Leukocyte Peptide Complex with Antibacterial and Anti-Inflammatory Effects

The aim was to study the inhibitory and bactericidal activity against bacteria of different systematic groups in vitro, as well as the anti-inflammatory activity of the leukocyte peptide complex in a model experiment of carrageenan edema.

Material and Methods. Leukocyte protein-peptide complex was obtained by ultrasound treatment of healthy donors' blood leukocytes with subsequent fractionation via high-performance liquid chromatography. Antibacterial activity of the isolated fraction was determined using the twofold dilution method. Anti-inflammatory activity was studied on the model of acute carrageenin edema caused by subplantar injection of carrageenin solution into the hind paw of white nonlinear rats.

Results. The leukocyte peptide complex consisting of peptides with molar mass less than 6.5 kDa demonstrated a pronounced antibacterial activity against Escherichia coli, Mycolicibacterium smegmatis, and Staphylococcus aureus. The minimum inhibitory concentrations of the complex were 0.5, 0.25, and 0.125 mg/mL, respectively. Intraperitoneal administration of leukocyte protein-peptide complex compared to the reference drug (nimesulide) provides a statistically significant inhibition of the inflammatory response, reaching 62.3%. The anti-inflammatory efficacy of the leukocyte protein-peptide complex exceeded that of nimesulide by more than 13%.

Conclusion. The possibility of creating an anti-inflammatory drug with pronounced antibacterial activity based on a peptide complex isolated from human blood leukocytes using a simple, fast, and effective method of ultrasound exposure has been shown.

1. Bindu S., Mazumder S., Bandyopadhyay U. Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective. Biochem Pharmacol. 2020; 180: 114147. doi: 10.1016/j.bcp.2020.114147.

2. Luo Y., Song Y. Mechanism of antimicrobial peptides: antimicrobial, anti-inflammatory and antibiofilm activities. Int J Mol Sci. 2021; 22 (21): 11401. doi: 10.3390/ijms222111401.

3. Volkova L. V., Grishina T. A., Volkov A. G. Method for fractionation of leukocyte proteins. Patent RF № 2737730; published 02.12.2020. (in Russion)

4. Volkov A. G., Kononova L. I., Korobov V. P., Volkova L. V. Study of molecular parameters and antibacterial activity of a peptide preparation derived from human leukocytes. Antibiot Khimioter = Antibiotics and Chemotherapy. 2023; 68 (9–10): 20–24. doi: https://doi.org/10.37489/0235-2990-2023-68-910-20-24. (in Russian

5. Grishina T. A., Volkov A. G., Volkova L. V. Cytotoxicity and toxicological characteristics of the new leukocitar polypeptide. Probl Bio Med Pharm Chem. 2020; 23 (5): 54–58. doi: https://doi.org/10.29296/25877313-2020-05-08. (in Russian)

6. Volkova L. V. Acute and chronic toxicity of antibacterial peptide complex. Rus J Biopharmaceuticals. 2022; 14 (1): 51–54. doi: https://doi.org/10.30906/20738099-2022-14-1-51-54. (in Russian)

7. European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2020) Version 8. http://www.eucast.org/

8. Trinus F. P., Klebanov B. M., Kondratyuk V. I. Methodological recommendations for experimental (preclinical) research of non-steroidal anti-inflammatory pharmacological substances. Moscow: Ministry of Health of the USSR. 1983; 16. (in Russian)

9. Chernov A. N., Orlov D. S., Shamova O. V. Peptides of the innate immunity as potential anticancer agents: pros and cons. Med Immunol. 2021; 23 (6): 1285–1306. doi: https://doi.org/10.15789/1563-0625-POT-2303. (in Russian)

10. Shamova O. V., Orlov D. S., Zharkova M. S., Balandin S. V., Yamshchikova E. V., Knappe D., Hoffmann R., Kokryakov V. N., Ovchinnikova T. V. Minibactenecins ChBac7.Nα and ChBac7.Nβ — Antimicrobial Peptides from Leukocytes of the Goat Capra hircus. Acta Naturae. 2016; 8 (30): 136–146. doi: https://doi.org/10.32607/20758251-2016-8-3-136-146. (in Russian)

11. Budikhina A. S., Pinegin B. V. Defensins are multifunctional human cationic peptides. Int J Immunopathology, Allergology, Infectology. 2008; 2: 31–40. (in Russian)

12. Identifier of bacteria Bergey. In 2 volumes: Transl. With. English. J. Hoult, N. Krieg, P. Sneath, J. Staley, S. Williams (eds.). Moscow: Mir, 1997; 800.

13. Korobov V. P., Shagdarova B. Ts., Varlamov V. P., Esaev A. L., Polyudova T. V. Inhibitory action of low-molecular chitosan on growth of bacteria with different tinctorial properties. Microbiology. 2023; 92 (2): 215–220. doi: doi.org/10.1134/S0026261722603347. (in Russian)

14. Tsvetkova E. V., Aleshina G. M., Shamova O. V., Kokryakov V. N., Leonova L. E., Lehrer R. I. α-Defensins from blood leukocites of the monkey Papio hamadryas. Biochemistry. 2006. 71 (8): 879–883. (in Russian)

15. Yuhnev V.A, Shartukova M. A., Lugovkina N. V., Kokryakov V. N., Shamova O. V. Search of novel antimicrobial peptidesof the cathelicidins and defensins families in moose (Alces alces) leucocytes. Bulletin of St. Petersburg University. Episode 3. Biology. 2014; 1: 115–131. (in Russian)

16. Selsted M. E., Harwig S. S., Ganz T., Schilling J. W., Lehrer R. I. Primary structures of three human neutrophil defensins. J Clin Invest. 1985. 76 (4): 1436–9. doi: 10.1172/JCI112121

17. Bensch K. W., Raida M., Magert H. J., Schulz-Knappe P., Forssmann W. G. hBD-1: a novel β-defensin from human plasma. FEBS Letters. 1995. 368: 331–335. doi: 10.1016/0014-5793(95)00687-5.

18. Lu S., Cao Y., Fan S. B., Chen Z. L., Fang R. Q., He S.M., Dong M. Q. Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures. Biophys Rep. 2018; 4 (2): 68–81. doi: 10.1007/s41048-018-0050-6.

19. Wu Z., Li X., Leeuw E., Ericksen B., Lu W. Why Is the Arg5-Glu13 Salt Bridge Conserved in Mammalian α-Defensins? J Biol Chem. 2005; 280 (52): 43039–43047. doi: 10.1074/jbc.M510562200

20. Sparks I. L., Derbyshire K. M., Jacobs W. R., Morita Y. S. Mycobacterium smegmatis: The vanguard of mycobacterial research. J Bacteriol. 2023; 205 (1): e0033722. doi: 10.1128/jb.00337-22.

21. Kang L., Han T., Cong H., Yu B., Shen Y. Recent research progress of biologically active peptides. Biofactors. 2022; 48 (3): 575–596. doi: 10.1002/biof.1822.

22. Panerai A. E. Lymphocytes as a source of hormones and peptides. J Endocrinol Invest. 1993; 16: 549–557. doi: 10.1007/BF03348904.