PVNOXT regulates acinar cell-mediated inflammatory response via DMVACh projections to the acinar cell

Figures

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  Fig. 1. Model Establishment. A Changes in body weight in HFD and normal groups (n = 10). B The changes of feed intake in HFD and normal groups (n = 10). Data are represented as mean ± SD. Significance was assessed using two-way analysis of variance (ANOVA) (*p < 0.05; **p < 0.01; ***p < 0.001). C Paraffin-embedded pancreas sections were stained with H&E. Areas of edema (solid arrowhead), inflammatory infiltrates (hollow arrow), hemorrhage (solid arrow), and necrosis (hollow arrowhead). The inset is a higher magnification of the boxed area (scale bar, 100 μm, 20 μm higher magnification inset) (n = 3 per group). D Representative columns show the pathological scores of the different groups (n = 3 per group). E-F Serum levels of TG, TC, AMY, and LPS (n > 3 per group). G Serum levels of IL-1β, IL-6, and TNF-α (n > 3 per group). H The protein expression of IL-1β, IL-6, and TNF-α in pancreas extracts prepared from control, HFD, and HTGP mice pancreas tissues (n > 3 per group). I Densitometry analysis of the intensity of the protein bands. Data are represented as mean ± SD. Significance was assessed using two-way ANOVA (**p < 0.01 vs. control; ***p < 0.001 vs. control; ##p < 0.01 vs. control;…

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  Fig. 2. EGFP signal in the pancreatic acinar cells and CNS after PRV administration. A-C Representative images of acinar cell-specific (amylase antibody staining, red) localization of Ba2001 (EGFP fluorescence, green) in Cela1-P2A-iCre mice at 12, 24, and 48 h after administration (scale bar, 50 μm) (n = 3 mice per condition, three images per mice). D-E Representative images of acinar cell-specific (amylase antibody staining, red) localization of Ba2001 or PRV-EGFP (EGFP fluorescence, green) in C57BL6 mice at 48 h after administration (scale bar, 50 μm) (n = 3 mice per condition, three images per mice). F-G Representative images of EGFP fluorescent labeling (green) shows the localization of Ba2001. All sections were counterstained with nuclear marker DAPI (blue); the inset is a higher magnification of the boxed area (scale bar, 250 μm, 25 μm higher magnification inset) (n = 3 per mice, three images per region)

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  Fig. 3. C-Fos activation of PVN and DMV in acinar cell-mediated HTGP. A Representative images show c-Fos expression in the PVN, DMV, Bar, LC, Gi, Amb, StHy, and Sol of the control and HTGP groups of mice (scale bar, 1 mm) (n = 3 per group). B Quantification of c-Fos+ cell numbers in nuclei of the control and HTGP mice. Data are represented as mean ± SD. Significance was assessed using two-way ANOVA (*p < 0.05; ***p < 0.001; ns, not significant). C Representative images of DMV sections showing c-Fos (green) and AChE immunostaining in HTGP mice (scale bar, 800 μm) (n = 3 per mice). D Quantification of the percentage of AChE+ & c-Fos+ neurons among total AChE+ neurons in the DMV. E Schematic representation of recording configuration in DMV slices of HTGP mice. F Representative samples of action potentials in the DMV from control and HTGP mice (control, n = 24 cells; HTGP, n = 19 cells). G Summary data for firing rate recorded in control and HTGP mice. The action potential frequency is increased in HTGP mice. The statistical analysis involved a two-way ANOVA followed by t-test (**p < 0.01, F(1, 656) = 115.5) (n = 5 per group). H Two-sample t-test was used to compare action potential…

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  Fig. 4. DMVACh neurons directly project to the acinar cells. A Schematic diagram for FG injection into the pancreas of HTGP mice. B FG-labeled neurons colocalized with ChAT immunofluorescence, the magnified image shows the left DMV and right DMVs (right, scale bar, 100 μm) (n = 5). C-E Experimental design and representative image of virus injection site (scale bar, 100 μm) (n = 8). F-G Serum levels of AMY, LPS, IL-1β, IL-6, and TNF-α (n > 3 per group). Data are represented as mean ± SD. Significance was assessed using two-way ANOVA (***p < 0.001 control vs. HTGP; ##p < 0.01 HTGP vs. HTGP + Gi; ###p < 0.001 HTGP vs. HTGP + Gi; $p < 0.05 HTGP vs. HTGP + Gq; $$p < 0.01 HTGP vs. HTGP + Gq; $$$p < 0.001 HTGP vs. HTGP + Gq; ns, not significant control vs. control + Gi; control vs. control + Gq). Dots represent individual mice

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  Fig. 5. DMVACh receives direct inputs from PVNOXT neurons. A Experimental design of the retrograde tracing from the DMV to the PVN. B-C Representative images showing the DMV injection site and EGFP+ neurons in the PVN (scale bar, DMV, 400 μm and PVN, 1 mm) (n = 5). D-G Representative images and summarized data showing that EGFP+ neurons within the PVN are colocalized with oxytocinergic (OXT), glutamatergic (Glu), and GABAergic (GABA) neurons (scale bar, 50 μm) (n = 3)

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  Fig. 6. Activation of the PVNOXT-DMVACh axis produces inflammatory response. A-C Experimental design and representative image of the virus injection site (scale bar, 100 μm) (n = 6). D-E Serum levels of AMY, LPS, IL-1β, IL-6, and TNF-α (n = 6). Data are represented as mean ± SD. Significance was assessed using two-way ANOVA (***p < 0.001 control vs. HTGP; ##p < 0.01 HTGP vs. HTGP + Gi; ###p < 0.001 HTGP vs. HTGP + Gi; $$p < 0.01 HTGP vs. HTGP + Gq; $$$p < 0.001 HTGP vs. HTGP + Gq; ns, not significant control vs. control + Gi; control vs. control + Gq). Dots represent individual mice. F-H Experimental design and representative image of virus injection site (scale bar, 100 μm) (n = 3). I Representative images of pancreatic resin slice observed by TEM. Zymogen granules (solid arrowhead), vacuoles (solid arrow), and ER (hollow arrowhead) (scale bar, 2 μm) (n = 5 per group). J The protein expression of IL-1β, IL-6, and TNF-α in pancreas extracts prepared from control and HTGP mice pancreas tissues (n > 3 per group). K Densitometry analysis of the intensity of the protein bands. Data are represented as mean ± SD. Significance was assessed using two-way ANOVA (***p < 0.001 control vs. HTGP;…

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  Fig. 7. Single-cell transcriptomic atlas of the PVN in HTGP. A Schematic diagram of the single-cell sequencing workflow. B Uniform Manifold Approximation and Projection (UMAP) embedding of single-cell RNA sequencing (scRNA-seq) data for all 20,995 cells. C The UMAP plots of the five cell types in HTGP and NS. D Bubble plot showing the expression of marker genes used to define the five major cell types. E Four principal neuronal subclusters were delineated by UMAP analysis. F Bubble plot for marker genes of neuronal subclusters. G-H Dot plot and bar graph displaying the KEGG and GO enrichment analysis results of differentially expressed genes in the HTGP group, respectively. I Representative differentially expressed Wdfy1 genes in the PVN region of the brain sections. The vertical axes of the violin plots indicate normalized gene expression values

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  Fig. 8. Wdfy1 is involved in the PVNOXT-DMVACh axis-mediated inflammatory response of acinar cells. A Schematic diagram of the real-time optical fiber photometry assay. B Representative image of the virus injection site in the PVN (scale bar, 100 μm) (n = 6 per group). C A typical image of GCaMP6s expression and track of the optical fiber in the DMV (scale bar, 50 μm) (n = 6 per group). D Example traces (left) and heatmaps (right) showing DMVACh neuronal calcium activity in rAAV-mOXT-shScramble-EGFP or rAAV-mOXT-shWdfy1-EGFP virus injection mice. The color scale on the right indicates ΔF/F (%). E Summary data for average ΔF/F (%) (left) and frequency (right) of spontaneous calcium events in DMVACh neurons in rAAV-mOXT-shScramble-EGFP or rAAV-mOXT-shWdfy1-EGFP virus injection mice. The statistical analysis involved a two-way ANOVA followed by t-test (**p < 0.01 D21 vs. D1; ****p < 0.0001 D21 vs. D1; ns, not significant) (n = 5 per group). F Example traces (left) and heatmaps (right) showing DMVACh neuronal calcium activity in rAAV-mOXT-hM3D(Gq)-EGFP + rAAV-mOXT-shScramble-EGFP virus injection mice or rAAV-mOXT-hM3D(Gq)-EGFP + rAAV-mOXT-shWdfy1-EGFP virus injection mice. The color scale on…