Protective effect of asiaticoside on radiation-induced proliferation inhibition and DNA damage of fibroblasts and mice death

2.1 Cell culture and drug treatment

Cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). For drug treatment, sub-confluent cells were treated with 5 J/m₂ radiation for 30 min followed by treatment with 400 μg/mL AC for indicated time. For the evaluation of survival rate, E.coli and fibroblasts were pretreated with 200 μg/mL AC before radiation.

2.2 MTT assay

6×10⁴ fibroblasts were seeded into 96-well plates according to the experimental grouping. After incubation for 48 h, 20 μL MTT solutions (5 g/L) was added to the cells for 4 h. 200 μL dimethyl sulfoxide was added to the cells after removing the supernatant and were vibrated for 10 min at low speed to make the crystals dissolve. Absorbance at the wavelength of 570 nm was read by microplate reader.

2.3 Animals

C57BL/6J mice (8-10 weeks old, male), which have been shown to be radiation sensitive, were housed five animals per cage under controlled conditions and received standard laboratory diet through the course of the experiment.

Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. Experiments were performed under protocols approved by the Committee on Animal Resources.

2.4 Irradiation treatment

The mice were randomly divided into four groups: Control, AC treatment, Radiation treatment, and AC plus Radiation treatment groups. For the irradiation, animals were restrained in plastic jigs and placed so that the thoraxes were located in the center of the field defined by the slit lead collimator and irradiated with 5 Gy to the thorax only. Treatment dose is based on the well-established threshold dose for the induction of fibrosis in the C57BL/6J strain, as previously reported using an identical exposure method. Animals were assigned to each treatment group for each time point examined. For AC treatment, AC-irradiated (5 Gy) mice received similar handling followed by administration of 25 mg/kg AC for 2 weeks on day 10 after radiation. After that, lung tissue of all mice was harvested and were used to detect histologic changes and protein/ mRNA levels of TGF-β1.

2.5 RT-PCR

RNA was extracted from frozen tissues with Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. Two micrograms of RNA were reverse transcribed according to the manufacturer’s specifications. Multiplex PCR was done according to the previous description. Complementary DNA was made from RNA with a PrimeScript™ RT reagent kit, followed by quantitative PCR analysis according to the SYBR® Premix Ex Taq™ manufacturer’s instructions. The reaction program was set as follows: 37°C for 15 min, 95°C for 30 s for the initial template denaturation and 40 cycles of denaturation at 95°C for 5 s, followed by annealing and extension at 60°C for 30 s.

2.6 Comet assay

The comet assay was performed as previously described [8]. Briefly, gels were lysed overnight at 4°C. After lysis, gels were transferred to an electrophoresis chamber filled with alkaline unwinding buffer for 40 min at 4°C. Electrophoresis was conducted with the same buffer at 4°C for 30 min 1 V/cm and 300 mA. Neutralization solution was used to neutralize ethylene-bromide for 1min and then rinsed with deionized water to remove the excess dye. The number of comet tail cells was observed under the fluorescence microscope.

2.7 T-AOC assay

Total antioxidant capacity (T-AOC) in the serum of the mice were detected by a biochemical method following the instructions provided with the reagent kits [9] (T-AOC, A015; malondialdehyde, A003) purchased from Nanjing Jiancheng Bioengineering Institute of China.

2.8 Statistical analysis

Two-tailed Student’s t-tests for statistical analyses were performed with the data from three independent biological replicates using GraphPad Prism 5 to compare parameters between groups. Statistical significance was determined as p<0.05.

3.1 AC increases cell viability of radiated or H₂O₂-treated Escherichia coli (E.coli)

It is confirmed that Escherichia coli lipopolysaccharide (LPS) can induce lung injury and release inflammatory factor NO [10]. In our study, 400 μg/mL AC inhibited proliferation and diminished the viability of E.coli, but there was no effect on the proliferation of E.coli when the concentration of AC was lower than 200 μg/mL (Figure 1A). However, we found that pretreatment with 200 μg/mL AC before radiation significantly increased survival rates of E.coli as compared to that treated with radiation alone at 50 J/m² doses (Figure 1B). Similarly, AC administration increased cell viability of H₂O₂-treated E.coli (Figure 1C). These data imply that the AC-mediated enhancement of E.coli cell viability might result in inhibition of LPS release, and subsequently the attenuation of the lung injury.

Figure 1

Effect of AC on cell viability of radiation or H2O2-treated E.coli. (A) The cytotoxicity of different concentrations of AC against E.coli.(B) The effect of AC on cell viability induced by 50 J/m² doses radiation. (C) Variation of E.coli survival pretreated with 200 μg/mL AC with the treatment of H₂O₂. **, P<0.01.

3.2 AC enhances cell viability of fibroblasts treated with radiation or H2O2

Fibroblasts, the pivotal cell type in lung tissue, have a key role in protecting against lung injury. Dysfunction of fibroblasts leads to lung injury [11]. As shown in Figure 2A, AC reduced proliferation and diminished the viability of fibroblasts when the concentration of AC was more than 400 μg/mL, whereas lower than 200 μg/ mL did not affect cell viability of fibroblasts. However, pretreatment of fibroblasts with 200 μg/mL AC before radiation significantly promoted survival rates compared to fibroblasts treated with radiation alone at 6 Gy doses (Figure 2B). Similarly, AC enhanced H₂O₂ treatment-mediated decline in fibroblast cell viability (Figure 2C). These data suggest that AC attenuates cell apoptosis in irradiated fibroblasts.

Figure 2

Effect of AC on cell viability of radiation or H2O2-treated fibroblasts. (A) The cytotoxicity of different concentrations of AC against fibroblasts. (B) The effect of AC on cell viability induced by 50 J/m² doses radiation. (C) Variation of fibroblasts survival pretreated with 200 μg/mL AC with the treatment of H₂O₂. **, P<0.01.

3.3 AC attenuates DNA damage in radiated fibroblasts

Next, we determined the effect of AC on DNA damage in fibroblasts. In our study, we discovered that irradiated fibroblasts resulted in an increase in the number of all comet tail moment, whereas pretreatment with AC before radiation clearly inhibited the increase of comet tail moment, indicating that AC had a protective effect on radiation-induced DNA damage (Figure 3A and 3B). In addition, AC attenuated the apoptosis of irradiated fibroblasts with a decrease in cleaved PARP compared to radiation treatment alone (Figure 3C).

Figure 3

Effect of AC on radiation-induced DNA damage in fibroblasts. (A) Representative micrographs of comet assay. (B) Relative comet tail rate (%). (C) Protein expression of full-length PARP, Cleaved PARP and Actin by WB. **, P<0.01.

3.4 AC improves survival rates of irradiated mice

We also evaluated the role of AC on radiation-evoked injuries in vivo. As shown in Figure 4A, lung tissue was severely damaged accompanied by increased fibrosis after radiation treatment. Once treated with AC, radiation-induced lung injury was significantly improved and the fibrosis level was obviously decreased (Figure 4A). Consistent with the previous study, we also observed that all irradiated mice died on day 10 after irradiation alone, while 50% of the mice survived day 10 after radiation when treated with 25 mg/kg AC (Figure 4B). These data show that AC improves the overall survival rates of irradiated mice.

Figure 4

Effect of AC on radiation-induced lung fibrosis and mice death. (A) Histologic results of lung tissue in different group measured by HE staining. Scale bar: 200 μm. (B) Survival rates of radiated mice with or without AC treatment for 15 days.

3.5 AC increases the levels of plasma T-AOC and inhibits the expression of TGF-β1

Radiation can induce lung injury and even pulmonary fibrosis. Elevated levels of TGF-β1 indicate the onset of pulmonary fibrosis [12]. Interestingly, we found that TGF-β1 mRNA was significantly increased with the passage of time under the condition of irradiation compared to the control, showing peak effect at 8 weeks which suggested that our in vitro model was successfully established (Figure 5A). To explore the role of AC on radiation-mediated elevation of TGF-β1, we also chose 200 μg/mL as the treatment concentration. Like previous results, protein and mRNA levels of TGF-β1 in mice was robustly boosted after irradiation, whereas it was significantly reduced followed by the treatment of 25 mg/kg AC (Figure 5B and 5C). Additionally, plasma T-AOC concentrations in the AC-treated group were significantly higher than those in the group treated with irradiation alone. Changes in the levels of T-AOC indicate the antioxidant potential of AC (Figure 5D).

Figure 5

Levels of Plasma T-AOC (a marker of oxidative stress) and the expression of TGF-β1. (A and B) The level of TGF-β1 mRNA. (C) Observation of the expression of TGF-β1 by WB. (D) Levels of plasma T-AOC. **, P<0.01.

In summary, AC administration improves irradiation-mediated DNA damage and growth inhibition of fibroblasts. At the same time, AC enhances the level of T-AOC and inhibits the level of TGF-β1 in mice after irradiation, thereby prolonging survival. It appears that AC could serve as an effective and safe radio-protective agent.