Effect of Curcumin Supplementation on Rat Skeletal Muscle Morphology and AMPK Levels: Study Protocol for a Randomized Controlled Trial

Abstract

Background: Curcumin has been investigated as a potential natural solution to prevent or even treat skeletal muscle decline. There are a number of externally sourced finished products (ie, those imported from other countries) containing curcumin, but high cost limits their usage. The emerging research and development of locally sourced curcumin is an opportunity to produce high-quality oral supplements comparable to existing imported products.

Objective: The primary purpose of this study is to determine the effects of oral administration of a local curcumin formulation on skeletal muscle using an animal model that similarly demonstrated the course of human sarcopenia.

Methods: Purpose-bred 11- to 12-week-old female Sprague Dawley (SD) rats will be used in this study. SD rats are extensively used in animal models of human diseases and conditions such as diabetes, obesity and sarcopenia. Female rats have been selected because they do not demonstrate more temperature or activity variance and have more stable behavior compared to males. To simulate sarcopenia in this animal model, the tail suspension (TS) method was utilized. The TS method involves decreased hind limb function by suspending the animal’s tail for the duration of treatment. The laboratory animals will be randomized to receive any of the four treatments: (1) low-dose curcumin + vehicle; (2) high-dose curcumin + vehicle; (3) vehicle only; and (4) control (distilled water). The interventions will be subdivided into two: 2-week treatment and 4-week treatment. The gastrocnemius muscles on both sides will be excised and weighed, and the muscle tissues subjected to rapid freezing in acetone-dry ice and sliced into 10 μm-thick sections for staining. Examination of muscle architecture and computation of atrophy factors will be performed. The presence of connective tissue, fat tissue and number of atrophic muscle cells will be determined. Accurate quantitative detection of the rat total AMP (adenosine monophosphate)-Activated Protein Kinase will be performed in the gastrocnemius muscle tissue utilizing the enzyme-linked immunosorbent assay kit. 

Keywords: curcumin, sarcopenia, animal model 

 

  1. Cruz-Jentoft AJ, Bahat G, Bauer J, Boirie Y, Bruyère O, Cederholm T, et al. Sarcopenia: revised European consensus on definition and diagnosis. Age Ageing [Internet]. 2019;48(4):601. Available from: http://dx.doi.org/10.1093/ageing/afz046 
  2. Chen L-K, Liu L-K, Woo J, Assantachai P, Auyeung T-W, Bahyah KS, et al. Sarcopenia in Asia: consensus report of the Asian Working Group for Sarcopenia. J Am Med Dir Assoc [Internet]. 2014;15(2):95–101. Available from: http://dx.doi.org/10.1016/j.jamda.2013.11.025 
  3. Chen L-K, Woo J, Assantachai P, Auyeung T-W, Chou M-Y, Iijima K, et al. Asian Working Group for sarcopenia: 2019 consensus update on sarcopenia diagnosis and treatment. J Am Med Dir Assoc [Internet]. 2020;21(3):300-307.e2. Available from: http://dx.doi.org/10.1016/j.jamda.2019.12.012 
  4. Talbot J, Maves L. Skeletal muscle fiber type: using insights from muscle developmental biology to dissect targets for susceptibility and resistance to muscle disease. Wiley Interdiscip Rev Dev Biol [Internet]. 2016;5(4):518–34. Available from: http://dx.doi.org/10.1002/wdev.230 
  5. Kim TN, Choi KM. Sarcopenia: definition, epidemiology, and pathophysiology. J Bone Metab [Internet]. 2013;20(1):1–10. Available from: http://dx.doi.org/10.11005/jbm.2013.20.1.1 
  6. Pascual-Fernández J, Fernández-Montero A, Córdova-Martínez A, Pastor D, Martínez-Rodríguez A, Roche E. Sarcopenia: Molecular pathways and potential targets for intervention. Int J Mol Sci [Internet]. 2020;21(22):8844. Available from: http://dx.doi.org/10.3390/ijms21228844 
  7. Bagherniya M, Mahdavi A, Shokri-Mashhadi N, Banach M, Von Haehling S, Johnston TP, et al. The beneficial therapeutic effects of plant-derived natural products for the treatment of sarcopenia. J Cachexia Sarcopenia Muscle [Internet]. 2022;13(6):2772–90. Available from: http://dx.doi.org/10.1002/jcsm.13057 
  8. Vargas-Mendoza N, Madrigal-Santillán E, Álvarez-González I, Madrigal-Bujaidar E, Anguiano-Robledo L, Aguilar-Faisal JL, et al. Phytochemicals in skeletal muscle health: Effects of curcumin (from Curcuma longa Linn) and sulforaphane (from Brassicaceae) on muscle function, recovery and therapy of muscle atrophy. Plants [Internet]. 2022;11(19):2517. Available from: http://dx.doi.org/10.3390/plants11192517 
  9. Sahebkar A, Serban M-C, Ursoniu S, Banach M. Effect of curcuminoids on oxidative stress: A systematic review and meta-analysis of randomized controlled trials. J Funct Foods [Internet]. 2015;18:898–909. Available from: http://dx.doi.org/10.1016/j.jff.2015.01.005 
  10. Hewlings SJ, Kalman DS. Curcumin: A review of its effects on human health. Foods [Internet]. 2017;6(10). Available from: http://dx.doi.org/10.3390/foods6100092 
  11. Lyu W, Kousaka M, Jia H, Kato H. Effects of turmeric extract on age-related skeletal muscle atrophy in senescence-accelerated mice. Life (Basel) [Internet]. 2023;13(4). Available from: http://dx.doi.org/10.3390/life13040941 
  12. Brower M, Grace M, Kotz CM, Koya V. Comparative analysis of growth characteristics of Sprague Dawley rats obtained from different sources. Lab Anim Res [Internet]. 2015;31(4):166–73. Available from: http://dx.doi.org/10.5625/lar.2015.31.4.166 
  13. Nemoto A, Goyagi T. Tail suspension is useful as a sarcopenia model in rats. Lab Anim Res [Internet]. 2021;37(1):7. Available from: http://dx.doi.org/10.1186/s42826-020-00083-9 
  14. Shu H, Huang Y, Zhang W, Ling L, Hua Y, Xiong Z. An integrated study of hormone-related sarcopenia for modeling and comparative transcriptome in rats. Front Endocrinol (Lausanne) [Internet]. 2023;14:1073587. Available from: http://dx.doi.org/10.3389/fendo.2023.1073587 
  15. Smarr B, Kriegsfeld LJ. Female mice exhibit less overall variance, with a higher proportion of structured variance, than males at multiple timescales of continuous body temperature and locomotive activity records. Biol Sex Differ [Internet]. 2022;13(1):41. Available from: http://dx.doi.org/10.1186/s13293-022-00451-1 
  16. Republic Act No. 3639: An Act Creating the Bureau of Animal Industry, Defining its Powers and Functions [Internet]. Philippine Veterinary Medical Association. [cited 2025]. Available from: https://www.pvma.com.ph/republic-acts#tab-1-5 
  17. Trivedi MK, Branton A, Trivedi D, Jana S. Prevention of aging and improvement of longevity and life-span in D-galactose induced aging rats after treatment with the biofield energy per se and biofield treated proprietary test formulation. J Ageing Res Healthc [Internet]. 2020;3(1):48–57. Available from: http://dx.doi.org/10.14302/issn.2474-7785.jarh-20-3425 
  18. Baek K-W, Jung Y-K, Kim J-S, Park JS, Hah Y-S, Kim S-J, et al. Rodent model of muscular atrophy for sarcopenia study. J Bone Metab [Internet]. 2020;27(2):97–110. Available from: http://dx.doi.org/10.11005/jbm.2020.27.2.97 
  19. Palus S, Springer JI, Doehner W, von Haehling S, Anker M, Anker SD, et al. Models of sarcopenia: Short review. Int J Cardiol [Internet]. 2017;238:19–21. Available from: http://dx.doi.org/10.1016/j.ijcard.2017.03.152 
  20. Manzoor M, Raza S. Proficient handling and restraint of the laboratory animal rat (rattus norvegicus) facilitate essential biochemical and molecular level studies in biomedical sciences. IOSR Journal of Pharmacy and Biological Sciences. 2013;6:21–33.
  21. ARRP Guideline 20: Guidelines for the Housing of Rats in Scientific Institutions [Internet]. Orange NSW, AU: Animal Welfare Branch, NSW Department of Primary Industries; 2008 [cited 2025]. Available from: https://www.animalethics.org.au/__data/assets/pdf_file/0014/222512/housing-rats-scientific-institutions.pdf 
  22. Turner PV, Brabb T, Pekow C, Vasbinder MA. Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci. 2011;50(5):600–13.
  23. Standard Operating Procedure #10. Standard Operating Procedures for Oral Gavage in Mice and Rats [Internet]. Washington State University Institutional Animal Care and Use Committee (WSU IACUC). 2021 [cited 2025]. Available from: https://iacuc.wsu.edu/documents/2016/06/wsu_sop_10.pdf/ 
  24. HEALTH EFFECTS. In: Toxicological Profile for Propylene Glycol [Internet]. Atlanta (GA): Agency for Toxic Substances and Disease Registry (US); 1997 [cited 2025]. p.7–65. Available from: https://www.ncbi.nlm.nih.gov/books/NBK598030/ 
  25. McMartin K. Propylene Glycol. In: Encyclopedia of Toxicology [Internet]. 3rd ed. Elsevier; 2014. p.1113–6. Available from: http://dx.doi.org/10.1016/b978-0-12-386454-3.01029-0 
  26. Robinson NB, Krieger K, Khan FM, Huffman W, Chang M, Naik A, et al. The current state of animal models in research: A review. Int J Surg [Internet]. 2019;72:9–13. Available from: http://dx.doi.org/10.1016/j.ijsu.2019.10.015
  27. Johnson PD, Besselsen DG. Practical aspects of experimental design in animal research. ILAR J [Internet]. 2002;43(4):202–6. Available from: http://dx.doi.org/10.1093/ilar.43.4.202 
  28. Sahin K, Pala R, Tuzcu M, Ozdemir O, Orhan C, Sahin N, et al. Curcumin prevents muscle damage by regulating NF-κB and Nrf2 pathways and improves performance: an in vivo model. J Inflamm Res [Internet]. 2016;9:147–54. Available from: http://dx.doi.org/10.2147/JIR.S110873 
  29. Rinkunaite I, Simoliunas E, Alksne M, Dapkute D, Bukelskiene V. Anti-inflammatory effect of different curcumin preparations on adjuvant-induced arthritis in rats. BMC Complement Med Ther [Internet]. 2021;21(1):39. Available from: http://dx.doi.org/10.1186/s12906-021-03207-3 
  30. Hocking AJ, Elliot D, Hua J, Klebe S. Administering fixed oral doses of curcumin to rats through voluntary consumption. J Am Assoc Lab Anim Sci [Internet]. 2018;57(5):508–12. Available from: http://dx.doi.org/10.30802/AALAS-JAALAS-17-000143 
  31. Guevara BQ. A Guidebook to Plant Screening; Phytochemical and Biological. Manila, Philippines: Research Center for the Natural and Applied Sciences, University of Santo Tomas; 2004.
  32. Animal welfare regulations require that the Attending Veterinarian (AV) shall provide guidance to the Principal Investigator (PI) and staff regarding euthanasia, and that the Institutional Animal Care and Use Committee (IACUC) must approve the method of euthanasia to be utilized for research animals. [Internet]. University of Nebraska Omaha (UNOMAHA) Institutional Animal Care and Use Committee. 2021 [cited 2025]. Available from: https://www.unomaha.edu/office-of-research-and-creative-activity/research-compliance/iacuc/iacuc-policies/policy-for-euthanasia-and-carcass-disposal.pdf 
  33. Sivula CP, Suckow MA. Euthanasia. In: Management of Animal Care and Use Programs in Research, Education, and Testing [Internet]. 2nd ed. Boca Raton : Taylor & Francis, 2018. CRC Press; 2017. p.827–40. Available from: http://dx.doi.org/10.1201/9781315152189-35 
  34. Ruehl-Fehlert C, Kittel B, Morawietz G, Deslex P, Keenan C, Mahrt CR, et al. Revised guides for organ sampling and trimming in rats and mice--part 1. Exp Toxicol Pathol [Internet]. 2003;55(2–3):91–106. Available from: http://dx.doi.org/10.1078/0940-2993-00311 
  35. Çinar İ, Bozoğlan M, Aytekin K, Esenyel D, Esenyel CZ. The histopathological effects of sleep disorders on striated muscle in rats. Saudi Med J [Internet]. 2023;44(4):355–62. Available from: http://dx.doi.org/10.15537/smj.2023.44.4.20220714 
  36. Rat AMPK (phosphorylated adenosine monophosphate activated protein kinase) ELISA kit [Internet]. FineTest ELISA Kit | FineTest Antibody | FineTest®. Wuhan Fine Biotech Co., Ltd.; 2019 [cited 2025]. Available from: https://www.fn-test.com/product/er0730/ 
  37. Kim T, Davis J, Zhang AJ, He X, Mathews ST. Curcumin activates AMPK and suppresses gluconeogenic gene expression in hepatoma cells. Biochem Biophys Res Commun [Internet]. 2009;388(2):377–82. Available from: http://dx.doi.org/10.1016/j.bbrc.2009.08.018 
  38. Thomson DM. The role of AMPK in the regulation of skeletal muscle size, hypertrophy, and regeneration. Int J Mol Sci [Internet]. 2018;19(10). Available from: http://dx.doi.org/10.3390/ijms19103125 
  39. Richter EA, Ruderman NB. AMPK and the biochemistry of exercise: implications for human health and disease. Biochem J [Internet]. 2009;418(2):261–75. Available from: http://dx.doi.org/10.1042/BJ20082055 
  40. Wojtaszewski JF, Nielsen P, Hansen BF, Richter EA, Kiens B. Isoform-specific and exercise intensity-dependent activation of 5’-AMP-activated protein kinase in human skeletal muscle. J Physiol [Internet]. 2000;528 Pt 1:221–6. Available from: http://dx.doi.org/10.1111/j.1469-7793.2000.t01-1-00221.x 
  41. Hamidie RDR, Shibaguchi T, Yamada T, Koma R, Ishizawa R, Saito Y, et al. Curcumin induces mitochondrial biogenesis by increasing cyclic AMP levels via phosphodiesterase 4A inhibition in skeletal muscle. Br J Nutr [Internet]. 2021;126(11):1642–50. Available from: http://dx.doi.org/10.1017/S0007114521000490 

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