Pretreatment with metformin protects mice from whole-body irradiation

Abstract

Metformin, a first-line oral drug for type II diabetes mellitus, not only reduces blood glucose levels, but also has many other biological effects. Recent studies have been conducted to determine the protective effect of metformin in irradiation injuries. However, the results are controversial and mainly focus on the time of metformin administration. In this study, we aimed to investigate the protective effect of metformin in BALB/c mice exposed to 6 Gy or 8 Gy of a ⁶⁰Co source of γ-rays for total body irradiation (TBI). Survival outcomes were assessed following exposure to 8 Gy or 6 Gy TBI, and hematopoietic damage and intestinal injury were assessed after exposure to 6 Gy TBI. Metformin prolonged the survival of mice exposed to 8 Gy TBI and improved the survival rate of mice exposed to 6 Gy TBI only when administered before exposure to irradiation. Moreover, pretreatment with metformin reduced the frequency of micronuclei (MN) in the bone marrow of mice exposed to 6 Gy TBI. Pretreatment of metformin also protected the intestinal morphology of mice, reduced inflammatory response and decreased the number of apoptotic cells in intestine. In conclusion, we demonstrated that pretreatment with metformin could alleviate irradiation injury.

INTRODUCTION

Radiation therapy (RT) is a major treatment strategy for malignant tumors, and more than 50% of patients with tumors receive RT [1]. The major limiting factor of RT is damage to the normal tissues [2]. The bone marrow and gastrointestinal tract are the organs most sensitive to ionizing radiation (IR). There are also some concerns about the danger of accidental exposure to radioactive sources and causing IR damage. In addition, the threat posed by the use of radioactive materials in war or terrorism is also a major global security concern. Therefore, the development of an effective radiation protection agent can not only reduce the side effects of RT, but can also allow for the use of higher doses of IR to more effectively kill tumor cells, increase the dose of IR to achieve the purpose of effectively killing tumor cells, provide alternative treatment choices for personnel involved in nuclear accident and provide protection to members of the nuclear accident rescue teams.

So far, amifostine is the only chemical radioprotective compound that has been approved by the US Food and Drug Administration (FDA) to mitigate IR-induced injuries in patients undergoing RT [3]. Since amifostine causes serious side effects such as toxicity, it can only be administered through limited routes and during a narrow time window, which tremendously limits its clinical use [4]. In view of this, new radioprotective agents that are nontoxic and easy to administer are still urgently needed. Searching effective radioprotective agents among drugs that have been approved for the treatment of various diseases is a new strategy to develop prevention and treatment of different forms of radiation damage [5]. The biggest advantage of this strategy is that there is no need to consider the toxicity of the drug itself when using it at known therapeutic concentrations. Based on this strategy, several studies have found that metformin has a certain radioprotective effect [6–8]. Metformin is the most commonly used type 2 diabetes drug in clinical practice. Recent studies have shown that, in addition to its hypoglycemic effect, metformin also possesses a variety of biological effects, including anti-cancer, anti-aging, neuro- and cardiovascular-protective effects [9]. Metformin has also been found to have some antioxidant effects and enhances the DNA repair capacity of cells [10]. Several studies have also confirmed that metformin possesses anti-inflammatory and anti-apoptotic properties [11, 12]. Moreover, clinical data have shown that metformin usage is positively correlated with the overall survival of patients with liver cancer post radiotherapy [13]. All these properties could potentially make metformin a promising radiation countermeasure.

Recently, an increasing number of studies have researched the radioprotective effects of metformin in mice or rats. In most of these studies, metformin was administered before and after irradiation [14–16]. However, the results of these studies are controversial. Some studies showed that metformin exhibited a radioprotective effect only when administered to mice after radiation exposure [7]. Therefore, the time at which metformin should be administered to generate a radioprotective effect still needs to be clarified. This can help determine whether metformin has protective or mitigative effects on irradiation induced injuries.

In this study, we aimed to investigate the protective effect of metformin administered before or after total body irradiation (TBI) based on survival in mice, as well as its influence on hematopoietic damage and intestinal injury after γ-ray irradiation.

MATERIALS AND METHODS

Mice

Male BALB/c mice weighing 18–22 g were obtained from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). The animals were housed in an air-conditioned room in a specific-pathogen-free facility under a 12 h light/dark cycle, with access to food and water ad libitum. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals, and the study protocols were approved by the Ethical Committee of the Fourth Military Medical University.

Total body irradiation and metformin administration

TBI was delivered using a ⁶⁰Co source of γ-rays at a dose rate of 100 cGy/min. For the survival experiments, mice were randomly divided into three groups as follows: (i) IR + vehicle (control, n = 9); (ii) metformin treatment before IR (n = 8); and (iii) metformin treatment after IR (n = 8). The mice in group 2 received daily an oral gavage of 200 mg/kg of metformin for 3 days before IR. The mice in group 3 received an oral gavage of 200 mg/kg of metformin immediately after irradiation and continued for 5 days. Animals in group 1 received an equivalent volume of PBS by oral gavage. All the mice were exposed to 8 Gy or 6 Gy of γ-rays. For the remaining experiments, mice (n = 10) were randomly divided into three groups as follows: (i) IR + vehicle (control); (ii) metformin treatment before IR; and (iii) metformin treatment after IR. The mice in group 2 received daily an oral gavage of 200 mg/kg of metformin for 3 days before IR. The mice in group 3 received an oral gavage of 200 mg/kg of metformin immediately after irradiation and continued for 5 days. The mice in group 1 received an equivalent volume of PBS by oral gavage. All the mice were exposed to 6 Gy of γ-rays and sacrificed at indicated time.

Micronucleus assay

The polychromatic erythrocytes (PCEs) in bone marrow of rodents is routinely used in micronucleus test, since when a bone marrow erythroblast develops into a PCEs, the main nucleus is extruded and any micronucleus that has been formed remains in the otherwise enucleated cytoplasm, which makes the micronucleated PCEs be easily visualized. The micronucleus test was performed using Schmidt’s method as previously reported [17]. The mice were sacrificed by using cervical dislocation on day 14 after irradiation. The bone marrow was flushed out from the hip bones with fetal calf serum, and the cell suspension was collected. The suspension was centrifuged at 1000 rpm (300 × g) for 7 min. The supernatant was discarded and the cells were resuspended in 100 μL fetal calf serum. Next, 20 μL of the solution was placed on a slide, which was kept for 24 h at room temperature and then fixated using methanol before staining. The samples were stained with May-Grunewald-Gyms. Once the slides were prepared, for each sample 1000 PCEs were counted along with nucleated cells, and the frequency of micronuclei (MN) in 1000 PCEs was calculated.

ELISA

Mouse serum was collected into tubes. Mouse blood was collected into a sterile tube from the orbital sinus by removing the eyeball, and allow it to clot by leaving the tube at room temperature for 30 min. Then, the serum was obtained by centrifuging 1500 × g for 10 min at 4°C. Intestinal tissues were homogenized with PBS using a homogenizer device. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum and intestine homogenates were detected using mouse IL-6 and TNF-α ELISA kits (4A BIOTECH, Beijing, China) according to the manufacturer’s instructions.

Histological analysis

Small intestine tissues were isolated at day 7 after 6 Gy of TBI. After fixing overnight in 10% neutral formalin, the tissues were embedded in paraffin. Then 3 μm-thick sections were cut, stained with hematoxylin and eosin, and observed under a light microscope (Olympus Corp., Tokyo, Japan). The villus height was determined from hematoxylin and eosin-stained sections using the CaseViewer software (version 3.3.6). At least 10 well-oriented, full-length crypt villus units per mouse were measured.

TUNEL assay

TUNEL staining was used to detect the degree of apoptosis in the small intestine. The TUNEL assay was performed using an in situ Cell Death Detection Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, paraffin sections were deparaffinized, rehydrated and incubated with the TUNEL reaction mixture at 37°C for 1 h. After counterstaining with DAPI, the sections were analyzed under a fluorescent microscope (Olympus Corp., Tokyo, Japan).

Statistical analysis

The data were analyzed by unpaired Student’s t-test or one-way ANOVA followed by a Tukey’s multiple comparisons post-test, appropriately. Differences were considered significant at P < 0.05. All the analyses were performed using GraphPad Prism 7 from GraphPad Software (San Diego, CA, USA).

RESULTS

Pretreatment with metformin prolonged the survival period of irradiated mice

Figure 1 shows the survival curves of mice exposed to γ-irradiation at a lethal dose of 8 Gy or a sublethal dose of 6 Gy. At a dose of 8 Gy, mice were found dead on the ninth day after irradiation in the control group; the survival rate was the same in the group where the animals were administered metformin after irradiation; however, in the group where mice were administered metformin 3 days before irradiation, a significant delay in the time of death was observed (Fig. 1a). Similarly, at a dose of 6 Gy, survival rate in the mice treated with metformin before irradiation was significantly improved from 30% to 70% compared to that in control mice, while the mice treated with metformin after irradiation were all dead (Fig. 1b). These results suggest that metformin may have a radioprotective effect but only when administrated before irradiation.

Fig. 1.

Effect of metformin on mice survival rate after TBI. (A) Time scheme of the experimental procedures, 200 mg/kg of metformin was administered for 3 consecutive days before irradiation or 7 consecutive days after irradiation. (B) The mice survival rate at a dose of 8 Gy (^**P < 0.01, n = 9 for control group, n = 8 for the other two group). (C) The mice survival rate at a dose of 6 Gy (^*P < 0.05, ^****P < 0.0001, n = 10 per group). p values were calculated by the log-rank test.

Pretreatment with metformin ameliorates hematopoietic damage caused by irradiation

We also evaluated the protective effects of metformin treatment between before and after TBI on irradiated mice at the sublethal dose of 6 Gy. Results showed that metformin pretreatment significantly improved the body weight of irradiated mice (Fig. 2a), as well as the reduced weight of the spleen and thymus caused by irradiation (Fig. 2b and c). Furthermore, the concentrations of the pro-inflammatory cytokines IL-6 and TNF-α in peripheral blood were lower in metformin-pretreated mice after irradiation, compared to the concentrations of the pro-inflammatory cytokines of animals treated with PBS or metformin after irradiation (Fig. 2d and e).

Fig. 2.

Pretreatment with metformin ameliorates irradiation caused hematopoietic damage. (A) Body weight were compared among mice treated with metformin before or after irradiation at day 14 after 6 Gy TBI. (B–C) The ratio of weight of dissected spleens (B) and thymuses (C) of mice to the body weight at day 14 after 6 Gy TBI. (D-E) The levels of IL-6 (D) and TNF-α (E) in peripheral blood of normal mice or mice pretreated with or without metformin at day 7 after 6 Gy TBI were detected by ELISA. The data are presented as mean ± SD (n = 5). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, using one-way ANOVA followed by a Tukey’s multiple comparisons post-test.

Formation of micronuclei in polychromatic erythrocytes

To assess the cytogenetic effects of γ radiation and the radio protective effects of metformin, we performed an MN test. The results of the MN analysis in the cells obtained from the bone marrow of the mice were consistent with the survival rates (Fig. 3a). The administration of metformin prior to irradiation lead to an increase in the number of nucleated cells in bone marrow polychromatocytes compared to the number of nucleated cells in the control mice or the mice treated with metformin after irradiation (Fig. 3b), as well as a significant decrease in the rate of MN formation in PCEs (Fig. 3c), which indicated that pretreatment with metformin could alleviate cytogenetic injuries caused by γ radiation.

Fig. 3.

Formation of MN in polychromatophilic erythrocytes. (A) Representative images of bone marrow cells stained with May-Grunewald-Gyms from normal mice and irradiated mice with or without metformin pretreatment at day 14 after 6 Gy TBI (Scale bar = 20 μm), the black arrows indicate the cells with typical MN. (B) Number of nucleated cells. (C) Frequency of micronucleated polychromatic erythrocytes (MNPCE). The data are presented as mean ± SD (n = 5). ^**P < 0.01, ^***P < 0.001, using unpaired Student’s t-test.

Pretreatment with metformin reduces irradiation-induced intestinal injury

As the gastrointestinal tract is also highly sensitive to IR, we next assessed the morphological changes in the small intestine of the mice. The villus height of the small intestine was significantly improved in metformin-pretreated mice compared to that in control mice or the mice treated with metformin after irradiation at day 7 after 6 Gy TBI (Figs 4a and b). ELISA analysis showed that the levels of pro-inflammatory cytokines IL-6 and TNF-α were also decreased in metformin-pretreated mice (Fig. 4c and d). These results demonstrated that pretreatment with metformin has a protective effect against intestinal damage after TBI.

Fig. 4.

Histopathological evaluation of the radioprotective effect of metformin in intestine. (A) Representative hematoxylin and eosin-stained section of mouse small intestine at day 7 after 6 Gy TBI or left untreated (scale bar = 200 μm). (B) Quantification of villus height, n = 5 in each group. (C) Intestinal IL-6 and TNF-α levels were measured in tissue homogenate obtained at day 7 after 6 Gy TBI. The data are presented as mean ± SD (n = 5). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, using one-way ANOVA followed by a Tukey’s multiple comparisons post-test.

Pretreatment with metformin impairs cell apoptosis in intestine

To test the effect of metformin on IR-induced cell death, we examined the apoptosis in the intestine tissue at day 7 after 6 Gy TBI. As shown in Fig. 5, the number of TUNEL-positive apoptotic cells was significantly lower in the metformin-pretreated group compared to the number of TUNEL-positive apoptotic cells in the control mice or the mice treated with metformin after irradiation group. Taken together, these results indicate that pretreatment with metformin inhibits radiation-induced apoptosis in the intestine.

Fig. 5.

Pretreatment with metformin reduces cell apoptosis in intestine. (A) Representative of bright-field (BF) and fluorescence images of TUNEL-stained intestinal sections at day 7 after 6 Gy TBI or left untreated. Cell nuclei (DAPI staining, blue), TUNEL-positive cells (green) (Scale bar = 50 μm). (B) Quantitative analysis of TUNEL-positive cells per field of view. The data are presented as mean ± SD (n = 5). ^***P < 0.001, using one-way ANOVA followed by a Tukey’s multiple comparisons post-test.

DISCUSSION

Previous studies have shown the radioprotective role of metformin in various animal models [6, 14, 18]. Recently, Bagheri et al. [19] reported that treatment with metformin 1 day before irradiation for 4 consecutive days could ameliorate radiation-induced intestinal toxicity in rats. However, they did not evaluate the radioprotective effect of metformin between administration before and after irradiation. In this study, we demonstrated that, compared to administration after irradiation, pretreatment with metformin prolonged the survival period of mice subjected to 8 Gy TBI and improved the survival rate of mice subjected to 6 Gy TBI. In the following sublethal dose of 6 Gy TBI, the irradiated mice pretreated with metformin showed less hematopoietic and intestinal injuries.

The bone marrow is one of the most sensitive organs to irradiation. Cheki et al. [20] revealed the radioprotective effect of metformin on human lymphocytes when used before irradiation. They showed that treatment with metformin before irradiation ameliorates MN formation in irradiated cells. In line with this study, our results further demonstrated that pretreatment with metformin alleviated hematopoietic damage of irradiated mice at a dose of 6 Gy. We also observed that pretreatment with metformin significantly reduced the loss of spleen and thymus weight caused by γ-rays, accompanied by lower levels of the pro-inflammatory cytokines IL-6 and TNF-α in peripheral blood. In addition, the mice pretreated with metformin after irradiation showed less MN formation in bone marrow cells. Our results, therefore, indicate that metformin could prevent chromosomal damage and alleviate inflammatory responses in the hematopoietic system, thus, decreasing the side effects commonly caused by irradiation.

Since intestinal epithelial cells have a rapid regeneration rate, the gastrointestinal tract is also very sensitive to irradiation. With the increasing numbers of patients with cancers affecting the pelvis and the abdomen, it is necessary and important to be concerned about the effects of irradiation on the gastrointestinal tract. Of note, it has been reported that metformin has a beneficial effect on intestinal maintenance after injury caused by chemical or biological toxins. Two recent studies found that metformin could protect against intestinal barrier dysfunction induced by dextran sodium sulphate (DSS) or lipopolysaccharide (LPS) [21, 22]. Intestinal inflammation is largely associated with barrier dysfunction. These two studies also reported that metformin decreased the levels of the pro-inflammatory cytokines IL-6 and TNF-α in the colon or intestine. However, it is still unclear whether metformin has protective effects on intestinal injuries caused by irradiation. In this study, we provided evidence suggesting that metformin pretreatment could alleviate the radiation-induced intestinal injury in mice, which was also recently confirmed by Chen et al. [23]. Our results showed that pretreatment with metformin ameliorates intestinal morphology in irradiated mice with higher villus, as well as reduce the production of the pro-inflammatory cytokines IL-6 and TNF-α in the intestine tissues. Moreover, the degree of apoptosis in intestinal cells was significantly reduced in metformin pretreated irradiated mice.

The potential mechanisms of radioprotective effects in metformin are mostly relied on its properties with antioxidant, anti-inflammatory, anti-apoptosis and enhancing DNA damage responses [10]. Metformin can be served as radical scavenger to reduce free radicals directly or via the stimulation of antioxidant enzymes [24]. Additionally, it also has ability to reduce reactive oxygen species (ROS) production and stimulate DNA damage responses via AMP-activated protein kinase (AMPK) [25, 26]. Xu et al. [14] revealed that treatment with metformin potently reduced IR-induced DNA damage and ROS production by decreasing expression of NADPH oxidase 4 (NOX4) and increasing activities of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase in mice hematopoietic stem cells. In human lymphocytes, metformin treatment reduced apoptosis induction via reducing BAX to Bcl-2 ratio after IR [27]. Although the liver is thought to be a major site of metformin pharmacodynamics, recent emerging evidence implicates the gut as an important site of action [28]. Notably, metformin levels in the jejunum are 30–300 times higher than in the plasma after it is taken orally [29]. Metformin has been found to benefit microbiota composition, promote gut barrier integrity and reduce inflammation in human and animal models of diabetes. Whether gut microbiota is involved in the radioprotective mechanisms of metformin is an interesting question, which deserves to be further studied in our future research.

An ideal radioprotective agent should possess the ability to provide significant protection to the normal cells against the effects caused by IR without minor influence on the radiosensitivity of the tumor cells. To date, although there are a number of promising agents emerging, there are still only a limited number of radioprotective agents used clinically to minimize the severity and duration of toxicities associated with radiotherapy. Antioxidants such as ascorbic acid (Vitamin C), α-tocopherol (Vitamin E) and β-carotene have been demonstrated to reduce various radiation-induced injuries [30]. Regrettably, the use of these nutritional antioxidants as radioprotectors has come under question due to concerns that these agents may also interfere with tumor treatment either through radioprotection or via an increase in the rate of second malignancies [31]. It has been reported that combined α-tocopherol and β-carotene supplementation given during and after radiation was also shown to increase the local recurrence rate of head and neck tumors [32]. These findings highlight the need to consider the possibility of tumor radioprotection carefully before radioprotective agents are used in clinical. Metformin, apart from its radioprotective effect on normal tissues, it is also reported to have anti-tumor activities [33]. Recent studies have shown that metformin inhibits cell proliferation and increases apoptosis in various cancer cells [34, 35]. Metformin could be as radiosensitizer that improves the sensitivity of several cancer cells to irradiation therapy [36, 37]. Moreover, recent clinical data showed that colorectal carcinoma (CRC) patients treated with metformin due to coexisting diabetes developed fewer distant metastases and survived longer compared to those not treated with metformin [38]. All these studies imply that metformin might be a promising radioprotective agent, with added anti-tumor effect.

Metformin has not significant adverse effects in normal clinical level, but it may cause severe lactic acidosis. Metformin, along with other biguanide class drugs, can increase plasma lactate levels in a plasma concentration-dependent manner by inhibiting mitochondrial respiration predominantly in the liver. As metformin is renally cleared, lactic acidosis usually occurs due to high levels in the plasma caused by drug overdose or renal dysfunction [39]. In addition, hypoglycemia may occur if metformin is used with other anti-diabetic drugs, drinking large amount of alcohol, doing heavy exercise, or not consuming enough calories from food [40]. So far, more than 10 case reports of hepatotoxicity, such as acute cholestatic jaundice, were reported to be associated with metformin administration [41, 42]. Therefore, it is necessary to consider the list of contraindications in the use of metformin.

In summary, in this study we showed that pretreatment with metformin prolonged the survival period of mice after 8 Gy TBI. It also reduced radiation-induced hematopoietic and intestinal injuries in mice after 6 Gy TBI, suggesting that metformin administrated before IR had a radioprotective effect in vivo. In conclusion, metformin serve as a radioprotective agent preventing damage caused by IR.

FUNDING

This study was financially supported founded by the Grant (no. 31770908) from the National Natural Science Foundation of China and a grant (no. 2019 M653964) from a project funded by the China Postdoctoral Science Foundation.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.