| Objective:Lung cancer is the malignant tumor with the highest incidence and mortality rate in China.Cancer cachexia is a multifactorial disease characterized by weight loss and skeletal muscle atrophy,which has a serious negative impact on the outcome,quality of life,and survival time of lung cancer patients.However,current therapeutic approaches for cancer cachexia muscle atrophy are still limited.Sirtuin1(SIRT1)has been shown to regulate a large range of biological processes that are associated with inflammation,oxidative stress,mitochondrial biosynthesis,muscle function,etc.Ursolic acid(UA)is a pentacyclic triterpenoid natural compound that has a wide range of biological functions such as anti-inflammatory and anti-tumor.UA can alleviate muscle atrophy in some disease models,but the role and mechanism in cancer cachexia are unknown.This study aimed to explore the expression of SIRT1 in lung cancer and its relationship with cancer cachexia and to study the role of UA on lung cancer cachexia and related mechanisms.Methods:The bioinformatics approach was used to analyze the differential expression of SIRT1 in pan-cancer,as well as the expression level of SIRT1 in lung cancer and the relationship with prognosis,and to explore the correlation between the muscle atrophyrelated proteins muscle ring finger 1(MuRF1)and F-box protein 32(FBXO32/MAFbx/Atrogin-1)and SIRT1 in normal muscle tissue.A clinical cross-sectional study was conducted to collect baseline information and diagnose and stage lung cancer patients who visited our oncology department from February 2021 to June 2021.Plasma was collected from patients to detect the level of SIRT1 and analyze its correlation with skeletal muscle index(SMI)and cancer cachexia stage.The C2C12 myotubular atrophy model induced by tumor-conditioned medium(TCM)and Lewis lung carcinoma(LLC)tumor-bearing cachexia mouse model were constructed.The effect of UA on the viability of C2C12 myotubes was detected by CCK-8 assay.C2C12 myotubes and LLC tumorbearing mice were treated with different concentrations of UA.The morphology of myotubules was observed by immunofluorescence staining and the diameter of myotubules was measured.The morphology of muscle fibers in the gastrocnemius muscle was visualized by HE staining and the cross-sectional area of the muscle fibers was measured.The expression levels of mRNA and protein in myotubes and gastrocnemius were measured by RT-PCR and Western blot.The interaction patterns of SIRT1 and UA were analyzed by molecular docking assay.The levels of tumor necrosis factor-α(TNF-α),interleukin 1β(IL1β),and interleukin 6(IL-6)in the plasma of mice were measured by ELISA.Furthermore,C212 myotubes and LLC tumor-bearing mice were treated with Ex-527,an inhibitor of SIRT1,to explore the effect of Ex-527 on the therapeutic effect of UA and to detect changes in the expression of related molecules.UA treatment was given to LLC tumor-bearing mice at different treatment time points to explore its effect on the efficacy of cancer cachexia.Results:Bioinformatics analysis revealed that SIRT1 expression was reduced in lung cancer tumor tissues and correlated with the prognosis of lung cancer patients,and MuRF1 and Atrogin-1 correlated with SIRT1 in normal muscle tissues.A total of 171 lung cancer patients were included in this study,and the levels of SIRT1 in the plasma of lung cancer cachexia patients were reduced,which were positively correlated with skeletal muscle index(SMI)and negatively correlated with the cancer cachexia stage.In cellular and animal experiments,UA dose-dependently increased the diameter of C2C12 myotubes,the weight of the gastrocnemius muscle,and the cross-sectional area of muscle fibers.RT-PCR and Western blot results showed that UA decreased the expression of MuRF1 and Atrogin-1,upregulated the expression of SIRT1,inhibited the expression of NADPH oxidase 4(NOX4),Forkhead box O1(FOXO1),and Forkhead box O3a(FOXO3a),and decreased the phosphorylation levels of NF-κB and STAT3.ELISA results showed decreased levels of TNF-α,IL-1β,and IL-6 in the plasma of LLC tumor-bearing mice.After the application of Ex-527,the alleviating effect of UA on muscle atrophy in C2C12 myotubes and LLC tumorbearing mice was inhibited,and the expression levels of the above molecules were altered.UA treatment given to LLC tumor-bearing mice at the early or late stage of cancer cachexia alleviated the symptoms of cachexia,but the anti-cachexia effect of UA gradually diminished with the delay of application.Conclusions:SIRT1 expression was reduced in lung cancer tumor tissues and correlated with the prognosis of lung cancer patients.MuRF1 and Atrogin-1 correlated with SIRT1 in normal muscle tissues.The level of SIRT1 in plasma was reduced in lung cancer cachexia patients,and the level of SIRT1 was positively correlated with SMI and negatively correlated with the cancer cachexia stage,indicating that SIRT1 was associated with cancer cachexia muscle atrophy.UA alleviates cancer cachexia through the activation of SIRT1 and inhibition of NF-κB and STAT3 pathways.The application of UA at the advanced stage of cancer cachexia still had a protective effect on LLC tumor-bearing mice.Part Ⅰ:The expression of SIRT1 in tumor tissue and plasma of lung cancer patientsObjective:Lung cancer is one of the malignant tumors with the fastest-growing morbidity and mortality rates and the greatest threat to the health and life of the population in China.With the development of medicine,the treatment methods for lung cancer have diversified,but the treatment effect,quality of life,and survival time of patients still need to be improved.Cancer cachexia is a multifactorial syndrome characterized by a persistent decrease in skeletal muscle(with or without fat loss),which cannot be completely reversed by conventional nutritional support and leads to progressive dysfunction.Cancer cachexia can affect the outcome of lung cancer patients,reduce the quality of life and shorten survival time.However,there are no effective drugs for cancer cachexia yet.Therefore,it is urgent to find effective drugs and treatment options against cancer cachexia.SIRT1 has been reported to have various biological functions such as anti-inflammatory,antioxidant,and anti-tumor,as well as important roles in muscle physiology and in balancing muscle cell differentiation and proliferation.The role of SIRT1 in muscle atrophy in cancer cachexia is also receiving increasing attention.This study aimed to explore the expression of SIRT1 in tumors and plasma of lung cancer patients and the relationship with cachexia.Methods:In this part of the study,the differential expression of SIRT1 in pan-carcinoma was first analyzed by bioinformatics methods,and the expression level and relationship between SIRT1 and prognosis in lung cancer were further studied,and then the correlation between MuRF1 and Atrogin-1 and SIRT1 in normal muscle tissue was analyzed.The clinical cross-sectional study was conducted to collect lung cancer patients who visited our oncology department from February 2021 to June 2021,and information about the patients was accessed and collected in the medical record system,including age,gender,height,weight,tumor pathological type,tumor stage,Eastern Cooperative Oncology Group(ECOG)score,and CT images of the abdomen at the level of the third lumbar vertebra.The crosssectional area of the third lumbar spine muscles was measured by ImageJ software.Lung cancer patients were diagnosed and staged for cachexia according to EPCRC-CSA criteria.The level of SIRT1 in the plasma of lung cancer patients was detected by ELISA experiment,and the correlation of plasma levels of SIRT1 with SMI and cachexia staging was analyzed.Results:Bioinformatics analysis revealed that SIRT1 expression was reduced in lung cancer tumor tissues.SIRT1 gene expression was correlated with the prognosis of lung cancer patients,and MuRF1 and Atrogin-1 were correlated with SIRT1 in normal muscle tissue.In this study,171 lung cancer patients were included and divided into the noncachexia group(n=43,25.15%)and cachexia group(n=128,74.85%),and the patients in the cachexia group were staged,including pre-cachexia stage(n=53,30.99%),cachexia stage(n=62,36.26%),and cachexia refractory stage(n=13,7.60%).The plasma SIRT1 levels of patients in the cachexia group were significantly lower than those in the noncachexia group,and plasma SIRT1 levels were positively correlated with SMI and negatively correlated with the cachexia stage.Conclusions:SIRT1 expression was reduced in lung cancer tumor tissues,and the expression of the SIRT1 gene was correlated with the prognosis of lung cancer patients.MuRF1 and Atrogin-1 were correlated with SIRT1 in normal muscle tissue.The levels of SIRT1 in plasma were reduced in the cachexia group of lung cancer patients.The plasma SIRT1 levels were positively correlated with SMI and negatively correlated with the cachexia stage.These results suggest that SIRT1 is associated with muscle atrophy in cancer cachexia.Part Ⅱ:Ursolic acid alleviates myotube atrophy via activating SIRT1 in vitroObjective:UA is a pentacyclic triterpenoid natural compound that can alleviate muscle atrophy and reduce muscle catabolism in some disease models.However,the role and molecular mechanism of UA in cancer cachexia are unclear.This part of the study aimed to explore the role of UA on cancer cachexia and related mechanisms in vitro.Methods:TCM-C2C12 myotube atrophy model was constructed.C2C12 myotubes were treated with different concentrations of UA for 24,48,and 72 hours.The effect of UA on the viability of C2C12 myotubes was detected by CCK-8 assay,and the concentration and time of UA application in experiments were explored.C2C12 my otubes were treated with different concentrations of UA,the morphology of myotubes was observed with immunofluorescence staining,and the diameter of myotubes was measured by ImageJ software.RT-PCR and Western blot were used to detect the expression of muscular atrophyrelated proteins MuRF1 and Atrogin-1 in C2C12 my otube,as well as the expression of myosin heavy chain(MyHC),SIRT1,NOX4,FOXO1,FOXO3a,and further detected the expression of NF-κB and STAT3 signaling pathways.The molecular docking experiment was used to analyze the interaction mode of SIRT1 and UA.UA-stimulated C212 myotubes were treated with Ex-527,an inhibitor of SIRT1,to observe the effect of Ex-527 on C2C12 myotube atrophy and to detect its effect on the expression of related molecules.Results:CCK-8 experiments showed that UA was non-toxic to C2C12 myotubes at concentrations of 0.5,1.0,2.5,and 5.0 μM,and UA at concentrations of 1.0,2.5,and 5.0μM were selected for 48 hours for follow-up experiments.Immunofluorescence staining results revealed that UA showed a dose-dependent improvement of TCM-C2C12 myotube atrophy,increasing the diameter of myotubes.RT-PCR and Western blot results showed decreased expression of MuRF1 and Atrogin-1,increased expression of MyHC and SIRT1,and decreased expression of NOX4,FOXO1,FOXO3a,and phosphorylation levels of p65 and STAT3 in C2C12 myotube.Molecular docking experiments showed that UA formed hydrogen bond binding with the ASN-147 domain of SIRT1,with bond lengths of 3.0 ? and 3.1 ?,respectively,and the binding energy was-8.5 kcal/mol.Ex-527 treatment weakened the alleviation effect of UA on C2C12 myotube atrophy and changed the effect of UA on the expression of the above molecules.Conclusions:UA dose-dependently alleviated TCM-C2C12 myotube atrophy in vitro.The application of Ex-527,an inhibitor of SIRT1,could attenuate the mitigating effect of UA on C2C12 myotube atrophy,demonstrating that UA alleviates C2C12 myotube atrophy by activating SIRT1 and inhibiting NF-κB and STAT3 signaling pathways.Part Ⅲ:Ursolic acid relieves lung cancer cachexia in vivoObjective:The second part of the study found that UA alleviated C2C12 myotube atrophy by activating SIRT1 and inhibiting NF-κB and STAT3 signaling pathways in vitro.This part of the study aimed to explore the effect of UA on LLC tumor-bearing cachexia mice and the related mechanisms in vivo.Methods:This study consists of three parts animal experiments using healthy male C57BL/6 mice at 6 weeks of age.The first part of the experiment was to explore the effect of UA on cachexia in LLC tumor-bearing mice,and the mice were divided into 4 groups randomly:(1)normal control group,(2)LLC tumor-bearing group,(3)LLC tumorbearing+100 mg/kg UA-treated group,(4)LLC tumor-bearing+200 mg/kg UA-treated group.LLC cells were subcutaneously inoculated in the right back of mice to construct the LLC tumor-bearing cachexia mouse model.From the 7th day of inoculation of LLC cells,mice were given different doses of UA gavage every day and sacrificed after 14 days of administration.The second part of the experiment was to explore the mechanism by which UA alleviates cachexia in LLC tumor-bearing mice,and the mice were divided into 5 groups randomly:(1)normal control group,(2)LLC tumor-bearing group,(3)LLC tumorbearing+200 mg/kg UA-treated(i.g.)group,(4)LLC tumor-bearing group+10 mg/kg Ex527-treated(i.p.)group,(5)LLC tumor-bearing+200 mg/kg UA-treated+10 mg/kg Ex-527treated group.From day 7 of inoculation of LLC cells,groups(3)and(5)were given UA gavage,and groups(4)and(5)were given Ex-527 intraperitoneal injection every day.The groups were released after 14 days of administration.The third part of the experiment was to explore the mitigating effect of UA on cachexia in LLC tumor-bearing mice at different treatment time points.Mice were divided into 5 groups randomly:(1)normal control group,(2)LLC tumor-bearing group,(3)LLC tumor-bearing+UA-D6 group,(4)LLC tumorbearing+UA-D10 group,(5)LLC tumor-bearing+UA-D14 group,that is,mice are given daily UA gavage starting on days 6,10,and 14 of subcutaneous injection of LLC cells.The study endpoint for all animal experiments was day 21 after the inoculation of LLC cells.Weight and tumor size of mice were measured every two days,tumors and other tissues were collected at the study endpoint,and plasma was collected to determine the levels of TNF-α,IL-1β,and IL-6 by ELISA assay.Part of the gastrocnemius muscle was stained for HE staining,and the cross-sectional area of the myofibers was observed and measured.Part of the gastrocnemius muscle was used to detect the expression of MuRF 1,Atrogin-1,SIRT1,NOX4,FOXO1,FOXO3a,and the phosphorylation level of p65 and STAT3.Results:UA dose-dependently alleviated the cachexia symptoms of LLC tumor-bearing mice.UA improved the reduction of body weight and tumor-free body weight,weakened the reduction of gastrocnemius muscle and epididymal fat weight,and increased spleen weight,but had no effect on tumor weight.HE staining showed that UA increased the crosssectional area of gastrocnemius muscle fibers.ELISA showed that UA could reduce the levels of TNF-α,IL-1β,and IL-6 in the plasma of LLC tumor-bearing cachexia mice.Western blot detected that UA reduced the expression of MuRF 1 and Atrogin-1,increased the expression of SIRT1,inhibited the expression of NOX4,FOXO1,and FOXO3a,and reduced the phosphorylation levels of p65 and STAT3.The application of Ex-527 weakened the alleviating effect of UA on cachexia symptoms in LLC tumor-bearing mice and changed the effect of UA on the expression of related molecules.UA treatment given to LLC tumorbearing mice at the early or late stage of cancer cachexia alleviated the symptoms of cachexia,but the anti-cachexia effect of UA gradually weakened with the delay of application time.Conclusions:UA alleviated cachexia in LLC tumor-bearing mice in vivo,and the anticachexia effect of UA was weakened after the application of SIRT1 inhibitor Ex-527.These results indicate that UA alleviates muscle atrophy and improves cachexia by activating SIRT1 and inhibiting NF-κB and STAT3 signaling pathways.The application of UA in advanced cancer cachexia still has a protective effect on LLC tumor-bearing mice. |
PDF Full Text Request