The Mechanism Of HMGB1 Regulating Muscle Wasting In Colon Cancer Cachexia

Part Ⅰ:The expression of HMGB1 in colon cancer tissues and plasma HMGB1 level in patients with colon cancer cachexiaObjective:Cancer cachexia is a metabolic wasting disease characterized by weight loss,muscle wasting,and systemic inflammation.However,there are currently no drugs that effectively treat cachexia muscle atrophy.A variety of inflammatory factors have been reported to be involved in the occurrence and development of cancer cachexia.HMGB1 is an inflammatory factor that participates in cancer,inflammation,and immunity.However,there are few studies on HMGB1 and cachexia.This part of the study explored the expression of HMGB1 in colon cancer tissue and the plasma HMGB1 level in colon cancer cachexia patients.Methods:The expression of HMGB1 in pan-cancer and its prognostic significance in colon cancer patients were explored using TCGA and GEO public databases.Patients diagnosed with colon cancer in our hospital from May 2020 to September 2020 were included.The baseline data,cachexia staging scale,ECOG performance status score,anorexia score,abdominal CT images within the past month and plasma were collected.Divide patients into cachexia group and non-cachexia group for analysis.Results:In TCGA COAD,GSE20916,GSE21510,GSE18105 and GSE39582 datasets,the HMGB1 mRNA was increased in colon cancer tissues compared with normal colon tissues.In TCGA COAD,GSE17538,GSE14333,and GSE39583 datasets with prognostic data,there is no prognostic difference between high and low HMGB1 groups.The levels of plasma HMGB1 between the colon cancer cachexia group and the non-cachexia group were compared.The plasma HMGB1 level in the cachexia group was higher than that in the non-cachexia group.Plasma HMGB1 level was negatively correlated with skeletal muscle index(R=-0.31,P<0.001),suggesting that HMGB1 may play a certain role in muscle atrophy.Conclusion:HMGB1 was elevated in colon cancer tissues and plasma HMGB1 was elevated in colon cancer cachexia patients.Plasma HMGB1 was negatively correlated with skeletal muscle index.Part Ⅱ:HMGB1 induces myotube atrophy by activating TLR4/NFκB signaling pathway in vitroObjective:The previous part of the study found that plasma HMGB1 was elevated in colon cancer cachexia patients and was negatively correlated with skeletal muscle index.Two important receptors of HMGB1,TLR4 and RAGE,were both reported to be involved in muscle atrophy,indicating that HMGB1 may be involved in muscle atrophy.To further explore the role of HMGB1 in cachexia,this part of the study explored the effect and related mechanism of HMGB1 on C2C12 myotube atrophy in vitro.Methods:Exosomes from CT26 cells were extracted and identified.The effect of CT26 derived exosomes on C2C12 myotubes was observed,and the expressions of atrophyrelated proteins Atrogin1 and MuRF1 were detected by Western blot;The level of HMGB1 in CT26 cells and CT26 derived EVs were detected.C2C12 myotubes were treated with different concentrations of EVs and different concentrations of recombinant HMGB1,respectively,to observe the shape of myotubes,measure the diameter of myotubes,and detect the expression of atrophy-related proteins Atrogin1 and MuRF1 by Western blot.HMGB1-stimulated C2C12 myotubes were treated with TLR4 inhibitor,and the expression levels of atrophy-related proteins Atroginl and MuRF1 were detected by Western blot.Using inhibitors of NF-κB signaling pathway and MAPK signaling pathway,we observed their effects on C2C12 myotube atrophy to explore their possible downstream pathways.Then,HMGB1-stimulated C2C12 myotubes were treated with HMGB1 inhibitor glycyrrhizin,and the expressions of atrophy-related proteins Atroginl and MuRF1 were detected by Western blot.Using HMGB1 inhibitor glycyrrhizin,its effect on EVs-induced myotube atrophy was observed.The CT26 cell line with low expression of HMGB1(shHMGB1 CT26)was constructed by lentivirus,the changes of HMGB1 level in EVs of this cell were detected,and the effect of shHMGB1 CT26 EVs on C2C12 myotubes was observed.Results:CT26 exosomes could be taken up by C2C12 myotube cells.A certain concentration of CT26 exosomes could cause muscle atrophy,and the expression of muscle atrophy-related proteins Atroginl and MuRF1 were increased,indicating that the cachexia model was successfully constructed.After depletion of exosomes in CT26 conditioned medium,C2C12 myotube atrophy was relieved,proving that exosomes are one of the factors of C2C12 myotube atrophy.Compared with normal colon epithelium,CT26 colon cancer cells have an increased level of HMGB1.A certain concentration of HMGB1 could cause myotube atrophy.After using glycyrrhizin,the myotube atrophy caused by HMGB1 and exosomes was alleviated.Myotube atrophy caused by HMGB1 was alleviated after using TLR4 inhibitor,suggesting that HMGB1 may cause muscle wasting through TLR4.The effect of CT26 exosomes on myotube atrophy was attenuated after HMGB1 knockdown.Conclusion:CT26-derived exosomes containing HMGB1 could induce muscle atrophy.Glycyrrhizin could alleviate HMGB1-induced myotube atrophy and HMGB1 may cause C2C12 myotube atrophy through TLR4/NF-κB signaling pathway.Part Ⅲ:Inhibition of HMGB1 can alleviate colon cancer cachexia in vivoObjective:The previous part of the study found that HMGB1 induces C2C12 myotube atrophy through the TLR4/NF-κB signaling pathway,and the use of HMGB1 inhibitor glycyrrhizin can alleviate C2C12 myotube atrophy induced by HMGB1 and EVs.This part of the study explores the effects of shHMGB1 CT26 tumor-bearing mice.And the effects of glycyrrhizin on CT26 tumor-bearing mice.Method:The CT26 cell line with stable low expression of HMGB1 was constructed(shHMGB1).Then 6w male BALB/C were randomly divided into five groups,namely control group,CT26 group,CT26 shCON group,CT26 shHMGB1 group,and CT26 shHMGB 1+CT26 EVs group.Observe body weight,tumor-free body weight,gastrocnemius muscle,epididymal fat weight,and skeletal muscle fiber cross-sectional area.Then we explored the effects of HMGB1 inhibitor glycyrrhizin on CT26 cachexia mice.6w male BALB/C were randomly divided into four groups:control group,CT26 group,CT26+10 mg/kg glycyrrhizin group and CT26+20 mg/kg glycyrrhizin group.Drug administration started from the seventh day after CT26 injection,and the mice were sacrificed 14 days after administration,Plasma,tumor,gastrocnemius muscle and epididymal fat were collected.The effects of glycyrrhizin on CT26 tumor-bearing mice were compared by tumor-free body weight,gastrocnemius muscle mass,epididymal fat mass,skeletal muscle fiber cross-sectional area and plasma inflammatory factor levels.Results:CT26 mice with low expression of HMGB1 showed alleviated cachexia,and cancer cachexia was aggravated after injection of CT26 EVs;HMGB1 inhibitor glycyrrhizin could alleviate the decrease in lean body mass,gastrocnemius muscle and epididymal fat,increase the cross-sectional area of gastrocnemius muscle fibers and reduce the levels of plasma inflammatory factors TNFa and IL-6.Western blot showed that glycyrrhizin reduced the expression of atrophy-related proteins Atroginl and MuRF1 and inhibited the expression of phosphorylated p65 and IκB.Conclusion:Knocking down of HMGB1 in CT26 cells can alleviate cachexia in CT26 tumor-bearing mice.HMGB1 inhibitor glycyrrhizin can alleviate muscle wasting in CT26 tumor-bearing mice by inhibiting NF-κB signaling pathway,and delaying the progression of cachexia.