Study of the effect of equine type I collagen preparation on the process of collagenesis in an experimental model using gene expression measurements and histological assessment of tissues

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Objective: Evaluation of the effect of equine type I collagen on the collagenesis process in a mouse model by measuring gene expression in mouse skin (genes: COL1, COL3, MMP1, MMP3, ELN, VCAN, IL-6, TNFα, TGFβ) and performing histological examination of tissues.

Materials and methods: The study used female BALB/C mice aged 6–8 weeks. The animals were kept under standard vivarium conditions at controlled temperature (22 ± 2 °C), humidity (50 ± 10%) and 12/12-hour light regime with access to water and food ad libitum. Experimental animals were shaved with two skin areas of ~3 cm² on the dorsal surface using a trimmer (day 0). On the next day (day 1), 100 μl of sterile saline (0.9% NaCl, control) were injected intradermally into one area, and 100 μl of equine collagen type I solution diluted in sterile saline according to the manufacturer’s instructions into the other ones. On day 1 of the experiment (24 hours after the first injection of the drug), one of the animals was sacrificed and skin areas in the injection areas were excised. Gene expression was measured using real-time polymerase chain reaction with reverse transcription (RT-rtPCR).

Histological analysis was performed on the 21st, 35th, and 53rd days.

Results: On day 1 of the experiment (24 hours after the first injection of equine collagen type I), a statistically significant increase in the expression of the COL1 and COL3 genes encoding collagen types I and III, the main structural proteins of the dermis, was observed in the tissues. This clearly indicates the initiation of new collagen synthesis by fibroblasts. Additionally, a significant increase in the TGFβ expression was recorded, which is one of the key regulators of fibroblast activity and differentiation, which is also known as a powerful inducer of collagenesis. Histological data confirm these molecular observations: on the 21st day, a higher ratio of fibroblasts to the total number of dermal cells and a denser organization of collagen fibers were found in the experimental samples than in the control group. Considering that fibroblasts are the main producers of extracellular matrix components, inclu-ding collagen, their high density in the dermis serves as an indirect sign of activation of the synthetic function of the skin under the influence of the drug.

A tendency towards an increase in the thickness of the dermis on the 35th day was also revealed in the experimental group, which may reflect the accumulation of a new matrix and structural reorganization of the tissue. At the same time, the structure of collagen fibers on the 35th and 53rd days remained compact and dense, which indicates stabilization of the remodeled matrix.

About the authors

Maria A. Morzhanaeva

Beauty Expert Medical; Melsytech Genetics Laboratory

Email: elene-elene@bk.ru

Cosmetologist, Medical Adviser

Russian Federation, Moscow; Nizhny Novgorod

Elena V. Svechnikova

Russian Biotechnological University; Polyclinic №1 of the Administrative Directorate of the President of the Russian Federation

Author for correspondence.
Email: elene-elene@bk.ru
ORCID iD: 0000-0002-5885-4872

Dr. Sci. (Med.), Professor, Department of Skin and Venereal Diseases; Head of the Department of Dermatovenereology and Cosmetology

Russian Federation, Moscow; Moscow

Elena Yu. Starkova

Beauty Expert Medical

Email: elene-elene@bk.ru

Plastic Surgeon, Dermatologist, Cosmetologist, Medical Adviser; International Class Trainer-methodologist, Physician-researcher

Russian Federation, Moscow

Yuri Yu. Babin

Melsytech Genetics Laboratory

Email: elene-elene@bk.ru

Molecular Biologist, Head of the Department for Technical Solutions in Genetics

Russian Federation, Nizhny Novgorod

Ulyana A. Mikheeva

National Research State University of Nizhny Novgorod named after N.I. Lobachevsky

Email: elene-elene@bk.ru

Biological

Russian Federation, Nizhny Novgorod

Ekaterina N. Gorshkova

Melsytech Genetics Laboratory; National Research State University of Nizhny Novgorod named after N.I. Lobachevsky

Email: elene-elene@bk.ru

PhD, Associate Professor, Department of Molecular Biology and Immunology

Russian Federation, Nizhny Novgorod; Nizhny Novgorod

Olga V. Starkina

Melsytech Genetics Laboratory

Email: elene-elene@bk.ru

Biotechnologist, Head of the Applied Genetics Department

Russian Federation, Nizhny Novgorod

Tatiana I. Kolegova

Melsytech Genetics Laboratory

Email: elene-elene@bk.ru

Biologist and Senior Researcher

Russian Federation, Nizhny Novgorod

Viktor V. Gladko

Russian Biotechnological University

Email: dr.gladko@mgupp.ru
ORCID iD: 0000-0003-3087-5038

Dr. Sci. (Med.), Professor, Head of the Department of Skin and Venereal Diseases with a Course in Cosmetology, Medical Institute of Continuous Education

Russian Federation, Moscow

Irina V. Izmaylova

Russian Biotechnological University

Email: izmajjlovaiv@mgupp.ru

Cand. Sci. (Med.), Associate Professor at the Department of Skin and Venereal Diseases with a Course in Cosmetology, Medical Institute of Continuous Education

Russian Federation, Moscow

References

  1. Fitzpatrick T.B. Dermatology in General Medicine. McGraw-Hill, Health Professions Division, 1993.
  2. Fisher G.J. et al. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997a;337(20):1419–1429.
  3. Bornstein P. The biosynthesis of collagen. Annu Rev Biochem. 1974;43(1):567–603. doi: 10.1146/annurev.bi.43.070174.003031
  4. Siegel R C. Lysyl oxidase/ Int Rev Connect Tissue Res. 1979;8:73–118.
  5. Fisher G.J. et al. Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med. 1997b;337(20):1419–1428.
  6. Fisher G.J., Varani J., Voorhees J.J. Looking Older: Fibroblast Collapse and Therapeutic Implications. Arch Dermatol. 2008;144(5):666–672.
  7. Jariashvili K. et al. Uv damage of collagen: Insights from model collagen peptides. Biopolymers. 2012;97(3):189–198.
  8. Liu J. et al. Mechanical stretching stimulates collagen synthesis via down-regulating SO2/AAT1 pathway. Sci Rep. 2016;6(1):21112.
  9. Varani J. et al. Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro. Am J Pathol. 2001;158(3):931–942. https://doi.org/10.1016/S0002-9440(10)64040-0
  10. Varani J. et al. Decreased collagen production in chronologically aged skin: roles of age-dependent alteration in fibroblast function and defective mechanical stimulation. Am J Pathol. 2006;168(6):1861–1868. https://doi.org/10.2353/ajpath.2006.051302
  11. Vandesompele J., De Preter K., Pattyn F. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034. https://doi.org/10.1186/gb-2002-3-7-research0034
  12. Моржанаева М.А., Свечникова Е.В., Бабин Ю.Ю., Старкина О.В. Исследование синтетической функции фибробластов после воздействия препаратов коллагена. Фарматека. 2024;31(9):106–111. [Morzhanaeva M.A., Svechnikova E.V., Babin Yu.Yu., Starkina O.V. Study of the synthetic function of fibroblasts after exposure to collagen preparations. Farmateka. 2024;31(9):106–111. (In Russ.)]. https://dx.doi.org/10.18565/pharmateca.2024.9.106-111
  13. Моржанаева М.А., Свечникова Е.В. Метод восстановления внеклеточного матрикса с помощью заместительной коллагенотерапии препаратом Linerase. Фарматека. 2024;31(5):92–101. [Morzhanaeva M.A., Svechnikova E.V. Method of extracellular matrix restoration using collagen replacement therapy with Linerase. Farmateka. 2024;31(5):92–101. (In Russ.)]. https://dx.doi.org/10.18565/pharmateca.2024.5.92-101

Supplementary files

Supplementary Files
Action
1. JATS XML
2. Figure 1. Scheme of excision of skin flaps in BALB/c mice

Download (94KB)
3. Fig.1

Download (39KB)
4. Fig.2

Download (57KB)
5. Fig.3

Download (64KB)
6. Fig.4

Download (68KB)
7. Fig.5

Download (53KB)
8. Fig.6

Download (45KB)
9. Fig.7

Download (47KB)
10. Fig.8

Download (43KB)
11. Fig.9

Download (45KB)
12. Fig.10

Download (46KB)
13. Fig.11

Download (53KB)
14. Fig.12

Download (53KB)
15. Figure 2. Microscopic images of a sagittal section of the skin of a BALB/C mouse at the injection site on the 21st day of the course of injections of equine collagen type I. stained with hematoxylin and eosin. A-В) control sample, C-D) experimental sample

Download (287KB)
16. Figure 3. Microscopic images of a sagittal section of the skin of a BALB/C mouse at the injection site on the 35th day of the course of injections of equine collagen type I. stained with hematoxylin and eosin. A-В) control sample, C-D) experimental sample

Download (310KB)
17. Figure 4. Microscopic images of a sagittal section of the skin of a BALB/C mouse at the injection site on day 53 of a course of injections of equine collagen type I. Hematoxylin and eosin staining. A-В) control sample, В-D) experimental sample

Download (288KB)
18. Figure 5. An example of an area of the dermis with neutrophilic Infiltration In the skin of a BALB/C mouse at a certain period after the start of a course of injections of equine collagen type I. A) In the experimental sample on day 21, B) in the control skin sample on day 35. Neutrophils are Indicated by white arrows. Hematoxylin and eosin staining

Download (248KB)
19. Figure 6. Areas of fibroblast accumulation in the skin dermis after the start of a course of injertions of equine collagen type I. A-B) in control samples on the 21 st day, В-D) in control samples on the 35th day, D) in the experimental sample on the 21 st day. The area with Increased fibroblast accumulation Is highlighted by the black line. Hematoxylin and eosin staining

Download (272KB)
20. Figure 7. The thickness of the dermis, hypodermis and epidermis of a BALB/C mouse at a certain period after a course of injections of the equine collagen preparation

Download (79KB)
21. Figure. 8. With the ratio of the number of fibroblasts to the total number of dermal cells in the skin of BALB/C mice at a certain time after a course of injections of equine collagen type I

Download (91KB)
22. Figure 9. Microscopic images of a sagittal section of mouse skin at the injection site on the 21st day of a course of injections of equine collagen type I. stained using the Masson method. A-В) control sample, C-D) test sample

Download (366KB)
23. Figure 10. Microscopic images of a sagittal section of mouse skin at the injection site on the 35th day of a course of injections of equine collagen type I. stained using the Masson method. A-В) control sample, C-D) test sample

Download (351KB)
24. Figure 11. Microscopic images of a sagittal section of mouse skin at the injection site on the 53rd day of a course of injections of equine collagen type I. stained using the Masson method. A-В) control sample, В-D) experimental sample

Download (354KB)
25. Figure 12. Relative expression of type I collagen, day 1 of the experiment

Download (33KB)
26. Figure 13. Relative expression of type III collagen, day 1 of the experiment

Download (38KB)
27. Figure 14. Relative expression of TGFß, day 1 of the experiment

Download (29KB)

Согласие на обработку персональных данных с помощью сервиса «Яндекс.Метрика»

1. Я (далее – «Пользователь» или «Субъект персональных данных»), осуществляя использование сайта https://journals.rcsi.science/ (далее – «Сайт»), подтверждая свою полную дееспособность даю согласие на обработку персональных данных с использованием средств автоматизации Оператору - федеральному государственному бюджетному учреждению «Российский центр научной информации» (РЦНИ), далее – «Оператор», расположенному по адресу: 119991, г. Москва, Ленинский просп., д.32А, со следующими условиями.

2. Категории обрабатываемых данных: файлы «cookies» (куки-файлы). Файлы «cookie» – это небольшой текстовый файл, который веб-сервер может хранить в браузере Пользователя. Данные файлы веб-сервер загружает на устройство Пользователя при посещении им Сайта. При каждом следующем посещении Пользователем Сайта «cookie» файлы отправляются на Сайт Оператора. Данные файлы позволяют Сайту распознавать устройство Пользователя. Содержимое такого файла может как относиться, так и не относиться к персональным данным, в зависимости от того, содержит ли такой файл персональные данные или содержит обезличенные технические данные.

3. Цель обработки персональных данных: анализ пользовательской активности с помощью сервиса «Яндекс.Метрика».

4. Категории субъектов персональных данных: все Пользователи Сайта, которые дали согласие на обработку файлов «cookie».

5. Способы обработки: сбор, запись, систематизация, накопление, хранение, уточнение (обновление, изменение), извлечение, использование, передача (доступ, предоставление), блокирование, удаление, уничтожение персональных данных.

6. Срок обработки и хранения: до получения от Субъекта персональных данных требования о прекращении обработки/отзыва согласия.

7. Способ отзыва: заявление об отзыве в письменном виде путём его направления на адрес электронной почты Оператора: info@rcsi.science или путем письменного обращения по юридическому адресу: 119991, г. Москва, Ленинский просп., д.32А

8. Субъект персональных данных вправе запретить своему оборудованию прием этих данных или ограничить прием этих данных. При отказе от получения таких данных или при ограничении приема данных некоторые функции Сайта могут работать некорректно. Субъект персональных данных обязуется сам настроить свое оборудование таким способом, чтобы оно обеспечивало адекватный его желаниям режим работы и уровень защиты данных файлов «cookie», Оператор не предоставляет технологических и правовых консультаций на темы подобного характера.

9. Порядок уничтожения персональных данных при достижении цели их обработки или при наступлении иных законных оснований определяется Оператором в соответствии с законодательством Российской Федерации.

10. Я согласен/согласна квалифицировать в качестве своей простой электронной подписи под настоящим Согласием и под Политикой обработки персональных данных выполнение мною следующего действия на сайте: https://journals.rcsi.science/ нажатие мною на интерфейсе с текстом: «Сайт использует сервис «Яндекс.Метрика» (который использует файлы «cookie») на элемент с текстом «Принять и продолжить».