according to an estimation of the world health organization ( who ) , more than 366 million people worldwide will have diabetes mellitus in 2030 ; the urban population in developing countries is projected to double between 2000 and 2030 .
diabetes mellitus ( dm ) is an endocrine and metabolic disease which affects almost all organs in our body .
although the lungs are not thought to be primarily affected by diabetes and lung physiological and structural abnormalities are happening in both type 1 and type 2 diabetes , the lung should be considered as a  target organ  .
chronic hyperglycemia is considered as the main reason for diabetes complications . in the process of prolonged
increased glucose concentration , reactive oxygen species ( ros ) overproduction may be one of the key factors which induce organ injury .
decreased antioxidative function and increased oxidative stress were seen in the lung of diabetic rats and rabbits [ 2 , 3 ] .
mitochondria dysfunction will cause oxidative stress injury which is one of the underlying factors for neurodegenerative diseases , diabetes , cardiovascular diseases , cancer , and so on .
physiological hypoxia triggers the changes of mitochondrial redox and increases superoxide generation at complex iii in alveolar epithelial cells .
mitochondrial aldehyde dehydrogenase 2 ( aldh2 ) is a nuclear - encoded mitochondrial enzyme that localizes in mitochondrial matrix ; aldh2 protein had been detected in liver , lung , heart , kidney , testis , esophagus , stomach , colon , and pancreas .
our previous study had reported that , in diabetic rats , ethanol ( etoh ) at low concentration offered myocardial protection through activation of aldh2 expression , and upregulation of aldh2 also plays the protective effect in myocardial ischemia and reperfusion injury , diabetes cardiomyopathy , and kidney and brain injury [ 912 ] ; meanwhile , some researchers reported the association between aldh2 and lung disease .
xu et al . reported that , in neonatal rat lung , after prolonged hyperoxic exposure , aldh2 was downregulated . in lung
epithelial cells , overexpression of aldh2 attenuated hyperoxia - induced cell death through reduction of ros , activation of erk / mapk , and pi3k - akt signaling pathways .
but it remains unknown whether activation of aldh2 expression can prevent diabetes induced lung injury .
so in this study , we mimic diabetes model by intraperitoneal injection of streptozotocin ( stz ) to observe the role of etoh at low concentration in diabetes induced lung injury and analyze the related mechanisms .
male sprague - dawley ( sd ) rats ( 200250  g ) obtained from the animal center of bengbu medical college were selected for the study .
all animal studies were approved by the animal ethics committee of bengbu medical college and performed in accordance with the ethical standards .
streptozotocin ( stz ) was purchased from sigma ( st . louis , mo , usa ) .
malondialdehyde ( mda ) and superoxide dismutase ( sod ) assay kits were from nanjing jiancheng bioengineering institute , china .
ethanol ( etoh , figure 1 ) was purchased from bengbu new chemical reagent factory , china .
the primers used were as follows : for aldh2 forward : 5-gtg ttc gga gac gtc aaa ga-3 and reverse : 5-gca gag ctt ggg aca ggt aa-3 and the product size was 187  bp ; for -actin forward : 5-gat ggt ggg tat ggg tca gaa gga c-3 and reverse : 5-gct cat tgc cga tag tga tga ct-3 and the product size was 630  bp .
mouse anti - aldh2 and anti--actin monoclonal antibodies were from santa cruz biotechnology ( ca ) .
stz at 55  mg / kg freshly dissolved in 0.1  mol / l sodium citrate buffer ( ph 4.5 ) was injected intraperitoneally to induce diabetic models in overnight fasted rats .
the rats in control group were injected with a similar volume of sodium citrate buffer alone .
the rats with plasma fasting blood glucose level higher than 16.7  mmol / l 72  h after injection were regarded as diabetic .
animals were randomly divided into control , diabetes ( dm ) , and etoh+dm groups , respectively ( n = 6 ) . in etoh+dm group , dm rats were fed with 2.5% etoh in their drinking water for one week to initiate drinking and then it was changed to 5% etoh continuous access through the 8 weeks .
the plasma fbg level and body weight ( bw ) were measured at the 8th week .
the ratio of lw / bw was calculated . at the end of the experimental period
the supernatant was collected after centrifugation for 20  min ( 2000  rpm ) .
malondialdehyde ( mda ) content and superoxide dismutase ( sod ) activity were detected according to the instruction manual .
for histological analysis by light microscope , the lung tissue was harvested and fixed in 4% paraformaldehyde for 24 hours , embedded in paraffin and cut into 5  m thick serial sections , and then stained with hematoxylin and eosin ( he ) for light microscope observation .
lung injury degree was evaluated according to mikawa 's scoring standards : ( 1 ) alveolar congestion , ( 2 ) hemorrhage , ( 3 ) infiltration or aggregation of neutrophils in airspace or vessel wall , and ( 4 ) thickness of alveolar wall / hyaline membrane formation .
each item was scored on a 5-point scale as follows : 0 : minimal damage , 1 : mild damage , 2 : moderate damage , 3 : severe damage , and 4 : maximal damage . the final lung injury score was the summation of the four items .
lung tissue was dissected and small pieces were fixed with 2.5% glutaraldehyde in 0.1  mol / l cacodylate buffer for 2  h and postfixed in 1% osmium tetroxide in 0.1  mol / l cacodylate buffer for 1  h. ultrathin sections were cut and contrasted with uranyl acetate followed by lead citrate and observed with jem-1230 transmission electron microscope ( jeol , japan ) .
briefly , total rna was extracted with trizol according to the manufacturer 's instructions . a total rna ( 2  mg )
quantification of the result was determined through measuring the optical density of the labeled bands ; the value was normalized to -actin intensity level .
anti - aldh2 ( 1  :  500 ) antibody was used . mouse anti--actin antibody ( 1  :  500 ) was used as an internal control .
the immunoblots were exposed to x - ray film and analyzed with a digital image system .
statistical comparisons were carried out by one - way variance analysis and the newman - keuls test .
in contrast to control group , fbg levels in dm and etoh+dm groups were increased significantly and fbg level in etoh+dm group was lower than in dm group ( table 1 ) . compared with control animals , body weight ( bw ) and lung weight ( hw ) were significantly decreased in dm and etoh+dm groups , and lw / bw was increased in dm group .
in contrast to dm group , bw was increased and lw / bw was decreased in etoh+dm group ( table 1 ) . in contrast to control rats , pulmonary sod activity was decreased in dm group while mda content was increased in dm and etoh+dm groups . in etoh+dm group ,
sod activity was higher while mda content was lower than in dm group ( table 2 ) . in control group , alveolar walls in lung tissue had unique shape and size , and hemorrhage and inflammatory infiltration were rare . in dm group ,
alveolar septum was thickened widely , infiltration of inflammatory cells and hemorrhage appeared , and the lung injury score was higher than in control group . the injury was ameliorated in etoh+dm group than in dm group ( figure 2 and table 1 ) . in control group
, many regular structure lamellar bodies appeared in type ii alveolar cell cytoplasm . in dm group ,
the structure of lamellar bodies was collapsed , discontinuous , and vacuolated . in etoh+dm group ,
the expressions of pulmonary aldh2 mrna and protein were decreased in dm group ; compared with dm group , pulmonary aldh2 mrna and protein expressions were increased in etoh+dm group ( figures 4 and 5 and table 3 ) .
in the present study , we observed that lung oxidative stress injury occurred in diabetic rats , which was indicated by the increase of pulmonary mda content and decrease of sod activity ; meanwhile , the decrease of pulmonary aldh2 mrna and protein expressions happened accompanying the happening of lung swelling , the destruction of pulmonary tissue , and type ii alveolar cell structure . when the diabetic rats were treated with etoh at low concentration which was reported to activate aldh2 expression [ 8 , 10 ] , lung aldh2 mrna and protein expressions were increased , lung oxidative stress injury and swelling degree were attenuated , the destruction of pulmonary tissue and type ii alveolar cell structure was alleviated , suggesting that downregulation of pulmonary aldh2 was likely to be correlated with oxidative stress injury in diabetic rats , and activation of aldh2 with etoh at low concentration attenuated diabetes induced lung injury and oxidative stress overload . in the development of diabetes , oxidative stress induced by chronic hyperglycemia plays a key role in the pathogenesis of diabetes - related complications including lung diseases .
diabetes induces impairment of defense system , which is associated with reduced antioxidant capacity , abnormal activity , or expression of antioxidant enzymes .
an increase of mda , a lipid peroxidation marker , accompanied by the depressing of sod activity ( one of the most important endogenous antioxidase enzymes ) , is assessed to imply oxidant damage . oxidative stress can destroy the pulmonary structure . in our study , we observed that , in diabetic rat , pulmonary mda content was increased with the decrease of sod activity , and the structures of lung tissue and type ii alveolar cell were destroyed , suggesting the unbalance of lung antioxidative system induced lung injury in diabetes rats . when the diabetic rat was treated with etoh at low concentration ,
pulmonary mda content was decreased and sod activity was increased accompanying the recovery of lung tissue and type ii alveolar cell structures in contrast to diabetic rat , suggesting that etoh protected the lung tissue through antioxidative stress role .
activation of aldh2 with alda-1 alleviated cardiac ischemic and reperfusion injury with the decrease of 4-hne in rodent models .
aldh2 overexpression rescued neuronal survival against 4-hne treatment in pc12 cells . in male c57bl/6 mice , activation of aldh2 with
etoh prevented renal ischemia and reperfusion injury with suppressed lipid peroxidation and increased sod activity .
but few reports focus on the relationship of pulmonary aldh2 and diabetes induced lung injury .
aldh2 overexpression attenuated hyperoxia - induced cell death in lung epithelial cells through reduction of ros , activation of erk / mapk , and pi3k - akt signaling pathways .
since diabetes induced lung injury accompanied with oxidative stress , while increasing aldh2 expression could decrease oxidative stress , so we investigated whether activating pulmonary aldh2 expression by etoh could decrease lung oxidative stress injury in diabetic rats .
the results displayed pulmonary aldh2 mrna and protein expressions were decreased in diabetic rats , while etoh treatment increased aldh2 expression and , meanwhile , decreased oxidative stress injury ; it suggested that , in diabetes induced lung injury , the aggravation of oxidative stress may be derived from the decrease of pulmonary aldh2 expression and improving pulmonary aldh2 expression could be against the happening of hyperglycemia induced oxidative stress .
it is worthwhile to note that , in our study , we selected etoh to promote aldh2 expression because there had been many papers reporting that etoh at a suitable concentration could activate aldh2 expression and play the protective effect [ 8 , 10 , 12 , 18 ] .
but in clinic , etoh is difficult to apply for patients because of its toxicity and addiction . so selecting an appropriate drug to activate pulmonary aldh2 expression may be beneficial for diabetic patients who had suffered from lung injury . in conclusion
, our results indicated that , in diabetes induced lung injury in rat model , pulmonary aldh2 expression was decreased .
treatment with etoh at low concentration can decrease diabetes induced lung injury through activating aldh2 expression .
 to observe the changes of lung injury when diabetic rats were treated with low concentration of ethanol ( etoh ) and analyze the related mechanisms , male sprague - dawley ( sd ) rats were divided into control , diabetic ( dm ) , and etoh+dm groups . 
 diabetic rat was mimicked by injection of streptozotocin intraperitoneally . fasting blood glucose ( fbg ) level , lung weight ( lw ) , body weight ( bw ) , and lw / bw were measured . 
 the changes of lung tissue and type ii alveolar cell were detected . 
 pulmonary malondialdehyde ( mda ) content and superoxide dismutase ( sod ) activity were measured ; meanwhile , aldh2 mrna and protein expressions were detected by rt - pcr and western blotting , respectively . compared with control group , in dm group 
 , sod activity was decreased ; fbg level , lw / bw , mda content , aldh2 mrna , and protein expressions were decreased . 
 compared with dm group , in etoh+dm group , sod activity , aldh2 mrna , and protein expressions were increased ; lw / bw and mda content were decreased . 
 the structures of lung tissue and lamellar bodies were collapsed in dm group ; the injury was attenuated in etoh+dm group . 
 our findings suggested that , in diabetic rat , pulmonary aldh2 expression was decreased accompanying lung injury . 
 etoh at low concentration decreased diabetes induced lung injury through activating aldh2 expression . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion