The Effect Of T-2 Toxin On Mice Testis Inflammatory Injury And The Regulatory Mechanism Of ROS/NLRP3 Inflammasome Activation

T-2 toxin can damage male reproductive function,reduce male mice fertility,but the mechanism is still unclear.Testicular inflammatory injury is the core mechanism of male reproductive dysfunction.ROS-mediated NLRP3 inflammasome activation plays a key role in regulating inflammatory response.However,it is unclear whether T-2 toxin causes male reproductive dysfunction via activating ROS-mediated NLRP3 inflammasome activation and resulting testicular inflammatory damage.Based on the relationship between testicular inflammatory injury,ROS and NLRP3 inflammasome activation,take"T-2 toxin causes inflammatory testicular damage and is regulated by ROS-mediated NLRP3 inflammasome activation"as the theoretical hypothesis and research entry point.The following three researches were performed.（1）Firstly,80 male Kunming mice,randomly divided into four groups,were orally administered 0（control group,CG）,0.5（low dose group,LG）,1（middle dose group,MG）,and 2 mg/kg B.W.（high dose group,HG）of T-2 toxin,the experiment period was 28 d,to establish subacute T-2 toxin intoxication mice model.Detect male mouse fertility,testicular structure（microstructure,ultrastructure,apoptosis and blood-testis-barrier（BTB））,testicular function（spermatogenesis and testosterone synthesis）,testicular inflammatory response,testicular ROS content and the key factor expression of NLRP3 inflammasome,to explore the effect of T-2 toxin on testicular injury,testicular inflammatory response and ROS-mediated NLRP3 inflammasome activation.（2）Then,TM4 cells（mice Sertoli cell line）were used as the test subject.The half-inhibitory concentration(IC₅₀)of T-2 toxin on TM4 cells was measured to determine the appropriate dose of T-2 toxin for subsequent cytotoxicity experiments.TM4 cells were treated with T-2 toxin at a final concentration of 0,2(1/4 IC₅₀),4(1/2 IC₅₀)or 6 nM(3/4 IC₅₀)to establish a T-2 toxin poisoning cell model.The cell activity,apoptosis rate,function markers（Ocln,ZO-1,Cx-43 and N-cad）expression of TM4 cell,inflammatory response,ROS content and the key factor expression of NLRP3 inflammasome were detected,to determine the dose-dependent effect among T-2 toxin,TM4 cells inflammatory injury and ROS-mediated NLRP3 inflammasome activation.Meanwhile,the appropriate dose of T-2 toxin was screened out for subsequent intervention experiments.（3）Finally,according to cell viability test,the appropriate intervention dose of ROS scavenger（NAC）and NLRP3 inflammasome activation inhibitor（MCC950）was defined.To verify the role of ROS-mediated NLRP3 inflammasome activation on T-2 toxin-induced inflammatory damage in TM4 cells,this intervention experiment chooses 5 mM NAC or 20 nM MCC950 combined with T-2 toxin（4 nM）to intervene TM4 cells.Comprehensive analysis the results of in vivo and in vitro tests to screen out therapeutic targets for male reproductive disorders caused by T-2 toxin.The experimental results are as follows:（1）The results of male mice treated with T-2 toxin are as follows:1)Male mice fertility:The mice body weight in toxin-treated groups were significantly lower than CG（P<0.05;P<0.01）;Compared with CG,the mating index and breeding index of HG mice were significantly reduced（P<0.05）;The embryo implantation and live births of female mice mated with MG and HG mice were significantly lower than CG（P<0.01）,the number of female mice with fetal resorptions significantly increased（P<0.05;P<0.01）,indicating T-2 toxin could inhibit mice growth and destroy male mice fertility.2)Testicular structure:the testis weight and volume of HG were significantly lower than CG（P<0.01）;T-2 toxin-treated groups damage the testis microstructure and ultrastructure.TUNEL staining showed that T-2 toxin exposure caused apoptosis of spermatogenic cells,Leydig cells and Sertoli cells in testicular tissue;The expression of BTB related proteins（Ocln,Cx-43,N-Cad and ZO-1）inMG and HG were significantly lower than those of CG（P<0.05;P<0.01）,indicating that T-2 toxin could cause BTB destruction and testicular structural damage.3)Testicular function:the daily sperm production and sperm density of MG and HG were significantly lower than those of CG（P<0.01）;the sperm deformity rate was significantly higher than CG（P<0.05,P<0.01）;sperm ultrastructure was destroyed in T-2 toxin-treated groups,indicating that T-2 toxin causes spermatogenesis disorders.The serum testosterone content of T-2toxin-treated groups were significantly lower than CG（P<0.05;P<0.01）;The expression of testosterone synthase（St AR,P450scc,P450c17,3β-HSD and 17β-HSD）inMG and HG was significantly lower than that of CG（P<0.05;P<0.01）,suggesting that T-2 toxin could cause testosterone synthesis disorder in mice.4)Testicular inflammation:the content of serum IL-1β,IL-6,IL-18 and TNF-αin T-2toxin-treated groups were significantly higher than those of CG,and the content of IL-10 in T-2toxin-treated groups was significantly lower than that of CG（P<0.05;P<0.01）;The content and m RNA expression of IL-6 and TNF-αin testes of T-2 toxin-treated groups were significantly higher than those of CG,and the content and m RNA expression of IL-10 were significantly lower than those of CG（P<0.05;P<0.01）,indicating that T-2 toxin could cause testicular inflammation.5)NLRP3 inflammasome activation:in testis,the expression of inflammasome key factors（NLRP3,ASC,Caspase-1,IL-1βand IL-18）,and the content of IL-1βand IL-18 inMG and HG were significantly higher than CG（P<0.05;P<0.01）,indicating T-2 toxin could activate NLRP3inflammasome in testis.6)ROS accumulation:the content of ROS and MDA inMG and HG was significantly higher than that of CG（P<0.01）,while the activity of CAT and SOD was significantly lower than that of CG（P<0.01）,indicating that T-2 toxin could cause ROS accumulation and oxidative stress in testis.（2）The results of TM4 cells treated with T-2 toxin are as follows:1)The IC₅₀ of T-2 toxin on TM4 cells is 8.10 nM.2)In 4 nM and 6 nM groups,cell activity and the expression of cell markers（Ocln,ZO-1,Cx-43 and N-cad）were significantly lower than those in the 0 nM group（P<0.05;P<0.01）,indicating that T-2 toxin could disrupt TM4 cell function.3)In the T-2 toxin-treated groups,TM4 cells apoptosis rate was significantly higher than that in the 0 nM group（P<0.01）,indicating that T-2 toxin induced TM4 cell apoptosis.4)In 4 nM and 6 nM groups,the content of IL-6 and TNF-αin cell culture supernatant was significantly higher than that in 0 nM group,and IL-10 content was significantly lower than that in0 nM group（P<0.05;P<0.01）,indicating T-2 toxin could induce inflammatory response in TM4cells.5)In 4 nM and 6 nM groups,the expression of NLRP3,ASC,Caspase-1,IL-1βand IL-18 in TM4 cells,IL-1βand IL-18 content in culture supernatant were significantly increased compared0 nM group（P<0.05;P<0.01）,indicating that T-2 toxin could activate NLRP3 inflammasome in TM4 cells6)In T-2 toxin-treated groups,ROS and MDA content in TM4 cells were significantly higher than that of 0 nM group,and the SOD and CAT activities were significantly lower than that of 0nM group（P<0.05;P<0.01）,indicating that T-2 toxin could cause ROS accumulation and oxidative stress in TM4 cells.（3）NAC and MCC950 combined with T-2 toxin to treat TM4 cell test results are as follows:1)5 mM NAC or 20 nM MCC950 significantly alleviated the decreasing of TM4 cell activity,TM4 cell marker factor（Ocln,ZO-1,Cx-43 and N-cad）m RNA expression and apoptosis caused by T-2 toxin（P<0.05;P<0.01）,indicating that ROS intervention or NLRP3 inflammasome activation intervention could alleviate the toxic effect of T-2 toxin on TM4 cells.2)5 mM NAC or 20 nM MCC950 significantly alleviated the increasing of IL-6 and TNF-αcontent and the decreasing of IL-10 content in the culture supernatant caused by T-2 toxin（P<0.05;P<0.01）,indicating that ROS intervention or NLRP3 inflammasome activation intervention could alleviate the inflammatory response of TM4 cells caused by T-2 toxin.3)5 mM NAC or 20 nM MCC950 significantly alleviated the increasing of NLRP3,ASC,Caspase-1,IL-1βand IL-18 expression in TM4 cells,IL-1βand IL-18 content in culture supernatant caused by T-2 toxin（P<0.05;P<0.01）,indicating that ROS intervention or NLRP3inflammasome activation intervention could alleviate NLRP3 inflammasome activation caused by T-2 toxin.4)5 mM NAC significantly alleviated ROS accumulation caused by T-2 toxin,indicating that ROS intervention could alleviate ROS accumulation caused by T-2 toxin.However,20 nM MCC950 had no significant effect on ROS accumulation,indicating that ROS-mediated NLRP3inflammasome activation.In summary,T-2 toxin caused testis and TM4 cells inflammatory injury by ROS-mediated NLRP3 inflammasome activation.Testicular inflammatory injury is manifested by testis structure damage（microstructure,ultrastructure,BTB damage and apoptosis）and functional disorders（spermatogenesis and testosterone synthesis）;TM4 inflammatory injury is manifested by the decreasing of cell activity and functional markers expression,and apoptosis.