Anat Cell Biol.  2023 Dec;56(4):526-537. 10.5115/acb.23.079.

Effect of Sofosbuvir on rats’ ovaries and the possible protective role of vitamin E: biochemical and immunohistochemical study

• Ebrahim NA
• Abdalla HA
• Yassin NAE
• Maghrabia AE
• Morsy AI

• KMID: 2548914
• DOI: http://doi.org/10.5115/acb.23.079

Abstract

Hepatitis C virus (HCV) infection is a major health problem worldwide and its eradication is mandatory. Direct acting HCV polymerase inhibitors, such as Sofosbuvir (SOF), is an effective regimen. However, it has some side effects like mutagenesis, carcinogenesis, and the impairment of testicular function. It is important to evaluate the safety of SOF on the ovary, as there are no studies yet. Increasing the production of Reactive Oxygen Species (ROS), causes oxidative stress, which affects ovulation process, female reproduction, and fertility. Accumulation of SOF in the cells was demonstrated to promote ROS generation. Vitamin E (Vit E) is an antioxidant agent that has an essential role in the female reproductive system, its deficiency can cause infertility. We explored the effect of SOF treatment alone and co-treated with Vit E on ovarian ROS level and ovarian morphology experimentally using biochemical and immunohistochemical studies. Significant changes in oxidative stress markers; nitric oxide and malondialdehyde lipid peroxidation, antioxidant enzymes; catalase, super oxide dismutase, and reduced glutathione, proliferating markers; proliferation cell nuclear antigen and Ki-67 antigen and caspase 3 apoptotic marker were demonstrated. It was shown that where SOF induced oxidative stress, it also aggravated ovarian dysfunction. The essential role of Vit E as an antioxidant agent in protecting the ovarian tissue from the effect of oxidative stress markers and preserving its function was also displayed. This could be guidance to add Vit E supplements to SOF regimens to limit its injurious effect on ovarian function.

Keyword

Ovary; Sofosbuvir; Oxidative stress; Apoptosis; Vitamin E

Figure

• 

  Fig. 1 Histograms showing the mean ovarian tissue oxidative stress markers (NO, MDA, SOD, GSH, and CAT). (A, B) Levels of NO and MDA in different experimental groups were demonstrated. The SOF-treated group shows a significant upregulation in the level of NO and MDA compared to the control group, ****P<0.00001. The oxidative stress markers’ levels are reversed considerably in the SOF+Vit E-treated group compared to the SOF group, ###P<0.0001, ####P<0.00001. (C–E) The ovarian tissue CAT, SOD, and GSH levels in different experimental groups. The SOF-treated group demonstrates a significant downregulation in CAT, SOD, and GSH levels compared to the control group, ***P<0.0001, ****P<0.00001. The oxidative stress markers are considerably altered in the SOF+Vit E co-treated group compared to SOF group, #P<0.01 and ###P<0.0001. One-way ANOVA was used to compare between all the groups, unpaired t-test was used to compare each group with the control, and the SOF+Vit E co-treated with SOF group. NO, nitric oxide; MDA, malondialdehyde; SOD, super oxide dismutase; GSH, reduced glutathione; CAT, catalase; SOF, Sofosbuvir; Vit E, vitamin E. *P<0.01 and **P<0.001.

• 

  Fig. 2 Micrographs of hematoxylin and eosin-stained rats’ ovarian sections (A–E). (A) Ovarian sections in the control group illustrate multiple growing GF, CL, vascularized M, PR with observable ovum. (B, C) The SOF-treated ovary demonstrating atretic follicile (T), congested B and multiple V. (D) The Vit E-treated ovary illustrates healthy follicles with multiple corpora lutea. (E) The SOF+Vit E co-treated ovary demonstrates health primary follicles with multiple corpora lutea and mildly vascularized medulla. (F–H) Histograms illustrate the morphometric study of the effect of SOF and Vit E on treated rats’ ovaries: (F) healthy follicles. (G) Atretic follicile and (H) corpora lutea. The SOF-treated group illustrates a significant reduction in the number of atretic follicles and corpora lutea compared to the control. While the number of atretic follicles was significantly increased in the SOF group compared to the control, *P<0.01, **P<0.001, ****P<0.00001. In the SOF+Vit E co-treated group, the number of healthy follicles and corpora lutea are significantly upregulated, while the atretic follicles are significantly downregulated, ####P<0.00001. One-way ANOVA was used to compare between multiple groups, unpaired t-test was used to compare between two groups. Scale bar: 50 μm. GF, graafian follicles; CL, corpus luteum; M, medulla; PR, primary follicle; SOF, Sofosbuvir; B, congested blood vessles; V, vacuolations; Vit E, vitamin E.

• 

  Fig. 3 Micrographs of rat’s ovaries immuno-histochemicaly stained with PCNA. PCNA-positive nuclei are localized in brown (A–D). (A) Control group, (B) SOF-treated group, SOF illustrates significant reduction in the PCNA-positive cells in comparison to the control group, (C) Vit E-treated group, and (D) the SOF+Vit E treated group, where PCNA-positive cells increased compared to the SOF group. (E) Histogram illustrates the quantitative analysis of the intensity of PCNA stained areas in different experimental groups. The SOF-treated group demonstrates a statistically significant reduction compared to the control group, ****P<0.00001. The SOF+Vit E co-treatment significantly reduces the PCNA immuno-staining in comparison with the SOF treated group, ####P<0.0001 (Bar=20 μm). PCNA, proliferation cell nuclear antigen; SOF, Sofosbuvir; Vit E, vitamin E.

• 

  Fig. 4 Micrographs of rat’s ovaries after Ki-67 immunohistochemical technique. Ki-67-strongly stains the cell nuclei (A–D). (A) The control group, granulosa cells and theca cells in the graafin folliciles (GF) are intensely stained. (B) The SOF-treated group demonstrates Ki-67-negativily stained cystic atretic follicles (CF), (C) The Vit E-treated group, and (D) the SOF+Vit E-treated group, where the number of Ki-67-positive cells localized in granulosa and theca cells of GF. (E) Histogram illustrates the quantitative analysis of the intensity of Ki-67 stain area percentage in different experimental groups. The SOF-treated group demonstrates a statistically significant reduction compared to the control group, *P<0.01, ****P<0.00001. The SOF+Vit E Co-treatment reduces Ki-67 immuno-staining significantly in comparison with the SOF treated group, ####P<0.0001 (Bar=20 μm). SOF, Sofosbuvir; Vit E, vitamin E.

• 

  Fig. 5 Micrographs immunolocalize caspase 3 in rat’s ovaries (A–D). (A) The control group demonstrates limited stain. (B) The SOF-treated group in which SOF significantly upregulates the caspase 3 positive cells, which are predominantly located in the cytoplasm of the cells, compared to the control group. The brown-stained cells concentrate at granulosa and luteal cells. (C) The Vit E-treated group, and (D) the SOF+Vit E treated group, where the number of caspase 3 positive cells are reduced as compared to the SOF group. (E) Histogram illustrates the quantitative analysis of the intensity of caspase 3 stain in different experimental groups. The SOF-treated group demonstrates a statically significant upregulation compared to the control group, ****P<0.00001. The SOF+Vit E co-treatment significantly reduces the caspase 3 immuno-staining in comparison with the SOF treated group, ####P<0.0001 (Bar=20 μm). SOF, Sofosbuvir; Vit E, vitamin E.

Reference

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