Atrial Natriuretic Peptide Attenuates Colitis via Inhibition of the cGAS-STING Pathway in Colonic Epithelial Cells

Chaoyue Chen, Ying Zhang, Meihui Tao, Xi Zhao, Qinyu Feng, Xiaoshang Fei, Yu Fu, Chaoyue Chen, Ying Zhang, Meihui Tao, Xi Zhao, Qinyu Feng, Xiaoshang Fei, Yu Fu

Abstract

Atrial Natriuretic Peptide (ANP) has known anti-inflammatory effects. However, the role of ANP in Ulcerative colitis (UC) remains unclear. This study aimed to explore the expression and function of ANP in UC, and its potential regulatory role in the stimulator of interferon genes (STING) pathway. Human colon biopsy and serum samples were collected between September 2018 and December 2019 at Wuhan Union Hospital. Levels of ANP and its receptors and STING pathway components were detected in people with UC and mice with dextran sulfate sodium (DSS)-induced colitis. These mice and HT-29 cells were treated with ANP and an agonist of the STING pathway. The level of inflammation, STING pathway, gut barrier, and endoplasmic reticulum (ER) stress-induced autophagy were measured. We found that the levels of ANP and its receptor decreased and the STING pathway activated statistically in people with UC and the mouse model of colitis. ANP treatment attenuated DSS-induced colitis and inhibited STING pathway phosphorylation in colonic tissue and epithelial cells. An interaction between cGAS and NPR-A was verified. ANP repaired the gut barrier and inhibited ER stress-induced autophagy via the STING pathway. ANP may thus alter colonic barrier function and regulate ER stress-induced autophagy as a promising therapy for UC.

Keywords: Atrial natriuretic peptide; Colonic epithelial cell; Endoplasmic reticulum stress-induced autophagy; Gut barrier; Inflammatory bowel disease; STING pathway.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1
Figure 1
Expression of atrial natriuretic peptide (ANP) and its receptor in colon and serum samples. (A) Relative mRNA levels of the ANP precursor (NPPA) and ANP receptors (NPR-A and NPR-C) in different organs of wild-type mice as determined by quantitative real-time polymerase chain reaction (qRT-PCR; n = 5 for each group). The Y axis represents the expression level difference between the cycle threshold (CT) value of the target gene and the β-actin, and the smaller the Y axis value, the higher the gene expression. (B) Mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 7 days. Relative expression of NPPA, NPR-A, and NPR-C in the colon of control and DSS-treated groups was detected by qRT-PCR. Results were normalized against the β-actin gene. (C) Level of ANP was determined in the serum of a DSS-induced colitis and control mice using an enzyme-linked immunosorbent assay (ELISA). (D) Relative expression of NPPA, NPR-A, and NPR-C in the colon of people with UC in activity (n = 15), UC in remission (n = 9) and control individuals (n = 45) was determined using RT-qPCR. (E) Level of ANP was detected in serum from UC patients with active disease (n = 12) and remission (n = 11) and control individuals (n = 27) using ELISA. (F) Correlation between murine serum ANP and percent weight loss. (G-I) Correlation between serum ANP and C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR) and neutrophilicgranulocyte (NE) level. (J) NPR-A expression was detected by immunohistochemical analysis in murine colonic tissue. (K) The level of adrenaline was determined using ELISA with the treatment of DSS and ANP or not. (L) The level of adrenaline was detected in serum from people with UC or controls using ELISA. (M) Mice with a DSS-induced experimental model of colitis (n = 6 in each group) were intraperitoneally injected with prazosin or PBS. Bodyweight curves were recorded. Statistical analysis was performed using T-tests: NS (not significant), P > 0.05; *P < 0.05; **P < 0.01; and ****P < 0.0001 are shown on the figure.
Figure 2
Figure 2
Atrial natriuretic peptide (ANP) ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. Mice with DSS-induced experimental colitis (n = 6-9 in each group) were intraperitoneally injected with ANP recombinant protein or phosphate buffered saline (PBS). Mice were sacrificed, and samples were collected on the 8th day. (A) Detailed method and dosing regimen for DSS and ANP. (B) Bodyweight curves. (C) Disease activity index (DAI) scores. (D) Typical image of a colon, from which length was measured. (E) Spleen weight. (F) Hematoxylin and eosin (H&E) staining of murine colonic slices and the histological activity index score for colon tissue. (G-H) Relative expression of tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), IL-6, interferon α (IFN-α), and IFN-β in colon tissue and colonic epithelial cells (CECs) were determined using quantitative real-time polymerase chain reaction (RT-PCR). (I) Levels of IFN-α and IFN-β were determined in serum samples using an enzyme-linked immunosorbent assay (ELISA). (J) Levels of ANP were determined in the serum by ELISA. (K) Relative expression of NPR-A in colon tissue and CECs was determined by qRT-PCR. Data are shown as mean ± standard error (SEM). n = 6-9 in each group. Statistical significance was calculated using one way analysis of variance (ANOVA). (Compared with the control group, *P < 0.05; ***P < 0.001; ****P < 0.0001. Compared with the DSS-treated group, NS, P > 0.05; #P < 0.05; ##P < 0.01; ###P < 0.001).
Figure 3
Figure 3
Stimulator of interferon genes (STING) pathway expression in colonic tissue of people with UC. (A) Relative expression of STING pathway related genes, including cGAS, STING, TBK1, and IRF3, in the colon of with UC patients in activity (n = 15), UC patients in remission (n = 9) and control individuals (n = 45), was tested using quantitative real-time polymerase chain reaction (qRT-PCR). Results were normalized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. (B-E) Expression levels of STING pathway related proteins in colonic tissue as determined by western blotting (n = 6 in each group). Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: (B) cGAS/GAPDH; (C) p-STING/STING; (D) p-TBK1/TBK1; (E) p-IRF3/IRF3. Statistical analysis was performed using T-tests. NS, P > 0.05; *P < 0.05; and **P < 0.01.
Figure 4
Figure 4
Atrial natriuretic peptide (ANP) promotes cGAS/NPR-A complex formation. HT-29 cells were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A) Localization of NPR-A (green fluorescence) and cGAS (red fluorescence) within HT-29 cells, as evaluated by immunofluorescence. (B) HT-29 cells were subjected to immunoprecipitation with cGAS or NPR-A antibodies, followed by western blotting using the indicated antibodies.
Figure 5
Figure 5
Atrial natriuretic peptide (ANP) inhibits stimulator of interferon genes (STING) pathway activation and repairs gut barrier damage in colonic epithelial cells (CECs). HT-29 cells were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A) Relative expression of interferon α (IFN-α), IFN-β, IFN-γ, cGAS, TBK1, and IRF3 in HT-29 cells was tested using quantitative real-time polymerase chain reaction (qRT-PCR. Results were normalized against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. (B-E) Expression levels of STING pathway related proteins in colon tissue, as identified by western blotting. Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: cGAS/GAPDH; p-STING/STING; p-TBK1/TBK1; and p-IRF3/IRF3. (F) Protein levels of ZO-1, E-cadherin, and occludin in HT-29 cells, as determined by western blotting (n = 5 for each group). Results were normalized against GAPDH. (G) Localization of E-cadherin (green fluorescence) and p-TBK1 (red fluorescence) within HT-29 cells, evaluated by immunofluorescence. (H) Localization of ZO-1 (red fluorescence) within HT-29 by immunofluorescence.
Figure 6
Figure 6
Atrial natriuretic peptide (ANP) inhibits endoplasmic reticulum (ER) stress-induced autophagy in colonic epithelial cells (CECs) via stimulator of interferon genes (STING) pathway activation. HT-29 were treated with DMXAA or ANP 24 h in advance (n = 5 for each group). (A-B) Relative expression of Atg5, Atg7, and Atg12 in primary CECs extracted from mice and in HT-29 cells. Results were normalized against the β-actin/ glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. (C) Expression levels of autophagy-related (LC3, free Atg12, Atg12-Atg5, Beclin1, P62) and ER stress-related (eIF2α, SAPK/JNK, BiP) proteins in HT-29 were identified using western blotting (n = 5 for each group). Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: LC3-II/LC3-I, Atg12-Atg5/GAPDH, Beclin1/GAPDH, P62/GAPDH, p-eIF2α/eIF2α, p- SAPK/JNK/SAPK/JNK, and Bip/GAPDH. (D-E) Localization of P62 (green fluorescence) in HT-29 cells, as evaluated by immunofluorescence. (F-H) The accumulation of StubRFP-SensGFP-LC3 in HT-29 cells treated with DMXAA or ANP was observed using confocal micrographs. Red arrows indicate autophagosomes and yellow arrows indicate autolysosomes.
Figure 7
Figure 7
Atrial natriuretic peptide (ANP) inhibits stimulator of interferon genes (STING) pathway activation and repairs gut barrier damage in a dextran sulfate sodium (DSS)-induced colitis mouse model. Mice with DSS-induced experimental colitis (n = 12 in each group) were intraperitoneally injected with ANP recombinant protein (2 μg in 400μL phosphate buffered saline [PBS] per mouse), DMXAA (0.1 mg in 400μL PBS per mouse) or 400μL PBS. Mice were sacrificed, and samples were collected on the 8th day. (A) Detailed method and dosing regimen for DSS, ANP and DMXAA. (B) Survival rates of mice with DSS-induced colitis. (C-F) Levels of STING pathway related proteins in colonic tissue, as identified by western blotting (n = 3 in each group). Representative blots are shown, and quantitative density data are shown as the intensity ratio of the target protein to relevant controls: cGAS/GAPDH; p-STING/STING; p-TBK1/TBK1; p-IRF3/IRF3. (G) Localization of E-cadherin (green fluorescence) and p-TBK1 (red fluorescence) within the colonic mucosa, as evaluated by immunofluorescence. (H) Localization of ZO-1 (green fluorescence) within colonic mucosa as evaluated by immunofluorescence. (I) Levels of ZO-1 and occludin in colonic tissue, as determined by western blotting (n = 5 in each group). Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (J) The levels of serum FD4 in different groups, reflecting colonic permeability. Statistical analysis was performed using one way analysis of variance (ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with group treated with DSS and DMXAA, #P < 0.05).

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