Research On Reproductive Injury And Improvement Effect Of Lutein Supplementation Induced By Long-term Excessive Drinking In Male Rats

Objective:Long-term excessive drinking can cause damage to the liver,brain,testis and other tissues and organs in human.This study aimed to establish an animal model of long-term excessive alcohol consumption on reproductive injury in male rats,and observe the effect of lutein supplementation with strong antioxidant activity on male reproductive injury caused by excessive alcohol.It is provided a reference for further revealing the harm of alcohol and reasonable dietary nutrition to reduce the injury.Methods:60 healthy male Wistar rats were randomly divided into 6 groups（n=10/group）after adaptive feeding for 2 weeks:normal control group（C）,lutein group（L）,alcohol model group（M）,alcohol+low dose lutein group（ML1）,alcohol+medium dose lutein group（ML2）and alcohol+high dose lutein group（ML3）.This study designed six groups in the animal experiments,which were divided into two parts:（1）the effects of long-term excessive drinking on reproductive injury of male rats were analyzed by using the experimental results of C and M groups;（2）the improvement effect and mechanism of lutein supplement were analyzed based on the experimental results of 6 groups.Experimental treatment of rats in each group was as follows:C group was given corn oil once a day;L group was given 24mg/（kg.bw.d）lutein（dissolved in corn oil）by intragastric administration;M group was given corn oil and56℃liquor 6ml/（kg.bw.d）,and the changes of body weight were closely observed.After the body weight stabilized,the alcohol dose was increased to 8,10 and 12ml/（kg.bw.d）;ML1,ML2 and ML3 groups were given lutein of 12mg/（kg.bw.d）,24mg/（kg.bw.d）and 48mg/（kg.bw.d）based on the M group,respectively.M group was given different doses of alcohol（8、10、12 ml/（kg.bw.d））by intragastric administration for 1h,and the blood was collected from orbit and the alcohol concentration was detected.The whole experiment lasted for 12weeks.At the end of the experiment,the rats were killed and the blood,testis,epididymis and other tissues were collected.Epididymis was used to detect sperm count,sperm motility and sperm deformity,and hematoxylin-eosin（H&E）staining was used to observe the histopathological changes of testis in each group.The kits were used to measure the activity of testicular marker enzymes,such as succinate dehydrogenase（SDH）,lactate dehydrogenase（LDH）,alkaline phosphatase（AKP）and acid phosphatase（ACP）;and serum sex hormones,such as testosterone（T）,follicle stimulating hormone（FSH）,luteinizing hormone（LH）and estradiol（E2）;and oxidative stress indicators,such as superoxide dismutase（SOD）,malondialdehyde（MDA）and glutathione peroxidase（GPH-Px）;and inflammatory factors,such as interleukin 1β（IL-1β）,interleukin 6（IL-6）and tumor necrosis factorα（TNF-α）.The protein levels of NF-κB（P65）,IKBα,Nrf2,HO-1,i NOS and the key proteins of mitochondrial mediated apoptosis pathway（Bax,Bcl-2,Cytc,Cleaved caspase-3,Caspase-3 and Caspase-9）in testis were detected by Western blotting to explore the mechanism of lutein,and the apoptosis of testicular germ cells was detected by fluorescent TUNEL.Results:Excessive drinking can cause obvious poisoning in rats.After 10 minutes of alcohol intragastric administration,the rats showed symptoms such as lethargy,posterior abdominal dragging,ataxia,lethargy,and the disappearance of the turning reflex,which lasted for 1～2hours.After 1 hour of intragastric administration of alcohol（12ml/（kg.bw.d））,the alcohol concentration of rats in blood reached 68.36mg/100m L,which was close to the concentration of normal drunkenness in human.However,excessive drinking for 12 weeks could cause chronic poisoning in rats.Compared with group C,the body weight and food intake of rats in group M increased slowly,and at the end of the 12^th week,the body weight and food intake of rats decreased by 20.29%and 43.5%,respectively（P<0.05）.Compared with C group,the arrangement of spermatogenic cells at all levels in testicular tissue of M group was uneven,and the number of cells was reduced and deleted.In addition,sperm motility of rats in M group decreased by 53.29%（P<0.05）,the sperm malformation rate increased by 63.95%（P<0.05）.The results of sex hormone and marker enzyme index showed that compared with C group,hormone FSH,T and E2 levels in M group were decreased by 27.27%,13.73%and 15.11%,respectively（P<0.05）.The results of activities of marker enzymes included SDH,AKP and ACP,which were decreased by 21.90%,34.10%and 20.72%,respectively（P<0.05）.The results of oxidative stress indexes and inflammatory factors showed that compared with C group,the level of MDA was increased by 57.00%and the level of GPH-Px was decreased by25.94%in M group（P<0.05）,serum IL-6 and TNF-αlevels were increased by 32.21%and17.64%in M group,respectively（P<0.05）.Western blot results showed that the protein expression levels of NF-κB（P65）and i NOS in M group were significantly higher than those in C group（P<0.05）,and the protein expression levels of IκBɑ,Nrf2 and HO-1 in M group were significantly lower than those in C group（P<0.05）.Compared with C group,the expression levels of Bax,caspase-3 cleaved,caspase-3,caspase-9 and Cyct protein were increased in M group（P<0.05）,the number of testicular germ cell apoptosis was increased by43.84%（P<0.05）.After 12 weeks of lutein supplementation,the results showed that the testis tissue structure of the rats in ML3 group was more complete than that in M group,the spermatogenic cells were arranged neatly,and the loss of cells was reduced.Compared with C group,the sperm count and motility in L group tended to increase,but there was no significant difference（P<0.05）.Compared with group M,the sperm quality of rats in ML2 and ML3 groups was significantly improved,in which the number of sperm in ML2 group was significantly increased by 50.59%（P<0.05）,and the sperm motility in ML3 group was significantly increased by 70.28%（P<0.05）.The rate of sperm deformity in ML2 group and ML3 group decreased by 50.7%and 53.62%respectively（P<0.05）.The results of sex hormone and marker enzyme indexes showed that compared with M group,the serum level of T in ML3 group was increased by 15.92%（P<0.05）,and the serum AKP and SDH levels were increased by 28.96%and 14.10%,respectively（P<0.05）.The results of oxidative stress indexes and inflammatory factors showed that the MDA level in ML3 group was reduced by 42.67%compared with M group（P<0.05）.The serum levels of TNF-αand IL-6 in ML3 group were significantly lower than those in M group（P<0.05）.Western blot results showed that after 12 weeks of lutein intervention,the protein expression levels of NF-κB（P65）and i NOS in ML3 group were significantly lower than that in M group（P<0.05）.The protein expression levels of IκBɑ,Nrf2and HO-1 in ML3 group were significantly higher than those in M group（P<0.05）.The expression levels of Bax,caspase-3,cleaved caspase-3 and Cytc in ML3 group were lower（P<0.05）.The number of testicular cell apoptosis in ML3 group was 33.65%compared with M group（P<0.05）.Conclusions:Long-term excessive drinking can increase the level of oxidative stress and cause male reproductive injury,especially the decrease of sperm motility and the increase of abnormal sperm count.Lutein supplementation can significantly improve the spermatogenic ability,sex hormone level and testicular tissue structure of rats,and the antioxidant effect of lutein may be related to NF-κB/Nrf2 pathway and Bax/Bcl-2-mediated apoptosis pathway.The results suggest that lutein,as an antioxidant nutrient,has a certain protective effect on reproductive injury caused by excessive alcohol.