Inhibition of type 3 deiodinase expression can improve mitochondrial function in skeletal muscle of sepsis by up-regulating peroxisome proliferator-activated receptor-γ coactivator-1α
Objective To investigate the protective effects and mechanisms of targeted inhibition of type 3 deiodinase(Dio3)on skeletal muscle mitochondria in sepsis.Methods ① In vivo experiments:adeno-associated virus(AAV)was employed to specifically target Dio3 expression in the anterior tibial muscle of rats,and a septic rat model was generated using cecal ligation and puncture(CLP).The male Sprague-Dawley(SD)rats were divided into shNC+Sham group,shD3+Sham group,shNC+CLP group,and shD3+CLP group by random number table method,with 8 rats in each group.After CLP modeling,tibial samples were collected and Western blotting analysis was conducted to assess the protein levels of Dio3,peroxisome proliferator-activated receptor-γ coactivator-1α(PGC1α),and silence-regulatory protein 1(SIRT1).Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was utilized to examine mRNA expression of genes including thyroid hormone receptors(THR α,THR β),monocarboxylate transporter 10(MCT10),mitochondrial DNA(mtDNA),and PGC1α.Transmission electron microscopy was employed to investigate mitochondrial morphology.② In vitro experiments:involved culturing C2C12 myoblasts,interfering with Dio3 expression using lentivirus,and constructing an endotoxin cell model by treating cells with lipopolysaccharide(LPS).C2C12 cells were divided into shNC group,shD3 group,shNC+LPS group,and shD3+LPS group.Immunofluorescence colocalization analysis was performed to determine the intracellular distribution of PGC1α.Co-immunoprecipitation assay coupled with Western blotting was carried out to evaluate the acetylation level of PGC1α.Results ① In vivo experiments:compared with the shNC+Sham group,the expression of Dio3 protein in skeletal muscle of the shNC+CLP group was significantly increased(Dio3/β-Tubulin:3.32±0.70 vs.1.00±0.49,P＜0.05),however,there was no significant difference in the shD3+Sham group.Dio3 expression in the shD3+CLP group was markedly reduced relative to the shNC+CLP group(Dio3/β-Tubulin:1.42±0.54 vs.3.32±0.70,P＜0.05).Compared with the shNC+CLP group,the expression of T3-regulated genes in the shD3+CLP group were restored[THR α mRNA(2-△△Ct):0.67±0.05 vs.0.33±0.01,THR βmRNA(2-△△Ct):0.94±0.05 vs.0.67±0.02,MCT10 mRNA(2-△△Ct):0.65±0.03 vs.0.57±0.02,all P＜0.05].Morphology analysis by electron microscopy suggested prominent mitochondrial damage in the skeletal muscle of the shNC+CLP group,while the shD3+CLP group exhibited a marked improvement.Compared with the shNC+Sham group,the shNC+CLP group significantly reduced the number of mitochondria(cells/HP:10.375±1.375 vs.13.750±2.063,P＜0.05),while the shD3+CLP group significantly increased the number of mitochondria compared to the shNC+CLP group(cells/HP:11.250±2.063 vs.10.375±1.375,P＜0.05).The expression of mtDNA in shNC+CLP group was markedly reduced compared with shNC+Sham group(copies:0.842±0.035 vs.1.002±0.064,P＜0.05).Although no difference was detected in the mtDNA expression between shD3+CLP group and shNC+CLP group,but significant increase was found when compared with the shD3+Sham group(copies:0.758±0.035 vs.0.474±0.050,P＜0.05).In the shD3+CLP group,PGC1α expression was significantly improved at both transcriptional and protein levels relative to the shNC+CLP group[PGC1α mRNA(2-△△Ct):1.49±0.13 vs.0.68±0.06,PGC1α/β-Tubulin:0.76±0.02 vs.0.62±0.04,both P＜0.05].② In vitro experiments:post-24-hour LPS treatment of C2C12 cells,the cellular localization of PGC1α became diffuse;interference with Dio3 expression promoted PGC1α translocation to the perinuclear region and nucleus.Moreover,the acetylated PGC1α level in the shD3+LPS group was significantly lower than that in the shNC+LPS group(acetylated PGC1α/β-Tubulin:0.59±0.01 vs.1.24±0.01,P＜0.05),while the expression of the deacetylating agent SIRT1 was substantially elevated following Dio3 inhibition(SIRT1/β-Tubulin:1.04±0.04 vs.0.58±0.03,P＜0.05).When SIRT1 activity was inhibited by using EX527,PGC1α protein expression was notably decreased compared to the shD3+LPS group(PGC1α/β-Tubulin:0.92±0.03 vs.1.58±0.03,P＜0.05).Conclusion Inhibition of Dio3 in skeletal muscle reduced the acetylation of PGC1α through activating SIRT1,facilitating nuclear translocation of PGC1α,thereby offering protection against sepsis-induced skeletal muscle mitochondrial damage.
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