Dachengqitang mitigates endoscopic retrograde cholangiopancreatography-induced pancreatitis

Abstract

BACKGROUND

Post-endoscopic retrograde cholangiopancreatography (ERCP) acute pancreatitis (PEP) is a severe postoperative inflammatory complication following ERCP, characterized by rapid onset and potentially life-threatening systemic manifestations. The integrity of the intestinal mucosal barrier plays a pivotal role in preventing secondary infections and systemic inflammatory response syndrome in the postoperative period. Traditional Chinese medicine has shown promising therapeutic potential in postoperative management and mucosal barrier protection.

AIM

To investigate the efficacy of Dachengqitang (DCQT) in ameliorating PEP through enhancement of intestinal mucosal barrier function and suppression of M1 macrophage polarization.

METHODS

Bioinformatics analysis was performed on differential gene expression and functional enrichment using the GEO database entry GSE54774. Subsequently, in vivo and in vitro experiments were conducted, including sham operation group, PEP model group, and DCQT + PEP model group, to evaluate intestinal mucosal integrity, inflammatory markers, and macrophage activation in the postoperative setting.

RESULTS

Differential analysis of GSE54774 identified 468 differentially expressed genes, with enrichment observed in blood microparticle, primary lysosome, and azurophil granule pathways. In vivo experiments demonstrated that compared to the PEP model group, DCQT treatment significantly enhanced the expression of intestinal mucosal barrier proteins ZO-1 and Occludin-1, reduced the population of CD86-positive pro-inflammatory macrophages, and promoted IL-10 secretion by M2 macrophages in the postoperative period. In vitro Caco-2 cell cultures showed that compared to the oxygen-glucose deprivation group, DCQT improved ZO-1 and Occludin-1 expression and enhanced mitochondrial membrane potential.

CONCLUSION

This study demonstrates that DCQT effectively enhances intestinal mucosal barrier function and reduces postoperative intestinal inflammation following ERCP-induced pancreatic inflammation, suggesting its potential as a postoperative therapeutic intervention for PEP management.

Key Words: Dachengqitang; Post-endoscopic retrograde cholangiopancreatography acute pancreatitis; Postoperative complications; Intestinal mucosal barrier; M1 macrophages; Inflammation

Core Tip: Post-endoscopic retrograde cholangiopancreatography (ERCP) acute pancreatitis is a serious complication involving intestinal mucosal barrier dysfunction and excessive inflammation. This study integrates bioinformatics analysis with in vivo and in vitro models to investigate the protective effects of Dachengqitang (DCQT). The results demonstrate that DCQT enhances intestinal barrier integrity by upregulating ZO-1 and Occludin-1, reduces M1 macrophage activation, and promotes M2 polarization with increased IL-10 expression. DCQT also improves mitochondrial membrane potential in epithelial cells. These findings highlight the therapeutic potential of DCQT in managing postoperative inflammatory responses following ERCP.

INTRODUCTION

Post-endoscopic retrograde cholangiopancreatography (ERCP) acute pancreatitis (PEP) constitutes one of the most significant complications following ERCP, a minimally invasive procedure extensively employed for biliary and pancreatic interventions. This complication is characterized by acute pancreatic inflammation developing within hours of the procedure, with clinical presentations ranging from mild self-limited disease to severe systemic inflammatory response syndrome (SIRS) and multi-organ dysfunction. The rapid progression to SIRS and organ failure significantly impacts patient outcomes, including prolonged hospitalization and increased morbidity. The pathophysiology of PEP encompasses procedure-induced pancreatic enzyme activation, mechanical trauma from endoscopic instrumentation, direct cellular injury, and subsequent amplification of inflammatory cascades[1-3].

Impairment of intestinal barrier function represents a critical pathogenic mechanism in PEP development. Disruption of this barrier facilitates bacterial translocation and systemic endotoxemia, thereby perpetuating and amplifying the inflammatory response. Understanding these pathophysiological mechanisms is essential for developing targeted therapeutic strategies to mitigate PEP incidence and severity while optimizing perioperative management.

Intestinal barrier integrity serves as a critical defense mechanism against translocation of luminal pathogens and toxins, maintaining intestinal homeostasis and preventing systemic inflammation following ERCP. Given that barrier dysfunction and increased intestinal permeability occur in the post-procedural period, therapeutic interventions aimed at preserving or restoring intestinal barrier function represent a promising strategy for PEP prevention and management.

Macrophages function as key effector cells in the post-procedural immune response, exhibiting phenotypic plasticity in response to environmental stimuli. M1-polarized macrophages, characterized by their proinflammatory profile, play a pivotal role in PEP pathogenesis through secretion of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and chemokines that amplify tissue inflammation. Consequently, inhibiting M1 macrophage polarization represents a rational therapeutic target for PEP management[4,5].

Dachengqitang (DCQT), a classical Chinese herbal formulation documented in the "Shanghan Lun" (Treatise on Cold Damage Disorders), has been prescribed for gastrointestinal dysfunction and abdominal distension for over 1800 years. Contemporary pharmacological investigations have identified multiple mechanisms through which DCQT promotes gastrointestinal health: (1) Enhancement of gastrointestinal motility and suppression of bacterial translocation; (2) Attenuation of intestinal inflammation via modulation of NF-κB signaling; and (3) Preservation of intestinal barrier integrity through regulation of tight junction protein expression in experimental colitis models. These mechanistic insights provide a robust scientific foundation for investigating DCQT's therapeutic potential in protecting intestinal barrier function during PEP. The constituent herbs possess documented anti-inflammatory properties[6,7], and emerging evidence suggests that DCQT exhibits immunomodulatory and antioxidant activities relevant to post-procedural care. However, the precise molecular mechanisms underlying DCQT's protective effects on intestinal barrier function in PEP remain to be fully elucidated.

This study explores the potential of DCQT to mitigate postoperative intestinal mucosal damage in PEP. We propose that DCQT operates through a dual mechanism: Enhancing mitochondrial membrane potential to restore mucosal integrity, while curbing intestinal macrophage activation and associated inflammation, ultimately leading to reduced disease severity and improved recovery.

MATERIALS AND METHODS

Analysis of the dataset GSE54774

Performing differential gene analysis on the data sourced from the GEO database GSE54774[8,9].

Gene set enrichment analysis

Using data sourced from the GEO database (GSE54774), we conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by applying R language packages. This process involved generating various visualizations, including bubble charts, circular charts, and bar charts[10,11].

Identification of hub genes

With visualization of these networks done using Cytoscape 3.6.1. In order to investigate the biological processes were analyzed further within the STRING database.

SAP mice model

The obstructive model of acute pancreatitis was developed by ligating the pancreatic duct where the gastric and duodenal lobes meet, while preserving the bile duct and its associated artery, as previously described and modified[12]. SAP model treated with DCQT (SAP + DCQT). The animals were fasted for 12 hours and deprived of water for 6 hours before surgery. The DCQT treatment group received a single gastric infusion of 10% DCQT solution (9.18 g/kg) 15 minutes before modeling, while the control group and SAP group received an equal amount of 0.9% sodium chloride solution. Anesthesia was induced by intraperitoneal injection of 10% chloral hydrate (300 mg/kg), followed by a midline abdominal incision. The bile duct and pancreatic duct opening at the duodenal papilla were closed with a non-damaging metal clip. A needle from a syringe was inserted into the pancreatic duct towards the duodenal opening, and 3.5% sodium taurocholate (0.1 mL/100 g body weight) was injected at a rate of 0.1 mL/min. The abdomen was closed 10 minutes later. In the control group, after opening the abdomen, the duodenum was turned over and the pancreas was palpated a few times before closing the abdomen. The survival of the rats was observed.

Macrophage depletion

Macrophage depletion was performed according to the following references[13,14]. Four groups of mice were randomly assigned, including Sham + PBS group (Sham; n = 4), SAP group (n = 4), SAP + DCQT group (SAD; n = 4), and SAP + DCQT + Cls group (MMP; n = 4). Intraperitoneal administration of CIs or control liposome (PBS) with a 24-hour interval before the SAP procedure was implemented.

Caco-2 cell culture and treatment

Caco-2 cells were sourced from the Cell Bank of the Chinese Academy of Sciences and randomly divided into four groups: The PBS group (n = 4), the oxygen-glucose deprivation (OGD) group (n = 4), and the OGD + DCQT group (n = 4).

The OGD model was selected based on its pathophysiological relevance to PEP. Severe acute pancreatitis, including PEP, is frequently accompanied by splanchnic hypoperfusion and intestinal ischemia due to systemic inflammatory response and microcirculatory dysfunction. Unlike single-cytokine models (TNF-α or IFN-γ alone), OGD simultaneously simulates the ischemic and metabolic stress conditions that occur in vivo during severe pancreatitis, providing a more comprehensive representation of the clinical scenario. Furthermore, this model serves as a complementary approach to our in vivo PEP model, allowing specific investigation of DCQT's direct protective effects on intestinal epithelial cells under stress conditions, independent of immune cell involvement.

Immunofluorescence

The research employed immunofluorescence on sections from both frozen and paraffin-embedded tissues. Samples, either cells or tissues, were secured onto glass slides or within centrifuge tubes and fixed with a 4% paraformaldehyde solution to preserve their morphology and protein localization. The primary antibodies used were against CD86, ZO-1, and Occludin. After the appropriate incubation with the secondary antibodies, the samples were examined using a fluorescence microscope[15,16].

Mitochondrial membrane potential measurement

Mitochondrial membrane potential was assessed according to the protocol provided with the assay kit. The cells were first extensively rinsed and then incubated with the JC-1 reagent for 20 minutes at a temperature of 37 °C. Subsequently, the dual emission fluorescence of JC-1 was recorded using a RuoChuang imaging system[17].

Statistical analysis

Analysis of the data was performed using GraphPad Prism 9.0. We assessed data distribution normality with the Kolmogorov-Smirnov test. For comparing multiple groups, a one-way ANOVA test followed by Tukey's HSD test was conducted. Data are expressed as the mean ± SD. A threshold for statistical significance was set at P < 0.05.

RESULTS

GSE54774 of differentially expressed genes

Examine the figures for representations of the heatmap and volcano plot highlighting differential gene expression. A sum of 468 genes were detected using the specified criteria. Notably, BMP4 exhibits increased expression in AP samples as depicted in Figure 1.

Figure 1 Illustrates the data processing and identification of differentially expressed genes. A: A heatmap displaying the sample-to-sample correlation. The diabetes group displayed a stronger intra-group correlation compared to the control group; B: A volcano plot generated using the limma R tool, showcasing all the differentially expressed genes in both the control and diabetes groups.

Comparison of inflammatory markers between control and experimental groups

Comparison of inflammatory markers in control and experimental group. most of these factors are associated with blood microparticles (blood mps), primary lysosomes, azurophil granules, and the vacuolar lumen (Figure 2).

Figure 2 Functional and pathway enrichment. A: Gene Ontology enrichment analysis showing significant enrichment in blood microparticle, primary lysosome, azurophil granule, and vacuolar lumen; B: Molecular function analysis displaying enrichment in endopeptidase inhibitor activity, peptidase inhibitor activity, endopeptidase regulator activity, and peptidase regulator activity.

GSE54774 were performed on the gene set enrichment analysis results

The enriched genes showed significant involvement in various biological processes, including interactions between lymphoid and non-lymphoid cells, reactome neutrophil degranulation, reactome signaling by interleukins, reactome cell cycle mitotic, wp retinoblastoma gene in cancer, KEGG hematopoietic cell lineage (Figure 3).

Figure 3 Functional and pathway enrichment analyses were performed on the gene set enrichment analysis results. A: Enrichment in lymphoid cell-related pathways; B: Enrichment in non-lymphoid cell-related pathways; C: Wiki pathways analysis showing extrafollicular B cell activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); D: Wiki pathways analysis displaying network map of SARS-CoV-2 signaling pathway; E: Kyoto Encyclopedia of Genes and Genomes pathway analysis revealing enrichment in cytokine-cytokine receptor interaction and chemokine signaling pathway.

The extent of AP-induced intestinal injury was found to be positively correlated with DCQT in mice

To investigate the impact of DCQT on injury, we delivered DCQT through intraperitoneal injections to mitigate the effects. The severity of injury was observed to be reduced in DCQT-administered mice, as evidenced by hematoxylin and eosin staining (Figure 4A-C). A substantial reduction in the intestinal injury marker was noted 24 hours post AP treatment via immunofluorescence analysis (Figure 4D-I). Additionally, treatment with DCQT led to a significant upregulation in the protein levels of occludin and ZO-1, as illustrated in the corresponding immunofluorescence photographs (Figure 4J and K).

Figure 4 The extent of AP-induced intestinal injury was found to be positively correlated with Dachengqitang in mice. A-C: Hematoxylin and eosin staining showed milder injury in mice treated with Dachengqitang (DCQT); D-I: Immunofluorescence analysis revealed a significant decrease in the intestinal injury marker following 24 hours of AP treatment; J and K: Furthermore, the protein expression of occludin and ZO-1 notably increased post-DCQT treatment, as illustrated in the immunofluorescence images. ^aP < 0.05. A-I features a scale bar of 50 μm. DCQT: Dachengqitang.

Clearing macrophages improved the function of intestinal epithelium and that was also true for in vivo experiments

As shown in Figure 5, the intestinal morphology and expression of tight junction protein ZO-1 and macrophage marker CD86 were examined across different experimental groups. H&E staining revealed that SAP induced significant intestinal mucosal damage compared to the control group, while DCQT treatment markedly improved intestinal tissue morphology (Figure 5A, D, G, and J). Immunofluorescence analysis demonstrated that ZO-1 expression was substantially decreased in the SAP group, whereas DCQT treatment significantly restored ZO-1 fluorescence intensity (Figure 5B, E, H, K, and M). Conversely, CD86 expression was markedly elevated in the SAP group compared to controls, indicating enhanced M1 macrophage polarization. DCQT treatment effectively suppressed CD86 expression (Figure 5C, F, I, L, and N). Notably, the addition of CI (claudin inhibitor) partially reversed the protective effects of DCQT on both ZO-1 and CD86 expression. Quantitative analysis confirmed these observations, with statistical significance observed among groups (^aP < 0.05). These findings suggest that DCQT treatment protects intestinal barrier integrity by upregulating tight junction protein expression and inhibiting M1 macrophage polarization in SAP mice.

Figure 5 In vivo experiments have demonstrated that clearance of macrophages can improve intestinal function and the integrity of the epithelium. A: Histological examination staining showing intestinal morphology in Control group; B: In Control group, immunofluorescence staining displaying ZO-1 expression in intestinal tissues; C: In Control group, immunofluorescence staining showing CD86 expression; D: Histological examination staining showing intestinal morphology in SAP group; E: In SAP group, immunofluorescence staining displaying ZO-1 expression in intestinal tissues; F: In SAP group, immunofluorescence staining showing CD86 expression; G: Histological examination staining showing intestinal morphology in SAP model treated with DCQT (SAP + DCQT) group; H: In SAP + DCQT group, immunofluorescence staining displaying ZO-1 expression in intestinal tissues; I: In SAP + DCQT group, immunofluorescence staining showing CD86 expression; J: Histological examination staining showing intestinal morphology in SAP + DCQT + CI group; K: In SAP + DCQT + CI group, immunofluorescence staining displaying ZO-1 expression in intestinal tissues; L: In SAP + DCQT + CI group, immunofluorescence staining showing CD86 expression; M: Quantitative analysis of ZO-1 fluorescence intensity; N: Quantitative analysis of CD86 fluorescence intensity. SAP significantly decreased ZO-1 expression and increased CD86 expression, while DCQT treatment restored ZO-1 levels and reduced CD86 expression. ^aP < 0.05. DCQT: Dachengqitang.

In vivo studies indicated that DCQT was able to increase the expression of Arg-1 and IL-10

In vivo experiments demonstrated that DCQT stimulates the expression of Arg-1 and IL-10, promoting the M2 macrophage phenotype. Following the induction of diabetes with streptozotocin, there was a marked decrease in the fluorescence intensity of Arg-1 (Figure 6A-I) and IL-10 (Figure 6J-T) as compared to the levels before treatment. The fluorescence intensity further diminished with DCQT administration.

Figure 6 In vivo studies have shown that Dachengqitang leads to the upregulation of Arg-1 and IL-10. A-J: Immunofluorescence staining showing Arg-1 expression in intestinal tissues across control, diabetes, and Dachengqitang (DCQT) treatment groups; K-T: Immunofluorescence staining displaying IL-10 expression levels across different experimental groups. Diabetes induction significantly decreased Arg-1 and IL-10 fluorescence intensity, while DCQT treatment restored their expression levels DCQT. The images are accompanied by a scale bar of 50 μm. ^aP < 0.05. DCQT: Dachengqitang.

In vitro models have also shown that OGD can deliver robust protection to the intestinal epithelium against injury

Fluorescence intensities of occludin (Figure 7A-C) and ZO-1 (Figure 7D-F) significantly decreased following OGD treatment compared to the baseline levels. Subsequent treatment with DCQT in the OGD group improved the fluorescence intensities of occludin (Figure 7G) and ZO-1 (Figure 7H).

Figure 7 In vitro models demonstrated that oxygen-glucose deprivation inhibits injury to the intestinal epithelium. A-C: Immunofluorescence staining showing decreased Occludin expression following OGD treatment; D-F: Immunofluorescence staining displaying reduced ZO-1 expression after OGD treatment; G: DCQT administration restored Occludin fluorescence intensity in OGD group; H: DCQT treatment improved ZO-1 fluorescence intensity in OGD group. ^aP < 0.05. Scale bar: 50 μm. OGD: Oxygen-glucose deprivation; DCQT: Dachengqitang.

In vitro models of Caco-2 cells were used to test the effect of DCQT on mitochondria

In this study, we assessed the effects of dexamethasone on mitochondrial dysfunction in OGD induced Caco-2 cells. Our results show that, compared to cells exposed only to high glucose conditions, the addition of remifentanil resulted in reduced JC-1 monomer levels and enhanced mitochondrial membrane potential. This indicates that dexamethasone may mitigate the mitochondrial damage induced by high glucose levels (Figure 8).

Figure 8 Dachengqitang induces mitochondrial impairment in Caco-2 cells in vitro. A-C: Mitochondrial membrane potential imaging in Control group after JC-1 staining; D-F: Mitochondrial membrane potential imaging in oxygen-glucose deprivation (OGD) group after JC-1 staining; G-I: Mitochondrial membrane potential imaging in Dachengqitang (DCQT) treatment group after JC-1 staining. Figure showing mitochondrial membrane potential across three groups; J: DCQT protects against OGD-induced mitochondrial membrane potential loss. DCQT treatment significantly reduced JC-1 monomer formation and improved mitochondrial membrane potential compared to OGD treatment alone. ^aP < 0.05. OGD: Oxygen-glucose deprivation; DCQT: Dachengqitang.

DISCUSSION

Post-ERCP pancreatitis represents a potentially life-threatening complication with acute onset and variable severity ranging from mild to severe disease[18-20]. This procedure-related pancreatitis may progress to SIRS and multi-organ dysfunction following endoscopic intervention. The pathophysiological mechanisms underlying PEP are multifactorial, encompassing procedure-induced pancreatic enzyme activation, mechanical trauma from endoscopic instrumentation, and amplification of inflammatory cascades[18-21]. A pivotal contributor to PEP progression is compromised intestinal barrier integrity. Barrier disruption facilitates bacterial translocation and endotoxin passage from the intestinal lumen into systemic circulation during the vulnerable peri-procedural period, thereby amplifying the inflammatory response. Elucidating these mechanistic pathways is essential for developing evidence-based therapeutic interventions to reduce PEP burden and optimize clinical outcomes.

The present study demonstrates the intricate relationship among intestinal barrier function, immune cell phenotype modulation, and inflammatory processes in PEP pathogenesis. The barrier-protective effects of DCQT are particularly significant, as intestinal barrier integrity is increasingly recognized as a critical determinant of systemic inflammatory responses following ERCP.

The intestinal mucosal barrier serves as a primary defense mechanism preventing bacterial and endotoxin translocation into systemic circulation, particularly during the peri-procedural period[22,23]. In PEP, barrier compromise results in increased intestinal permeability "leaky gut", which triggers or exacerbates systemic inflammation. Our findings demonstrate that DCQT enhances barrier function, potentially through upregulation of tight junction proteins and reduction of paracellular permeability. Restoration of barrier integrity may play a pivotal role in attenuating systemic inflammatory responses in PEP and facilitating recovery.

Macrophages exhibit remarkable phenotypic plasticity, polarizing toward M1 (proinflammatory) or M2 (anti-inflammatory) phenotypes in response to local microenvironmental cues[24,25]. The DCQT-induced shift toward M2 polarization, evidenced by increased expression of Arg-1 and IL-10, suggests that DCQT promotes an immunoregulatory phenotype, potentially mitigating PEP severity. This observation aligns with emerging evidence supporting immunomodulatory approaches for inflammatory complications.

Gene expression profiling and pathway analysis provide molecular insights into DCQT's mechanisms of action. Downregulation of inflammation-associated genes, coupled with enrichment of immunoregulatory pathways, indicates that DCQT modulates immune responses in PEP through specific signaling cascades. Based on our findings and existing pharmacological literature, we propose the following mechanistic hypotheses.

Direct epithelial effects

DCQT may activate PI3K/Akt signaling, which upregulates tight junction proteins (ZO-1, Occludin) and enhances barrier function. The observed improvement in mitochondrial membrane potential (JC-1 assay) suggests DCQT confers mitochondrial protection through antioxidant mechanisms, potentially involving Nrf2/HO-1 pathway activation. Bioactive compounds from key DCQT constituents-including emodin from rhubarb and magnolol from Magnolia officinalis-ave demonstrated tight junction stabilization via PKC-dependent mechanisms.

Macrophage polarization regulation

Our data showing reduced CD86⁺ M1 macrophages and increased Arg-1⁺/IL-10⁺ M2 macrophages suggest that DCQT inhibits NF-κB/STAT1 signaling while promoting STAT6/PPARγ activation-ey regulators of M1/M2 polarization. DCQT may modulate the intestinal cytokine milieu, shifting the balance from proinflammatory mediators (IL-6, TNF-α) toward anti-inflammatory cytokines (IL-10, TGF-β).

Integrated mechanism

We hypothesize that DCQT exerts barrier-protective effects through dual mechanisms: Direct cytoprotection via PI3K/Akt and Nrf2 pathway activation in epithelial cells, and indirect protection through immunomodulation of macrophage phenotypes via NF-κB/STAT signaling. Attenuated M1 macrophage activation may reduce local inflammatory burden, thereby further preserving epithelial barrier integrity.

Future validation studies will include: Western blot analysis of NF-κB, STAT1, STAT6, PI3K/Akt, and Nrf2 pathway components; pharmacological inhibitor studies to validate specific signaling cascade involvement; and transcriptomic profiling (RNA-seq) to comprehensively map DCQT-regulated genes and pathways.

The concordance between in vivo and in vitro findings strengthens the translational relevance of our results. DCQT's protective effects observed in the PEP animal model are recapitulated in intestinal epithelial cell cultures, demonstrating system-level improvements (enhanced ZO-1/Occludin-1 expression, reduced macrophage activation) paralleled by cellular-level benefits (improved intercellular tight junctions, preserved mitochondrial membrane potential).

Our findings have substantial clinical implications for PEP management. If DCQT's protective effects translate to human patients undergoing ERCP, it may offer a novel therapeutic approach, particularly for patients with severe complications or inadequate responses to conventional prophylactic strategies. Rigorously designed clinical trials are warranted to evaluate DCQT's safety and efficacy in PEP prevention and treatment. Furthermore, complete elucidation of the molecular mechanisms underlying DCQT's effects on intestinal barrier function and macrophage polarization may facilitate development of targeted pharmacological agents for pathway-specific interventions in PEP management.

CONCLUSION

In conclusion, our study conducted in pre-clinical rat models establishes the multifaceted therapeutic effect of DCQT in post-ERCP pancreatitis. DCQT alleviates the condition by enhancing the intestinal mucosal barrier, modulating macrophage polarization, and regulating specific inflammatory pathways, thereby demonstrating significant potential as a postoperative treatment.