Compensatory effect of IGF-I survival signaling is reduced by aging to accelerate apoptosis in cardiac cells exposed to second-hand smoke

Compensatory effect of IGF-I survival signaling is reduced by aging to accelerate

apoptosis in cardiac cells exposed to second-hand smoke

吳嘉平

_{*, 郭薇雯}

_{, 黃志揚}

Jia-Ping Wu

*, Wei-Wen Kuo

, Chih-Yang Huang

1.Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan.

2.Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.

3.Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan.

4.School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan.

Introduction: Secondhand smoke (SHS) exposure is associated with an

increased risk of coronary artery disease. Ageing is a physiological process

involving progressive impairment of normal heart functios, due to an

increasing vulnerability, which reduces the ability of survive. The aim of this

study was to examine secondhand smoke exposure in aging-related

death-survival balance. Methods:Rats were divided into two age groups, male

young adult and male old which were divided into two subgroups and

treated for 4 weeks SHS exposure as follows: Control (C), not exposed to

SHS. Secondhand smoke exposure (S), exposed to SHS. The rats were

placed in exposure chamber and exposed to 10 cigarettes for 30 min, twice

a day, five days/week for 1 month. Then, rats underwent morphological and

function study with echocardiography. Histopathologic of left ventricular

sections were stained with hematoxylin-eosin staining and related

death-survival protein expression by Western Blotting. Results:From

echocardiography results, we found EF (%) and FS (%) were apparently

decreased in young and aging rat under SHS two weeks exposure, and

more severe in aging but compensated occurred in young rats after four

weeks exposure. LVID, LVPW and IVS at systolic diameters were increased

in young SHS-exposed rats, and enhanced more in aging SHS exposed

rats. In addition, both upregulation of death receptor dependent apoptosis

pathways (TNFα/Fas-L-Fas/FADD-cleaved caspase 8) in young and aging

SHS exposure animals, and more enhanced in aging SHS exposed rats.

However, the Mitochondria apoptosis proteins, t-Bid, Bid, cytochrome c and

the ratio of Bad/Bcl 2 were only increased in aging and augumented

increased in aging SHS exposure rats. Similarly results were observed on

cleaved-caspase 9 and 3 proteins. Moreover, the survival pathways

(IGF-I/IGFIR-PI3K/p-Akt) was found only compensated in young SHS exposure,

but not in aging rats and even more decreased in aging SHS exposed rats.

Conclusion: Aging and SHS should be considered as risk factors for

cardiac dysfunction. Moreover, Aging reduces the IGF-I compensated

signaling and accelerate the cardiac apoptotic effects induced by

Second-hand Smoke.

Materials and Methods Animals

We purchased male Hamster rats (young age group: 6 weeks of age; body weight, 132.5 ± 4.61 g; old age group: 18 months of age, body weight, 145 ± 3.42 g) from the National Science Council Animal Center, Taipei, Taiwan. The animals were housed six in individual cages in an environmentally controlled animal room, and tap water was provided . All animals were handled according to the guidelines of the Taiwan Society for Laboratory Animals Sciences for the care and use of laboratory animals in

temperature- and humidity-controlled chambers.

Experimental groups and secondhand smoke (SHS) exposure

Rats were divided into 2 age groups, young adult and old, which were each divided into two subgroups and exposed to SHS for 4 weeks as follows: 1) control (C), 6

animals not exposed to cigarette secondhand smoke, and 2) secondhand smokers (S), 6 animals exposed to cigarette secondhand smoke (SHS). The rats were placed in

whole-body exposure chambers and exposed to 10 cigarettes. Filtered air was

introduced into the chamber at a low rate. Rats were exposed to cigarette smoke for 30 min, twice a day for five days per week for 1 month. Room temperature was

maintained at 22–25°C, and relative humidity was approximately 40%. Western blotting

We prepared the tissue extracts as described above. SDS-PAGE was carried out with polyacrylamide gels. The samples were electrophoresed at 100 V for 1 hr.

Electrophoresed proteins were transferred to nitrocellulose paper using a Hoefer

Scientific Instruments Transphor unit at 100 mA for 14 hr. We incubated nitrocellulose papers in blocking buffer for 2 hr at room temperature. Monoclonal antibodies were diluted 1:200 in antibody binding buffer (TBS). Incubations were performed at room temperature for 3.5 hr. We washed the immunoblots three times in 50 mL of blotting buffer for 10 min. The membranes were then immersed in the second antibody

solution containing alkaline phosphatase goat anti-rabbit IgG diluted 1,000-fold in

binding buffer for 1 hr. The membranes were then washed in blotting buffer for 10 min three times. Color development was performed by ECL chemiluminescence.

Statistical analysis

All data are expressed as the mean±standard error of the mean (SEM). For western blot analysis, quantitation was carried out by scanning and analyzing the intensity of the hybridization signals using the FUJIFILM Imagine program. Statistical analysis of the data was performed using SigmaStat software. Comparison between groups was conducted using a two-way analysis of variance (ANOVA). p values of less than 0.05 and 0.01 were considered to be statistically significant and highly statistically

significant, respectively.

Results

Echocardiography demonstrated that left ventricular function decreased in young and aging rats under a two-week SHS exposure and this decrease was more severe in aging

rats, while young rats exhibited compensation after four weeks of exposure.

Upregulation of death receptor dependent apoptosis pathways (TNFα/Fas-L-Fas/FADD-cleaved caspase 8) occurs in young and

aging SHS-exposed animals and was enhanced in aging SHS-exposed rats.

Mitochondria apoptosis proteins were only increased in aging rats, and the increase was augmented in aging SHS-exposed rats.

The cardiac survival pathway IGF-I/IGFIR-PI3K/p-Akt was found to be attenuated in young SHS exposed rats, but not in aging rats; especially those aging rats exposed to SHS.

Discussion

We believe SHS and aging both enhanced left ventricular hypertrophy. These results indicate that SHS exposure and aging induce upregulation of mitochondria-dependent and –independent

apoptosis signaling pathways and downregulation of survival signaling pathways. Moreover, aging reduces IGF-I compensated signaling and accelerates the cardiac apoptotic effects induced by

second-hand smoke. Aging and SHS should both be considered as risk factors for cardiac dysfunction.

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