Regulation Of Endoplasmic Reticulum Stress And Nrf2 Signaling Pathway In Cadmium-induced Toxic Effects In Rats

ObjectiveCadmium is an important production toxicant and environmental pollutant,and its toxicological mechanism has not been fully elucidated.Studies have shown that Nrf2 signaling factor acts as a central regulator of cellular antioxidant stress,which is activated by the action of the endoplasmic reticulum stress-related factor PERK,thereby initiating the antioxidant mechanism mediated by the Nrf2 signaling pathway.Other studies have indicated that activated Nrf2 can reduce the level of ERS response.However,in combination with endoplasmic reticulum stress and oxidative stress,the interaction mechanism between endoplasmic reticulum stress and Nrf2 signaling pathway in cadmium toxicity has not been reported at the animal level.Therefore,in this study,rat kidney,testis and ovary were used as target organs to investigate the effects of cadmium toxicity on oxidative stress,endoplasmic reticulum stress and Nrf2signaling pathway.Furthermore,the regulation of endoplasmic reticulum stress on Nrf2 signaling pathway was explored by up-regulating and down-regulating endoplasmic reticulum stress levels.The feedback regulation of Nrf2 on endoplasmic reticulum stress was explored by up-regulating and down-regulating the expression of Nrf2 signaling factors.Method120 SD rats,60 males and 60 females were selected.After quarantine,they were randomly divided into 20 groups according to body weight,with6 rats in each group.Group 1:blank control group;group 2-4:low,medium and high dose groups of CdCl₂;group 5:bacitracin reagent group;group 6-8:bacitracin+CdCl₂ low,medium and high dose groups;Group 9:TUDCA reagent group;Groups 10-12:TUDCA+CdCl₂ low,medium and high dose groups;Group 13:tBHQ reagent group;Groups 14-16:tBHQ+CdCl₂ low,medium and high dose groups;Group 17:luteolin reagent group;Group 18-20:luteolin+CdCl₂ low,medium and high dose groups.The doses of CdCl₂ were:5μmol/kg,10μmol/kg,20μmol/kg.Each group was exposed to 10 ml/kg body weight by intraperitoneal injection.The animals were sacrificed after 48h.The kidney,ovary and testis were dissected to determine the activity of SOD and GSH-Px,MDA content.mRNA and protein expression levels of ERS-related factors and Nrf2signaling pathway-related factors were determined using RT-PCR and Western blot.Results1.Oxidative stress results:After 48h treatment with cadmium,the activities of antioxidant enzymes SOD and GSH-Px in kidneys of rats decreased significantly at doses of 10 and 20μmol/kg Cd,MDA content was increased significantly.The activity of SOD and GSH-Px in testis and ovaries of rats decreased significantly at doses of 5 and 10μmol/kg,and MDA content increased significantly.2.ERS results:After treatment with cadmium at 5,10,20μmol/kg for48h,the mRNA expression levels of Bip,PERK,ATF4,and the protein expression of Bip and PERK in kidneys of rats were significantly increased.At high doses of cadmium（10,20μmol/kg）,the mRNA expression of Bip,PERK,ATF4,and the protein expression of Bip and PERK in testis and ovaries of rats were up-regulated significantly,especially at high doses.3.Nrf2 signaling pathway results:under the condition of 10μmol/kg dose,the expression of Nrf2 mRNA and protein in kidneys of rats increased significantly,and the expression of GST-P1 and GCLC mRNA also increased significantly.The expression level of Nrf2 protein was significantly decreased at high dose of cadmium.At 10,20μmol/kg doses,cadmium significantly up-regulated the expression of Nrf2 mRNA and protein in testis and ovaries of rats.At 10 and 20μmol/kg doses,the expression of GCLC and GST-P1 genes in rat testis of rats were significantly up-regulated,while rat ovary GCLC and GST-P1 were up-regulated at 20μmol/kg dose.The mRNA expression of GST-P1 and GCLC in ovaries of rats were significantly decreased at 5 and 10μmol/kg doses,respectively.4.Results of the regulation of Nrf2 signaling factors after ERS level up-regulation and down-regulation:at 20μmol/kg dose,the expression of PERK protein in baicalin+Cd group in kidneys of rats was significantly higher than that in the same-dose Cd treatment group.At this dose,the expression level of Nrf2 mRNA in baicalin+Cd group also increased significantly.At 5 and 10μmol/kg doses,the expression of PERK protein in TUDCA+Cd group in kidneys of rats was significantly lower than that in the same-dose Cd treatment group.However,the expression of Nrf2mRNA was not significantly changed at this dose.After treatment with bacitracin,the expression levels of Bip mRNA,PERK mRNA and PERK protein in testis and ovaries of rats were significantly higher than those in the same-dose Cd treatment group at 5μmol/kg dose.At this dose,the expression level of Nrf2 mRNA in testis and ovaries of rats did not increase.At 10μmol/kg dose,the expression levels of Bip protein,PERK mRNA and PERK protein in testis and ovaries of rats were significantly lower than those in the same-dose Cd treatment group.5.Results of the up-regulation and down-regulation of Nrf2 on the feedback regulation of cadmium-induced ERS:at 20μmol/kg dose,the expression of Nrf2 protein in tBHQ+Cd group in the kidneys of rats was significantly higher than that in the same-dose Cd treatment group.At this dose,the Bip mRNA and protein in tBHQ+Cd group in the kidneys of rats were significantly decreased.At the dose of 5μmol/kg,the expression of Nrf2 protein in luteolin+Cd group in the kidneys of rats was significantly lower than that in the same-dose Cd treatment group.At this dose,the expression levels of Bip protein and PERK mRNA in the kidneys of rats were also significantly decreased.At 10μmol/kg dose,the expression of Nrf2 protein in tBHQ+Cd group in the testis of rats was significantly higher than that in the same-dose Cd treatment group.At this dose,the ERS level in the testis of rats was not changed obviously.At the dose of20μmol/kg,the expression of Nrf2 protein in tBHQ+Cd group in the ovaries of rats was significantly higher than that in the same-dose Cd treatment group.At this dose,the expression level of Bip mRNA and protein,PERK mRNA in ovaries of rats were decreased significantly.At 5μmol/kg dose,the expression of Nrf2 mRNA and protein in luteolin+Cd group in the testis of rats decreased significantly,and the expression levels of Bip mRNA and PERK protein were decreased significantly.At 10 and20μmol/kg doses,the expression of Nrf2 mRNA and protein in luteolin+Cd group in the ovaries of rats was significantly lower than that in the same-dose Cd treatment group,but at this high dose,the expression levels of Bip protein and PERK protein in ovaries of rats also decreased significantly.Conclusion1.Under a certain doses,cadmium can induces OS and ERS in kidneys of rats,testis and ovaries,activate Nrf2,up-regulate the expression of phase II detoxification enzymes,and initiate Nrf2-mediated antioxidant mechanism.2.In the toxic effect of cadmum,there was a positive correlation between the expression of PERK and the expression of Nrf2 in kidneys,testis and ovaries of rats.The positive correlation between PERK and Nrf2in kidneys of rats was particularly obvious.3.Under these experimental conditions,the testis and ovaries of rats may be more sensitive to cadmium toxicity than the kidneys of rats,while the ovaries of rats may be more susceptible to cadmium toxicity than the testis of rats.4.In the process of cadmium toxicity,cadmium-induced ERS in kidneys of rats has a positive regulation effect on Nrf2,but has no obvious negative regulation effect on Nrf2.The increase of Nrf2 expression in kidneys of rats may attenuate ERS levels to a certain extent,but inhibition of Nrf2 expression does not feedbackly enhance ERS levels.5.Cadmium-induced ERS in testis and ovaries of rats had no significant positive regulation of Nrf2,but has significant negative regulation effect on Nrf2.The increase of Nrf2 expression in ovaries of rats can attenuate ERS levels,while the effect in testis of rats was not obvious.But the decrease of Nrf2 expression in testis of rats could increase the ERS levels to a certain extent,and the effect in ovaries of rats was not significant.