| Background:The latest research shows that in 57 countries including China cancer has overtaken cardiovascular disease as the disease with the highest fatality rate.According to the latest forecast,the number of new cancer cases in the world will be 28.4 million in 2040,a significant increase of 47%compared with 2020.The mortality and incidence of cancer still shows an increasing trend in China.Therefore,cancer is still an important disease that threatens the life safety of our people.Cancer cachexia(CC)is the main complication of cancer.CC is mainly characterized by weight loss,specifically manifested as anorexia,progressive decline in skeletal muscle(with or without loss of adipose tissue),which further leads to functional decline and shortened survival time.The mechanism involved in CC is extensive and complicated and many factors lead to muscle protein hydrolysis rate exceeding protein synthesis rate,which leads to muscle atrophy(MA)and cachexia.Skeletal muscle fibrosis(SMF)is an irreversible state of muscle sclerosis and muscle function decline caused by the excessive accumulation of extracellular matrix(ECM).In cancer cachexia,the degree of fibrosis is negatively correlated with survival.Therefore,inhibiting skeletal muscle fibrosis in patients with cachexia may be an effective strategy to delay cachexiaCollagen type I alpha 1(COL1A1)is a classic marker of skeletal muscle fibrosis.The high expression of COL1A1 in the muscle tissue of patients with cachexia leads to an increase in muscle fibrosis.In diseases such as aortic fibrosis and pulmonary fibrosis,Runt-related transcription factor 2(Runx2)can directly bind to the promoter region of COL1A1 mRNA,promote COL1A1 mRNA transcription and lead to tissue fibrosis.Deltex 3L(DTX3L)is the main E3 ubiquitin ligase in the Notch pathway.A study has shown that low levels of methyltransferase 3(METTL3)in the brain arteriovenous malformations can inhibit the heterodimerization of DTX3L and DTX1 by regulating the mRNA stability of DTX3L and promote gene expression downstream of Notch pathway.And Notch can promote the occurrence of SMF through Runx2.Therefore,we speculate that DTX3L may have the ability to regulate Runx2 through the Notch pathway.Standardized nutritional support in the early stage of cancer is considered to be one of the effective strategies to alleviate CC.Our group has focused on the role of L-carnitine(LC)in the treatment of cancer cachexia for a long time.L-carnitine is a biologically active low-molecular-weight class of amino acids.About 95%of LC is stored in cardiac muscle and skeletal muscle and its concentration in muscle is as high as 600mg/g.The level of carnitine in muscle and serum of CC patients decreased and the progress of CC was related to the low level of serum carnitine.Our group’s previous study proved that L-carnitine improved the skeletal muscle atrophy induced by cachexia.However,there is no research on L-carnitine improving skeletal muscle fibrosis caused by cancer cachexia.Here,we will investigate whether intervention with L-carnitine at physiological concentrations can improve skeletal muscle fibrosis in cancer cachexia and whether this effect is related to DTX3L/Runx2/COL1A1 pathway.Methods:1.In vivo experiments:Colon cancer MC38 cells and C57 aged mice were used to establish cancer cachexia model in vivo.Sixteen C57 mice were divided into non-tumor-bearing group(4),tumor-bearing+non-intervention group(6)and tumor-bearing+L-carnitine group(6).After 6 days of tumor implantation,5mg/kg L-carnitine was used to intervene for 14 days and the body weight and tumor were measured every 3 days.Before and after the intervention of L-carnitine,the grip strength of mice was measured to evaluate the effects of L-carnitine on the body weight,tumor-free body weight,gastrocnemius weight and grip strength of mice in the cancer cachexia state.After the intervention,the right gastrocnemius was taken for HE staining,Masson staining and Sirius red staining;total protein was extracted from the left gastrocnemius and the effects of L-carnitine on DTX3L,Runx2 and COL1A1 proteins were detected.2.In vitro experiments:After NIH 3T3 cells adhered to the wall,they were treated with 10ng/ml TGF-β1 for 24 hours to establish an in vitro fibrosis model.In vitro fibrosis model,the effects of L-carnitine at different doses and different times on α-SMA、DTX3L、Runx2 and COL1A1 protein levels and on DTX3L、Runx2 and COL1A1 mRNA levels were detected;The effect of L-carnitine on Runx2/COL1A1 pathway was detected by transfection of Flag-Runx2 in NIH 3T3 cells;The effects of L-carnitine on the ubiquitination of Runx2 were detected in combination with CHX,MG132 and immunoprecipitation;Transfection of siRNA-DTX3L in NIH 3T3 cells to verify the role of DTX3L in fibrosis;GFP-DTX3L and Flag-Runx2 plasmids were co-transfected in HEK 293 cells and the interaction between DTX3L and Runx2 was detected by immunoprecipitation,immunofluorescence and Duolink;The effect of L-carnitine on the DTX3L/Runx2 pathway and the interaction between DTX3L and Runx2 was examined by transfecting siRNA-DTX3L,GFP-DTX3L and Flag-Runx2 in NIH 3T3 cells.Results:1 L-carnitine relieved skeletal muscle fibrosis induced by cancer cachexia(1)The results from the in vivo cachexia model showed that the grip strength decreased by-46.59±21.24g in tumor-bearing+non-intervention group,while in the tumor-bearing+L-carnitine group it only decreased by-5.5±15.18g,indicating that L-carnitine can improve the decrease in grip strength induced by cachexia(P<0.05);(2)The results of HE staining of gastrocnemius showed that the cross-sectional area of non tumor bearing group,tumor-bearing+non-intervention group and tumor-bearing+L-carnitine group was 488.61±46.72μm2,207.46±54.55μm2 and 434.54±113.84μm2,respectively.Statistical analysis showed that the muscle cross-sectional area of tumor-bearing+L-carnitine group and non tumor bearing group was significantly higher than that of the tumor-bearing+non-intervention group(P<0.05).Masson staining showed that the collagenous fiber area ratio in the non tumor bearing group,tumor-bearing+non-intervention group and tumor-bearing+L-carnitine group was 0.039±0.009,0.097±0.016 and 0.055±0.012.Statistical analysis showed that the collagenous fiber area ratio in the tumor-bearing+L-carnitine group and non tumor bearing group was significantly lower than that in tumor-bearing+non-intervention group(P<0.05).The results of Sirius Red showed that the collagenous fiber area ratio in the non tumor bearing group,tumor-bearing+non-intervention group and tumor-bearing+L-carnitine group was 0.054±0.023,0.145± 0.029 and 0.061±0.013,respectively.Statistics showed that the tumor-bearing+L-carnitine group and non tumor bearing group was significantly lower than that in tumor-bearing+non-intervention group(P<0.05).The above results indicated that L-carnitine can improve the reduction of cross-sectional area of mice gastrocnemius caused by cachexia and reduce the collagenous fiber area ratio;(3)L-carnitine inhibited COL1A1 protein expression:The expression level of COL1A1 protein in gastrocnemius of mice in tumor-bearing+L-carnitine group and non tumor bearing group was significantly lower than that in tumor bearing+non intervention group(P<0.05).In vitro fibrosis model,treatment with 600mg/L L-carnitine for 60min inhibited the expression of COL1A1 protein(P<0.05).After treatment for 6h,it can suppress the expression of α-SMA protein(P<0.05).Treatment with L-carnitine at 300-1200mg/L for 60min or 6h inhibited COL1A1 and α-SMA protein in a dose-dependent manner.Real Time PCR results showed that treatment with 600mg/L L-carnitine for 30min inhibited the expression of COL1A1 mRNA(P<0.05).2 L-carnitine regulated COL1A1 through Runx2 to alleviate skeletal muscle fibrosis.(1)L-carnitine inhibited the expression of Runx2 protein:The expression level of Runx2 protein in gastrocnemius of mice in tumor-bearing+L-carnitine group and non tumor bearing group was significantly lower than that in tumor bearing+non intervention group(P<0.05).In vitro fibrosis model,L-carnitine at 600mg/L for 60min could inhibit the expression of Runx2 protein(P<0.05).The dose effect was similar to that of COL1A1 and the inhibition of Runx2 protein by L-carnitine for 60min was dose-dependent;(2)L-carnitine regulated COL1A1 through Runx2:In vitro fibrosis model,overexpression of Runx2 can improve the inhibitory effect of L-carnitine on α-SMA protein,COL1A1 protein and mRNA;(3)L-carnitine promoted the ubiquitination of Runx2:Compared with CHX alone,the protein level of Runx2 in CHX combined with L-carnitine decreased faster,showing a significant difference at 30min(P<0.05).MG132 treatment can improve the inhibitory effect of L-carnitine on Runx2.After Flag-Runx2 was transfected and treated with L-carnitine and MG132,L-carnitine promoted an increase in Runx2 ubiquitination levels.3 L-carnitine alleviated skeletal muscle fibrosis by regulating Runx2 through DTX3L(1)L-carnitine regulated DTX3L to restore it to normal level:The expression level of DTX3L protein in gastrocnemius of mice in tumor bearing+L-carnitine group and non tumor bearing group was significantly higher than that in tumor bearing+non intervention group(P<0.05).In vitro fibrosis model,TGF-β1 promoted the expression of DTX3L protein.After 60min treatment with 600mg/L L-carnitine,DTX3L returned to normal level.Similar to COL1A1 and Runx2 results,there was a dose dependence;(2)L-carnitine regulated fibrosis via DTX3L:In vitro fibrosis model,after inhibiting DTX3L,the inhibitory effect of L-carnitine on α-SMA protein,COL1A1 protein and mRNA can be improved;(3)In HEK 293 cells,DTX3L was confirmed to interact with Runx2 in both the nucleus and the cytoplasm;In vitro fibrosis model,L-carnitine promoted the interaction of DTX3L with Runx2;(4)L-carnitine regulated Runx2 via DTX3L:After inhibiting DTX3L,the downregulation effect of L-carnitine on Runx2 can be alleviated and the ubiquitination induction of Runx2 by L-carnitine can be reduced;Compared with transfection of Runx2 plasmid alone,co-transfection of Runx2 and DTX3L plasmid could restore the inhibitory effect of L-carnitine on α-SMA protein,COL1A1 protein and mRNA.Conclusion:L-carnitine at physiological concentration can improve the decrease in grip strength and cross sectional area of gastrocnemius in mice caused by cancer cachexia and alleviate skeletal muscle fibrosis;In vitro model,it is confirmed that DTX3L is the E3 ubiquitin ligase of Runx2 and L-carnitine regulates Runx2 ubiquitination through DTX3L resulting in a decrease in COL1A1,ultimately achieving the goal of alleviating fibrosis.Our research provides a theoretical basis for L-carnitine as a clinical drug against skeletal muscle fibrosis. |
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