Protective Effect and Mechanism of Action of Rosmarinic Acid on Radiation-Induced Parotid Gland Injury in Rats

Abstract

The parotid glands are damaged by oxidative stress and a series of pathophysiological changes after irradiation. Rosmarinic acid (RA) is a natural antioxidant that provides a radioprotective effect against harmful damage from ionizing radiation. The present study aims to explore the protective effects of RA on radiation-induced parotid gland injury and its underlying mechanism. Sprague-Dawley rats were irradiated with 15 Gy X-ray and treated with different concentrations of RA (30, 60, and 120 mg/kg) or amifostine (AMI, 250 mg/kg). Saliva secretion function, oxidative stress, apoptosis, the inflammatory response, and fibrosis were determined by the measurement of the salivary flow rate, enzyme-linked immunosorbent assay, transferase-mediated DUTP Nick end labeling, Western blot, quantitative real time polymerase chain reaction, and hematoxylin and eosin staining. Here, we show that RA treatment significantly attenuated reactive oxygen species by a direct hindrance effect and the indirect activation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha/nicotinamide adenine dinucleotide phosphate oxidase 4 signaling. Rosmarinic acid not only reduced apoptosis by inhibiting p53/jun N-terminal kinase activation but also reduced parotid gland tissue fibrosis by downregulating inflammatory factor levels. Compared to AMI, RA has the obvious advantages of late efficacy and convenient usage. Moreover, 60 mg/kg is the minimum effective dose of RA. Therefore, RA can potentially be applied as a therapeutic radioprotective agent to treat radiation-induced parotid gland injury in the future.

Keywords: apoptosis; fibrosis; oxidative stress; parotid gland; radiation injury; rosmarinic acid.

Figure 1.

Effect of rosmarinic acid on the salivary flow rate. Stimulated saliva was collected from each group 4 days before and 3, 10, 30, 60, and 120 days after irradiation (μL/min, n = 8 per group). AMI indicates amifostine; Ctrl, control; IR, irradiation; RA, rosmarinic acid; SFR, salivary flow rate.

Figure 2.

Effect of rosmarinic acid on the oxidative stress reaction. Reactive oxygen species and total antioxidant capacity levels were determined (A). PGC1-α and NOX4 were examined by Western blotting (B) and real-time polymerase chain reaction (C). * Values significantly (P < .05) differ from the Ctrl group. ^# Values significantly (P < .05) differ from the IR group. ^$ Values significantly (P < .05) differ from the IR+30 mg/kg RA group. AMI indicates amifostine; Ctrl, control; IR, irradiation; NOX4, nicotinamide adenine dinucleotide phosphateoxidase4; PGC1-α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; RA, rosmarinic acid.

Figure 3.

Effect of rosmarinic acid on apoptosis. The apoptosis rate was calculated as the ratio of the number of TUNEL positive cells relative to the number of total cells (%). * Values significantly (P < .05) differ from the Ctrl group. ^# Values significantly (P < .05) differ from the IR group. ^$ Values significantly (P < .05) differ from the IR+30 mg/kg RA group (A). Microscopic images (×400) showed apoptosis at days 30 postirradiation when the most significant difference among groups occurred. The arrows indicate apoptotic cells (B). The protein of p-JNK, JNK, p-c-Jun, c-Jun, MDM2, Bax, Bcl-2, p53, Caspase-3 were determined by Western blotting (C) and the mRNA expression of p53, MDM2, Caspase-3, Bax, Bcl-2 were determined by real-time polymerase chain reaction (D). AMI indicates amifostine; Bax, BCL2-associated X; Bcl-2, B-cell lymphoma 2; Ctrl, control; IR, irradiation; JNK, jun N-terminal kinase; MDM2, mouse double minute 2; mRNA, messenger ribonucleic acid; p-JNK, phosphorylated c-jun N-terminal kinase; p-c-Jun, phosphorylated c-Jun; RA, rosmarinic acid; TUNEL, transferase-mediated DUTP Nick end labeling.

Figure 4.

Effect of rosmarinic acid on interstitial hyperplasia. Interstitial fibrosis was observed at day 60 and 120 postirradiation (HE×200, A). Collagen Ⅰ and Collagen Ⅲ levels were determined by Western blotting (B). The mRNA levels of Col1a1, Col1a2, and Col3a1 were determined by real-time polymerase chain reaction (C). Tumor necrosis factor-alpha, IL-2 and IL-6 levels in the parotid glands were determined by enzyme-linked immunosorbent assays (D). * Values significantly (P < .05) differ from the Ctrl group. ^# Values significantly (P < .05) differ from the IR group. ^$ Values significantly (P < .05) differ from the IR+30 mg/kg RA group. ^& Values significantly (P < .05) differ from the IR+AMI group. AMI, amifostine; Ctrl, control; HE, hematoxylin and eosin; IL-6, interleukin-6; IL-2, interleukin-2; IR, irradiation; mRNA, messenger ribonucleic acid; RA, rosmarinic acid.