Research Article

The Effect of Black Cumin (Nigella sativa) on the Caspase-3 Expression and the Number of Testic Sertoli Cells in Balb/C Male Mouses Exposed to Diazinon

Galuh Chandra Agustina1*, Erwin Syahfitri1, Viski Fitri Hendrawan1, Siska Aditya1,2

1Faculty of Veterinary Medicine, Brawijaya University,Puncak Dieng Eksklusif, Kalisongo, Kec. Dau, Kab. Malang 65151; 2Research Group of Feed and Food Safety, Research Center for Food Technology Processing, The National Agency for Research and Innovation of The Republic of Indonesia, Jl. Jogja-Wonosari, Yogyakarta, Indonesia.

Received | January 13, 2023; Accepted | February 20, 2023; Published | April 01, 2023

*Correspondence | Galuh Chandra Agustina, Faculty of Veterinary Medicine, Brawijaya University,Puncak Dieng Eksklusif, Kalisongo, Kec. Dau, Kab. Malang 65151; Email: galuh.agustina@ub.ac.id

Citation | Agustina GC, Syahfitri E, Hendrawan VF, Aditya S (2023). The effect of black cumin (Nigella sativa) on the Caspase-3 expression and the number of testic sertoli cells in balb/c male mouses exposed to diazinon. Anim. Vet. Sci. 11(5): 791-794.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.5.791.794

ISSN (Online) | 2307-8316

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The utilization of pesticides is relatively high in agriculture sectors. According to BMC Public Health, about 720,000 cases of accidental pesticide poisoning, and approximately 44% of 840 million farmers worldwide experience pesticide poisoning throughout the year (Boedeker et al., 2020). Furthermore, diazinon is one of the pesticide frequently used due to its low cost. Nevertheless, this type of this pesticide could inhibit AChE in the blood, causing the disruption of blood circulation and neurotoxicity (Lesmana, 2013). Besides, about 4% of the cases of pesticide poisoning occurs in rice fields and dairy cows. Indeed, the contamination of pesticides in feed and food could cause residues in the body, subsequently hampering the health and reproduction aspects (Nining et al., 2019).

Furthermore, the consumption of antioxidant could enervate the harmful effect of diazinon when they entered the systemic circulation. Nowadays, there is high interest in developing therapeutic agents from various natural products to substitute the synthetic drugs due to their harmful effect (Danaei et al., 2018). Black cumin seeds are one of natural ingredients containing high antioxidant activity, like thymoquinone. Thymoquinone is the major bioactive compounds acting as antioxidants and powerful anti-inflammatory agents. Besides, this compound could positively affect the nerve system of the brain by the secretion of the hormone FSH and inhibit apoptosis in the cells of the reproductive organs (Forouzanfar et al., 2014). Therefore, this study was conducted to examine the effect of black cumin seeds powder on the number of Sertoli cells and the expression of caspase-3 in male mouse subjected diazinon.

MATERIALS AND METHODS

Animal and Experimental Design

The animal experiment was conducted at the Faculty of Veterinary Medicine, Brawjaya University (Malang-East Java, Indonesia). All procedures regarding the animal trial and treatments were approved by The Animal Ethics Committee of Brawijaya University (Malang-East Java, Indonesia). Besides, this experiment followed the animal research regulation of The Animal Welfare and Veterinary Public Health under The Republic of Indonesia (PP No.95/Tahun 2012/Kesehatan Masyarakat Veteriner dan Kesejahteraan Hewan).

A total of twenty Balb/c male mices (20-40 g; healthy condition; no inflammation) were selected for the experiment. The acclimation was done for 7 d prior to treatment. Furthermore, animals were divided into five different groups; K- (negative control, no treatment), K+ (positive control, 40 mg diazinon/kg BW); P1 (40 mg diazinon/kg BW + 200 mg black cumin/kg BW); P2 (40 mg diazinon/kg BW + 400 mg black cumin/kg BW); P3 (40 mg diazinon/kg BW + 600 mg black cumin/kg BW). The diazinon was orally submitted using gastric sonde daily for 8 wk. The mixture of feed and black cumin powder were given ad libitum. All the mices had an access to free water throughout the experiment.

Sampling, Preparation and Analyses

The edicular organs were collected at the end of experiment following previous protocols (Safithri, 2017). Furthermore, testicular immunohistochemical was done using the paraffin CPI procedures from The Cell Signaling Technology (Zulkarnain and Dicky, 2012). Meanwhile, the testicular histopathology was done using hematoxylin eosin staining (HE) following the prior procedures until further analyses and observation (Jusuf, 2009).

Statistical Analyses

The data was analyzed using the One Way Analysis of Variance (ANOVA) Test one-way pattern with a confidence level of 95% to find out the difference among groups. The significant difference among groups were considered with P < 0.05, meanwhile, P > 0.05 indicated no significant difference among groups.

RESULTS

Caspase-3 Testicles

The caspase-3 expression on testicular immunohistochemical are shown in Figure 1. Meanwhile, the average area of caspase-3 expression is pointed in Table 1. The data revealed that the supplementation of black cumin powder showing a decreased caspase-3 expression in mices subjected diazinon (P < 0.05), whereby the declined the average area of caspase-3 expression were more pronounced in P3 groups compared to their counterparts.

Table 1: Expression of caspase-3 on testicular immunohistochemical in mouses exposed to diazinon and treated with black cumin seed powder

Figure 2 shows The number of Sertoli cells of testicular in mice models. Meantime, the average number of Sertoli cells is summarized in Table 2. Overall, a significant difference was observed among the groups (P < 0.05). A more detailed comparison among groups showed the higher number of Sertoli cells was more pronounced in P3 groups compared to other treatment animals.

Table 2: Average number of Sertoli cells in testes mices exposed to diazinon and treated with black cumin seed powder

The caspase-3 expression in each treatment group showed differences in the brown area of expression due the reaction between DAB and the enzyme SA-HRP . This result indicated the presence of caspase-3 expression in cells which act as a mediator or executor of apoptosis associated in the destruction of cells. Furthermore, the apoptosis of testicular cells could trigger a decreased the quality and production of spermatozoa and induce infertility (Sari, 2018). Interestingly, the supplementation of black cumin powder therapy showed a decreased the average of caspase-3 expressions in mice exposed to diazinon. This result indicated the declined free radicals in the body, resulting the lower lipid peroxidation in endogenous membrane and apopotosis in testicular cells (Sari, 2018).

Besides, the decreased expression of caspase-3 in treatment groups might be due to the therapy of black cumin powder as a medicinal plant with thymoquinone content that has antioxidant effects. Indeed, thymoquinone could impair the negative impact on free radicals by donating electrons to free radicals, and subsequently stabilizing them and stopping the chain of free radical reactions to the formation of excess ROS. These circumtances could trigger oxidative stress of cells and create the apoptosis episodes (Fajrin, 2013). It is known that antioxidants are able to stabilize unpaired electrons and control the excessive oxidative stress to prevent apoptosis (Munawaroh, 2011).

Furthermore, we found the lower number of Sertoli cells in the seminiferous tubule area of mices subjected diazinon. Indeed, the induction of diazinon-type organophosphates could inhibit AChE which subsequently induce the incoordination of nerve system. Besides, these circumstances cause the inhibition of the GnRH release, then negatively affect the secretion of FSH and Sertoli cells (Osiana and Mayasari, 2017). Interestingly, mice subjected diazinon and supplemented black cumin powder showing a higher number of Sertoli cells. This result indicates that the black cumin powder positively affects the regulation of FSH and Sertoli cells. More details, an increased number of Sertoli cells from all three treatments might be due to the black cumin powder containing thymoquinone which play role as an antioxidant compounds. Hereinafter, this compound donates its electrons to the ACh binding free radical affecting a decreased AChEI. By these conditions, the neural synapse could deliver the impulses to the hypothalamus of the brain, subsequently triggers an increased GnRH in the anterior pituitary for FSH and Sertoli cells production (Osiana and Mayasari, 2017).

CONCLUSION

The data revealed that the administration of black cumin powder in mices subjected diazinon could decrease the expression of caspase-3 and increase the number of Sertoli cells. The data also pointed out that the black cumin powder with level 600 mg/kg BW show the strongest effects. Nevertheless, the further investigation concerning hormone profiles is suggested.

ACKNOWLEDGMENTS

The authors thank the Faculty of Veterinary Medicine, Brawijaya University-Indonesia for the financial support of this project (DPP/2020-Penelitian). The assistance of lab technician of Laboratory of Veterinary Reproduction-Brawijaya University is well appreciated.

conflict of interest

The author declares there is no conflict of interest in publishing this article.

novelty statement

This article has never been published and is not plagiarized from previous articles.

authors contribution

All authors contributed to the preparation of the article. The first author has the biggest contribution to compiling the article. A fourth author was also responsible for proofreading. So the correspondence was also carried out by the first writer.

REFERENCES

Boedeker W., M. Watts, P. Clausing, E.Marquez (2020). The Global Distribution of Acute Unintentional Pesticide Poisoning: Estimations Based on A Systematic Review. BMC Pub. Health. https://doi.org/10.1186/s12889-020-09939-0

Danaeia G., Hassan B., Memarb R., Ataeec dan M. Karamic (2018). Handbook for Protective Effect of Thymoquinone, The Main Component of Nigella sativa, Against Diazinon Cardio-Toxicity in Rats. Informa UK Limited, Taylor and Francis Group. https://doi.org/10.1080/01480545.2018.1454459

Dina M. S., Wahyuni S., Tr T. A. (2017). Decrease in The Number of Leydig Cells and White Mouse Sertoli Cells (Rattus norvegicus) Wistar Strain After Administration of Formalin. Jimvet., 1(2): 203–209.

Jusuf A.A. (2009). Handbook for Histoteknik Basic: Histology section of the Faculty of Medicine. University of Indonesia. Jakarta.

Fajrin F. A. (2013). Related to Cyclooxygenase (Cox)-2 To The Development of Cancer Therapy. J. K. G Unej, 10(1): 6 – 11

Forouzanfar, Fatemeh B.S.F., Bazzaz dan H. Hosseinzadeh (2014). Black Cumin (Nigella sativa) and Its Constituent (Thymoquinone): A Review on Antimicrobial Effects. Iranian J. Basic Med. Sci., 17(2): 930-938.

Lesmana, Listiya G., Diana A., Asus M. (2013). Observation of Taiwan’s Kijing Gastric Tissue (Anodonta woodiana Lea) Exposed to Diazinon 60 EC Pesticides At Some Concentrations. J. Exp. Life Sci. Vol. 3 No. 1. https://doi.org/10.21776/ub.jels.2013.003.01.07

Munawaroh L. (2011). Effect of Giving Black Cumin Seed Extract (Nigella sativa Linn.) Against Hepar Transaminase (GPT san SGPT) In Mice (Rattus norvegicus) Diabetes. State Islamic University (UIN) Maulana Malik Ibrahim Malang 2011, 16.

Nining E., R.S.S. Nazlib, Z.A. Mas’udc, Machfudd, Sobire (2019) Insecticide Residue Profile of Organophosphate in Onion Production Area (Allium Ascalonicum L.) Brebes Regency of Central Java. Bogor: Journal of Natural Resources and Environmental Management Bogor Agricultural Institute.

Osiana R., D. Mayasari (2017). Pesticides as One of the Risk Factors for Erectile Dysfunction. Medula, Vol. 7(5).

Son A., Kusuma B., Askandar, S. Mustokoweni (2015). Cleaved caspase-3 as Apoptosis Test in Cervical Cancer IIB Squamous Cell Type That Gets Neoadjuvan Cisplatin Chemotherapy. Obstet. Gynaecol. Magazine., 22(1): 22-27.

Safithri F. (2017). Potential of Black Cumin Seeds (Nigella sativa) In Endogenous Pancreatic Regeneration In Type-2 Diabetes Mellitus. Saintika Medika., 13(2): 76. https://doi.org/10.22219/sm.v13i2.5527

Sari L.M. (2020). Apoptosis: The Molecular Mechanism of Cell Death. Cakradonya Dent J., 10(2): 65-70 https://doi.org/10.24815/cdj.v10i2.11701

Sayuti K. (2015). Handbook for Natural Antioxidants and Sinetik. Field: Andalas University Press.

Zulkarnain, Dicky M.R. (2012). Expression of Caspase-3 Cell Leydig Mouse Sprague dawley Induced Low Dose Streptozotocin. J. Androl. Inonesia (MAI), No:43-44.