Protective Effects Of Rapamycin-induced Autophagy On Organ Injury Caused By Smoking And D-galactose

Mammalian target of Rapamycin(mTOR) is a serine/threonine protein kinase and a member of the phosphatidylinositol 3-kinase-related kinase family. mTOR is the catalytic subunit of two structurally distinct complexes: mTORC1 and mTORC2, and regulates cell growth, development, aging, metabolism, immunity and oncogenesis through regulation of protein synthesis, lipogenesis, energy metabolism, autophagy and cell cytoskeleton, etc. Our previous data in vitro have shown that autophagy activation could obviously alleviate cell senescence induced by hydrogen peroxide;autophagy inhibition increased the expression of cell cycle protein p53 and p21,induced the growth arrest and senescence of fibroblasts. All these data show that autophagy is involved in the aging process, but it is still unknown that whether the regulating effect of mTOR on aging is related to autophagic activity. Rapamycin is the first inhibitor of mTOR and mainly used as an immunosuppressive drug for treatment of rejection reaction after organ transplantation or autoimmune diseases. In order to enlarge application range of rapamycin, this study is going to explore the protective effects of rapamycin in aging models of organ injury and impaired memory induced by oxidative stress.Objective: To investigate the protective effects of rapamycin on organ injury induced by cigarette smoke and D-galactose in mice and its relationship with the level of autophagy.Methods: 70 male Kunming mice, weighing 20-22 g, were randomly divided into7 groups, 10 in each group. Group A was normal control group; group B was smoking group, the mice exposed to cigarette smoke 4 times a day, 20 minutes each time, six days a week for eight weeks; Group C was smoking+rapamycin group, the smokingmice were injected intraperitoneally with rapamycin(2mg/kg/d) from the fifth week for consecutive 4 weeks; Group D was smoking+ chloroquine(CQ) group, the smoking mice were injected intraperitoneally with CQ(50mg/kg/d) from the fifth week for consecutive 4 weeks; Group E was D-galactose(D-Gal) group, the mice received daily subcutaneous injection of D-galactose(80mg/kg/d) for continuous 8weeks; Group F was D-galactose + rapamycin(D-Gal+Rap) group, the mice received D-galactose and Rap; Group G was D-galactose+chloroquine(D-Gal+CQ) group, the mice received D-galactose and CQ. The mice in A, E, F and G groups received a six-day Morris water maze test to observe the learning and memory ability, then all the animals were sacrificed, and the tissues of lung, heart and brain were collected and stored in-80℃freezer. Another part of tissues were fixed with formaldehyde and HE staining was used to detect the morphological changes of lung, heart and brain;Superoxide Dismutase(SOD) activity and MDA content in tissues were assayed with xanthine oxidase method and thiobarbituric acid method, respectively; p62 protein expression was determined with western blot.Results:1. Morris water maze test showed that with the increase of training time and number, compared with control group, the average escape latency of mice to find the platform was significantly increased in D-Gal group; after the withdrawal of the platform, the number of crossing platform position was obviously reduced in D-Gal(P<0.05). Compared with D-Gal group, rapamycin treatment could markedly improve the impaired memory induced by D-Gal, but chloroquine intervention could further exacerbate the memory impairment caused by D-Gal(P<0.05).2. The morphology results showed that cardiac muscle fibers arranged neatly in control group while myocardial structure was in disorder, intercellular space and inflammatory cell infiltration were increased in smoking group and D-Gal group.Compared with smoking group and D-Gal group, CQ intervention induced more inflammatory cell infiltration and indistinct intercellular space, however, Rapamycin treatment could improved these changes, implying the protective effects of Rapamycin on myocardial injury induced by smoking and D-Gal. Hippocampal CA1 neurons stained uniformly, neatly arranged in control group. Smoking and D-Galgroups had less neurons, some abnormal neuronal morphology, thinner, longer,smaller cell body and nucleus as compared to control group, some neurons showed shrinkage, apparent degenerative changes; CQ treatment induced more serious phenomena, but rapamycin intervention alleviated neuronal loss and reversed above abnormal changes. Control mice showed lung clear tissue structure, complete alveoli and normal lung interval; the mice in smoking and D-Gal groups showed disorganized lung tissue structure, atrophic alveolar collapse, alveolar wall thickening, structure deletion and inflammatory cell infiltration. CQ intervention caused more disordered lung tissue structure and more inflammatory cell infiltrition, but rapamycin treatment significant improved disorder lung tissue structure, decreased inflammatory cell infiltration, and alleviated lung tissue injury.3. Compared with control group, cigarette smoke and D-Gal treatment significantly increased MDA content in lung, heart and brain tissue(P<0.05),obviously reduced SOD activity(P<0.05), and enhanced markedly the expression of p62 protein(P<0.05). Compared with smoke group or D-Gal group, chloroquine treatment could further induce MDA content elevation(P<0.05), SOD activity reduction(P<0.05), increase the expression of p62 protein(P<0.05). While rapamycin treatment inhibited the elevation of MDA content and the decrease of SOD activity induced by smoking or D-Gal(P<0.05), significantly reduced the expression of p62protein(P<0.05).Conclusion: Rapamycin can improve the impaired learning and memory in D-galactose-induced aging mice, alleviate the oxidant injury of brain, heart and lung tissues induced by smoking and D-galactose, and the mechanisms may be related with inhibition of mTOR activity and activated autophagy.