Russian Journal of PhysiologyRussian Journal of PhysiologyРоссийский физиологический журнал им. И.М. Сеченова0869-81392658-655XThe Russian Academy of Sciences65162410.31857/S0869813924080035BCOGVVEXPERIMENTAL ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИResearch ArticleApplication of lactoferrin for the prevention and restoration of bone tissue in Wistar rats under conditions of hindlimb unloadingПрименение лактоферрина для профилактики и восстановления изменений костной ткани у крыс Wistar в условиях опорной разгрузкиGordienkoK. V.ГордиенкоК. В.
Russian Federation
k.vl.gordienko@gmail.comLukichevaN. A.ЛукичёваН. А.
Russian Federation
k.vl.gordienko@gmail.comAkhmetzyanovaA. I.АхметзяноваА. И.
Russian Federation
k.vl.gordienko@gmail.comKolupaevA. K.КолупаевА. К.
Russian Federation
k.vl.gordienko@gmail.comSachenkovO. A.СаченковО. А.
Russian Federation
k.vl.gordienko@gmail.comBaltinaT. V.БалтинаТ. В.
Russian Federation
k.vl.gordienko@gmail.comSadchikovaE. R.СадчиковаЕ. Р.
Russian Federation
k.vl.gordienko@gmail.comVassilievaG. Yu.ВасильеваГ. Ю.
Russian Federation
k.vl.gordienko@gmail.comInstitute for Biomedical Problems of the Russian Academy of SciencesИнститут медико-биологических проблем РАНKazan (Volga region) Federal UniversityКазанский (Приволжский) федеральный университетInstitute of Gene Biology Russian Academy of SciencesИнститут биологии гена РАН1508202411081253126301022025Copyright ©; 2024, Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0869-8139/article/view/651624

The influence of gravitational unloading (antiorthostatic suspension) and subsequent recovery on the mineral density and mechanical properties of the femoral and tibial bones of Wistar rats was studied with oral administration of a biotechnological analog of human lactoferrin (200 mg/kg) derived from the milk of producer goats. Bone mineral density was determined by dual-energy X-ray absorptiometry, and strength and stiffness were assessed through three-point bending tests.

It was shown that gravitational unloading for 21 days led to a decrease in the mineral density of the tibial and femoral bones. The administration of lactoferrin did not significantly affect the mineral density or projected area of the studied bones. No statistically significant differences in mechanical stiffness were found between the experimental groups, but after readaptation, the ultimate strength was significantly higher in the groups that received lactoferrin. Thus, the obtained results may indicate the potential of lactoferrin preparations as prophylactic agents for maintaining bone strength. At the same time, maintaining bone mineral density under deficit-stimulating conditions requires consideration of alternative dosages and delivery methods of the drug.

Изучено влияние гравитационной разгрузки (антиортостатического вывешивания) и последующего восстановления на минеральную плотность и механические свойства бедренной и большеберцовой костей крыс Wistar при пероральном приеме биотехнологического аналога лактоферрина человека (200 мг/кг), выделенного из молока коз-продуцентов. Минеральную плотность костей определяли методом двухэнергетической рентгеновской абсорбциометрии, прочность и жесткость – исследованиями костей на трехточечный изгиб. Показано, что гравитационная разгрузка в течение 21 суток приводила к снижению минеральной плотности большеберцовой и бедренной костей. Применение лактоферрина значимо не повлияло на минеральную плотность и проекционную площадь исследуемых костей. Статистически значимых различий между экспериментальными группами по значению механической жесткости обнаружено не было, но при этом у животных после реадаптации предел прочности был значимо выше в группах, получавших лактоферрин. Полученные результаты могут свидетельствовать о перспективности применения препаратов лактоферрина в качестве средств профилактики для поддержания прочностных свойств. В то же время сохранение минеральной плотности костной ткани в дефицит-стимулирующих условиях требует рассмотрения альтернативных дозировок и способов доставки препарата.

rathindlimb unloadinglactoferrinfemurtibiaDXAthree-point bendingкрысаопорная разгрузкалактоферринбедренная костьбольшеберцовая костьостеоденситометриятрехточечный изгибМинобрнауки РоссииMinistry of Education and Science of Russia075–15–2021–668Правительство Российской ФедерацииGovernment of the Russian FederationFZSM–2023–0009Jiang S-D, Dai L-Y, Jiang L-S (2006) Osteoporosis after spinal cord injury. Osteoporos Int 17: 180–192. https://doi.org/10.1007/s00198–005–2028–8Aliprantis AO, Stolina M, Kostenuik PJ, Poliachik SL, Warner SE, Bain SD, Gross TS (2012) Transient muscle paralysis degrades bone via rapid osteoclastogenesis. FASEB J 26: 1110–1118. https://doi.org/10.1096/fj.11–196642Baran R, Wehland M, Schulz H, Heer M, Infanger M, Grimm D (2022) Microgravity-related changes in bone density and treatment options: a systematic review. IJMS23: 8650. https://doi.org/10.3390/ijms23158650Genah S, Monici M, Morbidelli L (2021) The Effect of Space Travel on Bone Metabolism: Considerations on Today’s Major Challenges and Advances in Pharmacology. IJMS22: 4585. https://doi.org/10.3390/ijms22094585LeBlanc A, Schneider V, Shackelford L, West S, Oganov V, Bakulin A, Voronin L (2000) Bone mineral and lean tissue loss after long duration space flight. J Musculoskelet Neuron Interact 1: 157–160.Оганов ВС, Бакулин АВ, Новиков ВЕ, Мурашко ЛМ, Кабицкая ОЕ, Моргун ВВ, Воронин ЛИ, Шнайдер В, Шейклфорд Л, Лебланк А (2005) Реакции костной системы человека в космическом полете: Феноменология. Авиакосм экол мед 39: 3–9. [Oganov VS, Bakulin AV, Novikov VE, Murashko LM, Kabitskaya OE, Morgun VV, Voronin LI, Schneider V, Shackelford L, LeBlanc A (2005) Reactions of the human skeletal system in space flight: phenomenology. Aviakosm Ekol Med 39: 3–9. (In Russ)].Leblanc AD, Schneider VS, Evans HJ, Engelbretson DA, Krebs JM (2009) Bone mineral loss and recovery after 17 weeks of bed rest. J Bone Miner Res 5: 843–850. https://doi.org/10.1002/jbmr.5650050807Kaplansky AS, Durnova GN, Burkovskaya TE, Vorotnikova EV (1991) The effect of microgravity on bone fracture healing in rats flown on Cosmos-2044. Physiologist 34: S196–S199.Капланский АС, Дурнова ГН, Сахарова ЗФ, Ильина-Какуева ЕИ (1987) Гистоморфометрический анализ костей крыс, находившихся на борту биоспутника «Космос-1667». Косм биол авиакосм мед 21: 25–31. [Kaplanskiy AS, Durnova GN, Sakharova ZF, Ilʹina-Kakueva EI (1987) Histomorphometric analysis of the bones of rats on board the Kosmos 1667 biosatellite. Kosm Biol Aviakosm Med 21: 25–31. (In Russ)].Hariom SK, Ravi A, Mohan GR, Pochiraju HD, Chattopadhyay S, Nelson EJR (2021) Animal physiology across the gravity continuum. Acta Astronautica 178: 522–535. https://doi.org/10.1016/j.actaastro.2020.09.044Дурнова ГН, Капланский АС, Логинов ВИ (2006) Динамика восстановления структуры большеберцовых костей крыс после остеопении, вызванной вывешиванием. Авиакосм экол мед 40: 29–31. [Durnova GN, Kaplanskiy AS, Loginov VI (2006) [Dynamics of restoration of the structure of the tibia bones of rats after osteopenia caused by hindlimb suspension]. Aviakosm Ekol Med 40: 29–31. (In Russ)].Дурнова ГН, Капланский АС (2003) Сравнительное гистоморфометрическое исследование большеберцовых костей у самцов и самок крыс при вывешивании за хвост в антиортостатическом положении. Авиакосм экол мед 37: 29–32. [Durnova GN, Kaplanskiy AS (2003) Comparative histomorphometric study of the tibia in male and female rats when suspended by the tail in an antiorthostatic position. Aviakosm Ekol Med 37: 29–32. (In Russ)].Metzger CE, Anand Narayanan S, Phan PH, Bloomfield SA (2020) Hindlimb unloading causes regional loading-dependent changes in osteocyte inflammatory cytokines that are modulated by exogenous irisin treatment. NPJ Microgravity 6: 28. https://doi.org/10.1038/s41526–020–00118–4Grano M, Mori G, Minielli V, Barou O, Colucci S, Giannelli G, Alexandre C, Zallone AZ, Vico L (2002) Rat hindlimb unloading by tail suspension reduces osteoblast differentiation, induces IL-6 secretion, and increases bone resorption in ex vivo cultures. Calcified Tissue Int 70: 176–185. https://doi.org/10.1007/s00223–001–2034–6Lang TF, Leblanc AD, Evans HJ, Lu Y (2006) Adaptation of the proximal femur to skeletal reloading after long-duration spaceflight. J Bone Mineral Res 21: 1224–1230. https://doi.org/10.1359/jbmr.060509Goldman IL, Georgieva SG, Gurskiy YaG, Krasnov AN, Deykin AV, Popov AN, Ermolkevich TG, Budzevich AI, Chernousov AD, Sadchikova ER (2012) Production of human lactoferrin in animal milk. Biochem Cell Biol 90: 513–519. https://doi.org/10.1139/o11–088Chernousov AD, Nikonova MF, Sharova NI, Mitin АN, Litvina ММ, Sadchikov PE, Goldman IL, Yarilin АА, Sadchikova ЕR (2013) Neolactoferrin as a stimulator of innate and adaptive immunity. Acta Naturae 5: 71–76. https://doi.org/10.32607/20758251–2013–5–4–71–76Naot D, Palmano K, Cornish J (2012) Lactoferrin – A Potential Anabolic Intervention in Osteoporosis. In: Dionyssiotis Y (ed) Osteoporosis. In Tech.Sachenkov O, Kharislamova L, Shamsutdinova N, Kirillova E, Konoplev Y (2015) Evaluation of the bone tissue mechanical parameters after induced alimentary Cu-deficiency followed by supplementary injection of Cu nanoparticles in rats. IOP Conf Ser: Mater Sci Eng 98: 012015. https://doi.org/10.1088/1757–899X/98/1/012015Bastos AR, Da Silva LP, Maia FR, Pina S, Rodrigues T, Sousa F, Oliveira JM, Cornish J, Correlo VM, Reis RL (2019) Lactoferrin-hydroxyapatite containing spongy-like hydrogels for bone tissue engineering. Materials 12: 2074. https://doi.org/10.3390/ma12132074Trybek G, Jedliński M, Jaroń A, Preuss O, Mazur M, Grzywacz A (2020) Impact of lactoferrin on bone regenerative processes and its possible implementation in oral surgery – a systematic review of novel studies with metanalysis and metaregression. BMC Oral Health 20: 232. https://doi.org/10.1186/s12903–020–01211–6Blais A, Malet A, Mikogami T, Martin-Rouas C, Tomé D (2009) Oral bovine lactoferrin improves bone status of ovariectomized mice. Am J Physiol Endocrinol Metabolism 296: E1281–E1288. https://doi.org/10.1152/ajpendo.90938.2008Grey A, Banovic T, Zhu Q, Watson M, Callon K, Palmano K, Ross J, Naot D, Reid IR, Cornish J (2004) The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells. Mol Endocrinol 18: 2268–2278. https://doi.org/10.1210/me.2003–0456Cornish J (2004) Lactoferrin promotes bone growth. Biometals 17: 331–335. https://doi.org/10.1023/B: BIOM.0000027713.18694.91Jia B, Xie L, Zheng Q, Yang P, Zhang W, Ding C, Qian A, Shang P (2014) A hypomagnetic field aggravates bone loss induced by hindlimb unloading in rat femurs. PLoS One 9: e105604. https://doi.org/10.1371/journal.pone.0105604He J, Feng X, Wang J, Shi W, Li H, Danilchenko S, Kalinkevich A, Zhovner M (2018) Icariin prevents bone loss by inhibiting bone resorption and stabilizing bone biological apatite in a hindlimb suspension rodent model. Acta Pharmacol Sin 39: 1760–1767. https://doi.org/10.1038/s41401–018–0040–8Smith JK (2020) Osteoclasts and microgravity. Life 10: 207. https://doi.org/10.3390/life10090207Lloyd SA, Morony SE, Ferguson VL, Simske SJ, Stodieck LS, Warmington KS, Livingston EW, Lacey DL, Kostenuik PJ, Bateman TA (2015) Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice. Bone 81: 562–572. https://doi.org/10.1016/j.bone.2015.08.021Hou J, Xue Y, Lin Q (2012) Bovine lactoferrin improves bone mass and microstructure in ovariectomized rats via OPG/RANKL/RANK pathway. Acta Pharmacol Sin 33: 1277–1284. https://doi.org/10.1038/aps.2012.83Guo HY, Jiang L, Ibrahim SA, Zhang L, Zhang H, Zhang M, Ren FZ (2009) Orally administered lactoferrin preserves bone mass and microarchitecture in ovariectomized rats. J Nutrition 139: 958–964. https://doi.org/10.3945/jn.108.100586Gao R, Watson M, Callon KE, Tuari D, Dray M, Naot D, Amirapu S, Munro JT, Cornish J, Musson DS (2018) Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis: local lactoferrin promotes bone regeneration. J Tissue Eng Regen Med 12: e620–e626. https://doi.org/10.1002/term.2348Ильин ЕА, Новиков ВЕ (1980) Стенд для моделирования физиологических эффектов невесомости в лабораторных экспериментах с крысами. Косм биол авиакосм мед 14: 79–80. [Il’in EA, Novikov VE (1980) Workbench for modelling the physiological effects of weightlessness in laboratory experiments with rats. Kosm Biol Aviakosm Med 14: 79–80. (In Russ)].Morey-Holton ER, Globus RK (2002) Hindlimb unloading rodent model: technical aspects. J Appl Physiol 92: 1367–1377. https://doi.org/10.1152/japplphysiol.00969.2001Akhmetzyanova AI, Sharafutdinova KR, Sabirova DE, Baltin ME, Gerasimov OV, Baltina TV, Sachenkov OA (2022) Assessing the effect of spinal cord injury severity on the mechanical properties of the hind limb bones of experimental rats. Russ J Biomechan 26: 38–46. https://doi.org/10.15593/RZhBiomeh/2022.4.04Sadchikov PE, Goldman IL, Razin SV, Chernousov AD, Alekseeva LI, Sadchikova ER (2016) The molecular mechanism of lactoferrin influence on bone formation. Osteopor Bone Dis 19: 12–22. https://doi.org/10.14341/osteo2016312–22Evans JD (1996) Straightforward statistics for the behavioral sciences. Brooks/Cole Pub. Co, Pacific Grove.Cunningham HC, West DWD, Baehr LM, Tarke FD, Baar K, Bodine SC, Christiansen BA (2018) Age-dependent bone loss and recovery during hindlimb unloading and subsequent reloading in rats. BMC Musculoskelet Disord 19: 223. https://doi.org/10.1186/s12891–018–2156-xKrause AR, Speacht TA, Steiner JL, Lang CH, Donahue HJ (2020) Mechanical loading recovers bone but not muscle lost during unloading. NPJ Microgravity 6: 36. https://doi.org/10.1038/s41526–020–00126–4Lloyd SA, Bandstra ER, Willey JS, Riffle SE, Tirado-Lee L, Nelson GA, Pecaut MJ, Bateman TA (2012) Effect of proton irradiation followed by hindlimb unloading on bone in mature mice: A model of long-duration spaceflight. Bone 51: 756–764. https://doi.org/10.1016/j.bone.2012.07.001Osterhoff G, Morgan EF, Shefelbine SJ, Karim L, McNamara LM, Augat P (2016) Bone mechanical properties and changes with osteoporosis. Injury 47: S11–S20. https://doi.org/10.1016/S0020–1383(16)47003–8Globus RK, Morey-Holton E (2016) Hindlimb unloading: rodent analog for microgravity. J Appl Physiol 120: 1196–1206. https://doi.org/10.1152/japplphysiol.00997.2015Wang B, Timilsena YP, Blanch E, Adhikari B (2019) Lactoferrin: structure, function, denaturation and digestion. Critical reviews in food science and nutrition 59: 580–596. https://doi.org/10.1080/10408398.2017.1381583Amidon GL, DeBrincat GA, Najib N (1991) Effects of Gravity on Gastric Emptying, Intestinal Transit, and Drug Absorption. J Clin Pharm 31: 968–973. https://doi.org/10.1002/j.1552–4604.1991.tb03658.xАфонин БВ, Гончарова НП, Карамышев ЮА (2007) Функциональное состояние желудка человека в эксперименте с 4-месячной антиортостатической гипокинезией. Авиакосм экол мед 41: 37–43. [Afonin BV, Goncharova NP, Karamyshev YuA (2007) Functional state of the human stomach in an experiment with 4-month antiorthostatic hypokinesia. Aviakosm Ekol Med 41: 37–43. (In Russ)].Shimano MM, Volpon JB (2009) Biomechanics and structural adaptations of the rat femur after hindlimb suspension and treadmill running. Braz J Med Biol Res 42: 330–338. https://doi.org/10.1590/S0100–879X2009000400004Ильин ЕА (1984) Исследование на биоспутниках «Космос». Косм биол авиакосм мед 18: 57–66. [Il’in EA (1984) Research on the Kosmos biosatellites. Kosm Biol Aviakosm Med 18: 57–66. (In Russ)].