Effect Of Long-term Exposure To Microcystin-LR On Blood-testis Barrier In Mice And The Potential Mechanism

ObjectiveMicrocystin-leucine arginine(MC-LR)is a common water environmental pollutant with potential toxicity to male reproductive system.The aim of the study is to investigate the effects of chronic MC-LR exposure to male reproductive system by treating male C57BL/6 mice and the role of Rho A/ROCK pathway-mediated cytoskeleton rearrangement in MC-LR-induced damage of blood-testis barrier(BTB).This study will further clarify the potential male reproductive toxicity and mechanism of chronic MC-LR exposure and provide theoretical basis for the prevention and treatment of male reproductive system damage and diseases caused by MC-LR.Methods1.The effects of chronic exposure to MC-LR on testicular histomorphology and BTB in mice were evaluated.In order to explore the effect of chronic exposure of MC-LR on the BTB,male C57BL/6 mice were randomly divided into 6 groups and exposed to different concentrations of MC-LR(0,1,30,60,90,120 μg/L)by drinking water.After exposure to MC-LR for 6,9 and 12 months,the mice were executed for testis tissue.Western blot was used to detect the MC-LR content in mouse testis.Hematoxylin and eosin(H&E)staining was used to observe the histopathological changes of testis.RT-q PCR was used to detect the m RNA expression of cell junction-related genes in mouse testis;Western blot and mmunohistochemistry was used to detect the expression of component proteins of BTB and the localization of adhesion junction-associated proteins β-Catenin and N-Cadherin.2.The changes of Rho A/ROCK pathway and the cytoskeleton after chronic MC-LR exposure were observed.Immunofluorescence staining was used to observe the distribution and expression of cytoskeletal proteins.The level of active-Rho A and the phosphorylated ROCK and in the testis of mice after chronic MC-LR exposure were detected by immunohistochemistry and Western blot.3.The effects of MC-LR and Rhosin on TM4 cell activity were evaluated,and the subsequent experimental concentrations were determined.TM4 cells were used as the model.After TM4 cells were exposed to MC-LR at different concentrations(0,0.1,0.5,1,5,10,20,30 μg/m L)and Rhosin at different concentrations(0,20,30,40,50,60,70,80 μM)for 24 h,the cell activity was detected by CCK-8 kit.Then,the subsequent experimental concentrations of MC-LR and Rhosin were determined.4.The role of Rho A/ROCK pathway-mediated cytoskeleton reorganization of TM4 cells in MC-LR-induced cell junction damage was clarified.After TM4 cells were exposed to MC-LR at different concentrations(0,1,5,10,15,20 μg/m L)with or without the pretreatment of Rhosin for 24 h,pull-down assay was performed to detect the active-Rho A and Western blot was used to detect the phosphorylation of ROCK.The cytoskeletal proteins(F-actin and α-Tubulin)were observed by phalloidin and immunofluorescence staining.The m RNA expression of adhesion junction-related genes was detected by RT-q PCR;the expression of component protein of BTB was detected by Western blot.Results1.Chronic MC-LR exposure induced pathological changes of testis and destruction of BTB in mice.With the increase of MC-LR exposure concentration and time,MC-LR was accumulated in mouse testis.H&E staining showed that the histopathological damage of testis in mice was gradually aggravated,including the enlargement of seminiferous tubule space,loose and disordered cell arrangement,exfoliated Sertoli cells and germ cells.Chronic exposure to MC-LR destroyed cell junctions in testis and decreased the expression of adhesion junction-related genes(N-cadherin,nectin2,α-catenin andβ-catenin).Meanwhile,the expression of component protein of BTB(ZO-1,Occludin,β-Catenin,N-Cadherin)were decreased with the increase of MC-LR concentration.2.Chronic MC-LR exposure can activate Rho A/ROCK pathway and induce abnormal cytoskeleton assembly.MC-LR exposure can up-regulate the relative expression of active Rho A and phosphorylated ROCK in a dose-dependent and time-dependent manner.After MC-LR exposure,the distribution of F-actin in testicular tissue changed from regular radial distribution to irregular distribution.The expression of α-tubulin decreased with the increase of MC-LR concentration.3.With the increase of MC-LR and Rhosin exposure concentration,TM4 cell activity decreased gradually.After exposed to MC-LR for 24 h,TM4 cell activity gradually decreased with the increase of MC-LR concentration.The half inhibitory concentration of MC-LR on TM4 cells was calculated to be 20 μg/m L.MC-LR concentration was selected to be10,7.5,5,2.5,1,0 μg/m L in subsequent experiments.After TM4 cells were treated with Rhosin for 24 h,the cell activity began to decrease significantly when the concentration reached 40 μM(p<0.05).In the follow-up experiment,30 μM was selected as the intervention dose.4.Rho A/ROCK pathway-mediated cytoskeleton reorganization plays a role in MC-LR-induced ell junction disruption in TM4 cells.With the increase of MC-LR concentration,the expression of component proteins of BTB(ZO-1,occludin,β-Catenin,N-Cadherin)in TM4 cells decreased.MC-LR down-regulated the expression of F-actin and α-tubulin and result in structure destruction of cytoskeleton in TM4 cells.Meanwhile,MC-LR dose-dependently activated Rho A and phosphorylated ROCK(p<0.05).With the pretreatment of Rhosin,the activation of Rho A pathway in TM4 cells induced by MC-LR was inhibited(p<0.05).Meanwhile,the destruction of cytoskeleton was alleviated.MC-LR-induced the down-regulation of proteins related to tight junction and adhesion junction was alleviated by Rhosin(p<0.05).Conclusions1.Chronic oral MC-LR exposure can cause damage to testicular cytoskeleton and blood-testis barrier in C57BL/6 mice.2.The Rho A/ROCK pathway-mediated cytoskeleton destruction participates in MC-LR-induced damage of BTB.