Expression of the apoptosis marker caspase-3 in toxic hepatitis caused by tetrachloromethane in the liver

Introduction. Despite significant attention to the study of the mechanisms of toxic liver damage in recent years, there are still very few effective treatment methods. Liver damage caused by carbon tetrachloride results in hepatocyte death, including apoptosis.

The purpose of the study was to study histological changes and immunohistochemical investigation of the expression of the apoptosis marker caspase-3 in a model of toxic liver injury induced by CCl₄ with hepatoprotective correction.

Materials and methods. The experiment involved forty five rats divided into 9 groups. CCl₄ oil solution was used as the toxicant for each rat group except the negative control groups. Hepatoprotective correction was performed using “Heptor”, “Mexidol”, and Oxymethyluracil. Correction was carried out twice (sacrifice after 24 hours) and four times (sacrifice after 72 hours) following a single CCl₄ injection. Liver tissues underwent standard histological processing with hematoxylin-eosin staining and immunohistochemical analysis for caspase-3. The number of caspase-3-positive cells was evaluated using a pre-trained YOLOv5 deep learning model.

Results. After 24 hours of intoxication, no statistically significant differences in the number of caspase-3-positive cells on microphotographs were found in the experimental groups (p=0.087). However, after 72 hours of CCl₄ administration, statistically significant differences were observed between the groups (p=0.020). Multiple comparisons showed statistically significant differences between the negative control and positive control groups (p=0.0076), as well as between the positive control group and the group corrected with Oxymethyluracil (p=0.0254).

Limitations. The limitations of the study include the evaluation of histological changes and the expression of the apoptosis marker caspase-3 only at 24 and 72 hours after intoxication without long-term studies, the use of a relatively small number of animals (45 rats), and the reliance solely on standard histological methods, immunohistochemical analysis, and deep learning techniques.

Conclusion: After 72 hours, the positive and negative control groups differed from each other, indicating an exacerbation of apoptosis processes after CCl₄ administration. The group corrected with Oxymethyluracil demonstrated fewer caspase-3-positive cells compared to the positive control group, suggesting the hepatoprotective effect of this drug.

Compliance with ethical standards. The study was approved by the Bioethical Commission of the Ufa Research Institute of Occupational Medicine and Human Ecology, conducted in accordance with the European Covention for the Protection of Vertebrate Animals Used for Experiments or for Other Scientific Purposes (ETS N 123), Directive of the European Parliament and the Council of the Eurpean Union 2010/63 /EC of 22.09.2010 on the protection of animals used for scientific purposes.

Contribution:
Baygildin S.S. – collection and processing of material, writing a text;
Repina E.F., Karimov D.O. – the concept and design of the study, editing;
Smolyankin D.A., Khusnutdinova N.Yu., Akhmadeev A.R. – collection and processing of material;
Kudoyarov E.R., Valova Ya.V. – collection and processing of material, statistical processing.
All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final version.

Conflict of interest. The authors declare no conflict of interest.

Acknowledgement. The work was carried out at the expense of subsidies for the implementation of a state task within the framework of the sectoral research program of Rospotrebnadzor “Hygienic scientific substantiation of minimizing risks to the health of the population of Russia” for 2016–2020 on topic 3.5, state no. registration AAAA-A16-116022610045-4. The synthesis of the 5-hydroxy-6-methyluracil composition was carried out in accordance with the research plan of the Ufa Institute of Chemistry of the Ufa Federal Research Center of the Russian Academy of Sciences (State Registration No. AAAA-A19-119011790021-4).

Received: March 13, 2024 / Revised: April 26, 2024 / Accepted: October 2, 2024 / Published: March 7, 2025