The Roles Of Thioredoxin Intercacting Protein(TXNIP) In Oxidative Stress In Aged Rats And Senile Patients With Chronic Obstructive Pulmonary Disease

Objective: To define the aged rats.Then to study the expression and change of thioredoxin binding protein(TXNIP) in aged rats and senile patients with chronic obstructive pulmonary disease(COPD). Further to investigate whether the inbalance expression of TXNIP and thioredoxin1(TRX1) participated in the inbalace of oxidation and anti-oxidation in senile patients with COPD. Therefore, it provide a new theoretical basis for the prevention and treatment of COPD.Methods: The study was composed of two parts: animal experiment and clinical trial.(1)animal experiment: 1)Forty adult male SpragueDawley(SD) rats were divided into three month、six month、twelve month、eighteen month、twenty-four month group in terms of age and every group had 8 male SD rats. To measure echocardiography、electroencephalogram(EEG) and lung function of rat in every group. To observe the rat tissue morphology of heart, liver, lung, kidney, brain. And then count the ration of organ to body weight. Rat peripheral blood was collected to measure rat biochemical index of cardiac function, liver function, renal function, lipids, glucose, hemorrheology and coagulation function. Enzyme-linked immunosorbnent assay(ELISA) was used to measure the protein expression of anti-aging factor sirtuin(Sirt1) of rat every organ tissue, peripheral blood and bronchoalveolar lavage fluid(BALF). Western blot was used to test the protein expression of TXNIP in rat lung tissue. Therefore, they were aimed at defining the aged rats. 2)Sixty-four male SD rats were randomly divided into two groups: adult(6 month) group and aged group. Then each group were divided into normal control group, copd model group, telmisartan-treated group, suberoylanilide hydroxamic acid(SAHA) treated group and every group had 8 male SD rats. The lung function measurements were carried out and the pathological changes were observed. ELISA was used to measure the protein expression of reactive oxygen species(ROS) in rat peripheral blood and BALF. Protein expression of TXNIP and TRX1 in clinical lung tissue were observed by immunohistochemistry and western blot.(2) clinical trial : Clinical peripheral lung tissue samples were obtained from thirty-seven patients undergoing resection for lung cancer, and all patients were measured lung function prior to they were operated. Those patients were divided into adult group(which was less than 65 years old) and aged group(which was 65 and over). And then each group were divided into normal control group and copd model group. Pathological changes of lung tissue were observed in every group. Immunohistochemistry and western blot were used to test the protein expression of TXNIP and TRX1 of every patient lung tissue. Collecting 60 COPD patients peripheral venous blood and they were divided into adult group and aged group. Otherwise, forty normal peripheral venous blood were collected and then were divided into adult group and aged group similarly. Protein expression of ROS、TXNIP and TRX1 in clinical peripheral blood were observed by ELISA. The forty collected aged COPD patients was classified based on the Chinese Medical Association established standards in 2013 and they were divided into mild to moderate group and severe group. Protein expression of ROS、TXNIP、TRX1 in clinical peripheral blood were observed by ELISA.Results:1. Animal experiments:(1)With age increased, the morphology of lung tissue、 kidney tissue and brain hippocampus structure in every group had significant change. But the rat heart tissue、liver tissue of every group in morphology had no significant difference. In 24 month group, the rat functional examination index included echocardiography、EEG and lung function was much lower than in the other group(P<0.05);(2)Compared to other groups, the protein expression of Sirt1 in rat lung、brain、kidney、BALF and peripheral blood serum was decreased.(P<0.05).The protein expression of TXNIP in 24 month group compared to other groups was increased by western bolt(P<0.05);(3)The results of rats pulmonary function showed: forced expiratory volume in first 0. 3 second(FEV0.3)、percentage of forced expiratory volume in first 0. 3 second to forced vital capacity(FEV0.3/FVC%) and peak expiratory flow(PEF) were all obviously decreased in COPD group compared with control group(P<0.05) and which were all improved in telmisartan-treated group compared with COPD model group(all P<0.05). Meanwhile, the lung function index were decreased in SAHA-treated group compared with COPD model group(P < 0.05). Compared with adult groups, the FEV0.3 、FEV0.3/FVC% and PEF in aged groups were all decreased but had no statistical significance(P>0.05). Lung pathological changes in COPD model group conformed morphological character of COPD, however, pathological changes of rats lung tissue in telmisartan-treated group markedly reduced and in SAHA-treated group was increased compared with COPD model group. In a word, the pathological changes in aged group was markedly increased compared with adult groups.(4)In COPD model group, ROS levels was raised compared to controls(P<0.05), whereas in telmisartan-treated group, ROS levels was reduced compared to COPD model group(P<0.05)and was increased in SAHA-treated group compared with COPD(P<0.05). ROS levels was increased in aged group compared with adult group.(5)Immunohistochemistry showed that TXNIP was mainly located in alveolar epithelial cells and lung interstitial cells. Which protein expression was mainly found in nucleus and cytoplasm. Western blot showed that protein expression of TXNIP and TRX1 in rat lung tissues were significantly higher in COPD model group than those in controls(P<0.05). Compared with COPD model group, protein expression of TXNIP in telmisartan-treated group was reduced(P<0.05) and the TRX1 protein expression was increased(P<0.05). For another, the protein expression of TXNIP in SAHA-treated group was increased compared with COPD group(P<0.05), while the protein expression of TRX1 was decreased compared with COPD model group(P<0.05). And the protein expression of TXNIP in aged group was higher than adult group(P<0.05), but the expression of TRX1 in aged group was reduced compared with adult group(P<0.05).(6)ELISA showed that the proteins expression of TXNIP and TRX1 in BLAF and serum were increased in COPD model groups compared with control groups(P<0.05). In the one hand, compared with COPD model group, the protein expression of TXNIP in telmisartan-treated group was decreased(P<0.05), while the expression of TRX1 was increased(P<0.05). In anther hand, the protein expression of TXNIP in SAHA-treated group was increased compared with COPD group(P<0.05), while the protein expression of TRX1 was decreased compared with COPD model group(P<0.05). And the protein expression of TXNIP in aged group was higher than adult group(P<0.05), but the expression of TRX1 in aged group was reduced compared with adult group(P<0.05).2. Clinical experiments:(1)Immunohistochemistry showed that TXNIP was mainly located in alveolar epithelial cells and lung interstitial cells in lung of patients. And the protein expression of TXNIP was mainly found in nucleus and cytoplasm of alveolar epithelial cells and lung interstitial cells. Western blot showed that protein expression of TXNIP and TRX1 in patient lung tissues were higher in COPD group than those in controls(P<0.05). And the protein expression of TXNIP in aged group was higher than adult group(P<0.05), but the expression of TRX1 in aged group was reduced compared with adult group(P<0.05).(2)ELISA showed that the proteins expression of ROS、TXNP and TRX1 in peripheral blood were increased in COPD groups compared with control groups(P<0.05). And the protein expression of ROS and TXNIP in aged group was higher than adult group(P<0.05), but the expression of TRX1 in aged group was reduced compared with adult group(P<0.05).(3)ELISA also showed that the proteins expression of ROS、TXNP and TRX1 in peripheral blood were increased in aged severe COPD group compared with aged mild to moderate COPD group(P<0.05). But the expression of TRX1 in aged severe COPD group was reduced compared with aged mild to moderate COPD group(P<0.05).(4)Linear correlation analysis showed that the protein expression of TXNIP was positively correlated with FEV1(%)and the protein expression of ROS(P<0.05). And the protein expression of TXNIP had negative correlation with protein expression of TRX1.Conclusions:1.The aged rats was 24 month explicitly.2.TXNIP levels was increased with increasing age and COPD severity. Antioxidant enzyme TRX1 may be compensatory up-regulate to against oxidative stress in the early stage of COPD, but which maybe a progressive decompensation with increasing age and COPD severity. To some extent, it can reflect the activity of disease.3.The activation of TXNIP and TRX1 possibly gave rise to the inbalance of oxidation and anti-oxidation in senile patients with COPD. In oxidative stress conditions such as smoking could be made the protein expression of TXNIP increased and TRX1 reduced. Then the inbalance relationship may increase the ROS content and further aggravated the oxidative damage in the body.4.Monitoring the level of TXNIP and TRX1 was important for the diagnosis and the the assessment of severity of aged COPD.