The Effects Of IRE1 Signal Pathway Of Endoplasmic Reticulum Stress On Fluoride-induced Reproductive Lesion In Rats

Objects The present study was designed to investigate the effect of fluoride exposure on reproductive system induced by endoplasmic reticulum stress,and to evaluate the interaction of NAC and Na F.Then this study could provide some new ideas for the possible mechanism of toxicity and the prevention of male reproductive injury caused by fluoride exposure.Methods1.Animal groups and treatmentsSixty healthy male Sprague-Dawley(SD)rats(4 weeks old)with SPF,weighing about 100-140 g,were divided into 4 groups randomly,including control group,NAC group,Na F group and Na F+ NAC group.Each group had 15 rats.The control group was treated with 0.9% sodium chloride.The Na F group and NAC group were respectively treated with 25mg/kg/d Na F solution and 150mg/kg/d NAC solution.The Na F+ NAC group was given 150mg/kg/d NAC at first,after half an hour 25mg/kg/d Na F solution was given.And each group was administrated with gastric perfusion for 7 weeks,during that every week the rats were continuously treated by gavage for 6 days and stopped the last day.2.Detection of relevant effect indicators of reproductive injuryThe testis and epididymis of rats were weighted for calculating the corresponding organ coefficient.In addition,the fluorine content of testis tissue was measured with fluoride ion selective electrode,and the levels of serum testosterone(T),follicle stimulating hormone(FSH)and luteinizing hormone(LH)were measured by ELISA.The apoptosis of testis cells was detected by TUNEL method.3.Detection of The endoplasmic reticulum stress related molecularq-PCR method was used to measure the m RNA expression of IRE1,XBP1 and JNK.The protein expression levels of GRP78,IRE1,p-IRE1,XBP1 u,XBP1s,JNK and p-JNK in testis was assayed by Western Blot.Results1.Effect index results of male reproductive functionCompared with control group,testicular fluorine content was increased significantly in group of Na F and NAC + Na F(P<0.05).The weight of body,testis and epididymis in Na F group were decreased(P<0.05).But there was no difference observed from the testicular and epididymal viscera coefficient.After detecting the serum reproductive hormone levels,results showed that compared with the control group,serum testosterone level in Na F group and NAC + Na F group was significantly decreased(P<0.05),and the level of serum follicle-stimulating hormone and luteinizing hormone were significantly increased(P<0.05).While compared with Na F group,treatment with NAC,serum follicle-stimulating hormone and luteinizing hormone were markedly decreased(P<0.05).2×2 factorial analysis of variance showed that the interaction of NAC and Na F had an impact on follicle-stimulating hormone and luteinizing hormone content(P<0.05).2.Apoptosis analysis resultsTUNEL method detecting apoptosis results showed that compared with the control group,testicular cell apoptosis index of Na F and NAC+Na F group were significantly enhanced(P<0.01).Compared with Na F group,NAC intervention inhibited testicular cell apoptosis and markedly suppressed apoptosis index(P<0.01).3.The results of m RNA and protein expression of endoplasmic reticulum stress markersq-PCR was used for detection of m RNA expression of endoplasmic reticulum stress related factors.Compared with control group,the fluoride exposure significantly increased the m RNA expression of IRE1,XBP1 and JNK(P<0.01).NAC+Na F group significantly reduced the m RNA expression level of IRE1,XBP1 and JNK(P<0.01).Factorial analysis of variance showed that the interaction of NAC and Na F had effect on the m RNA expression of IRE1,XBP1 and JNK(P<0.05).Western Blot was used to analysis protein expression of endoplasmic reticulum stress related markers.It showed that Na F group markedly elevated the protein expression of GRP78,XBP1 s and p-JNK(P<0.01).In addition,p-IRE1 protein expression in Na F group and NAC + Na F group were significantly raised(P<0.05).However,treatment with NAC significantly inhibited the increase of protein expression of GRP78,XBP1 s and p-JNK(P<0.01).Factorial analysis of variance showed that the interaction of NAC and Na F had effect on the protein expression of GRP78,p-IRE1,XBP1 s and p-JNK(P<0.05).Conclusion1.Fluoride exposure damaged the reproductive system in rats may through activating the endoplasmic reticulum stress IRE1-XBP1 signal pathway and IRE1 mediated JNK apoptosis pathway.2.NAC nay antagonize endoplasmic reticulum stress,suppress the reproductive cell apoptosis and play a major role in protecting the male reproductive system.