Cellular expression of Bcl-2 and Bax in atrophic submandibular glands of rats

Abstract

In submandibular gland atrophy, most acinar cells disappear by apoptosis, while many duct cells remain. The present study aimed to establish whether Bcl-2 and Bax, members of the Bcl-2 gene family, regulating the signalling pathway of apoptosis were involved in duct cell survival and acinar cell death in atrophic submandibular glands. The excretory duct of rat submandibular gland was doubly ligated with metal clips from 1 to 14 days to induce atrophy to the gland. The expressions of Bcl-2 and Bax in the atrophic submandibular gland were examined using immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemically, Bcl-2 expression was identified in duct cells in the experimental glands at all time points. Some acinar cells showed Bax positivity 1 day after excretory duct ligation, and there were more Bax-positive acinar cells on days 3 and 5 when many apoptotic acinar cells were observed. Analysis by RT-PCR showed that the expression of mRNA for Bcl-2 became stronger as the glandular atrophy progressed and that Bax mRNA strongly expressed on days 1 and 3. These observations suggest that Bcl-2 inhibits duct cell apoptosis and Bax promotes apoptosis of acinar cells during atrophy of submandibular glands.

Figure 1

Bcl-2 immunohistochemistry. Bar = 50 μm (a) Control. Striated duct cells are positive. Intercalated duct cells (arrows) and acinar cells are negative. (b) Day 1: Cells in dilated ducts show positive reaction. (c) Day 5: The remaining duct cells are immunoreactive to Bcl-2 antibody. There are some positive (arrow) and many negative (arrowhead) acinar cells in the atrophic gland. (d) Day 14: Most residual duct cells are positive and remaining acinar cells are negative (arrows). Inset: higher magnification of (d). Bar = 20 μm. The spindle-shaped (myoepithelial-like) cell is immunopositive (arrowhead).

Figure 2

Bax immunohistochemistry. Bar = 50 μm (a) Control. No immunoreaction is identified in normal submandibular glands. (b) Day 1: There are some positive acinar cells (arrows). (c) Day 3: Many acinar cells are Bax immunoreactive. (d) Day 14: The remaining acinar cells are reactive with the anti-Bax antibody (arrow).

Figure 3

RT-PCR analysis of mRNA expression of Bcl-2, Bax and GAPDH. C, control (normal submandibular gland); 1d, 1 day after duct ligation; 3d, day 3 and 14d, day 14. The bands correspond to 612 (Bcl-2), 361 (Bax) and 306 bp (GAPDH).