Effects Of N-Hexane On Nrf2 Signal Pathway And Estrogen Receptor In Female Rats And Intervention Of Lycium Barbarum Polysaccharides

ObjectiveTo explore the effects of n-hexane toxicity on Nrf2 signal pathway,sex hormone level and estrogen receptor in female rats,and the intervention effect of Lycium barbarum polysaccharide(LBP)on n-hexane,so as to reveal the reproductive toxicological mechanism of n-hexane and the reproductive protective effect of LBP.Method48 Sprague Dawley(SD)female rats were randomly divided into 8groups according to weight: control group,and low,middle and high dose(675,1350,2700 mg/kg)n-hexane groups,50 mg/kg LBP group,and LBP+low,middle and high dose n-hexane groups.On the first day,n-hexane groups and LBP+n-hexane groups were intraperitoneally injected with 675,1350,2700 mg/kg n-hexane(10 mg/kg)solution once,control group and LBP group were intraperitoneally injected with physical saline(10 mg/kg).From day 2 to day 4,control group and n-hexane groups were given physical saline [10 mg/(kg�day)] by gavage,LBP group and LBP+n-hexane groups were given LBP(50 mg/kg)solution [10mg/(kg�day)] by gavage.On day 5,the animals were sacrificed to obtain the Serum and ovaries.Superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content were detected by hydroxylamine method,enzymatic reaction method and thiobarbituric acid method,respectively.The expression of estradiol(E2),follicle stimulating hormone(FSH),luteinizing hormone(LH),progesterone(PROG)were measured by enzyme linked immunosorbent assay,the mRNA expression of Nrf2,glutamate cysteine ligase catalytic subunit(GCLC),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1),estrogen receptor ?(ER?),estrogen receptor ?(ER?)and G protein coupled estrogen receptor 1(GPER1)were measured by real-time PCR and Western Blotting was applied to measure the protein expression of Nrf2,ER?,ER? and GPER1.Results(1)Effects of n-hexane and LBP combined with n-hexane on oxidative stress in rats: In the serum of rats,the activity of SOD was significantly decreased with low dose n-hexane(P<0.05),the activities of SOD and GSH-Px were significantly decreased,but the content of MDA was significantly increased with middle and high dose n-hexane(P<0.05).In the ovaries of rats,the activity of GSH-Px was significantly decreased with low dose n-hexane(P<0.05),the activities of SOD and GSH-Px were significantly decreased with middle and high dose n-hexane(P<0.05),but the content of MDA was significantly increased with high dose n-hexane(P<0.05).And the decrease of GSH-Px activity and the increase of MDA content in serum were more significant,the oxidative stress was more serious.Compared with the same dose groups without adding LBP,in the serum of rats,the activity of GSH-Px was significantly increased with middle dose n-hexane+LBP(P<0.05),the activity of SOD was significantly increased with high dose n-hexane+LBP(P<0.05);In the ovaries of rats,the activity of SOD was significantly increased with high dose n-hexane+LBP(P<0.05).In LBP group,there was no significant change in the activity of SOD,GSH-Px and the content of MDA in serum and ovary.(2)Effects of n-hexane and LBP combined with n-hexane on Nrf2 signal pathway in rats: The mRNA expression of Nrf2,HO-1,NQO1,GCLC and the protein expression level of Nrf2 were significantly increased in the middle and high dose n-hexane groups(P<0.05).Compared with the same dose groups without adding LBP,the mRNA expression level of Nrf2,HO-1,NQO1,GCLC and the protein expression of Nrf2 were significantly decreased in the middle and high dose n-hexane+LBP groups(P<0.05),but the expression level of Nrf2 protein and HO-1,NQO1,GCLC gene were significantly increased in LBP group(P<0.05).Not only in the n-hexane groups,but also in the LBP+ n-hexane groups,there was a high correlation between the transcription level of HO-1,NQO1,GCLC gene and the expression level of Nrf2 gene and protein.(3)Effects of n-hexane and LBP combined with n-hexane on sex hormone levels in rats: The expression of FSH,E2 and PROG in serum of rats increased at first and then decreased in high dose n-hexane group,the expression of LH decreased at first and then increased in medium dose n-hexane group,but there were no significant difference.There was a correlation between the expression of FSH,E2 and PROG and the dose of n-hexane,which increased at first and then decreased with the increase of the dose,and the level was the lowest at the high dose(from the control group to the middle dose group: the correlation coefficients of E2 and PROG were 0.376 and 0.407,P<0.05;and from the middle dose group to the high dose group: the correlation coefficients of FSH and PROG were-0.484 and-0.484,P<0.05).Compared with the same dose groups without adding LBP,the expression of LH in middle dose n-hexane+LBP group and the expression of FSH and PROG in high dose n-hexane+LBP group were up-regulated.(4)Effects of n-hexane and LBP combined with n-hexane on estrogen receptor in rats: The protein expression of ER?,GPER1 and the mRNA expression of ER? were significantly increased but the protein expression of ER? and the mRNA expression of ER?,GPER1 were significantly decreased in midde and high dose n-hexane groups(P<0.05).Compared with the same dose groups without adding LBP,the protein expression of ER? and GPER1 were significantly decreased in low dose n-hexane+LBP group(P<0.05),the expression of ER? protein was significantly increased in middle dose n-hexane+LBP group(P<0.05),and the expression of GPER1 mRNA and protein were significantly decreased in the high dose n-hexane group(P<0.05).Correlation analysis showed that the expression of Nrf2 protein was positively correlated with the expression of ER? and GPER1 protein,but negatively correlated with the expression of ER? protein under the toxicity of n-hexane(P<0.05).And there was a low positive correlation between the expression of Nrf2 protein and the expression of GPER1 protein in n-hexane+LBP group(P<0.05).Conclusion(1)A certain exposure dose(1350,2700 mg/kg)of n-hexane can induce the oxidative stress in female rats,activate Nrf2 signaling pathway,and up-regulate the expression of downstream phase? detoxification enzymes.LBP can alleviate the n-hexane-mediated oxidative stress and antagonize the activation of Nrf2 and downstream phase? detoxification enzymes.(2)Under the experimental conditions,n-hexane exposure did not interfere with estrogen secretion,but the expression of FSH,E2 and PROG were related to the dose of n-hexane.LBP can alleviate the down-regulation of FSH and PROG induced by high dose of n-hexane and protect the hypothalamus-pituitary-ovary axis to a certain extent.(3)A certain dose(1350,2700 mg/kg)of n-hexane can lead to disorder of ovarian and reproductive system by up-regulating the expression of estrogen receptor transcription factors ER? and GPER1 and down-regulating the expression of ER?.LBP plays a role in reproductive protection by affecting the sensitivity of GPER1 and alleviating its overexpression.(4)Under n-hexane-induced oxidative stress,the up-regulated expression of ER? and GPER1 regulates the activation of Nrf2 signal pathway.LBP may play an antioxidant role by down-regulating the expression of GPER1,resulting in the decrease of the expression of Nrf2 signaling factors.LBP can activate Nrf2 signal pathway and regulate the expression of ER?,ER? and GPER1.