Ameliorative Effects Of Nrf2/ARE Pathway And The Influence Of N-acetylcysteine On Fluoride-induced Male Reproductive Damage

ObjectiveThrough establishing fluoride-treated rat model and using NAC as intervention agent, the purpose of this study was to explore whether NaF induce oxidative stress and DNA damage in rats’ testis, and examine whether NAC exhibit the protective effects on the development of testicular dysfunction under such conditions through activating Nrf2/ARE signal transduction pathway. This research provided theoretical basis and experimental basis for revealing the pathogenetic mechanism of male reproductive toxicity induced by NaF and exploring the possible antidotes relieving the toxicity of fluoride.Methods 1. Animal grouping and treatmentA total of 60 SPF(Specific pathogen free) SD male rats that were 28 days old, weighing 100–150g, License No: SCXK(Yu) 20100002. The rats were randomly divided into four different treatment groups(15 rats per group) by weight, which were normal saline control group, NaF treatment group, NAC treatment group and NAC + NaF treatment group. The corresponding treatment factor and dose were 0.9% physiological saline(10ml/kg), NaF(25mg/kg/d), NAC(150mg/kg/d) and NaF(25mg/kg/d) +NAC(150mg/kg/d), the exposure time is 7 weeks. 2. Detection of reproductive related indicatorsThe organ coefficients of testis and epididymis were calculated, rat sperm count, sperm motility ratio and sperm abnormality rate were detected by the computer-assisted sperm analysis system. ELISA method was adopted to detect serum testosterone content. The structure of testicular seminiferous tubule was observed under optical microscope by HE(Hematoxylin and Eosin) staining method. Detect the testis fluoride content by fluoride selection electrode. 3. Detection of oxidative stress and DNA damage related indicatorsMDA content was determined by using glucosinolates barbituric acid colorimetric method. Ammonium molybdate colorimetry was used to determine the CAT activity. SOD activity was detected by xanthine oxidase. Serum and testicular 8-OHdG contents were detected by ELISA method. Immunohistochemical technique(S-P method) was used to detect the expression of 8-OHdG in testes. 4. Detection of Nrf2/ARE pathway related signal moleculeFluorescence quantitative PCR and western blot method were used to determine Nrf2 and downstream factors NQO1 and HO-1 mRNA and protein expression in the testis, respectively. Results 1. The results of male reproductive function indexCompared with control group, testicular fluorine content was significantly elevated, body weight, testis and epididymis wet weight, sperm quantity and quality, serum testosterone levels were significantly reduced, organ coefficients of testis and epididymis didn’t show significant difference, the morphological structure of rat testis seminiferous tubule were destructed in NaF treatment group. Compared with NaF treatment group, testis fluorine content wasn’t significantly decreased, body weight, testis and epididymis wet weight, sperm quantity and quality, serum testosterone content was not significantly increased in NAC + NaF treatment group. Although, there was no significant difference, however the improvement trend could be seen. Moreover, NAC pretreatment significantly improve the morphological structure of rat testis seminiferous tubule compared with NaF treatment group. 2. The analysis results of oxidation and DNA damageCompared with control group, serum MDA content, serum and testicular 8-OHdG levels, the testicular 8-OHdG immune staining intensity were significantly elevated, SOD and CAT activity were significantly reduced in NaF treatment group. Immunoreaction positive products were mainly distributed in the cytoplasm, characterized by diffuse cytoplasmic brown-yellow or brown particles. Compared with NaF treatment group, serum MDA content, 8- OHDG levels and the intensity of 8-OHdG immune staining were significantly reduced, serum SOD and CAT activity didn’t show significant difference in NAC + NaF treatment group. 3. The results of Nrf2/ARE pathways expressionCompared with control group, the mRNA expression levels of Nrf2 and its downstream factors NQO1 and HO-1 in rat testis were significantly increased in NaF treatment group and NAC treatment group. Compared with NaF treatment group, the mRNA expression levels of Nrf2 in NAC + NaF treatment group were significantly reduced with no significant difference in HO-1 and NQO1. Compared with control group, the nucleus Nrf2 and cytoplasm HO-1 and NQO1 protein levels in rat testis were significantly increased with no significant difference in cytoplasm Nrf2 protein in NaF treatment group and NAC treatment group. Compared with NaF treatment group, Nrf2 in nucleus and cytoplasm and HO-1 protein levels were significantly decreased with no significant difference in NQO1 protein levels in NAC + NaF treatment group. ConclusionExcessive fluoride exposure could induce oxidative stress in rats, hence caused DNA damage and male reproductive damage, meanwhile, excessive fluoride exposure might activate Nrf2/ARE signal transduction pathway. NAC pretreatment could partly promote the expression of Nrf2/ARE pathway to resist fluorosis to some extent and eventually partly protect male reproductive function, nevertheless,NAC pretreatment could not completely restore the male reproductive dysfunction induced by excessive fluoride exposure.