diabetes mellitus ( dm ) is one of the major public health problems around the world . according to the compiled data of the world health organization ( who ) , approximately 150 million people have diabetes mellitus worldwide , and this number of diabetic patients may be doubled by the year 2025 . alongside established lifestyle factors , such as smoking , adiposity , and diet ,
ethanol is a very commonly used chemical substance and also has dose - related effect on cardiac events .
possible cardioprotective effects of ethanol consumption continue to be hotly debated in the medical literatures and popular media .
systematic reviews and meta - analyses have addressed that there is a curvilinear relationship between ethanol consumption and cardiovascular disease , with a protective effect of moderate ethanol consumption and a detrimental effect of large amounts intake [ 37 ] .
importantly , epidemiological studies suggest that light to moderate alcohol consumption decreases the risk of cardiovascular events , that is , 1 - 2 drinks per d or 39 drinks per wk ( one beer , one glass of wine , or one glass of spirit was approximated to one standard drink defined as 1.5  cl or 12  g of pure ethanol ) .
moderate drinking can activate metabolism of ethanol by acetaldehyde dehydrogenase-2 generation   to reduce the incidence of diabetic cardiomyopathy [ 8 , 9 ] .
diabetic cardiomyopathy is defined as the ventricular dysfunction that occurs in diabetic patients independent of another cause , such as coronary artery disease or hypertension .
diabetic cardiomyopathy is a common complication of diabetes which has become a major cause of diabetes - related morbidity and mortality .
mitochondrial acetaldehyde dehydrogenase-2 ( aldh2 ) is a key enzyme which plays an important role in the metabolism of acetaldehyde and other toxic aldehydes .
furthermore , in transgenic mice , overexpression of aldh2 has been shown to be protective against streptozotocin - induced diabetic cardiomyopathy .
our previous results had indicated that aldh2 expression was further decreased accompanying the development of diabetes . meanwhile , we investigated the effects of low - dose ethanol feeding in diabetic rats which could promote myocardial protection through activation of aldh2 expression .
and more , we also found that upregulation of aldh2 played a protective effect in myocardial ischemia and reperfusion injury and diabetes cardiomyopathy .
aldh2 may be an endogenous cardiac protective factor in myocardial injury [ 2 , 8 , 14 ] .
several biological mechanisms have been proposed to explain that myocardial fibrosis is one of the main pathological changes of diabetic cardiomyopathy .
studies have found c - jun n - terminal kinase ( jnk ) signaling pathway played a key role in the process of myocardial fibrosis [ 1517 ] .
evidence had shown that activation of the jnk pathway was involved in the progression of diabetes induced myocardial fibrosis and that such a pathway could be a therapeutic target for diabetic heart injury and cardiomyopathy [ 18 , 19 ] . however , whether the jnk pathway is involved in the cardioprotective effect of low - dose ethanol on diabetic rats has not been fully elucidated .
so in this study , we mimic diabetes model by intraperitoneal injection of streptozotocin ( stz ) in combination with low - dose ethanol ; the purpose of the present study is as follows : ( 1 ) to investigate the mechanism of low - dose ethanol which alleviates myocardial fibrosis in diabetic cardiomyopathy ; ( 2 ) to clarify whether low - dose ethanol mediated protection is associated with downregulating the jnk signaling pathway .
this study might shed some light on low - dose ethanol as an effective therapeutic in the treatment of diabetic cardiomyopathy .
adult male sprague - dawley rats ( 200 to 250  g ) were obtained from bengbu medical college animal administration center .
all animal studies were approved by the animal ethics committee of bengbu medical college and performed in accordance with the ethical standards .
trizol was purchased from invitrogen ( usa ) ; hydroxyproline ( hp ) was purchased from nanjing jiancheng bioengineering institute ( china ) .
ethanol ( etoh ) was purchased from bengbu new chemical reagent factory ( china ) .
-actin antibodies were purchased from santa cruz biotechnology ( usa ) , rabbit c - jun n - terminal kinase ( jnk ) and phosphorylated jnk ( p - jnk ) were purchased from anbo biotechnology ( usa ) . chemiluminescence reaction ( ecl )
all primers were purchased from shanghai sangon biotech ( china ) . as previously described by our laboratory , 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 .
all rats were randomly divided into four groups : normal control group ( con ) , diabetes at 4 weeks group ( dm4w ) , diabetes at 8 weeks group ( dm8w ) , and ethanol + diabetes at 8 weeks group ( etoh + dm8w ) , respectively ( n = 6 ) . in con group , rats were fed with standard rat chow and received an intraperitoneal injection of the same volume of citrate buffer for 8 weeks . in etoh + dm group , dm rats were fed with 2.5% etoh in their drinking water for one week to initiate drinking then , it was changed to 5% etoh continuous access through the remaining 7 weeks .
male sd rats were anaesthetized by use of chloral hydrate ( 100  mg / kg ) through intraperitoneal injection . throughout the experiment ,
systolic pressure ( sp ) , diastolic pressure ( dp ) , mean arterial pressure ( map ) , and heart rate ( hr ) were determined by invasive hemodynamic evaluation methods for 30  min . at the end of the experimental period , hearts were excised rapidly , placed in ice - cold krebs - henseleit ( k - h ) buffer , and weighed and the ratio of heart weight / body weight ( h / b ) was calculated . heart tissue ( 100  mg )
the supernatant was collected after centrifugation for 20  min ( 2000  rpm ) .
left ventricular tissue obtained from all groups was stained with masson 's trichrome for the quantification of collagen .
the histological sections were taken in 10% neutral formalin - fixed , dehydrated , paraffin embedded sections .
then , the samples underwent the dehydration of gradient ethanol , neutral resin embedding , and masson trichrome staining .
myocardial collagen was quantified at a final magnification of 200x with a polarized microscope connected to a video camera .
myocardial collagen volume fraction ( cvf ) was analyzed using image pro analysis software , expressed as the mean percentage of collagen area to the total area of each microscopic field .
five visions under microscope of each sample were randomly chosen and the average of them was taken for analysis .
total rna was extracted from the left anterior myocardium using trizol according to the manufacturer 's instructions .
total rna ( 3  g ) was reversely transcribed to cdna , and pcr was performed by a routine method .
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-gag acc ttc aac acc cca gcc-3 and reverse : 5-ggc cat ctc ttg ctc gaa gtc-3 and the product size was 312  bp .
the pcr condition was as follows : predenaturing at 95c for 3  min and then 40 cycles ( 50  s denaturation at 95c , 50  s annealing at 62.5c , and 60  s extension at 72c ) , followed by a final step at 72c for 10  min .
myocardium tissues ( 100  mg ) from each group were collected and homogenized in a lysis buffer .
homogenates were sonicated and centrifuged at 12,000  g for 30  min at 4c .
the protein concentration was determined using the bicinchoninic acid ( bca ) protein assay kit .
total protein ( 80  g ) was separated by sds - polyacrylamide gel electrophoresis ( page ) and transferred electrophoretically to a polyvinylidene difluoride ( pvdf ) filter membrane .
the membranes were blocked with 5% nonfat milk in tris - buffered saline tween ( tbst ) for 2  h , and then they were incubated at 4c overnight with the corresponding primary rabbit jnk antibody ( 1  :  1000 ) , rabbit p - jnk antibody ( 1  :  1000 ) , and mouse -actin antibody ( 1  :  500 ) .
all membranes were incubated for 1  h with corresponding secondary antibody hrp - linked anti - mouse igg or hrp - linked anti - rabbit igg .
one - way analysis of variance ( anova ) followed by student - newman - keuls ( snk ) was used for multiple comparisons .
in contrast to con group , there was no statistical difference about hemodynamic parameters in dm4w groups , but systolic pressure ( sp ) , diastolic pressure ( dp ) , mean arterial pressure ( map ) , and heart rate ( hr ) were decreased significantly in dm8w groups .
compared with dm8w group , sp , dp , map , and hr were rather increased significantly in etoh + dm8w group .
( table 1 ) compared with con group , there was no significant difference about the ratio of heart weight to body weight ( h / b ) and hydroxyproline ( hp ) content in myocardial tissue in diabetic rat in dm4w group ; however , with extended duration , h / b ratio and myocardial hp content were increased in dm8w group , and the difference was statistically significant ( table 2 ) . compared with dm8w group , h / b ratio and myocardial hp content were significantly deceased in etoh + dm8w group ( p < 0.05 ) . in masson trichrome staining ,
however , collagen tissue was increased markedly and disrupted in some area in diabetic group . in dm4w group , myocardial cells were approximately arranged well , collagen fibers were sparsely distributed , and interstitial collagen was dyed a little blue .
compared with control group , myocardial cells were in a disordered arrangement , the interstitial collagen were edematous , and collagen fibers were unevenly distributed and increased markedly in dm8w group . compared with dm8w group ,
myocardial cells were arranged neatly , and collagen fibers were significantly reduced in etoh + dm8w group . 
 quantitative analysis results .
as displayed in table 2 , compared to control group , the contents of collagen volume fraction ( cvf ) were significantly increased in dm4w group ( p < 0.05 ) , and with the development of diabetes , cvf were further increased in dm8w groups ( p < 0.01 ) .
but in contrast to dm8w group , the contents of cvf were markedly decreased in etoh + dm8w group ( p < 0.01 , figure 1 , table 2 ) .
real - time pcr revealed that , compared with control group , the expression of aldh2 mrna level was reduced in dm4w ( p < 0.05 ) . with the development of diabetes , aldh2 mrna
in contrast to dm8w group , the expression of aldh2 mrna was increased in etoh + dm8w group ( p < 0.01 , table 3 ) . on western blot analysis ,
compared with control group , the ratio of p - jnk / jnk protein expression was increased in dm4w ( p < 0.05 ) , and with the development of diabetes , the ratio of p - jnk / jnk was further increased in dm8w groups ( p < 0.01 ) . compared with dm8w group , the ratio in etoh + dm8w group was decreased ( p < 0.01 , figure 2 , table 3 ) .
diabetes mellitus is becoming an epidemic health threat and represents one of the most prevalent chronic noncommunicable disorders .
cardiovascular disease is a serious complication of diabetes and is responsible for 80% of the deaths among diabetics .
diabetic cardiomyopathy is one of the most common complications of diabetes ; its major pathological characteristics are hypertrophy or hyperplasia of cardiac myocytes .
excessive deposition of myocardial interstitial collagen and myocardial fibrosis often leads to cardiac hypertrophy and decrease of heart function , which play a vital role in the occurrence and development of diabetic cardiomyopathy . in the present study
, our results demonstrated that , with the progression of diabetes , systolic pressure , diastolic pressure , mean arterial pressure , and heart rate were declined significantly in dm8w group compared with con group , but the ratio of heart weight / body weight , hydroxyproline content , and cvf were markedly increased .
meanwhile , the mrna expression of aldh2 was decreased and the ratio of p - jnk / jnk was increased . when the dm rats were treated with ethanol at low concentration ,
hemodynamic parameters were improved and hydroxyproline content and cvf were decreased , accompanied with the increase of myocardial aldh2 mrna expression and decrease of p - jnk / jnk protein expression .
the results suggested that with low - dose ethanol intervention , enhanced aldh2 expression can antagonize the happening of myocardial fibrosis in diabetic cardiomyopathy , which may be relevant with downregulating the jnk pathway .
mitochondrial aldehyde dehydrogenase 2 ( aldh2 ) is a kind of aldehyde oxidase that is involved in ethanol metabolism , which is closely related to human alcohol drinking behavior .
heavier alcohol consumption is associated with increased risk of developing diabetes mellitus , hypertension , and cardiovascular and cerebrovascular disease .
nevertheless , there are increasing reports showing a critical role for light to moderate alcohol ingestion could protect against myocardial injuries
. researches indicated a u - shaped relationship between alcohol consumption and cardiovascular disease mortality [ 6 , 26 ] .
statistics manifested the mortality of drinking alcohol up to or over 6 cups ( 355  ml / cup ) per day is 1.6 times as high as control , while light - to - moderate drinking of 1~2 cups alcohol per day [ fewer than 2 cups or 1 ounce ( 28.3495  g ) ] reduced mortality from cardiovascular disease .
in addition , clinical data showed that moderate drinking can protect metabolic syndrome ( cardiovascular disease and diabetes ) via influencing various factors to reduce the diabetes prevalence , such as glycosylated hemoglobin , high density lipoprotein cholesterol , and fibrinogen .
after continuously feeding c57bl/6 mice with 18% of ethanol for 12 weeks , the myocardial mechanics were restored and the expression and activation of pkc and akt were increased , which established that etoh feeding causes cardiac expression of activated pkc- .
sun et al . observed that ischemic injury could result in downregulation of mitochondrial aldh2 in mice hearts inducing an elevation of aldehyde 4-hydroxy-2-nonenal ( 4-hne ) , leading to cardiomyocyte apoptosis through downregulation of hsp70 and activation of jnk and p53 .
aldh2 detoxifies 4-hne , a mediator of programmed cell death events , by transmitting a mitochondrial aldh2 signal to elicit a cytosolic response through the jnk / p53 pathway .
however , using transgene technology to increase the expression of aldh2 can contribute to antagonizing heart failure and decreasing heart function .
furthermore , our previous research found that there was a close relationship between aldh2 and oxidative damage . with the progression of diabetes ,
the myocardial antioxidant ability of diabetic rats was decreased , which exacerbated the progression of myocardial fibrosis [ 8 , 13 , 31 ] . in this study
, we found that the ratio of heart weight / body weight , hydroxyproline content , and cvf were markedly increased in diabetic 4 and 8 weeks ' rats , which suggested diabetic induced myocardial fibrosis . when the diabetic rat was treated with low doses of ethanol to induce aldh2 activity ,
the hemodynamic parameters were increased and hydroxyproline content and cvf were decreased , which indicated that increasing aldh2 expression can attenuate the happening of myocardial fibrosis and the destroying of myocardial injuries .
mitogen - activated protein kinases ( mapks ) play an important role in the signal transduction pathways from the membrane to intracellular compartments including the nucleus .
they regulate the functions of many gene products and therefore affect cell growth , differentiation , and apoptosis .
oxidative stress , inflammation , endoplasmic reticulum stress , and autophagy defect can activate mapks signaling pathway in the progression of diabetes complications .
li et al . observed that production of a large number of ros was thought to be an important contributing factor , concomitant with activation of jnk , p38 mapk , and tgf- in the development and the progression of diabetic cardiomyopathy .
c - jun nh2-terminal kinase ( jnk ) is one of the major members of mapks , and jnk activation is also implicated in cardiac fibrosis [ 33 , 34 ] .
jnk signaling pathway plays a key role in the growth of cardiac fibroblasts induced by high glucose .
studies of diabetic rats had demonstrated that myocardial fibrosis was developed ; meanwhile , jnk mrna expression level and activity were upregulated .
furthermore , disruption of the jnk protein kinase decreased the occurrence and development of diabetic myocardial fibrosis [ 18 , 35 ] .
it is worthwhile to note that , in our experiment , we found that , with the progression of diabetes , the expression of myocardial aldh2 at mrna level was decreased and the ratio of p - jnk / jnk at protein level was increased , which suggested aldh2 and jnk signaling pathway both participated in the occurrence and development of diabetic cardiomyopathy .
moreover , further enhancing activation of aldh2 expression of low doses of ethanol for 8 weeks in diabetic rats , accompanied with the high - expression of aldh2 , p - jnk / jnk were decreased in contrast to diabetes rats , which suggested activation of aldh2 expression might be associated with downregulating the jnk signaling pathway to relieve myocardial fibrosis and myocardial injuries .
we only observed that the protection of low - dose ethanol was relevant with downregulating the jnk pathway in diabetic cardiomyopathy .
to better understand the mechanisms involved , we will adopt the activation or inhibition of jnk to investigate the downstream signaling molecules in the follow - up experiments . to address this issue
, we will use jnk inhibitor to observe whether inhibiting jnk pathway can play the cardiovascular role in diabetes rats .
moreover , we further explore whether enhanced activation of jnk signaling pathway can antagonize the cardioprotective effect of activation of aldh2 by low - dose ethanol . through these experiments , we want to verify low - dose ethanol could attenuate myocardial fibrosis via downregulating the jnk pathway in diabetic cardiomyopathy . in summary ,
our results demonstrate that with the progression of diabetes , aldh2 expression was decreased accompanied with the happening of myocardium fibrosis .
treatment with ethanol at low concentration can protect the heart by upregulating aldh2 and downregulating the jnk signaling pathway against myocardial fibrosis in diabetic cardiomyopathy .
 
 objective . to investigate the effects of low dose ethanol feeding in diabetic rats and analyze its underlying mechanisms 
 . methods . 
 male sprague - dawley rats were divided into 4 groups : control ( con ) , diabetes at 4 weeks ( dm4w ) , diabetes at 8 weeks ( dm8w ) , and etoh + dm8w . 
 after 8 weeks , hemodynamic parameters were recorded and heart weight / body weight ( h / b ) and hydroxyproline ( hp ) content in myocardium were measured . 
 morphology of collagen in myocardial tissue was observed with masson 's trichrome staining method and collagen volume fraction ( cvf ) was analysed . 
 the mrna expression of aldh2 was assessed with real - time pcr . 
 the protein expressions of p - jnk and jnk were evaluated using western blot . 
 results . 
 in contrast to con group , there was no difference in hemodynamic parameters in dm4w group , but mean arterial pressure and heart rate were decreased in dm8w group , and the ratios of h / b , hp , and cvf were markedly increased . 
 aldh2 mrna expression was decreased , while the ratio of p - jnk / jnk were increased . 
 compared with dm8w group , the above indexes were improved in etoh + dm8w group . conclusion . with low dose ethanol intervention , enhanced aldh2 expression 
 can antagonize the happening of myocardial fibrosis in diabetic rats , which may be relevant with downregulating the jnk pathway . 
1. Introduction
2. Methods
3. Results
4. Discussion