Therapeutic effect of psoralen on muscle atrophy induced by tumor necrosis factor-α

Document Type: Original Article


1 Intensive Care Unit, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510405, China

2 Department of Spleen-Stomach, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510405, China

3 The First Clinical School, Guangzhou University of Chinese Medicine, Guangzhou, 510405, China

4 Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou, 510405, China


Objective(s): To observe and determine the effect and mechanism of psoralen on tumor necrosis factor-α (TNF-α)-induced muscle atrophy.
Materials and Methods: Three sets of C2C12 cells, including blank control, TNF-α (10 or 20 ng/ml) treatment and a TNF-α (10 or 20 ng/ml) plus psoralen (80 μM) administration were investigated. Cell viability was assessed using Cell Counting Kit-8 (CCK-8) assay. Western blot analysis was used to detect protein expression of atrophic markers. Flowcytometry was used to observe the effect of psoralen on apoptosis. A quantitative real-time PCR (qRT-PCR) assay was performed to detect the mRNA level of miR-675-5P.
Results: TNF-α (1, 10, 20 and 100 ng/ml) treatment inhibited C2C12 myoblast viability (P<0.001), while 24 hr of psoralen administration increased the viability, and lowered TNF-α cytotoxicity (P<0.001). MURF1, MAFbx, TRIM62 and GDF15 expressions were significantly increased in TNF-α (10 ng/ml or 20 ng/ml)-treated group (P<0.001), and psoralen could significantly decrease the expression of these proteins (P<0.001). Apoptotic rate of C2C12 myoblasts was increased after TNF-α (10 ng/ml and 20 ng/ml) treatment, and was significantly decreased after psoralen treatment (P<0.001). miR-675-5P was increased in TNF-α-treated C2C12 myoblasts compared to control group, and it was significantly decreased after psoralen treatment.
Conclusion: Psoralen could reduce TNF-α-induced cytotoxicity, atrophy and apoptosis in C2C12 myoblasts. The therapeutic effect of psoralen may be achieved by down-regulating miR-675-5P.


1. Acharyya S, Guttridge DC. Cancer cachexia signaling pathways continue to emerge yet much still points to the proteasome. Clin Cancer Res 2007; 13:1356-1361.
2. Jackman RW, Kandarian SC. The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 2004; 287:C834-843.
3. Li H, Malhotra S, Kumar A. Nuclear factor-kappa B signaling in skeletal muscle atrophy. J Mol Med (Berl) 2008; 86:1113-1126.
4. Hudson MB, Smuder AJ, Nelson WB, Bruells CS, Levine S, Powers SK. Both high level pressure support ventilation and controlled mechanical ventilation induce diaphragm dysfunction and atrophy. Crit Care Med 2012; 40:1254-1260.
5. McClung JM, Judge AR, Powers SK, Yan Z. p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting. Am J Physiol Cell Physiol 2010; 298:C542-9.
6. Roberts-Wilson TK, Reddy RN, Bailey JL, Zheng B, Ordas R, Gooch JL, et al. Calcineurin signaling and PGC-1alpha expression are suppressed during muscle atrophy due to diabetes. Biochim Biophys Acta 2010; 1803:960-967.
7. Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, et al. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. Faseb j 2004; 18:39-51.
8. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, et al. Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. Faseb J 2007; 21:140-155.
9. Powers SK, Smuder AJ, Judge AR. Oxidative stress and disuse muscle atrophy: cause or consequence? Curr Opin Clin Nutr Metab Care 2012; 15:240-245.
10. Price SR, Gooch JL, Donaldson SK, Roberts-Wilson TK. Muscle atrophy in chronic kidney disease results from abnormalities in insulin signaling. J Ren Nutr 2010; 20:S24-28.
11. Chen CH, Hwang TL, Chen LC, Chang TH, Wei CS, Chen JJ. Isoflavones and anti-inflammatory constituents from the fruits of Psoralea corylifolia. Phytochemistry 2017; 143:186-193.
12. Panno ML, Giordano F. Effects of psoralens as anti-tumoral agents in breast cancer cells. World J Clin Oncol 2014; 5:348-358.
13. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116:281-297.
14. Kukreti H, Amuthavalli K, Harikumar A, Sathiyamoorthy S, Feng PZ, Anantharaj R, et al. Muscle-specific microRNA1 (miR1) targets heat shock protein 70 (HP70) during dexamethasone-mediated atrophy. J Biol Chem 2013; 288:6663-6678.
15. Wada S, Kato Y, Okutsu M, Miyaki S, Suzuki K, Yan Z, et al. Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy. J Biol Chem 2011; 286:38456-65.
16. Wang XH, Hu Z, Klein JD, Zhang L, Fang F, Mitch WE. Decreased miR-29 suppresses myogenesis in CKD. J Am Soc Nephrol 2011; 22:2068-2076.
17. Dey BK, Pfeifer K, Dutta A. The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration. Genes Dev 2014; 28:491-501.
18. Guller I, Russell AP. MicroRNAs in skeletal muscle: their role and regulation in development, disease and function. J Physiol 2010; 588:4075-4087.
19. Spate U, Schulze PC. Proinflammatory cytokines and skeletal muscle. Curr Opin Clin Nutr Metab Care 2004; 7:265-269.
20. Tracey KJ, Cerami A. Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol 1993; 9:317-343.
21. Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The role of the ubiquitin-proteasome pathway. N Engl J Med 1996; 335:1897-1905.
22. Mitch WE, Medina R, Grieber S, May RC, England BK, Price SR, et al. Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. J Clin Invest 1994; 93:2127-2133.
23. Price SR, Bailey JL, Wang X, Jurkovitz C, England BK, Ding X, et al. Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription. J Clin Invest 1996; 98:1703-1708.
24. Wing SS, Goldberg AL. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting. Am J Physiol 1993; 264:E668-76.
25. Zhang L, Rajan V, Lin E, Hu Z, Han HQ, Zhou X, et al. Pharmacological inhibition of myostatin suppresses systemic inflammation and muscle atrophy in mice with chronic kidney disease. Faseb J 2011; 25:1653-1663.
26. Li YP, Reid MB. NF-kappaB mediates the protein loss induced by TNF-alpha in differentiated skeletal muscle myotubes. Am J Physiol Regul Integr Comp Physiol 2000; 279:R1165-1170.
27. Li YP, Schwartz RJ, Waddell ID, Holloway BR, Reid MB. Skeletal muscle myocytes undergo protein loss and reactive oxygen-mediated NF-kappaB activation in response to tumor necrosis factor alpha. Faseb J 1998; 12:871-880.
28. Srivastava AK, Qin X, Wedhas N, Arnush M, Linkhart TA, Chadwick RB, et al. Tumor necrosis factor-alpha augments matrix metalloproteinase-9 production in skeletal muscle cells through the activation of transforming growth factor-beta-activated kinase 1 (TAK1)-dependent signaling pathway. J Biol Chem 2007; 282:35113-35124.
29. Langen RC, Schols AM, Kelders MC, Wouters EF, Janssen-Heininger YM. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB. Faseb j 2001; 15:1169-1180.
30. Miller SC, Ito H, Blau HM, Torti FM. Tumor necrosis factor inhibits human myogenesis in vitro. Mol Cell Biol 1988; 8:2295-2301.
31. Cai D, Frantz JD, Tawa NE, Jr TN, Melendez PA, Oh BC, Lidov HG, et al. IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 2004; 119:285-298.
32. Acharyya S, Villalta SA, Bakkar N, Bupha-Intr T, Janssen PM, Carathers M, et al. Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy. J Clin Invest 2007; 117:889-901.
33. Hunter RB, Kandarian SC. Disruption of either the Nfkb1 or the Bcl3 gene inhibits skeletal muscle atrophy. J Clin Invest 2004; 114:1504-1511.
34. Mourkioti F, Kratsios P, Luedde T, Song YH, Delafontaine P, Adami R, et al. Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration. J Clin Invest 2006; 116:2945-2954.
35. Li YP, Reid MB. NF-kappaB mediates the protein loss induced by TNF-alpha in differentiated skeletal muscle myotubes. Am J Physiol Regul Integr Comp Physiol 2000; 279:R1165-1170.
36. Stasko SA, Hardin BJ, Smith JD, Moylan JS, Reid MB. TNF signals via neuronal-type nitric oxide synthase and reactive oxygen species to depress specific force of skeletal muscle. J Appl Physiol (1985) 2013; 114:1629-1636.
37. Glass DJ. Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell Biol 2005; 37:1974-84.
38. Ventadour S, Attaix D. Mechanisms of skeletal muscle atrophy. Curr Opin Rheumatol 2006; 18:631-635.
39. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, et al. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 2001; 294:1704-1708.
40. Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci U S A 2001; 98:14440-14445.
41. McNab FW, Rajsbaum R, Stoye JP, O’Garra A. Tripartite-motif proteins and innate immune regulation. Curr Opin Immunol 2011; 23:46-56.
42. Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, et al. The tripartite motif family identifies cell compartments. Embo J 2001; 20:2140-2151.
43. Langhans C, Weber-Carstens S, Schmidt F, Hamati J, Kny M, Zhu X, et al. Inflammation-induced acute phase response in skeletal muscle and critical illness myopathy. PloS one 2014; 9:e92048.
44. Uchil PD, Hinz A, Siegel S, Coenen-Stass A, Pertel T, Luban J, et al. TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. J Virol 2013; 87:257-272.
45. Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, et al. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A 1997; 94:11514-11519.
46. Johnen H, Lin S, Kuffner T, Brown DA, Tsai VW, Bauskin AR, et al. Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1. Nat Med 2007; 13:1333-1340.
47. Tsai VW, Husaini Y, Manandhar R, Lee-Ng KK, Zhang HP, Harriott K, et al. Anorexia/cachexia of chronic diseases: a role for the TGF-beta family cytokine MIC-1/GDF15. J Cachexia Sarcopenia Muscle 2012; 3:239-243.
48. Welsh JB, Sapinoso LM, Kern SG, Brown DA, Liu T, Bauskin AR, et al. Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum. Proc Natl Acad Sci U S A 2003; 100:3410-3415.
49. Chung HK, Ryu D, Kim KS, Chang JY, Kim YK, Yi H, et al. Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J Cell Biol 2017; 216:149-165.
50. Kalko SG, Paco S, Jou C, Rodriguez MA, Meznaric M, Rogac M, et al. Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies. BMC genomics 2014; 15:91.
51. Ito T, Nakanishi Y, Yamaji N, Murakami S, Schaffer SW. Induction of Growth Differentiation Factor 15 in Skeletal Muscle of Old Taurine Transporter Knockout Mouse. Biol Pharm Bull 2018; 41:435-439.
52. Zhou YW, Zhang H, Duan CJ, Gao Y, Cheng YD, He D, et al. miR-675-5p enhances tumorigenesis and metastasis of esophageal squamous cell carcinoma by targeting REPS2. Oncotarget 2016; 7:30730-30747.