage fluid of group C, D and E were significantly lower than those of group B (P 0.05).
4. Pulmonary histopathological results: HE staining results, group B and group A compared, bronchial and perivascular lymphocytes and eosinophils infiltration of inflammatory cells; bronchial epithelial cells more exfoliated, basement membrane can be seen slightly thickened, bronchial smooth muscle proliferation, small bronchioles can be seen more mucus thrombus and inflammatory infiltration. Inflammatory cell infiltration was also observed in the pulmonary interstitial and alveolar cavities. Compared with the model group, the infiltration of pulmonary interstitial inflammatory cells was significantly reduced in C.D.E group.
5. Compared with group A, the expression levels of pJNK. CHOP, GPR78 and IRE1 in lung tissues of mice in group B were significantly increased, but the expression of JNK was not significantly different, indicating that the lung had endoplasmic reticulum stress reaction during asthma onset. The expression levels of BAX/Bcl 2 and Caspase 12 in lung tissue of mice in group B were significantly higher than those in group A. The expression levels of BAX/Bcl 2 and Caspase 12 in glutamine treatment group were significantly lower than those in group A.
6. Cytotoxic MTT assay showed that the concentration of Chlamycin treated A549 was lug/m1, and the inhibition rate of cell proliferation was 51.3% at 12 hours, closest to 50%. Therefore, the concentration of Chlamycin was lug/ml and the action time was 12 hours.
7. MTT assay was used to detect the proliferation inhibition rate of A549 cells at different time points (12,24,48 hours) induced by different concentrations of glutamine (2,4,8,12,16 mmol/1). The results showed that the proliferation inhibition rate of A549 cells at different time points (12,24,48 hours) was 48.9% after 12 hours of glutamine 8 mmol/l treatment, which was the closest to 50%. Degree and time.
8. In vitro cell culture test, the expression levels of pJNK. CHOP and GPR78 in the Chlamydia group were significantly higher than those in the blank control group, but there was no significant difference in the expression of JNK, indicating that Chlamydia induced endoplasmic reticulum stress in A549 cells. Amides have protective effects on endoplasmic reticulum stress.
Conclusion:
1. In asthma, the levels of IL-4, IL-6, IgE and TNFalpha increased, while the levels of IL-12 in serum decreased. Glutamine treatment can reduce the release of pro-inflammatory factors and increase the levels of anti-inflammatory factors, indicating that glutamine plays a protective role in airway injury of asthma by inhibiting the inflammatory reaction of asthma.
2. In asthma, endoplasmic reticulum (ER) stress occurs in lung tissues, and there is apoptosis and inflammation. Glutamine has protective effect on ER stress, and further inhibits apoptosis and airway inflammation, thus has therapeutic effect on asthma.
3. Cell culture in vitro showed that glutamine could protect A549 cells from endoplasmic reticulum stress.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.6;R-332
本文编号：2219925
[Abstract]:BACKGROUND & OBJECTIVE: The incidence of asthma is increasing all over the world, and the incidence of asthma in China is also increasing year by year. Therefore, the study of the pathogenesis of asthma and the search for effective and less side-effect drugs have become a hot spot. Endoplasmic reticulum stress (ERS) is a protective stress response of eukaryotic cells, including viral infection, oxidative stress, ischemia and hypoxia, undernutrition and so on. Calcium imbalance can cause ERS, so ERS is involved in many pathological and physiological processes, including liver function damage, neurodegenerative diseases, circulatory system diseases, tumor occurrence and development. There are many reports about the relationship between ERS and COPD in respiratory diseases, but there are few reports about the relationship between ERS and asthma in China, and the essence of asthma is chronic airway inflammation, so it is speculated that ERS is also involved in the pathogenesis of asthma. Glutamine is a non-essential amino acid in the body, mainly used as a gastric mucosal protective agent in the treatment of chronic gastritis, ulcers, etc., but many scholars have found that glutamine has anti-inflammatory, immune regulation, protection of mucosal barrier and other effects, and the incidence of asthma is related to abnormal T cell immune function, and to Chronic airway inflammation is its characteristic, and glutamine may be used in the treatment of asthma. This paper mainly discusses the changes of endoplasmic reticulum stress and inflammatory factors in asthmatic mice model, and observes the therapeutic effect of glutamine on asthma and its possible mechanism after intervention with glutamine.
Method:
1.6-8 weeks old BALB/C mice were sensitized with ovalbumin and aluminium hydroxide and then inhaled with aerosolized ovalbumin to induce airway asthma in mice. Amido treatment group: 1.5g/kg.
2. serum levels of IL-6, IL-4, IgE, TNF alpha and IL-12 were measured by ELISA in each group of mice.
3. Diff-quik staining was used to detect the number of leukocytes and eosinophils in BALF-centrifuged cells.
4. the supernatants of BALF from each group were determined by ELISA, IL-6, IL-4.
5. one lung tissue of mice was fixed with formaldehyde, then HE staining was used to observe the changes of lung inflammation.
6. on the other side of the lung, the expression of JNK, pJNK, CHOP, GRP78IRE-1, BAX, Bcl2 and Caspase12 in lung tissue were detected by Western Blot.
7. Establishment of cell ERS model with chlamydia: Human lung adenocarcinoma cell line A549 was cultured in vitro. The inhibition rate of proliferation of A549 cells treated with Chlamydia at different concentrations (0.1ug/ml, 1ug/ml, 10ug/ml) for different time (6, 12, 24, 48h) was detected by MTT method.
8. MTT assay was used to detect the inhibitory rate of proliferation of A549 cells at different time points (12,24,48 hours) induced by different concentrations of glutamine (2,4,8,12,16 mmol/1).
9. Cell culture was divided into 5 groups, blank control group: A549 cells and Chlamycin group: A549 cells and Chlamycin 1ug/ml were added into the culture medium, and divided into 3 groups: GLN4 group: A549 cells, Chlamycin lug/ml and 4 mMmmol/1 glutamine were added into the culture medium; GLN8 group: A549 cells were added into the culture medium. GLN12 group: A549 cells were added into the culture medium, and the expressions of JNNK, pJNK.CHOP and GPR78 were detected by Western Blot method 12 hours after culture.
Result:
1. The serum levels of IL-6, IL-4, IgE and TNF-alpha in group B were significantly higher than those in group A (control group), and IL-12 was significantly lower (P 0.05). The serum levels of IL-6, IL-4, IgE and TNF-alpha in group C, D, E were significantly lower than those in group B (P 0.05).
2. Diff-quik staining results: Group B (model group) alveolar lavage fluid white blood cell and eosinophil counts were significantly higher than group A (normal group) difference was significant (p0.05); C, D, E alveolar lavage fluid white blood cell and eosinophil counts were significantly lower than the model group, but higher than the normal group, the difference was significant (P 0.05);
3. The levels of IL-6 and IL-4 in alveolar lavage fluid of group B (model group) were significantly higher than those of group A (control group) (P 0.05). The levels of IL-6 and IL-4 in alveolar lavage fluid of group C, D and E were significantly lower than those of group B (P 0.05).
4. Pulmonary histopathological results: HE staining results, group B and group A compared, bronchial and perivascular lymphocytes and eosinophils infiltration of inflammatory cells; bronchial epithelial cells more exfoliated, basement membrane can be seen slightly thickened, bronchial smooth muscle proliferation, small bronchioles can be seen more mucus thrombus and inflammatory infiltration. Inflammatory cell infiltration was also observed in the pulmonary interstitial and alveolar cavities. Compared with the model group, the infiltration of pulmonary interstitial inflammatory cells was significantly reduced in C.D.E group.
5. Compared with group A, the expression levels of pJNK. CHOP, GPR78 and IRE1 in lung tissues of mice in group B were significantly increased, but the expression of JNK was not significantly different, indicating that the lung had endoplasmic reticulum stress reaction during asthma onset. The expression levels of BAX/Bcl 2 and Caspase 12 in lung tissue of mice in group B were significantly higher than those in group A. The expression levels of BAX/Bcl 2 and Caspase 12 in glutamine treatment group were significantly lower than those in group A.
6. Cytotoxic MTT assay showed that the concentration of Chlamycin treated A549 was lug/m1, and the inhibition rate of cell proliferation was 51.3% at 12 hours, closest to 50%. Therefore, the concentration of Chlamycin was lug/ml and the action time was 12 hours.
7. MTT assay was used to detect the proliferation inhibition rate of A549 cells at different time points (12,24,48 hours) induced by different concentrations of glutamine (2,4,8,12,16 mmol/1). The results showed that the proliferation inhibition rate of A549 cells at different time points (12,24,48 hours) was 48.9% after 12 hours of glutamine 8 mmol/l treatment, which was the closest to 50%. Degree and time.
8. In vitro cell culture test, the expression levels of pJNK. CHOP and GPR78 in the Chlamydia group were significantly higher than those in the blank control group, but there was no significant difference in the expression of JNK, indicating that Chlamydia induced endoplasmic reticulum stress in A549 cells. Amides have protective effects on endoplasmic reticulum stress.
Conclusion:
1. In asthma, the levels of IL-4, IL-6, IgE and TNFalpha increased, while the levels of IL-12 in serum decreased. Glutamine treatment can reduce the release of pro-inflammatory factors and increase the levels of anti-inflammatory factors, indicating that glutamine plays a protective role in airway injury of asthma by inhibiting the inflammatory reaction of asthma.
2. In asthma, endoplasmic reticulum (ER) stress occurs in lung tissues, and there is apoptosis and inflammation. Glutamine has protective effect on ER stress, and further inhibits apoptosis and airway inflammation, thus has therapeutic effect on asthma.
3. Cell culture in vitro showed that glutamine could protect A549 cells from endoplasmic reticulum stress.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.6;R-332
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