| Object:Titanium dioxide nanoparticles（Ti O2NPs）have toxic effects,but whether they can induce calcium overload in vivo and in vitro remains controversial.In this study,meta-analysis was used to explore whether Ti O2 NPs could induce calcium overload in mammals in vitro and in vivo.Cell toxicity experiments were conducted to investigate whether Ti O2 NPs could cause calcium overload in testicular sertoli cells（TM4）via oxidative stress,and to identify the role of Ca2+/p38/AKT/m TOR signaling pathway in TM4 cell apoptosis induced by Ti O2 NPs,in order to provide a supplement for the study of biological toxic effects of Ti O2 NPs.Methods:Through literature retrieval and screening,Revman 5.4 and Stata 15.0 software were used to perform systematically review and meta-analyze to investigate whether Ti O2 NPs cause calcium overload in mammals in vivo and in vitro.And a toxic model of Ti O2 NPs with different concentrations（0,50,100,150 and 200μg/m L）was established based on TM4 cells.After Ti O2 NPs exposed for 24 h,cell morphology was observed by inverted phase contrast microscope,and cell viability was determined by CCK-8 assay.Cell reactive oxygen species（ROS）levels,Ca2+levels and Ca2+-ATPase activity were detected by the kits,and cell apoptosis rate was detected by flow cytometry.The expressions of signal pathway-related proteins（p38,p-p38,AKT,p-AKT,m TOR and p-m TOR）and apoptosis-related proteins（Bcl-2,Bax,Caspase 3,Caspase 9 and p53）were determined by Western Blot.ROS inhibitors（NAC）were used to investigate whether Ti O2 NPs induce calcium overload in mouse TM4 cells through oxidative stress.Intracellular Ca2+chelating agent（BAPTA-AM）was used to verify the regulatory role of Ca2+/p38/AKT/m TOR signaling pathway in TM4 cell apoptosis induced by Ti O2 NPs.Results:1.Meta-analysis showed that Ti O2 NPs exposure could significantly increase the level of Ca2+in vivo and in vitro,and significantly decrease the activity of Ca2+-ATPase.Species,particle size and exposure time of Ti O2 NPs had significant effects on calcium homeostasis in vivo.Cell type,particle size and exposure dose of Ti O2 NPs had significant effects on calcium homeostasis in vitro.2.After TM4 cells treated with Ti O2 NPs for 24 h,compared with the control group,most of the cells in the high-dose Ti O2 NPs treatment group changed from spindle to irregular shape,and the cell space increased and the number of cells decreased.The CCK-8 results showed that,compared with the control group,the cell viability decreased with the increase of Ti O2 NPs exposure dose,and the significant difference started from 100μg/m L Ti O2 NPs treatment group（P<0.05）.3.The ROS levels of TM4 cells increased with the increase of Ti O2 NPs exposure dose,and starting from 100μg/m L Ti O2 NPs group,the difference was significant compared with the control group（P<0.01）.4.Compared with the control group,the levels of intracellular free Ca2+increased with the increase of Ti O2 NPs exposure dose in dose-dependent manner,and the significant difference started from 100μg/m L Ti O2 NPs group（P<0.01）.The activity of Ca2+-ATPase decreased with the increase of Ti O2 NPs exposure dose in dose-dependent manner,and the significant difference started from 100μg/m L Ti O2 NPs group（P<0.05）.5.After TM4 cells treated with Ti O2 NPs for 24 h,compared with the control group,the protein expression level of p-p38 showed an increasing trend with the increase of Ti O2 NPs exposure dose,and the significant difference started from 150μg/m L Ti O2 NPs treatment group（P<0.05）.The protein expression levels of p-AKT and p-m TOR were decreased with the increase of Ti O2 NPs exposure dose,and the significant differences started from 100μg/m L Ti O2 NPs group（P<0.05）.6.The cell apoptosis rates increased with the increase of the Ti O2 NPs exposure dose,and the significant difference started from 100μg/m L Ti O2 NPs group,compared with the control group（P<0.05）.The protein expression levels of Caspase 3,p53,Bax and Caspase 9 increased with the increase of Ti O2 NPs dose.The protein expression levels of Caspase 3 and p53 showed significant difference between Ti O2 NPs treatment groups and the control group,starting from 150μg/m L Ti O2 NPs group（P<0.05）;and the protein expression levels of Bax and Caspase 9 showed significant difference between Ti O2 NPs treatment groups and the control group,starting from 100μg/m L Ti O2 NPs group（P<0.05）.The expression level of Bcl-2 protein decreased with the increase of Ti O2 NPs dose,and the significant difference started from 100μg/m L Ti O2 NPs group（P<0.01）.7.Compared with 150μg/m L Ti O2 NPs group,ROS level and Ca2+level in NAC+Ti O2 NPs（150μg/m L）combined treatment group were significantly decreased（P<0.01）,and Ca2+-ATPase activity was significantly increased（P<0.01）.8.Compared with 150μg/m L Ti O2 NPs group,the protein expression levels of p-p38 in BAPTA-AM+Ti O2 NPs（150μg/m L）combined treatment group were significantly decreased（P<0.01）,and the protein expression levels of p-AKT and p-m TOR were significantly increased（P<0.01）,the apoptosis rate was significantly decreased（P<0.01）,the protein expression level of Bcl-2 was significantly increased（P<0.01）,and the protein expression levels of p53,Bax,Caspase 3 and Caspase 9 were significantly decreased（P<0.01）.Conclusion:Ti O2 NPs can induce calcium overload in mammals in vivo and in vitro;and Ti O2 NPs can reduce viability of TM4 cells,induce oxidative stress,calcium overload,thus inducing cell apoptosis;the Ca2+/p38/AKT/m TOR signaling pathway play an important regulatory role in the induction of TM4 cell apoptosis by Ti O2 NPs. |
PDF Full Text Request