The Role Of PI3K/AKT/HIF-1α Pathway In The Effect Of Nano-TiO₂ On Lactate Production In TM4 Cells

Objective:The present study aimed to assess the effects of titanium dioxide nanoparticles（nano-TiO₂）on lactate production in Mouse Sertoli cell line（TM4 cells）by establishing the in vitro toxic model of nano-TiO₂,and to investigate whether PI3K/AKT pathway or ROS level regulates HIF-1α and thus affecting lactate production in TM4 cells.This study expected to provide a necessary supplement for the study of the influence of nano-TiO₂ on glucose metabolism process and related mechanisms.Methods:TM4 cells were treated with nano-TiO₂ at different concentrations（0,50,100,150,200μg/mL）for 24 h and 48 h,respectively.The morphologic of TM4 cells were observed through inverted phase contrast microscope and the cell viability was detected using CCK-8 kit.The content of glucose and lactate in TM4cells was determined with glucose and lactate detection kits,respectively.Western Blot was performed to examine the expression level of GLUT3 and key enzymes（HK1,HK2,PFKM,ENO1 and LDH）during lactate production.Since no significant change in the content of glucose in TM4 cells was observed after exposure to nano-TiO₂ for 24 h,the subsequent exposure duration was 48 h.After the exposure of nano-TiO₂for 48 h,the expression level of PI3K/AKT pathway proteins and HIF-1α protein in TM4 cells was detected through Western Blot.ROS level was analyzed using ROS detection kit.In addition,the agonist of PI3K（IGF-1）and the inhibitor of ROS（NAC）were added to compare and explore whether nano-TiO₂ regulated HIF-1α through PI3K/AKT pathway or ROS level and thereby affecting the production of lactate in TM4 cells.Results:1.After the treatment of TM4 cells with different concentrations of nano-TiO₂ for 24 h,the cell viability decreased in a concentration-dependent manner when compared with the control group,and the decrease was statistically significant starting from the concentration of nano-TiO₂ was 100μg/mL（P<0.05）;there was no significant change in intracellular content of glucose when compared with the control group,while nano-TiO₂ decreased the intracellular content of lactate in TM4 cells in a concentration-dependent manner,and the difference was statistically significant starting from the concentration of nano-TiO₂ was 150μg/mL（P<0.01）.When the TM4 cells were exposed to nano-TiO₂ with different concentrations for 48 h,the cell viability decreased in a concentration-dependent manner as compared with the control group,and the difference was statistically significant from the concentration of nano-TiO₂ was 50μg/mL（P<0.05）;the intracellular content of glucose showed a concentration-dependent increase,with a statistically significant difference starting at a concentration of 100μg/mL of nano-TiO₂（P<0.01）,while the intracellular content of lactate decreased in a concentration-dependent manner,with a statistically significant difference starting at the concentration of 50μg/mL of nano-TiO₂（P<0.01）.2.The expression levels of HK1,HK2,PFKM,ENO1,LDH and GLUT3 showed concentration-dependent decrease after the TM4 cells were treated with nano-TiO₂ at different concentrations.Specifically,when the cells were exposed to nano-TiO₂ for 24 h and compared with the control group,the expression of HK1 decreased statistically significantly（P<0.01）starting from the concentration of nano-TiO₂ at 50μg/mL,and the expression of HK2,PFKM and LDH began to decrease significantly（P<0.05）from the exposure group with a concentration of 150μg/mL,while ENO1 and GLUT3 showed a significant decrease in the 200μg/mL nano-TiO₂ exposure group（P<0.05）.After the TM4 cells were exposed to nano-TiO₂ for 48 h,the expression levels of HK1 and PFKM in TM4 cells showed a statistically significant difference compared with the control group starting at the concentration of 50μg/mL of nano-TiO₂（P<0.05）,and the expression levels of HK2,ENO1,LDH and GLUT3 decreased with a statistically significant difference from the concentration of nano-TiO₂ at 100μg/mL when compared with the control group（P<0.05）.3.The expression levels of p-PI3K,p-AKT,p-m TOR,p-p70s6k and HIF-1α in TM4 cells treated with different concentrations of nano-TiO₂ for 48 h decreased in a concentration-dependent manner compared with the control group.The expression levels of p-PI3K and p-AKT decreased significantly from the concentration of nano-TiO₂ at 50μg/mL（P<0.01）,and the expression levels of p-m TOR,p-p70s6k and HIF-1α decreased with a statistically significant difference starting at the concentration of 100μg/mL of nano-TiO₂（P<0.05）.After treating cells with different concentrations of nano-TiO₂ for 48 h,the level of ROS in TM4 cells increased in a concentration-dependent manner,and the increase of ROS level was statistically significant from the concentration of nano-TiO₂ at 50μg/mL（P<0.05）.4.When TM4 cells were treated with nano-TiO₂ and IGF-1,the expression level of HIF-1α was significantly increased（P<0.05）,the intracellular content of glucose was significantly decreased（P<0.01）,the intracellular content of lactate was reduced significantly（P<0.01）and the expression levels of HK2,PFKM and LDH were significantly increased（P<0.05）when compared with nano-TiO₂ treated group.5.When TM4 cells were treated with nano-TiO₂ and NAC,the expression level of HIF-1α was not significantly changed,as well as the contents of glucose and lactate in the cells were not significantly changed when compared with the nano-TiO₂ treated group,and there was also no significant change in the expression levels of HK1,HK2,PFKM,ENO1,LDH and GLUT3.Conclusion:Nano-TiO₂ down-regulated the expression levels of proteins and enzymes related to lactate production in TM4 cells by inhibiting PI3K/AKT pathway and subsequently reducing HIF-1α ,thus leading to the decrease of lactate production in TM4 cells.The increase of ROS induced by nano-TiO₂ was not involved in the stabilization of HIF-1α and production of lactate in TM4 cells.