Fig 1.
Submandibular gland ductal ligation followed by deligation results in reversible acinar cell atrophy, immune cell infiltration and glandular fibrosis.
(A, D, G) Control SMG, (B, E, H) 7 day duct-ligated SMG and (C, F, I) 7 day duct-ligated SMG followed by deligation and recovery for 28 days were subjected to immunofluorescence staining with (A-C) antibodies to the acinar cell marker AQP5 (green) and Hoechst nuclear stain (blue), (D-F) antibodies to the pan-immune cell marker CD45 (red) and Hoechst nuclear stain (blue) and (G-I) hematoxylin and eosin. Results indicate that ligation of the main SMG excretory duct induces (B) loss of acinar cells, (E) substantial immune cell infiltration and (H) atrophy of acinar cells (white arrow) and loss of secretory granules (pink) within ductal cells (black arrow) that is reversed by subsequent deligation (C, F, I). Images are representative of results from at least 3 independent experiments and scale bar = 20 μm.
Fig 2.
Upregulation of TGF-β1, TGF-β3 and TGF-β R1 in response to SMG duct ligation is reversible upon deligation.
(A) Western analysis of whole gland lysates shows upregulation of pro-TGF-β1/2/3 expression (top) in response to 7 day SMG duct ligation that is reversible following deligation and recovery for 28 days. Ponceau S staining (bottom) shows equal amounts of total protein in each well. (B) RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of TGF-β1 and TGF-β3, but not TGF-β2, mRNA expression after 7 days of SMG excretory duct ligation (grey bars), as compared to contralateral control glands (white bars). When ducts were ligated for 7 days then deligated for 28 days (black bars), expression levels of TGF-β1 and TGF-β3 mRNA return to control levels, whereas TGF-β2 mRNA levels remain unchanged. Data represent means ± S.E.M. (n = 7 for control and 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where *P<0.05 and **P<0.01 indicate significant differences in mRNA expression, as compared to control. (C) Dual-immunofluorescence analysis of 8 μm frozen SMG sections for control, 7 day ligation and 7 day ligation followed by deligation and recovery for 28 days reveals that TGF-β R1 (red) expression is upregulated primarily in acinar cells (marked by residual AQP5 expression; green) after a 7 day SMG duct ligation, whereas little staining is visible in ductal cells. After a 7 day duct ligation followed by deligation and a 28 day recovery, TGF-β R1 expression levels are similar to control. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.
Fig 3.
Upregulation of Smad2/3, TAK1 and TAB1 in response to SMG duct ligation is reversible upon duct deligation.
(A) Western blot analysis of whole gland lysates from unligated control SMG, SMG after a 7 day duct ligation or a 7 day ligation followed by deligation and a 28 day recovery. Duct ligation increases Smad2/3 expression (top) and phospho-Smad2/3 levels (middle) that return to control levels after deligation and recovery. Ponceau S staining (bottom) shows equal amounts of total protein in each well. Immunofluorescence analysis revealed that increases in (B) Smad2/3 (red) expression and (C) p-Smad2/3 (red) levels after a 7 day duct ligation are restricted to acinar cells (marked by residual AQP5 expression; green), where p-Smad2/3 is localized to the nucleus, as determined by colocalization with Hoechst nuclear stain (blue). Smad2/3 and p-Smad2/3 returned to control levels after deligation of SMG ducts and recovery. Images are representative of results from at least 3 independent experiments and scale bar = 20 μm. (D) RT-PCR analysis of whole gland lysates shows increased TAK1 and TAB1 mRNA expression following a 7 day duct ligation, which was reversed to control levels following deligation and a 28 day recovery. Data represent means ± S.E.M. (n = 6 for control, n = 8 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where *P<0.05 and **P<0.01 indicate significant differences in mRNA expression, as compared to control SMG.
Fig 4.
Upregulation of Snail and Slug in response to SMG duct ligation is reversible upon duct deligation.
RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of (A) Snail and (B) Slug mRNA after a 7 day duct ligation, which is reversed to control levels after deligation and a 28 day recovery. Data represent means ± S.E.M. (n = 4 for control, n = 8 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where ***P<0.001 and *P<0.05 indicate significant differences in mRNA expression, as compared to control SMG. (C) Immunofluorescence analysis reveals that Snail (red) expression is primarily upregulated in acinar cells after a 7 day duct ligation, and returns to control levels after deligation and recovery. (D) Dual-immunofluorescence staining confirmed the colocalization of Snail (red) with the acinar marker AQP5 (green) following a 7 day duct ligation. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.
Fig 5.
E-cadherin upregulation in response to SMG duct ligation is reversible after duct deligation.
(A) RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of E-cadherin mRNA after a 7 day duct ligation (grey bar), which is reversed to control levels (white bar) after deligation and a 28 day recovery (black bar). Data represent means ± S.E.M. (n = 8 for control, n = 7 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where ***P<0.001 indicates a significant difference in mRNA expression, as compared to control SMG. (B) Immunofluorescence analysis reveals that E-cadherin (red) was significantly upregulated in SMG ducts after a 7 day ligation, and returned to control levels after deligation and recovery. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.
Fig 6.
Glandular fibrosis following SMG duct ligation is resolved following deligation.
SMGs from unligated controls (A, D), after a 7 day duct ligation (B, E) or a 7 day ligation followed by deligation and a 28 day recovery (C, F) were subjected to Masson’s trichrome staining to analyze collagen deposition (blue). A 7 day duct ligation resulted in blue staining of collagen fibers around blood vessels and large saliva collecting ducts (B), as compared to controls (A). At 400X magnification, heavy deposition of collagen fibers can be seen in the interstitial area around acinar and ductal cells after a 7 day duct ligation (E), as compared to controls (D). Following ductal deligation and a 28 day recovery (C, F), collagen deposition returns to levels similar to control SMGs (A, D). Images are representative of results from at least 3 independent experiments. (A-C) scale bar = 1,000 μm, (D-F) scale bar = 20 μm. (G) RT-PCR analysis of cDNA prepared from whole SMGs shows extensive upregulation of collagen 1 and fibronectin mRNAs after a 7 day duct ligation (grey bars), which is reversed to control levels (white bars) after deligation and a 28 day recovery (black bars). Data represent means ± S.E.M. (n = 8 for control, n = 7 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where ***P<0.001 indicates a significant difference in mRNA expression, as compared to control SMGs.
Fig 7.
TGF-β R1 inhibitors SB431542 and GW788388 attenuate duct ligation-induced upregulation of fibrosis markers.
RT-PCR analysis of 7 day ligated and contralateral unligated control SMGs shows significant attenuation of 7 day duct ligation-induced collagen 1 and fibronectin mRNA upregulation in mice treated with either SB431542 (20 mg/kg mouse weight) or GW788388 (2 mg/kg mouse weight), as compared to DMSO-treated controls. Data represent means ± S.E.M. (n = 6 for DMSO, n = 6 for SB431542, n = 5 for GW788388), where *P<0.05 and ***P<0.001 indicate significant differences in mRNA expression, as compared to DMSO-treated controls.