Radioprotective effect of heat shock protein 25 on submandibular glands of rats

Abstract

Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.

Figure 1

Bioluminescence imaging (BLI) and immunolocalization of HSP25 and HSP70i in HSP25 and HSP70i transferred rat. A: BLI of luciferase expression in a living rat at 24 hours after gene transfer into salivary gland. Active luminescence was detected in bilateral submandibular glands of the rat. B: Expression of HSP25 and HSP70i during the days after IR. Representative photographs show expression changes of HSP25 and HSP70i in rats after transfer of control adenoviral vector (A–F) and HSP70i-expressing (G–L) and HSP25-expressing (M–R) adenovirus and pretreatment with amifostine (S–X). HSP25 was expressed in acinar cells and HSP70i in granular convoluted cells. C: Distributions of HSP25 and HSP70i during experimental periods. The graphs indicate the percentage (mean ± SD) of HSP25- or HSP70i-positive cells against the unit area (200 × 200 μm², ×200) of submandibular gland at 24 hours after gene transfer and 40 and 90 days after 17.5 Gy IR using image analyzer. Each group consisted of three rats, and five fields were obtained from each animal. Original magnifications, ×200.

Figure 2

Total body weight and submandibular gland weight of rats. Measurements were obtained 40 and 90 days after 17.5-Gy IR of head and neck. A: Changes of total body weight during experimental periods. Radiation significantly reduced body weight of animals, and amifostine significantly prevented the loss of body weight induced by IR. B: The weights of submandibular glands. HSP25 and amifostine significantly attenuated the loss of glandular weight compared with each controls. Differences of body weight and glandular weight, compared with controls, are indicated by •, compared with normal control; ♦; compared with vector transferred control; ▾, compared with vector transferred IR control; and ▿, compared with radiation control (P < 0.05).

Figure 3

Histopathological analysis of parenchymal changes in damaged salivary gland by radiation. H&E staining of nonirradiated control (A–D), 40 days (E–H) and 90 days (I–L) after 17.5-Gy IR. Nonirradiated control groups are composed of acinar (a), intercalated duct (i), granular convoluted duct (g), and secretory duct (d). At 40 days after 17.5-Gy IR in the vector control group, severe vacuolization (squares), some pyknotic nuclei (arrows), and lysis of acinar or granular convoluted ducts (l) are seen. At 90 days after IR, most of the parenchymal structures in the vector control group are destroyed, with severe fibrosis and some inflammatory infiltration. During the days after IR, HSP25 (F, J)-, HSP70i (G, K)-, and amifostine (H, L)-pretreated salivary glands show clearer lobular structures, more acinar and granular convoluted cells, and fewer vacuoles than vector-transferred IR control (E and I). It was more severe at 90 days than at 40 days after IR. Original magnifications, ×400.

Figure 4

Quantitative analysis of apoptotic cell, active caspase-3, and cleaved PARP. A: Apoptotic index of different cell types (mean ± SD) in control and irradiated submandibular gland at 1 day, 40 days, and 90 days after IR. •, compared with normal control; ♦, compared with vector transferred control; ▾, compared with vector transferred IR control; and ▿, compared with radiation control denote statistical significance of P < 0.05. B: Immunoblot of active caspase-3 and cleaved PARP in damaged submandibular gland at 1 day after 17.5-Gy IR. Activation of caspase-3 and PARP by radiation was inhibited in HSP25-, HSP70i-, and amifostine-pretreated salivary gland. Representative image of two independent animals from each group is shown.

Figure 5

Immunolocalization of AQP5 in irradiated rat salivary gland. A: Immunohistochemical analysis for AQP5 was performed at 40 and 90 days after IR. Nuclei were counterstained with autohematoxylin. A–D: AQP5 is located at apical membrane of secretory cells, and AQP5-positive cells are abundant in nonirradiated salivary glands. E and I: In vector transferred IR control, most of AQP5 activity has disappeared. J: HSP25-transferred submandibular glands have AQP5-positive cells until 90 days after IR. B: Distribution of AQP5 in salivary gland at 40 and 90 days after IR. The graph indicates the positive signal ratio of AQP5 against normal submandibular gland. There were 10 fields (60 × 50 μm², ×1000) per rat in each group. *Significance compared with control group (P < 0.05). Original magnifications, ×1000.