The Effect And Mechanism Of 8 Weeks Aerobic Exercise On The Regulation Of Sarcoplasmic Reticulum Calcium In Nrf2 Knockout Mice

Objective:As an important nuclear transcription factor in vivo,Nrf2 is a key regulator of cell redox balance.Ca²⁺ is a critica signal of skeletal muscle contraction.This study was to investigate the role of Nrf2 in the regulation of Ca²⁺ in skeletal muscle sarcoplasmic reticulum and to establish a aerobic exercise training model to study the effect and mechanism of Nrf2 on the regulation of sarcoplasmic reticulum Ca²⁺ in skeletal muscle of mouse.Methods: In vitro experiments,the mouse skeletal muscle C2C12 cell line was used as the research object,respectively,using Nrf2 agonist t-BHQ and Nrf2 inhibitor Brusatol intervention.The levels of ROS in C2C12 cells were detected by fluorescence colorimetric assay.After Fluo-4 AM loaded on C2C12 cells,The changes of free Ca²⁺([Ca²⁺]i)in C2C12 cells were measured by laser scanning confocal microscopy with 100?M H₂O₂ oxidative stress stimulation.The expression of Nrf2,Ry R1,SERCA1,SERCA2,FKBP12,Ca M,Ca MK? and PKA protein in C2C12 cells were detected by Western Blot.8-week-old Nrf2 knockout mice and WT mice were divided into four groups randomly:wild control group?WC?,wild exercise group?WE?,Nrf2 KO control group?KC?and Nrf2 KO exercise group?KE?,10 mice each group.WE group and KE group were motor treadmill trained in aerobic exercise protocal for 8 weeks.The program was 12m/min,60min/day,5day/week.Fluorescence colorimetric assay was used to detect ROS levels in skeletal muscle of mice.Visible light colorimetric detected the tissue calcium concentration([Ca²⁺])of skeletal muscle.The expression of Nrf2,NQO1,HO-1 and CAT m RNA in skeletal muscle of mice were determined by RT-PCR.The expression of Nrf2,NQO1,HO-1,CAT,Ry R1,SERCA1,SERCA2,FKBP12,Ca M,Ca MK? and PKA protein were detected by Western Blot.Results:?1?Compared with the control group,the ROS level of C2C12 cells in Nrf2 inhibitor group was significantly increased;?2?Compared with the control group,the [Ca²⁺]i of the C2C12 cells in the Nrf2 inhibitor group was significantly increased after 185.4 seconds,while the [Ca²⁺]i of the Nrf2 agonist group was significantly decreased after 61.8 seconds;?3?Compared with the control group,the protein expression of Nrf2,SERCA1 and SERCA2 in the Nrf2 inhibitor group was significantly lower,and FKBP12 and PKA were significantly increased,while the expression of protein in Nrf2 agonist group did not change;?4?Compared with the WE group,the ROS level of the KE group was significantly decreased;?5?Compared with the WC group,the expression of Nrf2,NQO1 and CAT m RNA in the KC group was significantly decreased,while the Nrf2,NQO1 and HO-1 m RNA in the WE group were significantly increased.The expression of Nrf2,NQO1,HO-1 and CAT m RNA were significantly decreased;?6?Compared with WC group,the expression of Nrf2,PKA and SERCA1 protein in KC group was decreased,while the expression of Ry R,FKBP12 and Ca M increased,the expression of 1 decreased.Compared with WE group,the expressions of Nrf2,NQO1,HO-1 and CAT in KE group were decreased,while the expression of PKA and Ca MK?is increased significantly.Campared with KC group,the expression of PKA,Ca MK? and SERCA1 were lower significantly.?7?There was no significant difference among groups in skeletal muscle [Ca²⁺].Conclusion:?1?Nrf2 plays a protective role in the process of oxidative stress-induced skeletal muscle abnormal [Ca²⁺]i increase.?2?The deficient of Nrf2 increases the function of sarcoplasmic reticulum calcium release in mouse skeletal muscle,while the function of calcium recovery is weakened.8 weeks aerobic exercise training can against the decrease of sarcoplasmic reticulum calcium absorb function due to Nrf2 deficient.