Effects Of T-2 Toxin On Bone Formation And The Regulatory Mechanism Of Parkin-mediated Mitophagy

T-2 toxin is a type A trichothecene mycotoxin produced by the Fusarium genus of fungi,which mainly accumulates in bone tissue and leads to bone development disorders after entering the body.The inhibition of bone formation is the main pathological basis.Mitochondrial damage and apoptosis in osteoblasts are the core mechanisms leading to bone formation disorders.Parkin-mediated mitophagy can relieve osteoblast injury and promote bone formation by clearing damaged mitochondria,alleviating apoptosis,and regulating the Wnt pathway.But the effect of T-2 toxin on bone formation and its mechanism are still unclear.Therefore,this study hypothesizes that"T-2toxin inhibits bone formation and is regulated by Parkin-mediated mitophagy."The following research was conducted:（1）Firstly,4-week-old male wild-type C57BL/6N mice were orally administered with 0,0.5,1,and 2 mg/kg B.W.T-2 toxin for 28 d.The bone formation,apoptosis,Wnt pathway,and mitophagy key indicators were detected to clarify the effect and dose-effect relationship of T-2 toxin on bone formation and mitophagy,as well as screen the optimal dose for subsequent Parkin^-/-mouse experiments.（2）Mouse embryonic osteoblasts（MC3T3-E1）were used to determine the half-maximal inhibitory concentration(IC₅₀)of T-2 toxin on cells.Then,0,2(1/20IC₅₀),4(1/10 IC₅₀)and 8 n M(1/5 IC₅₀)T-2 toxin were added based on the IC₅₀,and the cells were cultured for 24 h.The bone formation,apoptosis,Wnt pathway,and mitophagy key indicators were detected to clarify the effect and dose-effect relationship of T-2 toxin on bone formation and mitophagy in MC3T3-E1 cells,as well as screen the optimal dose for subsequent siParkin cell experiments.（3）Normal and siParkin MC3T3-E1 cells were used,and the cells were divided into blank control group,T-2 toxin poisoning group,siParkin transfection group,and siParkin control group.The cells were cultured with 0 or 4 n M T-2 toxin for 24 h.The mitophagy,apoptosis,Wnt pathway,and the expressions of key factors related to bone formation were detected to clarify the role of Parkin-mediated mitophagy in T-2 toxin-induced bone formation disorders in MC3T3-E1cells.（4）Wild-type and Parkin^-/-C57BL/6N mice were used,and they were divided into wild-type control group,wild-type poisoning group,Parkin^-/-poisoning group,and Parkin^-/-control group.The mice were administered with 0 or 1 mg/kg B.W.T-2 toxin for 28 d.The mitophagy,apoptosis,Wnt pathway,and the expressions of key factors related to bone formation in the femur were detected to clarify the role of Parkin-mediated mitophagy in T-2 toxin-induced bone formation disorders.The experimental results are as follows:（1）Results of T-2 toxin poisoning experiments in mice1)After T-2 toxin treatment,mice showed varying degrees of depression and delayed reaction,and their body weight decreased,indicating that T-2 toxin inhibited mouse growth and development.2)After T-2 toxin treatment,the density and length of the femur were decreased,and the femur showed varying degrees of reduction and disorder of bone trabeculae,reduction of bone cells and fat degeneration,indicating that T-2 toxin caused structural damage to the femur.3)After T-2 toxin treatment,the number of alkaline phosphatase（ALP）positive cells was decreased,and the protein expressions of osteocalcin（OCN）and Osterix（OSX）were decreased,indicating that T-2 toxin inhibited bone formation in mouse femur.4)After T-2 toxin treatment,the rate of apoptosis was increased,the protein expression of Bcl-2 was decreased,the protein expression of Bax and the activities of Caspase 3 and Caspase 9 were increased,indicating that T-2 toxin caused apoptosis in mouse femur.5)After T-2 toxin treatment,the protein expressions of Wnt andβ-catenin were decreased,the protein expression of GSK-3βwas increased,and the expressions of Wnt pathway target genes were decreased,indicating that T-2 toxin inhibited Wnt pathway in mouse femur.6)After T-2 toxin treatment,the mitochondrial membrane potential（MMP）and content of triphosadenine（ATP）were decreased,and the content of reactive oxygen（ROS）was increased,indicating that T-2 toxin caused mitochondrial damage in mouse femur.7)After T-2 toxin treatment,the protein expressions of Parkin,LC3-II and p62 were increased,indicating that T-2 toxin activated mitophagy in mouse femur.（2）Results of T-2 toxin poisoning experiments in MC3T3-E1 cells1)The IC₅₀ of T-2 toxin for MC3T3-E1 cells is 41.8 n M.2)After T-2 toxin treatment,the cells showed varying degrees of reduction in number and decreased vitality,indicating that T-2 toxin caused cell damage.3)After T-2 toxin treatment,the contents of ALP and mineralized nodules were decreased;the protein expressions of OCN and OSX were decreased,indicating that T-2 toxin inhibited the osteogenic function of the cells.4)After T-2 toxin treatment,the rate of apoptosis was increased,the protein expression of Bcl-2 was decreased,and the protein expression of Bax,as well as the activities of Caspase 3 and Caspase 9 were increased,indicating that T-2 toxin caused apoptosis.5)After T-2 toxin treatment,the protein expressions of Wnt andβ-catenin were decreased,the protein expression of GSK-3βwas increased,and the expressions of Wnt pathway target genes were decreased,indicating that T-2 toxin inhibited Wnt pathway.6)After T-2 toxin treatment,the MMP and content of ATP were decreased and the content of ROS was increased,indicating that T-2 toxin caused mitochondrial damage.7)After T-2 toxin treatment,the protein expressions of Parkin,LC3-II,and p62 were increased,indicating that T-2 toxin activated mitophagy.（3）Results of T-2 toxin treatment in siParkin MC3T3-E1 cells1)After Parkin silencing,the protein expressions of Parkin and LC3-II were decreased and the protein expression of p62 was increased in T-2 toxin treated cells,indicating that siParkin inhibits mitophagy.2)After Parkin silencing,the MMP and content of ATP were decreased and the content of ROS was increased in T-2 toxin treated cells,indicating that siParkin exacerbates T-2 toxin-induced mitochondrial damage in cells.3)After Parkin silencing,the rate of apoptosis was increased,the protein expression of Bcl-2 was decreased,and the protein expression of Bax,as well as activities of Caspase 3 and Caspase 9 were increased in T-2 toxin treated cells,indicating that siParkin exacerbates T-2 toxin-induced apoptosis.4)After Parkin silencing,the protein expressions of Wnt andβ-catenin were decreased and the protein expression of GSK-3βwas increased in T-2toxin treated cells indicating that siParkin exacerbates Wnt pathway inhibition induced by T-2 toxin.5)After Parkin silencing,the cell viability was decreased,the contents of ALP and mineralization nodules were decreased,and the protein expressions of OCN and OSX were decreased in T-2 toxin treated cells,indicating that siParkin exacerbates T-2 toxin-induced inhibition of osteogenic function.（4）Results of T-2 toxin treatment in Parkin^-/-mice experiment1)After Parkin knockout,the protein expressions of Parkin and LC3-II were decreased and the protein expression of p62 was increased in T-2 toxin treated mouse femur,indicating that Parkin^-/-inhibits mitophagy.2)After Parkin knockout,the MMP and content of ATP were decreased and the content of ROS was increased in T-2 toxin treated mouse femur,indicating that Parkin^-/-exacerbates mitochondrial damage caused by T-2 toxin.3)After Parkin knockout,the rate of apoptosis was increased,the protein expression of Bcl-2 was decreased,and the protein expression of Bax,as well as activities of Caspase 3 and Caspase 9 were increased in T-2 toxin treated mouse femur,indicating that Parkin^-/-exacerbates apoptosis caused by T-2 toxin.4)After Parkin knockout,the protein expressions of Wnt andβ-catenin were decreased and the protein expression of GSK-3βwas increased in T-2 toxin treated mouse femur,indicating that Parkin^-/-exacerbates the inhibition of the Wnt pathway caused by T-2 toxin.5)After Parkin knockout,the bone density and length were decreased,the damage of trabecular bone and bone cells were worse,and the protein expressions of OCN and OSX were decreased in T-2 toxin treated mouse femur,indicating that Parkin^-/-exacerbates the inhibition of femoral bone formation caused by T-2 toxin.In summary,T-2 toxin leads to bone formation disorders in both mouse femur and MC3T3-E1cells,induces apoptosis,inhibits Wnt pathway,damages mitochondria,and activates Parkin-mediated mitophagy.Parkin-mediated mitophagy can antagonize the inhibition of T-2 toxin-induced bone formation by inhibiting apoptosis and activating the Wnt pathway.After Parkin intervention,mitophagy of cells and bone tissue induced by the T-2 toxin is inhibited,which further aggravates the apoptosis and Wnt pathway inhibition caused by T-2 toxin,as well as aggravates the damage of cells and bone tissue,indicating that mitophagy plays the protective role in the inhibition of bone formation by T-2 toxin.