Effect of Lactobacillus species on apoptosis-related genes BCL2, BAX, and caspase 3 in the testes of gamma-irradiated rats

Abstract

OBJECTIVE:

Ionizing radiation has various applications, including uses in medicine, industry, agriculture, and research. However, ionizing radiation is accompanied by side effects in normal radiosensitive tissues. Probiotics as natural radioprotective agents can protect normal tissues from ionizing radiation. In this regard, this study aimed to investigate the effect of Lactobacillus species on apoptosis-related genes BCL2, BAX, and caspase 3 (CASP3) in the testes of gamma-irradiated rats.

METHODS:

A total of 30 male Wistar rats were involved in this study. The animals received the whole- body radiation with the dose rate of 2 Gy gamma-ray and were orally gavaged with 0.2 mL of 1×10¹⁰ Lactobacillus species in phosphate-buffered saline (PBS) for 4 weeks. Then, the relative gene expression levels of BCL2, BAX, and CASP3 in the testis were assessed by using the quantitative real-time polymerase chain reaction (qRT-PCR).

RESULTS:

Compared with the control group, radiation significantly downregulated the BCL2 and upregulated the BAX and CASP3 genes (p<0.0001). However, Lactobacillus species significantly reversed these effects.

CONCLUSION:

All in all, according to our results, employing Lactobacilli probiotics as a natural radioprotector may protect radiosensitive tissue from damage.

KEYWORDS:
Gamma radiation; Apoptosis; Probiotics; Lactobacillus species

INTRODUCTION

It has been estimated that about 50–70% of all oncology patients undergo radiation therapy or a combination of chemotherapy and radiation therapy¹. Although radiotherapy has turned into one of the most common treatments for cancer, the detrimental effects on the radiosensitive normal tissues limit the radiation exposure amount that can be applied². The radiation damages radiosensitive normal tissues through various mechanisms³. Reactive oxygen species (ROS) are the major cause of cell apoptosis and DNA damage through increasing BAX and caspase 3 (CASP 3) levels and decreasing BCL2 levels in radiation-exposed normal tissues⁴. Therefore, the administration of certain antioxidants as radioprotective agents is a critical procedure to attenuate the radiation-related harmful effects on normal tissues.

Recently, probiotics, as a natural radioprotector, have attracted scientific interest. Probiotics, especially lactic acid bacteria (LAB), are live nonpathogenic microorganisms that have health benefits when consumed in adequate quantity⁵. They can increase antioxidant capacity through different ways such as scavenging the excess free radicals and producing various metabolites such as glutathione (GSH), butyrate, and folate⁶. Some studies have also shown that probiotics, especially Lactobacillus species, exert their radioprotective effect through the regulation of the nuclear factor kappa B (NF-κB) pathway⁷,⁸. Additionally, the anti-apoptosis and anti-inflammation effects of Lactobacillus species on the irradiated normal tissues were reported by other studies⁹,¹⁰.

Since the testis is one of the most radiosensitive tissues, this study aimed to investigate the effect of Lactobacillus species on apoptosis-related genes BCL2, BAX, and CASP 3 in the testes of gamma-irradiated rats.

METHODS

Irradiation

The animals received the whole- body radiation with the dose rate of 2 Gy gamma-ray (⁶⁰Co) and 100 cGY/min and a source-to-skin distance (SSD) of 100 cm. At the same time, the field of view (FOV) was set at 36×36, and SSD was 80 cm. The single 2 Gy dose of X-ray irradiation was conducted in Rad Source Model RS2000 Irradiator with a 0.3-mm copper filter and X-ray tube settings of 160 kVp and 24 mA (Rad Source Technologies, USA).

Preparation of probiotic strains

Lactobacillus casei (LC) and Lactobacillus acidophilus (LA) isolates were provided from the Iranian Biological Resource Center (IBRC). The bacteria were aerobically grown in de Man, Rogosa, and Sharpe (MRS) medium (Sigma-Aldrich, UK) at 37°C for 16 h. The live bacteria were harvested by centrifugation (10 min, 4000×g, room temperature). Thus, plate bacteria were mixed with phosphate-buffered saline (PBS) at the desired concentration. During the experiment, the rats received 0.2 ml of PBS containing 1×10¹⁰ colony-forming unit (CFU) of the probiotics daily.

Animals and treatment

The male Wistar rats (6–8 weeks old) weighing 220±20 g were purchased from the Animal House of Tehran University of Medical Sciences. They were housed under standard laboratory conditions (12-h light/dark cycle at 22±1°C temperature and 55±10% humidity).

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Tehran University of Medical Sciences (the ethical code: 34613).

The rats were divided into six groups (five rats in each group) and treated as it follows:

• Group 1 (healthy control): the animals only received PBS.

• Group 2: the animals only received radiation using the dose rate of 2 Gy gamma- rays.

• Group 3: the animals were orally gavaged with 0.2 mL of 1×10¹⁰ LC in PBS.

• Group 4: the animals were orally gavaged with 0.2 mL of a suspension of 1×10¹⁰ CFU LC in PBS, and their whole body was exposed to radiation.

• Group 5: the animals were orally gavaged with 0.2 mL of a suspension of 1×10¹⁰ CFU LA in PBS.

• Group 6: the animals were orally gavaged with 0.2 mL of a suspension of 1×10¹⁰ CFU LA in PBS, and their whole body was exposed to radiation.

After 4 weeks, the rats were anesthetized intraperitoneally (i.p.) with 250 mg/kg 2,2,2-tribromoethanol (TBE; Avertin^®, Sigma-Aldrich) and sacrificed by cervical dislocation. Then, the rats’ testes were isolated and immediately frozen in liquid nitrogen and then stored at -80°C for the subsequent analyses.

Quantitative analysis of real-time PCR (qRT-PCR)

The total RNA was extracted from the rat’s testis using TRIzol^®LS (Invitrogen Corp., USA) reagent according to the manufacturer’s directions. Then, the single-stranded complementary DNA (cDNA) was synthesized from equal amounts of RNA using the Prime Script cDNA Synthesis Kit (Takara Bio, Japan), following the manufacturer’s directions. The relative expression levels of the target gene were measured by qRT-PCR using SYBR Green with the following primer sets: BCL2 (forward, 5'-GGTGAACTGGGGGAGGATTG-3'; reverse, 5'-GCATGCTGGGGCCATATAGT-3') (product size: 197 bp), BAX (forward, 5'-GGCGATGAACTGGACAACAA-3'; reverse, 5'-CAAAGTAGAAAAGGGCAACC-3') (product size: 151 bp), CASP3 (forward, 5'-AGCTGGACTGCGGTATTGAG-3'; reverse, 5'-ATGGCGCAAAGTGACTGGAT-3') (product size: 189 bp), and Hypoxanthine-guanine phosphoribosyl transferase (HPRT) (forward, 5'-TCAGTCAACGGGGGACATAAA-3'; reverse, 5'-GGGGCTGTACTGCTTAACCAG-3') (product size: 142 bp). The relative amounts of BCL2, BAX, and CASP3 mRNA were normalized against the endogenous control, HPRT, and calculated with the 2^−ΔΔCt formula.

Statistical analysis

The graphs and the statistical analysis of the data were performed using SPSS 16. The results were represented as the mean±SD. One-way analysis of variance (ANOVA) was followed by the Tukey’s post hoc test for multiple comparisons. p≤0.05 was considered statistically significant.

RESULTS

Effect of Lactobacillus species on the BCL2 and BAX gene expression in the testes of gamma-irradiated rats

As shown in Figure 1, radiation significantly downregulated the BCL2 gene expression in the testis tissues in comparison with the control group (p<0.0001). Conversely, Lactobacillus spp. administration significantly reversed this effect (p<0.01). Moreover, the mRNA level of BCL2 slightly decreased in the LC and LA groups compared with the control group.

Figure 1
Effect of Lactobacillus species on the BCL2 gene expression in the testes of gamma-irradiated rats.

The qRT-PCR results also showed that radiation significantly upregulated the BAX gene expression in the testis tissues in comparison with the control group (p<0.0001) (Figure 2). However, in the ionization radiation-treated rats and LC (IR+LC) and (ionization radiation-treated rats and LA (IR+LA) groups compared with the irradiated group, Lactobacillus spp. significantly decreased the expression of the BAX gene (p<0.05) (Figure 2). Additionally, our results showed that the mRNA level of BAX did not change between Lactobacillus spp. (LC and LA) treated groups and control groups.

Figure 2
Effect of Lactobacillus species on the BAX gene expression in the testes of gamma-irradiated rats.

Effect of Lactobacillus species on the CASP3 gene expression in the testes of gamma-irradiated rats

Similar to the BAX gene, the mRNA level of CASP3 significantly increased in the irradiated group compared with the control group (p<0.0001) (Figure 3). However, this effect was significantly reversed by treatment with Lactobacillus spp. in the IR+LC and IR+LA groups compared with the irradiated group (p<0.01) (Figure 3). We also found that the mRNA level of CASP3 did not change in the LC and LA groups compared with the control group.

Figure 3
Effect of Lactobacillus species on the CASP3 gene expression in the testes of gamma-irradiated rats.

DISCUSSION

Ionizing radiation plays a critical role in medical diagnosis and cancer-related therapy. It has been shown that the testis is one of the most radiosensitive organs because very low doses of radiation lead to abnormalities in spermatogenesis by mutagenesis, apoptosis, and necrosis¹¹. Exposure of the testes to radiation leads to the induction of apoptosis in the radiosensitive normal cells, which may result in temporary or permanent infertility¹². Additionally, Mingote et al. reported that changes in the brain lipid’s intensities are early tissue responses to radiation exposure¹³{Mingote, 2020 #1}. The development of natural radioprotective agents with less toxicity and high effectiveness is attractive and interesting. Therefore, this study pursued the goal to determine the probiotic treatment effects on modulating apoptosis-related genes BCL2, BAX, and CASP3 in the testes of gamma-irradiated rats. The probiotics such as LAB are the beneficial bacteria employed as an adjunct to reduce the adverse effects of ionizing radiation through several mechanisms¹⁴. Some studies indicated that certain probiotics modulate the activation of signaling pathways in radiation therapy⁸. The cell survival and death are regulated by the equilibrium of pro-apoptotic and the anti-apoptotic BCL2 family proteins and BAX/BCL2 ratio determines the cell susceptibility to apoptosis¹⁵. Our qRT-PCR results revealed that radiation downregulated the BCL2 and increased the expression of the BAX gene in the normal testicular cells. The reaction of ionizing radiation with the cellular contents such as the small molecules of water in the normal testicular cells leads to the generation of ROS¹⁶. The high ROS level induces apoptosis via controlling the phosphorylation and ubiquitination of BCL2 family proteins, which results in the upregulation of pro-apoptotic genes (e.g., BAX) and the downregulation of anti-apoptotic (e.g., BCL2)⁴.

Some studies indicated that probiotics modulate the cellular signaling pathway in mammals by direct attachment to the cell surface¹⁷. For instance, Lutfi et al. showed that probiotic Lactobacillus rhamnosus negatively regulates appetite markers possibly through melatonin receptors¹⁸. In this study, we showed that Lactobacillus spp. significantly upregulates the BCL2 gene and downregulates the BAX and CASP3 genes in the testes of irradiated rats. In the human body, LAB such as LA and LC are part of the normal microbiota or microflora. The protective effects of probiotics against radiation were reported by many studies. Liu et al. demonstrated that the probiotic Lactobacillus Plantarum 299v reduced gastrointestinal injury and inflammation in the rats that were locally irradiated with 10 Gy¹⁹. In addition, it was shown that L. rhamnosus GG ATCC 53103 reduced intestinal epithelial apoptosis and improved crypt survival following whole-body gamma radiation at a dose of 12 Gy⁸. Intestinal bacteria also lowered the negative effect of radiation on intestinal barrier integrity by regulating the expression of tight junction-related proteins and restoring intestinal permeability²⁰. A recently published study reported that probiotics improved the testes’ function by neutralizing the toxins, improving sperm quality and testosterone levels, and modulating the immune system²¹. Researchers suggested that probiotics reduced the ROS activation evoked by radiation via the production of antioxidant enzymes such as superoxide dismutase, GSH peroxidase, GSH reductase, and catalase²²,²³. Shokri et al. demonstrated that melatonin with its antioxidant property can decrease oxidative damage induced by radiofrequency electromagnetic radiation (RF-EMR) of mobile phones on testis tissue²⁴. Moreover, melatonin has been reported to have an important anti-apoptotic action by attenuating the production of ROS and pro-apoptotic proteins, such as BAX²⁵. As above mentioned probiotics affect the melatonin pathway, therefore, it seems that melatonin might also be involved in the radioprotective effects of probiotics on testis tissue.

Certain limitations should be noted in this study. Primarily, immunohistochemical studies or tunnel analysis were not used to exactly evaluate the apoptosis induction in the testis tissue. However, in view of these findings, it is probable, therefore, that Lactobacillus species protect the testes of gamma-irradiated rats by modulating apoptosis-related genes BCL2, BAX, and CASP3. This study is the first report, to the best of our knowledge, indicating the modulatory effect of Lactobacillus species on apoptosis-related genes BCL2, BAX, and CASP3 in the testes of gamma-irradiated rats.

CONCLUSION

In summary, we concluded that Lactobacillus spp., particularly LC protects the testicular tissue against high- dose radiation (2 Gy) through modulating the BAX and BCL2 genes expression, which play a significant role in the activation of apoptosis.

• Funding: this study was financially supported by the Tehran University of Medical Sciences.

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Publication Dates

• Publication in this collection
  13 Dec 2021
• Date of issue
  Nov 2021

History

• Received
  23 Aug 2021
• Accepted
  24 Aug 2021