Introduction

We earlier published evidence of relaxation of human gut muscle contraction by GLP1 and analog ROSE-010 in surgically isolated biopsies (1). Pharmacological experiments pointed to dependency on functional nerves. Immunohistochemistry (IHC) revealed presence of GLP1 receptor (GLP1R) in a subset of cells within myenteric plexus. Exact cell types were not established. A hypothetical model was proposed in which GLP1R activation at nNOS expressing nitrergic myenteric neurons triggers NO production, then driving muscle relaxation.

Aims & Methods

This hypothesis was pursued by identifying FOX3 and nNOS expressing myenteric plexus neurons relative GLP1 and ROSE-010 binding. Paraffin sections from human gut transmural biopsies were, by segment (n): cardia (6), fundus (2), corpus (6), jejunum (2), ileum (5), unspecified small intestine (6), colon (7), sigmoid (2), rectum (3). GLP1R was first mapped using a new antibody (clone D-6, sc-390774, Santa Cruz Biotech, USA) by ordinary IHC with DAB. This targets amino acids 91-145 of N-terminal extracellular domain of human GLP-1R. Mapping was verified in serial sections using biotinylated GLP1 (B-GLP1, 1 µM) or ROSE-010 (B-ROSE, 5 µM) synthesized with biotinyl-AEEAc tag at amino terminal histidine. To visualize this 1:1 stoichiometry, a signal amplification system was developed using streptavidin-HRP, biotinylated goat anti-streptavidin antibody and a second round of streptavidin-HRP followed by DAB staining. For triple labelling on other slides, streptavidin tagged fluorophores were instead used after FOX3 staining of neuronal nuclei using monoclonal antibody (Biolegend, USA) and HRP-DAB; nNOS was visualized by antibodies (mouse sc-5302 FITC, Santa Cruz Biotech or goat NB100-858, Novus Biologicals). Although unusual, amplification and triple labelling only required off-the-shelf reagents (Vector Labs, USA).

Results

Across the gut, approx. half of myenteric plexus cells were positive using GLP1R antibody; this number being variable. Many had clear neuronal morphology. Intensity was scored between 2 and 3 (max was 4). The amplification system introduced a small background, but B-GLP1, B-ROSE and nNOS stained far above this. Myenteric plexus cells most strongly staining for B-GLP1 and B-ROSE were in some cases negative for nNOS. Triple labelling further revealed peptide positive cells within myenteric plexus that were nNOS- with FOX3+ nuclei. Myenteric plexus GLP1R and nNOS distribution was confirmed by ordinary IHC of serial sections.

Conclusion

This arrives at the existence of GLP1R+ myenteric neurons that are not nitrergic as well as GLP1R- nitrergic neurons. Support was found for the hypothesis in which GLP-1 and ROSE-010 act on nitrergic neurons (i.e., GLP1R+/nNOS+/FOX3+). However, the other permutations of GLP1R relative nNOS expressing neurons indicate that GLP1 and ROSE010 act simultaneously on other types of neurons (as of now undefined), whereas not all nitrergic neurons are equipped to respond to these peptides. The net effect of these peptides to induce muscle relaxation arises from selective signaling at a distinct subset of neurons that collectively are only definable as “GLP1R+”.

References

1. Halim, Md.A., et al. J. Clin. Endocrinol. Metab. 103(2):575–585. 2018

Disclosure

ROSE Pharma provided financial support.

Introduction

Gene therapy holds great promise for the treatment of various diseases. AAVpan mediated SPINK1 overexpression has shown considerable promise in alleviating pancreatitis ^[1]. However, in the case of severe pancreatitis induced by high-dose caerulein administration, the protective effect was not markedly evident possibly due to the insufficient expression of SPINK1. To ensure therapeutic levels of hSPINK1 expression within the safe dosage range of AAV, we introduced the short intron 1 (mini intron1) sequence of hSPINK1 gene to explore whether mini-intron could enhance hSPINK1 expression, and validated the therapeutic efficacy of the AAVpan-hSPINK1-mini intron1 in pancreatitis models.

Aims & Methods

The first intron of human SPINK1 (1.8 kb) conforms to significantly enhance gene expression ^[2]. Considering limited packaging capacity of AAV (4.7 kb), we introduce the first and last 50 base pairs (which do not affect splicing) into hSPINK1 cDNA to construct hSPINK1-mini intron1. First, we investigated whether mini intron could enhance hSPINK1 overexpression: the hSPINK1 expression levels in AAVpan-hSPINK1 and AAVpan-hSPINK1-mini intron1 group were compared using qRT-PCR, Western blot, and trypsin-inhibition assays. The stability of hSPINK1 mRNA in both groups were evaluated following in vitro transcriptional inhibition with actinomycin D. Then, we evaluated the safety of mini intron-mediated hSPINK1 overexpression by detecting hepatic and renal function as well as pancreatic histology. Further, we constructed cerulein-induced severe acute pancreatitis model (250 μg/kg/h) to determine the protective effectiveness of mini intron-mediated overexpression.

Results

The AAVpan-hSPINK1-mini intron1 demonstrated substantially enhanced hSPINK1 expression compared to the AAVpan-hSPINK1. Quantitative analysis revealed a remarkable 30-fold increase in hSPINK1 mRNA levels, 7-fold elevation in protein expression, and 1.5-fold improvement in trypsin-inhibition activity in the AAVpan-hSPINK1-mini intron1 group than those in AAVpan-hSPINK1 group. The addition of actinomycin D resulted in continuous decay of hSPINK1 mRNA in AAVpan-hSPINK1 group, whereas the hSPINK1 mRNA levels in AAVpan-hSPINK1-mini intron1 group remained stable, indicating that mini intron1 effectively stabilized hSPINK1 mRNA. In safety analysis, the histological analysis of liver and pancreas, hepatic and renal function, as well as serum amylase remained within normal levels in both groups for up to 8 weeks post-injection. In the AAVpan-hSPINK1 group, mice with high-dose caerulein-induced SAP exhibited typical pancreatitis phenotypes. In contrast, the AAVpan-hSPINK1-mini intron1 attenuated SAP, implying a dose-dependently protective effect of hSPINK1 overexpression.

Conclusion

The enhanced mini intron could safely enhanced hSPINK1 expression level and trypsin-inhibition activity by improving mRNA stability, representing a promising strategy for optimizing transgene expression and opening up new possibilities for the treatment of severe pancreatitis and potentially other diseases in the field of gene therapy.

References

[1] Wang YC, Mao XT, Sun C, et al. Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice [J]. Gut, 2024, 73(7): 1142-55.
[2] Kereszturi E, Király O, Sahin-Tóth M. Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis [J]. Gut, 2009, 58(4):545-9.

Introduction

Pancreatic fibrosis is the main pathological feature of chronic pancreatitis and stroma of pancreatic cancer, which is the result of excessive deposition of extracellular matrix of collagen fibers. Activated pancreatic stellate cells (PSCs) are the main effector cells of pancreatic fibrosis. The characteristics of heterogeneity and subgroup of PSCs may help to determine the therapeutic target of pancreatic fibrosis. The purpose of this study was to investigate the function of JUND in the activation of PSCs, evaluate the role of JUND in glutamine metabolism during pancreatic fibrosis, and explore the molecular regulated mechanism between JUND and PIWIL1/mTOR signaling pathway.

Aims & Methods

When inducing pancreatic fibrosis in C57BL/6 mice, 20% L-arginine (250 mg/100g) was injected intraperitoneally twice a week, with an interval of 1 hour, lasting for 8 weeks, and then PSCs were isolated. Use RNA interference (RNAi) constructed by lentivirus and vectors constructed based on over-expression vectors to knock out and over-express JUDN and PIWIL1 Protein. In order to explore the effects of JUND on PSC activation, EMC secretion, actin cytoskeleton, invasion and migration ability, as well as glutamine metabolism, ELISA, western blotting, wound healing test, transwell and phalloidin staining were used respectively. To explore transcriptional molecular mechanism between JUND and PIWIL1, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were applied. Hematoxylin-eosin, Masson and Sirius red staining were used to analyze the effect of JUND on histological changes and collagen deposition.

Results

In LV-JUND group, the level of α-SMA, Col I and Col III were significantly increased compared with si-JUND group. However, the expression of PIWIL1, desmin and p-mTOR were reduced in LV-JUND group. Meanwhile, the synthesis and metabolism of glutamine decreased significantly under the LV-JUND treatment. Wound healing test, transwell indicated that JUND promoted the migration and invasion ability of PSCs. Furthermore, JUND directly binded to the PIWIL1 promoter and then activated its transcription. In addition, as fibrosis aggravated over time, the levels of JUND significantly increased.

Conclusion

Our findings suggest that JUND-regulated glutamine metabolism in PSCs promotes pancreatic fibrosis via PIWIL1/mTOR signaling pathway. JUND could become a potential target for the treatment of pancreatic fibrosis.

Disclosure

This work was supported by grants from the Fund for Fostering Young Scholars of Peking University Health Science Center (BMU2021PY010), National Natural Science Foundation of China (No. 82002461), Beijing Municipal Hospital Management Center "Youth" Talent Plan (QML20231104) and Medjaden Academy and Research Foundation for Young Scientists (MJR20211110).

Introduction

Type 1 diabetes is a disease of the endocrine pancreas, but it also affects the exocrine part, especially the ductal cells. Several studies have shown that secretin-induced ductal HCO₃^- secretion is reduced in diabetes, however, the underlying mechanism and the effect of diabetes on basal secretion is less known.

Aims & Methods

Our aim is to investigate the effect of diabetes on pancreatic exocrine function. Diabetes was induced in wild type and cystic fibrosis transmembrane conductance regulator (CFTR) knock out mice by i.p. administration of streptozotocin and disease development was confirmed by fasting blood glucose level measurement. Pancreatic ductal fluid and HCO₃^- secretion were measured by fluid secretion measurements and fluorescence microscopy, respectively. Expression of ion transporters were investigated by real-time PCR and immunohistochemistry, whereas TEM was used for the morphological characterization of the pancreas.

Results

​Basal fluid and HCO₃^- secretion are significantly elevated in diabetes. Acute or chronic glucose treatment did not affect HCO₃^- secretion, but inhibition of CFTR significantly reduced it in both normal and diabetic mice. Cl^- efflux and the expression of CFTR, ANO1, NHE-1 and AQP1 increased in diabetes. Secretin-stimulated fluid secretion was also significantly higher in diabetic mice.

Conclusion

Our results show that diabetes increases fluid and HCO₃^- secretion in ductal cells both under basal and stimulated conditions in which the increased function of ion and water transporters, especially CFTR, plays central role.
This study was supported by the National Research, Development and Innovation Office Grant (SNN134497 to VV) and the CFRD-SRC Grant (No.: SRC 007).