| ObjectiveIn this study,4-nonylphenol(4-NP)and bisphenol A(BPA)were used as test substances to research the effects of 4-NP and BPA combined exposure on the reproductive function of F1 male rats and its mechanism.Methods1.Using a 2×2 factorial design method,48 SD rats(12 males and 36 females)were randomly divided into 4 groups(control group,4-NP group(5 mg/kg),BPA group(0.5mg/kg),4-NP + BPA group(5 mg/kg + 0.5 mg/kg)).After the adaptive breeding,the rats were administered with the drug every other day(5 ml / kg).Until the rats were 55 d who were in estrus,male and female rats mating 1:3 in the same group.Female rats who found a lot of sperm through vaginal smear microscopy and were in estrus were judged to be pregnant.Separated and fed separately,and continued to gavage until the end of lactation.During the period of time,pregnant rats were administrated with gavage and F1 male mice were randomly selected 6 from each group who were reared to 60 days and then took the corresponding tissues.2.Hematocyte counting method was used to count sperm.HE staining was used to observe the structure of testis tissue,transmission electron microscope(TEM)was used to observe the occurrence of autophagy.Colorimetric method was used to measure the corresponding index of oxidative stress,and RT-PCR was used to detect the relative m RNA expression levels of Akt,m TOR,4EBP1,p70S6 K.The expression of Akt,p-Akt,p-4EBP1,p-p70S6 K and LC3 proteins were measure by Western blot.Results1.Weight change and organ coefficientCompared with the control group,the weight loss of the 4-NP group was statistically significant(P<0.05);the weight of the combined group was smaller than that of the4-NP group(P<0.05).At the same time,in the analysis of testicular weight and testicular coefficient,the decrease in testicular weight in the 4-NP group,the BPAgroup and the combined group was different from that in the control group(P<0.05),and there was also a difference between the combined group and the individual exposure group(P<0.05).The testicular coefficients of the 4-NP group and the BPA group were different from those of the control group(P<0.05),and the combination group was only statistically significant with the 4-NP group(P<0.05).There was no statistical difference between the other groups(P>0.05).By factorial analysis of variance,the interaction of combined exposure on rat weight and testicle weight showed an antagonistic effect(P<0.05).2.Sperm countsCompared with the control group,the sperm count of the BPA group and the combined group had a statistically significant decrease(P<0.05).Factorial analysis of variance showed no specifi ccombined effect(P> 0.05).3.HE staining resultsIn the 4-NP,BPA,and combined groups,the tissue cells at all levels are not as compact as the control group,the gap between the tissues is increased,and the arrangement of the tissues is chaotic in a small amount of tissues,and the number of sperm in the lumen is small.4.Oxidative stress in testicular tissueThe activity of SOD and GSH-PX in the testis tissues decreased compared with the control group.Among the changes in GSH-PX activity,compared with the control group,only the BPA group had a difference(P<0.05),and there was a difference between the combined group and the BPA group(P<0.05).In the change of SOD activity,the BPA group and the combined group were different from the control group(P<0.05),while the combined group and the 4-NP group were also statistically different(P<0.05);The MDA content was higher than that of the control group and there was a difference between the combination group,the control group and each single exposure group(P<0.05).Factorial results showed that the combined exposure had an antagonistic effect on the change of GSH-PX activity(P<0.05).5.The relative expression of Akt,mTOR,4EBP1,and p70S6 K m RNA in testisThe expressions of Akt,mTOR and p70S6 K in testis were lower than those in control group,while the expression of 4EBP1 was increased.Among them,the expression ofAkt was different between the 4-NP group and the control group,the combined group(P<0.05).The expression of mTOR was different between the BPA group and the control group,the combined group(P<0.05).Among the changes in the expression of p70S6 K,only the 4-NP group was different from the control group(P<0.05);the expression of 4EBP1 was statistically different between each exposure group and the control group(P<0.05).There was no statistical difference between the other groups(P>0.05).Factorial analysis showed that the combined exposure to Akt and 4EBP1 was antagonistic(P<0.05).6.The relative expression of Akt,p-Akt,p-p70S6 k,p-4EBP1,and LC3 proteins in testisThe relative expressions of Akt,p-p70S6 K,and p-4EBP1 proteins were all lower than those of the control group.Among them,each exposure group had a statistically significant difference compared with the control group(P<0.05).The combination group was also statistically significant compared with the individual exposure groups(P<0.05).The expression of autophagy protein LC3-I did not change significantly,but the expression of LC3-II increased slightly compared with the control group(P<0.05).The ratio of LC3-II/LC3-I showed an upward trend,and the exposure groups were statistically significant compared with the control group(P<0.05).Factorial analysis showed that the combined effects of Akt,p-p70S6 K,p-4EBP1,and LC3-II / LC3-I ratios were statistically different(P<0.05)and the type of their combined effect was antagonistic.Conclusion1.4-NP and BPA combined exposure could reduce the weight of F1 male rats,destroy the normal tissue structure of testes,reduce the number of sperm,and affect normal male reproductive function.2.4-NP and BPA combined exposure could cause F1 male rats to produce oxidative stress,inhibit Akt/m TOR pathway,and induce autophagy.3.Based on all factor analysis,it could be preliminarily judged that the combined effect of 4-NP and BPA on male reproduction of offspring male rats is mainly antagonistic. |
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