| Background and Objectives:1.Definition and epidemiology of cachexiaCachexia is composed of two Latin roots,Kakos(bad)and Hexia(state),indicating the extremely poor state of the patients experiencing this condition.Cachexic patients usually present with rapid weight loss,extreme muscle loss,anorexia,nausea,weakness,poor physical fitness,and ultimately,multiple organ dysfunction syndromes.It has been thought that cachexia is the end of many diseases,frequently signaling the end of life.With the deepening research about cachexia,it is expected to that the early stages of cachexia will be identified,providing opportunities for clinical intervention.The definition and classification of cancer cachexia published in Lancet Oncology in 2011 pointed out that cachexia is a multifactorial syndrome with a progressive decline in skeletal muscle mass,with or without fat loss,and cannot be supported by conventional nutrition.[1].Diseases which cause cachexia include malignant tumors,chronic heart failure,chronic renal failure,AIDS,severe trauma,malabsorption,and severe sepsis.Among them,malignant tumors are the most common cachexia,which is called tumor cachexia.Pathogenic factors activate various signal pathways in cells and secretion of various negative cytokines in the body through various pathways,which cause complex negative changes in the metabolism of the body.At the same time,the patient’s anorexia is obvious,and the various nutrients are digested and absorbed,leading to further metabolic disorders in the body and entering the state of cachexia.Patients with digestive tract cancer have a high incidence of cachexia,and the advanced cancers such as lung cancer,ovarian cancer,prostate cancer,and breast cancer also exhibit a state of cachexia.2.Skeletal muscle loss is the core feature of cachexiaIt has been reported that about 60%-80% of patients with advanced tumors will develop cachexia.[2].In fact,cachexia can occur even in the early stages of tumor progression.Fearon et al.[3] indicated that cancer cachexia can be divided into three phases: precachexia,cachexia,and refractory cachexia.Muscle loss is the core feature of cachexia.However,because accurate measurement of the body composition is not commonly performed in most clinics,weight loss is the most important clinical manifestation of cachexia.3.Molecular mechanism of muscle atrophy of cachexiaAdult skeletal muscle is always in a balance between muscle protein synthesis and degradation,while muscle loss in patients with cachexia is mainly due to the reduction or acceleration of muscle protein synthesis and degradation.Body skeletal muscle protein degradation is mediated by three main signaling pathways: the lysosomal protease,calcium-dependent protease,and ATP-ubiquitin-proteasome pathways.Studies have shown that MuRF1(muscle ring finger-containing protein 1,also known as TRIM63)and MaFbx(muscle atrophy F-box protein,also known as atrogin-1,FBXO32)in the ATP-ubiquitin-proteasome pathway are closely related to the degradation of skeletal muscle protein.Patients with cachexia produce many pro-inflammatory cytokines,including tumor necrosis factor-α(TNF-α),proteolysis-inducing factor(PIF),interleukin-1(IL-1),interleukin-6(IL-6)and γ-interferon(interferon-γ,IFN-γ).These cytokines act directly on skeletal muscle cells,bind to their corresponding receptors,and activate multiple signaling pathways within the cell,further promoting protein degradation via the ATP-ubiquitin-proteasome pathway.Another important signaling pathway associated with skeletal muscle protein synthesis and degradation is the insulin growth factor-1(IGF-1)pathway.PI3K/AKT is the most prominent molecule downstream of IGF-1,and PI3K/AKT signaling leads to the activation of glycogen synthase kinase 3 beta(GSK-3β),mammalian target of rapamycin(mTOR),p70S6,etc.,which also promote muscle synthesis,as well as FOXOs family molecules and E3 enzyme-related ubiquitin,which promote muscle cell breakdown and muscle atrophy.In addition,some scholars have found that myostatin plays a negative regulatory role in muscle cell growth and differentiation.Myostatin causes muscle atrophy and muscle protein degradation in a similar way to the IGF-1-PI3K-AKT pathway.It mainly activates the two ubiquitination molecules of MuRF1 and MaFbx by regulating molecules such as AKT and FOXOs downstream of PI3 K,promoting muscle protein degradation and muscle reduction.In addition,some scholars have found that myostatin plays a negative regulatory role in muscle cell growth and differentiation.Myostatin causes muscle atrophy and muscle protein degradation in a manner similar to the IGF-1-PI3K-AKT pathway.It mainly activates the two ubiquitination molecules(MuRF1 and MaFbx)by regulating molecules such as AKT and FOXOs downstream of PI3 K,promoting muscle protein degradation and leading to muscle reduction.At present,there is no effective treatment that can reverse cachexia.Clinically,the main goals are to increase anabolism,reduce muscle degradation,correct inflammation and provide nutritional support,helping to control cachexia to improve the body’s functions to extend the patient’s life expectancy.4.Potential effects of L-carnitineL-carnitine is an amino acid whose main function is to promote the β-oxidation of long-chain fatty acids.It also plays an important role in cell energy metabolism.Some research has shown that L-carnitine can improve protein metabolism,promote protein synthesis,improve heart function and myocardial ischemia,clear free radicals,and protect the liver.In addition,L-carnitine also has antioxidant effects.L-carnitine deficiency can cause symptoms such as fatigue,weakness,angina,and arrhythmia.Therefore,L-carnitine is often used in the treatment of diseases affecting the cardiovascular system,nervous system,liver,and kidneys,but few studies have explored the effects of L-carnitine on the development of cachexia in cancer patients.Here,we show that L-carnitine has beneficial effects against cancer cachexia,and suggest the possible mechanism by which it exerts these effects in vitro.Methods:1.To examine whether L-carnitine can promote mouse skeletal muscle C2C12 cell differentiation.An in vitro model of cachexia was established by treat C2C12 cells with 100 ng/ml TNF-α during C2C12 cell differentiation.C2C12 cells were treated with L-carnitine with or without TNF-α.The morphology of myotubes was observed to determine the effects of L-carnitine on cell differentiation.Crystal violet staining was performed to observe the differentiation of C2C12 myoblasts.2.To verify the mechanism(s)by which L-carnitine ameliorates muscle atrophy via the AKT/p70S6 and AKT/FOXO3 a pathways.C2C12 cells were treated with L-carnitine with or without TNF-α during differentiation.After protein extraction,the expression levels of AKT/p70S6 K and AKT/FOXO3 a pathway-related molecules were detected by Western blotting analyses.3.The mechanism and verification of carnitine that regulates muscle protein degradation via the pathway of AKT/p70S6 and AKT/FOXO3aThe diameter and morphology of C2C12 myotubes were examined following treatment with L-carnitine with or without si-RNA against AKT.Then,the CPT1,AKT,P-AKT,p70S6 K,p-p70S6 K,FOXO3a,p-FOXO3 a,MAFbx and MuRF1 protein levels were determined by Western blotting.The specific experimental methods are as follows:A.C2C12 cells were cultured and induced to differentiate using basic cell culture techniques;B.Crystal violet staining to observe the degree of differentiation of C2C12 myoblasts;C.The effect of different concentrations of carnitine on the morphology of myocytes was observed by inverted microscope;D.Constructing inhibitor of AKT-siRNA;E.Changes in the degree of cell differentiation were observed after the addition of AKT inhibitor siRNA;F.The effects of carnitine on the expression of CPT1,AKT,P-AKT,S6,P-S6,FOXO3 a,P-FOXO3 a,MuRF1,MaFbx and Myostatin were detected by Western Blotting technique.Results:1.L-carnitine promotes myocyte hypertrophy and inhibits TNF-α-induced muscle fiber atrophyMouse skeletal muscle C2C12 cells were treated with TNF-α to generate an in vitro research model of skeletal muscle atrophy.Our experimental results showed that the induced skeletal muscle myotubes showed different morphological changes after the induction of C2C12 cells for 3 days followed by treatment with TNF-α and L-carnitine for 48 hours.Of note,the TNF-α-treated group had the smallest muscle fiber diameter,while the largest diameters were found in the control group,the 100 μg/mL L-carnitine with TNF-ɑ group and the 1000 μg/mL L-carnitine with TNF-ɑ group.Western blotting showed that the protein expression levels of MuRF1 and MaFbx were significantly lower in the 1000 μg/mL L-carnitine intervention group(L-carnitine + TNF-α)than in the untreated cachexia(TNF-α)group.2.L-carnitine induces the activation of the AKT/p70S6 K pathway,decreases FOXO3 a protein expression,and promotes FOXO3 a phosphorylationThe thickening of skeletal muscle myotubes is mainly related to the activation of the AKT/p70S6 K pathway in myocytes.Our study showed that the expression levels of P-AKT,P-p70S6 and P-FOXO3 a in the upstream and downstream of the AKT pathway were higher than those in the cachexia group after treatment with either 100 μg/mL or 1000 μg/mL L-carnitine.The ratios of P-AKT/AKT,P-p70S6K/p70S6 K,and P-FOXO3a/FOXO3 a were increased,and the increase in the group treated with the higher dose of L-carnitine(1000 μg/mL)was more obvious.Phase-phase studies of the cells with the 1000 μg/mL L-carnitine intervention showed that L-carnitine promoted the expression of some molecules in the AKT/p70S6 K within 24 hours.Of note,the increase was time-dependent,and the peak induction of P-AKT and P-p70S6 protein expression was reached 24 hours after the L-carnitine intervention.3.L-carnitine inhibits TNF-ɑ-induced muscle fiber atrophy via the AKT pathwayTNF-ɑ-induced C2C12 cells were treated with an AKT inhibitor,siRNA-AKT,and then treated with L-carnitine at 1000 μg/mL.The results showed that: in the L-carnitine intervention group,the diameter of the muscle fibers was significantly larger than that of the non-intervention group;the muscle fibers of the siRNA-AKT-treated group were thinner than those of the blank plasmid control group.Western blotting showed that the expression of the corresponding proteins downstream of the AKT/p70S6 K and AKT/FOXO3 a pathways was reduced in the siRNA-AKT-treated experimental group.In the cells treated with siRNA plus L-carnitine,the expression of molecules that promote muscle fiber thickening(such as p70S6K)was increased,and the expression of molecules that inhibit muscle fiber synthesis(such as FOXO3a)was decreased.Conclusions:L-carnitine inhibit muscle fiber atrophy in differentiated C2C12 cells simulating cachexia,and the AKT/p70S6 and AKT/FOXO3 a pathways are involved in these protective effects of L-carnitine on cachexia.However,these findings are preliminary,and the role of L-carnitine deserves further study. |
