| Background and Objective:The whole world asthma incidence increased gradually, in China, patients with asthma increased year by year also, so studies on the pathogenesis of asthma and further to seek effective medications and with little side effects had become hot points. Endoplasmic reticulum stress(ERS) is a protective response to stress in eucaryote cells. Virus infection, oxidative stress, hypoxia ischemia, inadequate nutrition and calcium imbalance can cause ERS, so ERS is involved in many pathophysiological processes and involved in the occurrence and development of many diseases including atherosclerosis, diabetes, liver injury, neurodegenerative diseases, the incidence of heart disease, cancer development and et al.ERS and inflammation related through many pathways,so ERS plays an important role in the body’s inflammatory response and involved in many inflammatory diseases.For respiratory diseases, most researches were on the relation between ERS and chronic obstructive pulmonary disease, but on asthma had little reported in the domestic. The characteristic of asthma is chronic airway inflammation, so we guess that ERS is also involved in the pathogenesis of asthma.For asthma, glucocorticoid and bronchodilator are the mainly treatment drugs at present, but long-term use of these drugs can cause many side effects, also have certain influence on growth of children. Glutatmine is an important non-essential amino acid in the body,in clinical, chiefly used as gastric mucosal protective agent for the treatment of chronic gastritis and ulcers.But there were also many scholars founded that glutamine had anti-inflammatory, immunity-regulating, protection of mucosal barrier function and et al effects.Asthma was characterized with T cell function abnormalities and chronic airway inflammation,so it is speculated that glutamine can be used for the treatment of asthma.Here our objection is to survey the change of endoplasmic reticulum stress in the mice with asthma, and observe the treatment effect of glutamine on asthma and the possible mechanism.Methods:1. Select6-8-week-old Balb/C mice, sensitized with ovalbumin and aluminum hydroxide, then inhaled ovalbumin to stimulate the airway to make mouse asthma model. Mice were randomly divided into5groups, with10rats in each group:A:normal control group, B:asthmatic model group; C:low dose glutamine treatment group:0.4g/kg; D:middle dose glutamine treatment group:0.75g/kg; E:high dose glutamine treatment group:1.5g/kg.2.Detect the serum levels of IL-6, IL-4, IgE, TNF a, IL-12with ELISA method.3.Detect the numbers of white blood cell and eosinophile granulocytes of BALF with Diff-quik staining.4.Measured IL-6, IL-4in BALF by ELISA method in each group.5.One side of Lung tissues of mice were fixed with formaldehyde and stained by HE to observe pulmonary inflammation.6.The other side of lung tissues were used to detect JNK, pJNK, CHOP, GRP78, IRE-1, BAX, Bcl2, Caspase12expression by Western Blot.7.Used tunicamycin to make endoplasmic reticulum stress cell model:cultured human lung adenocarcinoma cells-A549and administrated with different concentrations (0.1ug/ml,1ug/ml,lOug/ml) of tunicamycin for different time (6,12,24,48h),and cell proliferation inhibition rate were tested by MTT to select appropriate time and concentrations of the tunicamycin administrated on A549cells.8. Detected the inhibitory rate of cell proliferation to explore the roles of different concentrations (2,4,8,12,16mmol/1) of glutamine on the A549cells with endoplasmic reticulum stress after different time (12,24,48hours).9.Cells were cultured and divided into5groups, blank control group:only A549cells in the culture medium; tunicamycin group:Cultured A549cells and tunicamycin lug/ml in the culture medium; different concentrations of glutamine groups,including3groups:GLN4group: cultured A549with lug/ml of tunicamycin and4mmol/1glutamine; GLN8group:cultured A549cells with lug/ml of tunicamycin and8mmol/1glutamine; GLN12group:cultured A549cells with lug/ml tunicamycin and12mmol/1glutamine. Detected by Western Blot the JNK, pJNK.CHOP, GPR78expression levels of A549cells with endoplasmic reticulum stress after intervened by different concentrations of glutamine (4,8,12mmol/1) for12hours.Results:1. Serum IL-6, IL-4, IgE, TNFa levels were significantly increased in B group (model group) than that of A group (control group),while IL-12decreased significantly, there were significant differences (P<0.05).Serum IL-6, IL-4, IgE, TNFa of C, D, E were lower than that of B group,while IL-12was higher, with statistical significance (P＜0.05).2.The results of Diff-quik staining:the number of white blood cells and eosinophile granulocytes in bronchoalveolar lavage fluid of B group (model group) were higher than that in group A (normal group),there were significant differences (P＜0.05);. but in C, D, E, those were significantly decreased than in B(model group), but higher than those in the normal group,there were significant differences (P<0.05);3.IL-6, IL-4levels in bronchoalveolar lavage fluid of B group (model group) were significantly increased than in A group (control group), with significant differences(P<0.05).IL-6and IL-4in BALF of C, D, E groups were lower than those of B group, there were statistical significances(P<0.05).4.The lung tissue pathological results:HE staining of lung tissues showed that B group compared with A group,there were large amount of inflammatory cells infiltration,such as lymphocytes,eosinophils et al in the surroundings of bronchial and vascular; bronchial epithelial multiple shed, mild thickening of the basement membrane, bronchial smooth muscle proliferation, mucous plug and inflammatory exudate in the small endobronchial, inflammatory cells infiltration in pulmonary interstitial and alveolar cavity; C.D.E group compared with B group, inflammatory infiltration were decreased in the surroundings of bronchial and vascular, pulmonary interstitial, bronchial epithelial shedding diminished, alveolar inflammatory cells decreased.5.B group compared with A group,the expression levels of pJNK,CHOP,GPR78,IRE1in lung tissues of mice increased significantly, while no significant differences of JNK expression, it showed that the endoplasmic reticulum stress occured in the lung of asthma. pJNK.CHOP, GPR78, IRE1expression levels of glutamine treatment groups were significantly lower than the model group, there were significant difference, which indicated that glutamine had protective effects on the endoplasmic reticulum stress;B group compared with A group, the ratio of BAX/Bcl2expressions of Caspasel2in lung tissues of mice were increased significantly, and the values of glutamine treatment groups decreased, which indicated that glutamine inhibited the cell apoptosis through inhibiting of endoplasmic reticulum stress;6.The detection results of cytotoxic with MTT method:Treated A549cells with tunicamycin, when the concentration was1ug/ml, reaction for12hours,the cell proliferation inhibition rate was51.3%,which was most close to50%, so select the tunicamycin concentration as1ug/ml, the react time is12h.7.Detection results of the inhibit rate of cell proliferation with MTT method response to different concentrations of glutamine (2,4,8,12,16mmol/1) treated A549cells which had produced endoplasmic reticulum stress for different time points (12,24,48hour):Cell proliferation inhibit rate resulted for8mmol/1of glutamine concentration,12hours of reaction time was48.96%, the most close to50%, so select this concentration and time for the appropriate concentration and time.8. In vitro test, the expression levels of pJNK.CHOP, GPR78increased significantly in tunicamycin group than the control group, while the expression of JNK had no significant difference, which indicated tunicamycin induced A549cells to produce the endoplasmic reticulum stress. pJNK.CHOP, GPR78expression level was decreased in glutamine+tunicamycin group than that of tunicamycin group, there were significant differences, which indicated that glutamine had protective effect on the endoplasmic reticulum stress.Conclusion:1.Proinflammatory factors such as IL-4, IL-6, IgE and TNFa increased and serum anti-inflammatory cytokine IL-12levels decreased in asthma; Glutamine treatment can reduce the release of pro-inflammatory factors, increase the anti inflammatory factors, so it plays a protective role in the asthmatic airway injury through inhibiting the inflammation of asthma.2. Endoplasmic reticulum stress produced in the lung of asthma,also there appeared apoptosis and inflammation. Glutamine had protective effect on the endoplasmic reticulum stress, and further inhibited the apoptosis and inflammation, which had effect on the treatment of asthma.3. In vitro cell culture test results supported that at the cellular level, glutamine had protective effects on A549cells with endoplasmic reticulum stress. |
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