Experimental study of the protein profile of the bone tissue from the proximal femoral epiphysis in the development of aseptic necrosis

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Abstract

BACKGROUND: The development of aseptic necrosis of the femoral head is accompanied by complex disturbances of the molecular and cellular regulation of bone metabolism. Existing conservative treatment options are not always effective, and surgical techniques are not directly aimed at inhibiting bone destruction. Thus, investigating alterations in intercellular interactions in the development of aseptic necrosis offers the prospect of introducing targeted therapy to normalize bone remodeling signaling.

AIM: To study the protein profile of cancellous bone from the proximal femoral epiphysis in the development of aseptic necrosis in an experimental animal model.

METHODS: We analyzed the protein profile of cancellous bone samples from the proximal femoral epiphysis in Wistar rats after surgical induction of aseptic necrosis and in healthy control animals. Proteomic profiling was performed using high-performance liquid chromatography with mass spectrometry on both the aseptic necrosis side and the healthy contralateral side. Proteins were identified using FragPipe software with MSFragger, IonQuant, and Philosopher modules on the Windows 11 operating system with preinstalled Java and AMD64 architecture. Protein identification was considered reliable with a false discovery rate of less than 1% and the presence of at least two unique peptides. The rat protein database (Rattus norvegicus) SwissProt and the common Repository of Adventitious Proteins (cRAP) contaminant database were used for analysis.

RESULTS: A total of 1288 proteins were isolated in 12 samples, of which 989 were common to both control and aseptic necrosis samples, 114 were found only in control samples, and 82 only in aseptic necrosis samples. Gene Ontology cellular component analysis showed that proteasomes accounted for the largest number of associated proteins. By biological function, these included proteins involved in coagulopathy, fibrinolysis, glycolysis, gluconeogenesis, catabolic processes, and cellular response to interleukins 7, 1, 4, and 6; by molecular function, these included NAD-binding proteins, ADP-binding proteins, antigen-interacting proteins, and P-type calcium transporter proteins. Bioinformatic analysis of the protein profile revealed the importance of uncontrolled inflammatory responses and endothelial dysfunction in the breakdown of compensatory mechanisms in the development of aseptic necrosis of the femoral head.

CONCLUSION: Analysis of signaling pathway interaction networks in the development of aseptic necrosis confirmed the role of endothelial dysfunction and dysregulation of compensatory mechanisms at the molecular and cellular level. Moreover, the development of uncontrolled inflammation may play a leading role in progressive bone destruction.

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About the authors

Nikita A. Shabaldin

Kemerovo State Medical University

Author for correspondence.
Email: shabaldin.nk@yandex.ru
ORCID iD: 0000-0001-8628-5649
SPIN-code: 6283-2581

MD, Cand. Sci. (Medicine), Assistant Professor

Russian Federation, Kemerovo

Egor A. Repkin

St. Petersburg State University

Email: st049553@student.spbu.ru
ORCID iD: 0000-0002-8599-3173
SPIN-code: 1429-8914
Russian Federation, Saint Petersburg

Anton G. Kutikhin

Research Institute for Complex Issues of Cardiovascular Diseases

Email: antonkutikhin@gmail.com
ORCID iD: 0000-0001-8679-4857
SPIN-code: 4527-8939

MD, Dr. Sci. (Medicine)

Russian Federation, Kemerovo

Vladimir M. Kenis

H. Turner National Medical Research Center for Children’s Orthopedics and Trauma Surgery

Email: kenis@mail.ru
ORCID iD: 0000-0002-7651-8485
SPIN-code: 5597-8832

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Saint Petersburg

Anna V. Sinitskaya

Research Institute for Complex Issues of Cardiovascular Diseases

Email: annacepokina@mail.ru
ORCID iD: 0000-0002-4467-8732
SPIN-code: 3195-7252

Cand. Sci. (Biology)

Russian Federation, Kemerovo

Alexander D. Stepanov

Research Institute for Complex Issues of Cardiovascular Diseases

Email: sasste@mail.ru
ORCID iD: 0009-0009-7947-5917
SPIN-code: 8462-8235
Russian Federation, Kemerovo

Andrey V. Shabaldin

Kemerovo State Medical University; Research Institute for Complex Issues of Cardiovascular Diseases

Email: weit2007@yandex.ru
ORCID iD: 0000-0002-8785-7896
SPIN-code: 5281-0065

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Kemerovo; Kemerovo

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Supplementary files

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2. Fig. 1. Venn diagram showing the distribution of proteins: common proteins, proteins unique to the healthy limb (ctrl), and proteins identified after induction of aseptic necrosis (path).

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3. Fig. 2. Distribution of proteins classified by function in the control and aseptic necrosis groups using partial least squares discriminant analysis (PLS-DA).

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4. Fig. 3. Signaling pathway enrichment using the Kyoto Encyclopedia of Genes and Genomes (KEGG) in the development of aseptic necrosis of the femoral head. ECM, extracellular matrix.

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