Dose Effects And Molecular Mechanisms Of Cadmium Exposure Affecting The Biological Functions Of BTB In Male Prepubertal Rats

Cadmium（Cd）is a heavy metal environmental pollutant with biological toxicity.Human activities have contributed to the global spread of Cd pollution,and our country is one of the most Cd-contaminated countries in the world due to its developed manufacturing industry.Human exposure due to persistent Cd contamination increases health risks in populations,including young children,but there is a lack of mechanistic studies on this preadolescent health risk.Population studies have identified correlations between Cd exposure and human disease and have identified multiple organs affected by Cd exposure,including the male reproductive system,while there is a scientific gap in the mechanism by which Cd exposure affects the Blood-Testis Barrier（BTB）in prepubertal males.In this study,we used SD rats and Sertoli cell line TM4 to investigate the dosage effects and molecular mechanisms of Cd exposure affecting the biological function of BTB.This thesis includes three parts:1.Effect of Cd exposure on BTB in prepubertal male rats.To investigate the effects of Cd exposure on testicular structure,BTB integrity,tight junctions,adherens junctions,and the rictor/mTOR2 complex in prepubertal male rats.A Cd exposure model was established in prepubertal male rats using a single intraperitoneal injection of CdCl₂.Twenty-four SD male rats were randomly divided into a blank control group(0mg·kg^-1),low-dose group(0.5mg·kg^-1),medium-dose group(1.0mg·kg^-1),and high-dose group(2.0mg·kg^-1)according to the dose of Cd expousre,with 6 rats in each group.The morphological structure of testicular seminiferous tubules was observed by HE staining.The barrier function of BTB was observed by diffusion of small molecule fluorescent markers.The contents of tight junction key protein occludin,adhesion junction key protein E-cadherin,actin filament binding protein ZO-1,rictor,and the phosphorylation levels of mTOR and Akt were detected by Western blotting.The results showed that compared with the blank control group,the low-dose and medium-dose groups showed no obvious damage to the testicular morphological structure and BTB barrier function.However,the high-dose group showed obvious testicular structural damage,disorganized arrangement of constituent cells of the seminiferous epithelium,and the appearance of heterochromatic cells,accompanied by disruption of BTB barrier function,as evidenced by the diffusion of fluorescent markers from the interstitial space of the seminiferous tubules into the internal lumen.Cd exposure decreased the protein levels of occludin,E-cadherin,rictor,Phospho（Ser473）-Akt（p-Akt）and Phospho（Ser2448）-mTOR（p-mTOR）in the testis in a dose-dependent manner,with the high-dose group decreasing by 77.1%,65.1%,68.7%,81.3%,and 81.6%,respectively,when compared to the blank control group（all P<0.001）,accompanied by an abnormal distribution of ZO-1 in the periphery of the seminiferous epithelium.Collectively,Cd exposure can affect the biological function of BTB in prepubertal male rats in a dose-dependent manner,and this effect may be associated with abnormalities in BTB-associated connexin and the rictor/mTOR2 complex in the testis.2.The effect of Cd exposure on connexin and rictor/mTOR2 complex in Sertoli cell line.To investigate the effects of Cd exposure on Sertoli cell tight junctions,adherens junctions,the rictor/mTOR2 complex,and the relevance of these effects to intracellular adaptive responses,an in vitro Cd exposure model of Sertoli cell was established using a CdCl₂-treated TM4 cell line.The effect of CdCl₂ treatment on cellular activity was analyzed by the CCK8 method.The effect of Cd exposure on the protein level of occludin,E-cadherin,ZO-1,rictor,p-mTOR,and p-Akt was analyzed by Western blotting.The effect of Cd exposure on mRNA levels of genes involved in endoplasmic reticulum stress,mitochondrial unfolding protein response,mitochondrial dynamics,Nrf2-mediated antioxidant response,autophagy,and metallothionein expression,was analyzed by Real-time fluorescence quantitative PCR（q PCR）.The cellular ROS level and mitochondrial morphology were analyzed by Immunofluorescence staining.The intracellular MDA level was determined by TBA assay.The expression and distribution of p62 were detected by Cell immunofluorescence.The results showed that CdCl₂（0-15μM）reduced the cellular activity of TM4 in both dose-dependent and time-dependent manners.Treatment with CdCl₂（5μM）for 24h exerted no effects on the protein levels of ZO-1,occludin,E-cadherin,rictor,p-Akt,and p-mTOR,while the protein levels of these proteins were reduced by 20.5%（p<0.05）,49.2%,42.1%,57.1%,81.5%and 47.0%（all p<0.001）when compared to the blank control group.During the treatment of CdCl₂（1μM and5μM）for 24h,the transcripts of genes,which are intimately involved in endoplasmic reticulum stress（PERK,XBP1,CHOP,GRP）,mitochondrial unfolded protein responses（Sirt1,Sirt3,PGC1a,Tfam,NRf1,Htra2）,mitochondrial dynamics（Opa1,Mfn1,Mfn2,Fis1,Mff）,Nrf2-mediated anti-oxidative response（Nfe212,Homx1,Nqo1,Gsta1,Gclc,Gclm,Cat,Sod1,Sod2,Sod3）,autophagy（Ulk1,Ulk2,Wdr45,Atg13,Atg7,Nbr1）,and metallothionein（Mt1,Mt2）,showed dynamic changes.After treatment with CdCl₂（5μM）for 12h,CHOP,Homx1,Mt1,and Mt2 showed significant and persistent high expression,and their mRNA levels were up-regulated5.3-fold,125.8-fold,26.1-fold,and 404.0-fold（all p<0.001）,respectively,compared to the blank control group;while the expression of Mfn1,Sod2,Sod3,and Ulk2 was persistently suppressed,which decreased to 36.3%,55.0%,33.7%and 58.5%（all p<0.001）of the blank control group.However,except for metallothionein,the transcript levels of the remaining adaptive response genes tended to return to normal after 24h.CdCl₂（5μM）treatment for 12h also altered the morphology of mitochondria,showing a shift from a grid-like distribution within the cytoplasm to a dot-like distribution around the nucleus.The treatment also induced upregulation of p62 expression and a scattered distribution within the cytoplasm.Collectively,Cd exposure affected TM4 intracellular connexins and mTOR complexes as well as adaptive responses in a dose-dependent manner.Among them,low-dose Cd exposure failed to affect cellular connexins and the mTOR2 complex,which may be related to the general upregulation of Nrf2-mediated antioxidant response,high expression of metallothionein,and inhibition of autophagy.3.The role of oxidative stress-induced autophagy in Cd exposure affecting BTB.The objective of this part was to investigate the effects of oxidative stress and autophagy on BTB tight junctions,adherens junctions,and the rictor/mTOR2complex during Cd exposure.Based on the in vivo Cd exposure model of prepubertal male rats established in Part I,the intra-testicular MDA content was detected using the TBA assay.The protein level of LC3 in the testis was detected by Western blotting.Based on the in vitro Cd exposure model of the Sertoli cell established in the second part,the effect of Cd exposure on intracellular MDA content was analyzed using the TBA assay.Western blotting was used to determine the protein levels of HO1,p62,LC3,Atg5,Atg7,occludin,E-cadherin,ZO-1,rictor,and p-mTOR protein levels.The autophagic activity was determined by MDC assay,and the p62 was detected by cell immunofluorescence.The results showed that in in vivo experiments,Cd exposure increased the testicular content of MDA and the protein levels of LC3-Ⅰ/Ⅰin a dose-dependent manner,with the protein levels of IL3-Ⅰbeing upregulated 5.27-fold in the high-dose group compared to the blank control group（p<0.001）.In in vitro experiments,CdCl₂ increased the protein levels of HO1,MDA content,and ROS production in both a dose-dependent and time-dependent manner.After co-treatment with the antioxidant NAC（3 m M）and CdCl₂（10μM）for 24h,the protein levels of occludin,ZO-1,E-cadherin,rictor,p-Akt,and p-mTOR were increased 2.66-fold,1.32-fold（all p<0.05）,1.26-fold,3.66-fold,1.84-fold and 1.92-fold（all p<0.05）,respectively,compared to the CdCl₂-treated group.After treatment with CdCl₂（10μM）for 24h,the protein levels of LC3-II,p62,and Atg7 were upregulated 1.73-fold,3.01-fold（all p<0.001）and 1.31-fold（p<0.05）,respectively,compared to the blank control group,while enhancing autophagic activity.After co-treatment with the immunosuppressant chloroquine（50μM）and CdCl₂（10μM）,the protein levels of occludin,E-cadherin,rictor,and p-mTOR were increased 1.19-fold,2.20-fold,2.19-fold and 1.65-fold（all p<0.05）,respectively,compared to the CdCl₂-treated group.Collectively,high-dose Cd exposure can affect connexin and the rictor/mTOR2 complex through aberrant activation of oxidative stress-autophagy in the Sertoli cell.This mechanism is involved in the process by which high-dose Cd exposure impairs the biological function of prepubertal BTB.