The Role And Mechanism Of Endoplasmic Reticulum Stress In Sepsis-induced Muscle Atrophy

Objective: Endoplasmic reticulum(ER)stress is involved in muscle atrophy in various conditions,but the role of ER stress in sepsis-induced muscle atrophy is not yet clear,our study aims to explore the role and mechanism of ER stress in sepsis-induced muscle atrophy.Methods: WT mice and CHOP knockout(KO)mice were selected to conduct CLP surgery to establish the mouse model of sepsis.C2C12 myoblasts were differentiated into myotubes with 2% horse serum and then stimulated with LPS.4-PBA and TUDCA were used to inhibit ER Stress;si RNA was used to silence CHOP;Thap was used to induce ER stress;WP1066 and SB431542 were used to inhibit STAT3 pathway and Smad3 pathway.Serum inflammatory factors were detected by enzyme-linked immunosorbent assay(ELISA);body weight of mice was weighed daily and muscle strength was assessed by wire hang test;the mice were sacrificed at 1,3,7 days after surgery,and then the tibialis anterior,gastrocnemius and extensor digitorum longus were separated and weighed;immunofluorescence staining was used to assess the cross-sectional area(CSA)of myofibers and the diameter of myotubes;real-time quantitative PCR was used to measure the m RNA expression of Atrogin-1,Mu RF1 and CHOP;Western Blot was used to detect the protein expression of E3 ubiquitin ligases Atrogin-1 and Mu RF1,poly-ubiquitinated proteins,ER stress biomarkers,p-STAT3/STAT3 and p-Smad3/Smad3.Results: In WT mice,the levels of serum inflammatory factors increased significantly after CLP surgery,and the body weight,muscle mass and strength,and CSA of myofibers in the CLP group both decreased significantly compared to the Sham group.The m RNA and protein expression of E3 ubiquitin ligases Atrogin-1 and Mu RF1 were both increased distinctly and the levels of poly-ubiquitinated proteins were upregulated after CLP.LPS stimulation also increased the levels of E3 ubiquitin ligases and poly-ubiquitinated proteins and induced remarkably myotube atrophy in vitro.The above results indicating that sepsis activates UPS,increases protein degradation and induces severe muscle atrophy.At the same time,sepsis activated ER stress.ER stress inhibitors and knockdown of CHOP both suppressed the activity of E3 ubiquitin ligases and markedly alleviated LPS-induced myotube atrophy.CHOP KO significantly reduced UPS-mediated protein degradation,increased muscle weight and strength and the CSA of myofibers in septic mice,thus improved sepsis-induced muscle atrophy.Furthermore,inhibition of ER stress and CHOP KO could significantly suppress the phosphorylation of STAT3 and Smad3 during sepsis,and inhibition of STAT3 and Smad3 reduced ER stress-induced protein degradation.Conclusions: ER stress activates UPS-mediated protein degradation and promotes sepsis-induced muscle atrophy,which is achieved by activating STAT3 and Smad3.Part I: The relationship between UPS-mediated protein degradation and sepsis-induced muscle atrophyObjective: To explore the relationship between UPS-mediated protein degradation and sepsis-induced muscle atrophy.Methods: Fifty WT male mice were randomly divided into six groups,including the Sham group(Day 1: n=6,Day 3: n=6,Day 7: n=6)and the CLP group(Day 1: n=8,Day 3: n=12,Day 7: n=12).C2C12 myoblasts were differentiated into myotubes with 2% horse serum and then stimulated with LPS.Serum inflammatory factors were detected by ELISA;the body weight of mice was weighed daily and the muscle weight was weighed at 1 day,3 days and 7 days after operation;muscle strength was assessed by wire hang test;immunofluorescence staining was used to assess the CSA of myofibers and the diameter of myotubes;real-time quantitative PCR was used to measure the m RNA expression of Atrogin-1 and Mu RF1;Western Blot was used to detect the protein expression of Atrogin-1,Mu RF1 and poly-ubiquitinated proteins.Results: Compared to the Sham group,the levels of serum inflammatory factors increased significantly in the CLP group,and the body weight,muscle mass and strength,and CSA of myofibers in the CLP group both decreased significantly.The m RNA and protein expression of E3 ubiquitin ligases Atrogin-1 and Mu RF1 were both increased distinctly and the levels of poly-ubiquitinated proteins were upregulated after CLP.LPS stimulation also increased the levels of E3 ubiquitin ligases and poly-ubiquitinated proteins and induced remarkably myotube atrophy in vitro.Conclusions: Sepsis activates UPS,increases protein degradation and induces severe muscle atrophy.Part II: The role of ER stress in sepsis-induced muscle atrophyObjective: To explore the role of ER stress in sepsis-induced muscle atrophy.Methods: Fifty CHOP KO male mice were randomly divided into six groups,including the Sham group(Day 1: n=6,Day 3: n=6,Day 7: n=6)and the CLP group(Day 1: n=8,Day 3: n=12,Day 7: n=12)and then compared with the WT mice.C2C12 myotubes were stimulated with LPS,4-PBA and TUDCA were used to inhibit ER stress and si RNA was used to silence CHOP.The body weight of mice was weighed daily and the muscle weight was weighed at 1 day,3 days and 7 days after operation;muscle strength was assessed by wire hang test;immunofluorescence staining was used to assess the CSA of myofibers and the diameter of myotubes;real-time quantitative PCR was used to measure the m RNA expression of CHOP;Western Blot was used to detect the protein expression of Atrogin-1,Mu RF1,poly-ubiquitinated proteins and ER stress biomarkers.Results: In WT mice,the protein expression of ER stress biomarkers and the m RNA expression of CHOP in the CLP group were both increased compared to the Sham group,and LPS stimulation also caused elevation of ER stress biomarkers in vitro,indicating that sepsis activates ER stress.ER stress inhibitors and knockdown of CHOP suppressed the activity of E3 ubiquitin ligases and markedly alleviated LPS-induced myotube atrophy;and CHOP KO significantly reduced UPS-mediated protein degradation,increased muscle weight and strength and the CSA of myofibers in septic mice,thus improved sepsis-induced muscle atrophy.Conclusions: ER stress is involved in promoting sepsis-induced muscle atrophy.Part III: The mechanism of ER stress in sepsis-induced muscle atrophyObjective: To explore the mechanism of ER stress in sepsis-induced muscle atrophy.Methods: Western Blot was used to detect the phosphorylation of STAT3 and Smad3 in muscles of WT mice and CHOP KO mice.C2C12 myotubes were stimulated with LPS,4-PBA was used to inhibit ER Stress and si RNA was used to silence CHOP,and then the phosphorylation of STAT3 and Smad3 was detected by Western Blot.Thap was used to induce ER stress in vitro and WP1066 and SB431542 were used to inhibit STAT3 pathway and Smad3 pathway,Western Blot was then used to detect the protein expression of E3 ubiquitin ligases Atrogin-1 and Mu RF1.Results: The phosphorylation of STAT3 and Smad3 were significantly increased in the muscles of septic mice,and CHOP KO could significantly suppress the activation of STAT3 and Smad3.The same result was observed in vitro,the activation of STAT3 and Smad3 induced by LPS stimulation were suppressed remarkably after inhibition of ER stress or knockdown of CHOP with si RNA.The expression of Atrogin-1 and Mu RF1 increased notably after activation of ER stress,and then decreased after inhibition of the phosphorylation of STAT3 and Smad3.Conclusions: ER stress activates UPS-mediated protein degradation and promotes sepsis-induced muscle atrophy,which is achieved by activating STAT3 and Smad3.