Study On The Interaction Mechanism Of PGC-1α-NF-κB In The Anti-inflammatory/pro-inflammatory Effect Switching Of IL-6 Synthesized By Myoblast

Objective:IL-6 is the first recognized muscle factor that can be released into the blood by contracting skeletal muscle to mediate beneficial effects.However,a large body of literature reports that IL-6 exerts pro-inflammatory effects often in association with NF-κB.ROS are unavoidable products of mitochondrial quality control processes,with high levels of ROS causing oxidative stress,while low levels of ROS function as signaling molecules.Since PGC-1αand NF-κB are redox-sensitive factors,they interact with each other in addition to ROS.It is speculated that different levels of ROS may affect the dual effect of IL-6 by modulating the interactive antagonism of both PGC-1α-NF-κB.In this study,we used two H₂O₂concentrations in C2C12 cells to simulate the level of ROS under exercise/oxidative stress,and then observed whether the interaction antagonism of PGC-1α-NF-κB was related to the dual effect of IL-6,and explored the relationship between different levels of ROS,PGC-1α-NF-κB interaction antagonism and the dual effect of IL-6.The relationship between the dual effect of ROS,PGC-1α-NF-κB and IL-6 was investigated.Methods:The experimental subjects of this study were C2C12 cells,and the study was divided into 3 parts:the first part mapped the drug concentrations and grouped into:control group（C）,low concentration hydrogen peroxide incubation group（LCH）;control group（C）,high concentration hydrogen peroxide incubation group（HCH）.different drug concentration gradients were set up to intervene in C2C12 myogenic cells in the LCH and HCH groups,respectively,and DCFH-DA was used to probe assay to detect the level of ROS production;JC-1 probe assay to detect the mitochondrial membrane potentialΔΨto reflect the mitochondrial function;and tetramethylazole salt assay（CCK-8）to detect C2C12 cell activity to reflect the level of cell proliferation and cytotoxicity.The second part verifies whether ATF5-siRNA reduces anti-inflammatory factor expression by affecting PGC-1α-NF-κB interaction antagonism,grouped as control（C）,low concentration hydrogen peroxide incubation group（LCH）,and low concentration hydrogen peroxide incubation+ATF5-siRNA group（L+S）.The third part verified whether overexpression of ATF5 decreased the expression of pro-inflammatory factors by affecting PGC-1α-NF-κB interaction antagonism,grouped as high concentration hydrogen peroxide incubation group（HCH）,and high concentration hydrogen peroxide incubation+ATF5 overexpression group（H+OE）.The protein expression levels of mitochondrial UPRmt pathway ATF5,CLPP,HSP60;oxidative stress pathway PGC-1α,Nrf-2,NF-κB;inflammatory pathway IL-1β,IL-10,IL-6 were detected by Western-blotting assay.Results:（1）H₂O₂regulates ROS,membrane potential levels in C2C12 cells.The LCH group was incubated with different concentrations of H₂O₂（0μmol/L,1μmol/L,5μmol/L,10μmol/L,20μmol/L,40μmol/L,50μmol/L）in C2C12 cells with an intervention time of 15 min.compared with the C group,the ROS was significantly higher in the LCH group（p<0.01）,and the mitochondrial membrane potential levels were significantly increased（p<0.05）.Determine the H₂O₂concentration of 10μmol/L for simulating low level of ROS in the experiment and the intervention time of 0.25h;In the high concentration group,C2C12 cells were incubated with different concentrations of H₂O₂（0μmol/L,100μmol/L,200μmol/L,500μmol/L,750μmol/L,1000μmol/L,2000μmol/L）for 1 h.Compared with group C,the HCH group showed a significant increase in ROS level（p<0.01）,and the level of mitochondrial membrane potential was significantly lower（p<0.05）.The H₂O₂concentration to simulate high levels of ROS was determined to be 500μmol/L and the intervention time was 1h.（2）ATF5-siRNA reduces anti-inflammatory factor expression by affecting PGC-1α-NF-κB interaction antagonism Compared with the control group（C）,PGC-1αand Nrf-2 protein expression was significantly increased（p<0.05）,IL-10 and IL-6 protein expression was significantly increased（p<0.01）,and NF-κB protein expression was significantly decreased（p<0.01）IL-1βprotein expression was significantly decreased（p<0.05）in the low-concentration H₂O₂group（LCH）;compared with the low-concentration H₂O₂group,PGC-1αand IL-10 protein expression was significantly decreased（p<0.01）,IL-6 and Nrf-2 protein expression was significantly decreased（p<0.05）,protein expression of NF-κB was significantly increased（p<0.01）and IL-1βprotein expression was significantly increased in the ATF5-siRNA+low concentration of H₂O₂intervention group（L+S）compared to the low concentration of H₂O₂group（p<0.05）.（3）Overexpression of ATF5 reduces the expression of pro-inflammatory factors by affecting PGC-1α-NF-κB interaction antagonismCompared with the control group（C）,PGC-1αand IL-10 protein expression was significantly lower（p<0.05）,Nrf-2 protein expression was significantly lower（p<0.01）,and NF-κB,IL-6,and IL-1βprotein expression was significantly higher（p<0.01）in the high concentration H₂O₂group（HCH）;compared with the high concentration H₂O₂group（HCH）,ATF5 overexpression expression+high concentration of H₂O₂intervention group（H+OE）PGC-1α,Nrf-2,IL-10 protein expression was significantly higher（p<0.01）and NF-κB,IL-6,IL-1βprotein expression was significantly lower（p<0.01）.Conclusion:（1）Low levels of ROS increase the expression of IL-6 and its co-factor IL-10 by activating PGC-1αand inhibiting NF-κB to mediate the anti-inflammatory effect of IL-6.（2）High level ROS increased the expression of IL-6 and its co-factor IL-1βby activating NF-κB and inhibiting PGC-1α,and mediated the pro-inflammatory effect of IL-6.