Abstract

   Local pancreatic necrosis and systemic inflammatory response caused by
   acute pancreatitis (AP) are closely related to the disease prognosis
   and severity. This study aimed to explore whether pentraxin 3 (PTX3)
   regulates AP pancreatic necrosis and reveals the underlying mechanism.
   By using AP time gradient transcriptomics, proteomics and liquid phase
   chip analysis, we found a close association between PTX3 and AP.
   Subsequently, caerulein (CAE) induced in vivo AP model and CCK induced
   in vitro acinar cell damage model were constructed to determine the
   expression of PTX3 and its regulation of AP. The results showed that
   PTX3 was highly expressed in the CAE-induced AP model. And more severe
   pancreatic tissue damage and increased serum amylase were observed
   after the intervention of recombinant protein PTX3, which was strongly
   linked to the decreasing in mitochondrial membrane potential,
   increasing in reactive oxygen species, and regulation by oxidative
   phosphorylation pathway. PTX3 could exacerbate acinar cell damage in AP
   by mediating the process of oxidative phosphorylation.

   Keywords: Acute pancreatitis, Pentraxin 3, Acinar cell injury,
   Oxidative phosphorylation, Reactive oxygen species

   Subject terms: Biomarkers, Gastrointestinal diseases

Introduction

   Acute pancreatitis (AP) is a serious gastrointestinal disorder
   characterized by acinar cell injury caused by the pathological
   activation of trypsinogen^[44]1. Approximately 20% of individuals with
   AP experience extensive pancreatic or peripancreatic necrosis, leading
   to a mortality rate ranging from 30 to 39% in cases of infectious
   necrosis^[45]2. Pancreatic necrosis profoundly impacts the prognosis
   and quality of life of patients with AP, creating substantial economic
   and social burdens. Presently, clinical drug trials for AP are
   restricted, and effective preventive and treatment strategies for
   pancreatic necrosis remain elusive^[46]3. Thus, investigating the
   primary mediators and molecular pathways of AP is essential for
   addressing clinical challenges.

   In a prior investigation, we obtained plasma samples from 70 acute
   pancreatitis patients and identified pentraxin 3 (PTX3) as a promising
   marker for early detection of severe acute pancreatitis^[47]4. Prior
   studies have demonstrated that Pentraxins are an evolutionarily
   conserved superfamily of pattern recognition molecules (PRMs) in the
   innate immune system. This superfamily is divided into two subclasses:
   the classical short-chain protein C-reactive protein and the long-chain
   protein PTX3, which plays a crucial role in modulating innate immunity
   and inflammation^[48]5. For instance, in a model of ischemic acute
   kidney injury, the homozygous knockout of PTX3 in mice ameliorated
   renal damage^[49]6, whereas overexpression of PTX3 exacerbated the
   severity of ventilator-induced lung injury^[50]7. Studies have shown
   that PTX3 plays an important role in endoplasmic reticulum stress
   induced by tunicamycin. This indicates that in retinal pigment
   epithelial cells, endoplasmic reticulum stress can up-regulate the
   expression of PTX3^[51]8.

   However, previous studies have not investigated the role of PTX3 in
   acinar cell damage and its associated molecular mechanisms. Through a
   comprehensive multi-omics analysis in this study, we identified PTX3 as
   a potential key player in acinar cell necrosis. Our research applied
   proteomic and molecular biology techniques to thoroughly explore how
   PTX3 exacerbates acinar cell necrosis in AP.

Materials and methods

Animals and ethics

   The C57BL/6J strain mice (25–30 g) were procured from the Model Animal
   Research Center of Nanjing University (Nanjing, China). The mice were
   housed in specific pathogen-free (SPF) facilities and fed a standard
   rodent diet. The lighting regime was set at a 12-hour light-dark cycle,
   while the temperature in the housing area was maintained at
   25 °C ± 2 °C. This study received approval from the Science and
   Technology Commission of the Medical College of Yangzhou University
   (YXYLL-2024-074) and strictly adhered to the guidelines for
   experimental animal care (NIH Publication No.85 − 3, revised in 1996).

Acute pancreatitis model induced by caerulein (CAE)

   The C57BL/6J mice were randomly allocated into three groups: control,
   model, and rPTX3 groups (5, 10, 20 ug/kg). In the model group, mice
   received intraperitoneal injections of CAE (150 ug/kg, 1-hour interval,
   10 times). One hour after CAE injection, the rPTX3group received the
   injection of PTX3 recombinant protein, while the control group was
   administered an equivalent volume of PBS.

Pancreatic histopathology evaluation

   Following fixation, dehydration, and embedding in wax, pancreatic
   tissues were stained with hematoxylin and eosin. Subsequently, we
   evaluated the histomorphology features, such as necrosis of acinar
   cells, infiltration of inflammatory cells, and edema in the
   tissue^[52]9.

Determination of serum enzymology by ELISA

   The Mouse Pentraxin 3/TSG-14 Quantikine ELISA Kit (Catalog No. MPTX30,
   R&D Systems) was employed to determine the serum concentration of PTX3
   in mice. Meanwhile, serum amylase (Biotechnology&Science Inc.), lipase
   (Nanjing Jiancheng Corp.) levels were measured.

Isolation and processing of pancreatic acinar cells

   Collagenase I (Sigma-Aldrich) was injected into the pancreatic tissue
   through the main pancreatic duct to digest and extract mouse pancreatic
   acinar cells. Subsequently, the isolated cells were incubated at 37 °C
   in HEPES (Sigma-Aldrich) buffer. Utilizing CCK stimulation for 6 h, we
   established an in vitro model for acinar damage^[53]10. Finally, both
   the cells and the supernatants were harvested for subsequent analysis.

LDH release assay

   Mouse pancreatic acinar cells were evaluated following the experimental
   protocols outlined by the manufacturer of the LDH release kit (LDH
   Cytotoxicity Assay Kits, Beyotime Biotechnology).

Calcein-AM/PI double staining method

   Pancreatic acinar cells were double-stained with Calcein-AM (2mM,
   strong green fluorescence for live cells) and PI (1.5mM, red
   fluorescence for dead cells). The staining working solution was
   prepared by mixing Calcein-AM solution, PI solution, and 5 mL of 1×
   Assay Buffer. The collected acinar cells were centrifuged to remove the
   supernatant, washed with 1× Assay Buffer, and resuspended in 200 µL of
   1× Assay Buffer. The staining working solution was added, and the cells
   were incubated at 37 °C for 15 min. After incubation, the cells were
   observed and photographed using a laser confocal microscope.

Mitochondrial membrane potential assessment

   The mitochondrial membrane potential determination was conducted
   following the protocol specified by the manufacturer of the
   Mitochondrial Membrane Potential Kit (JC-1 Mitochondrial Membrane
   Potential Assay Kit, HY-K0601, MCE).

Multi-omics combined analysis

RNA sequencing, quality control, and analysis

   Pancreatic tissue samples collected at 0 h, 3 h, 6 h, and 12 h induced
   by CAE were subjected to RNA-Seq analysis. RNA integrity and quantity
   were precisely determined using the Agilent 2100 bioanalyzer,
   initiating the RNA analysis from total RNA. Illumina sequencing was
   employed for generating 150 bp paired-end reads after the library was
   prepared.

   Quality control procedures included converting sequenced image data
   into fastq format, filtering out reads with adaptors, those containing
   uncertain bases (N), and low-quality reads (bases with Qphred < = 20
   contributing to over 50% of the read length). The clean data’s quality
   was evaluated by calculating parameters such as Q20, Q30, and GC
   content. Subsequently, the reference genome index was constructed using
   HISAT2 v2.0.5 to align the paired-end clean reads.

   Each gene’s read count was determined using feature Counts, followed by
   the calculation of FPKM values. Differential expression analysis
   between various comparison combinations was carried out using the
   DESeq2 program package in RStudio (p-value<0.05, |log2FC|≥1), utilizing
   a negative binomial distribution model for identifying differentially
   expressed genes. To control false positive rates, P-values were
   adjusted using the Benjamini.

KEGG and GO pathway enrichment analysis

   We conducted pathway enrichment analysis of the Gene Ontology (GO) and
   Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways
   utilizing the DAVID database.

Short time -series expression miner (STEM) analysis

   The STEM program was utilized to examine trends in gene expression over
   time. The “STEM Clustering Method” served as the primary clustering
   algorithm, with a maximum of 50 models and a maximum variation of 2
   between time points.

Suspension liquid chip data analysis

   The plasma samples from patients with AP were analyzed on the
   Luminex100™ l suspension liquid chip platform utilizing data from
   previous publications4. Initially, thirty cytokines linked to the
   severity of acute pancreatitis were carefully chosen for subsequent
   analysis.

Common targets

   Through the integration of RNA-Seq and liquid microarray data, genes
   displaying a progressive expression pattern over time were identified
   using STEM analysis and compared with 30 cytokines linked to the
   severity of AP.

   Data analysis and visualization were conducted using the bioinformatics
   platform available at [54]https://www.bioinformatics.com. The CN online
   platform was specifically utilized for data analysis and visualization
   to generate graphical representations.

Statistics

   Statistical analysis was conducted using GraphPad Prism 8.0, and the
   outcomes were depicted as mean ± standard deviation (SD). Group
   distinctions were assessed using a t-test for two groups and a one-way
   ANOVA for more than two groups. Statistical significance was defined as
   a P-value < 0.05.

Results

Transcriptomic analysis of AP combined with suspension liquid chip identified
PTX3

   To investigate the gene expression profiles during the progression of
   AP, we analyzed the RNA-Seq data under the time gradient CAE induced AP
   model. Specifically, we presented the number of up-regulated and
   down-regulated genes at 3 h, 6 h, and 12 h time points (Fig. [55]1A).
   Meanwhile, a volcano plot was drawn (Fig. [56]1B), and a Venn diagram
   was utilized to illustrate the intersection of differential genes
   (Fig. [57]1C). Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia
   of Genes and Genomes (KEGG) pathway enrichment analyses were conducted
   on the common genes (Fig. [58]1D, F), and the KEGG pathways were
   further classified and annotated^[59]11-^[60]13 (Fig. [61]1E). Next,
   through STEM analysis, three clusters of genes with an upward
   expression trend were screened out. The expression changes of each gene
   within these clusters at different time points were visualized using a
   heat map (Fig. [62]2A), along with the corresponding GO analysis
   results (Fig. [63]2B). Finally, the intersection was taken between 30
   plasma cytokines related to the severity of AP disease and 4194
   up-regulated genes to display the interaction relationships among
   plasma factors (Fig. [64]2C), leading to the identification of PTX3 and
   MIF (Fig. [65]2D). Notably, PTX3 exhibited a significant differential
   expression in transcriptomics.

Fig. 1.

   [66]Fig. 1
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   Transcriptomic analysis of AP. (A) AP transcriptomic gene
   up-down-regulation histogram. (B) AP transcriptomic volcano map
   (Screening criteria: FC≥1 P value> 0.05). (C) Wayne diagram of
   overlapping differential genes at different time periods. (D) KEGG
   pathway analysis of differential genes. (E) KEGG annotation
   classification histogram. (F) GO analysis histogram of common
   differential genes.

Fig. 2.

   [68]Fig. 2
   [69]Open in a new tab

   Transcriptomic analysis of AP combined with suspension liquid chip
   identified PTX3. (A) Heat map and trend map of short time series
   expression mining (STEM) analysis. (B) GO chord plot of gene clusters
   1,2,3 and significant enriched GO terms. (C) Interaction of AP severity
   related proteins. (D) Common genes of liquid chip and rising trend gene
   group.

   In conclusion, through the combined analysis of AP transcriptomics and
   liquid chip technology, it was clearly demonstrated that there is a
   close association between PTX3 and AP disease.

Expression levels of PTX3 in the AP model

   Immunofluorescence and quantitative analysis revealed notable PTX3
   overexpression in pancreatic tissue of the AP model (Fig. [70]3A, B).
   Additionally, PTX3 mRNA levels exhibited a substantial increase in
   pancreatic tissue of AP mice compared to wild-type (WT) mice
   (Fig. [71]3C). Furthermore, serum analysis confirmed a significant
   elevation in PTX3 levels (Fig. [72]3D). These findings strongly
   suggested a pronounced upregulation of PTX3 in the AP model induced by
   CAE.

Fig. 3.

   [73]Fig. 3
   [74]Open in a new tab

   Expression levels of PTX3 in the AP model. (A) Representative
   immunofluorescence images of PTX3 expression in pancreatic tissue. (B)
   Quantitative analysis of immunofluorescence=4, ***p < 0.001. (C) mRNA
   level of PTX3 in pancreatic tissue. (D) Serum PTX3 level determined by
   ELISA; **p < 0.01, ***p < 0.001.

PTX3 exacerbates pancreatic tissue Injury in AP

   In order to investigate the impact of PTX3, recombinant PTX3 protein
   (rPTX3) was intraperitoneally injected to evaluate its effects on AP
   mice, classified into control, AP, and rPTX3 dose gradient groups (5,
   10, 20 ug/kg). The outcomes demonstrated that varying doses of rPTX3
   exacerbated pathological injury in AP, evident in tissue edema,
   heightened neutrophil infiltration, and increased acinar cell necrosis
   (Fig. [75]4A-E), with the 10 ug/kg dose exacerbating pancreatic injury
   to a greater extent. Notably, the rPTX3 groups displayed elevated serum
   amylase levels, and serum lipase was not statistically significant
   (Fig. [76]4F, G). These findings indicated that rPTX3 significantly
   contributes to the aggravation of pancreatic tissue injury in AP.

Fig. 4.

   [77]Fig. 4
   [78]Open in a new tab

   PTX3 exacerbates pancreatic tissue injury in AP. (A) Representative H&E
   staining of pancreatic tissue at 100X and 400X magnification. Scale
   bars, 200/50μm, N=5. (B-E) Histopathological scores of pancreatic
   tissues, N=5. (F-G) Serum amylase and lipase levels. *p < 0.05, **p <
   0.01, ***p < 0.001.

PTX3 exacerbates acinar cell damage in AP

   Noting that acinar cells constitute over 90% of pancreatic tissue, as
   cited in literature^[79]14, we explored the direct impact of PTX3 on
   acinar cells. The vitro experiments revealed that rPTX3 intensified
   primary acinar cell death detected by acinar cells PI/AM double
   staining and LDH release (Fig. [80]5A, B). Previous studies have
   highlighted the regulation of PTX3 expression by oxidative stress and
   reactive oxygen species^[81]15,^[82]16. Consistent with this, our
   findings indicated elevated levels of reactive oxygen species in rPTX3
   groups, with the most substantial increase observed at 50 ng/ml rPTX3
   stimulation (Fig. [83]5C). Considering the excessive ROS to disrupt
   mitochondrial membrane potential and exacerbate cellular injury^[84]17,
   we further analyzed the alteration in mitochondrial membrane potential
   in pancreatic acinar cells. Remarkably, a notable decrease was
   observed, particularly following the administration of 50 ng/ml
   recombinant PTX3 (Fig. [85]5D, E). These findings showed the ability of
   recombinant PTX3 to worsen acinar cell mortality.

Fig. 6.

   [86]Fig. 6
   [87]Open in a new tab

   Proteomics identifies oxidative phosphorylation pathways. (A) KEGG
   pathway enrichment analysis. (B) GO analysis. (C) KEGG classification
   annotation. (D) Oxidative phosphorylation pathway differential gene
   heat map. (E) Oxidative phosphorylation pathway differential gene
   correlation coefficient analysis.

Fig. 5.

   [88]Fig. 5
   [89]Open in a new tab

   PTX3 exacerbates acinar cell damage in AP. (A) Acinar cell PI/AM live
   and dead cell double staining, N = 3. (B) Percentage of LDH release in
   acinous cells (%), N = 5, **p < 0.01, ***p < 0.001. (C) Average
   fluorescence intensity produced by ROS in acinous cells, N = 8,
   ***p < 0.001. (D) Changes in mitochondrial membrane potential of
   acinous cells after recombinant protein PTX3 intervention. (E)
   Quantitative fluorescence analysis of mitochondrial membrane potential,
   N = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

PTX3 mediates the oxidative phosphorylation pathway to exacerbate acinar cell
damage

   To investigate the mechanism of PTX3 in exacerbating AP damage, KEGG
   enrichment analysis and classification annotation were conducted
   (Fig. [90]6A, C), highlighting the prominent involvement of the
   oxidative phosphorylation pathway as a notable focus (Fig. [91]6A). The
   results of GO analysis on differential genes revealed cellular
   localization within mitochondria, associated with biological functions
   of mitochondrial translation and mitochondrial small ribosomal subunit
   (Fig. [92]6B). Additionally, the cluster heatmap illustrated genes with
   the most significant alterations in the oxidative phosphorylation
   pathway (Fig. [93]6E), along with correlation coefficient calculations
   and gene associations (Fig. [94]6D). Furthermore, the mRNA-level of
   ATP6V1D, NDUFA11,ATP5F1D, ATP6V0A2,NDUFS4,SDHC gene expression
   verification was performed (Fig. [95]7A-F). The results showed that
   except for ATP5F1D and ATP6V0A2, the mRNA expression levels of other
   genes were decreased, which was consistent with the results of omics.

Fig. 7.

   [96]Fig. 7
   [97]Open in a new tab

   mRNA levels of validated gene with significant changes in oxidative
   phosphorylation pathways. (A-F) mRNA levels of differential genes, N=4,
   *p < 0.05, **p < 0.01, ***p < 0.001.(G) Percentage of LDH release in
   acinous cells after oxidative phosphorylation inhibitor rotenone
   intervention, N=5，**p < 0.01, ***p < 0.001

   Furthermore, to further clarify the changes in the oxidative
   phosphorylation pathway, we stimulated pancreatic acinar cells by
   Rotenone, one of inhibitors targeting the oxidative phosphorylation
   pathway. Rotenone effectively blocks the electron transport chain,
   inhibiting the oxidative phosphorylation process and leading to the
   obstruction of intracellular ATP synthesis. As shown in Fig. [98]7G,
   after blocking the oxidative phosphorylation pathway and intervening
   with recombinant protein PTX3, we observed that intervention with
   recombinant protein PTX3 combined with oxidative phosphorylation
   inhibitor had no significant difference in damage to pancreatic acinic
   cells compared with intervention with inhibitor alone (Fig. [99]7G).
   This finding showed that the oxidative phosphorylation pathway plays a
   critical role in PTX3-induced acinar cell damage.

Discussion

   This study initially focused on the role of PTX3 in acinar cell injury
   within AP. As previous clinical data indicated a significant elevation
   in PTX3 levels in the peripheral blood of AP patients, the multi-omics
   data and experimental validation in our study also highlighted the
   heightened expression of PTX3 in pancreatic tissue in AP model induced
   by CAE. Moreover, both the experiments in vitro and in vivo experiments
   demonstrated that recombinant PTX3 intensifies damage to pancreatic
   tissue and acinar cells. And, there was potential for the oxidative
   phosphorylation pathway as the underlying mechanism for PTX3-mediated
   acinar cell injury in AP.

   PTX3 exhibited an increasing trend in the early 24 to 48 h period and
   proved to be a more effective serum marker for predicting the severity
   of severe acute pancreatitis (SAP) compared to conventional scores4. A
   combination of nutritional support and insulin therapy significantly
   reduced the serum PTX3 levels in patients suffering from diabetic
   ketoacidosis and AP^[100]18. These findings signified the close
   association of PTX3 with AP, yet the underlying biological
   functionalities and mechanisms warrant further exploration. Our
   research demonstrated markedly elevated levels of PTX3 in the pancreas
   and serum. Additionally, vitro and vivo investigations unveiled that
   intervention with PTX3 recombinant protein could exacerbate pancreatic
   tissue and acinar cell damage in AP. This aligned with prior research
   indicating the detrimental effects of PTX3, where PTX3 overexpression
   aggravated LPS-stimulated proptosis and NLRP3 inflammasome activation
   in human periodontal ligament stem cells^[101]19. Furthermore, PTX3 was
   found to escalate the inflammatory response in psoriasis-like
   dermatitis^[102]20.

   Intracellular calcium overload and reduced ATP production in
   mitochondria contribute to pancreatic necrosis in AP^[103]21. Calcium
   overload or elevated reactive oxygen species levels can trigger the
   opening of the mitochondrial permeability transition pore, resulting in
   diminished mitochondrial membrane potential, cellular swelling, and
   ultimately rupture^[104]22. Our observations demonstrated a reduction
   in mitochondrial membrane potential and an increase in reactive oxygen
   species production in acinar cells through fluorescent staining.
   Meanwhile, the proteomic KEGG analysis in this study suggested that
   PTX3 may play a role in modulating the oxidative phosphorylation
   pathway in AP, thereby exacerbating pancreatic injury.

   Oxidative phosphorylation is the process where energy from oxidation
   drives the phosphorylation of ADP to generate ATP^[105]23. PTX3 may
   disrupt this process by inhibiting the electron transport chain. It can
   cause electron leakage, leading to the production of ROS, such as
   superoxide anions. These ROS can further convert into other reactive
   species, increasing intracellular ROS levels^[106]24. Additionally,
   PTX3-induced electron transfer blockage disrupts the mitochondrial
   membrane potential, impairing mitochondrial function and ATP synthesis
   efficiency, ultimately triggering mitochondrial dysfunction^[107]25.

   However, there were several limitations should be noted. Firstly, this
   study did not further investigate the effect of endogenous PTX3
   deficiency on AP pancreatic injury in mice. The lack of validation
   using PTX3-KO mice may somewhat limit the broader applicability of our
   study. Additionally, we did not explore the challenges related to
   therapeutic translation targeting PTX3 such as studies on PTX3
   inhibitors or small interfering RNA intervention. Finally, a
   comprehensive investigation of PTX3’s involvement in mediating the
   oxidative phosphorylation pathway in AP was needed.

   Currently, there is no product of PTX3 inhibitors. In the transition
   from basic research to clinical application, the future development of
   PTX3 inhibitors may serve as a potential therapeutic target for
   intervening in pancreatic necrosis associated with AP. By modulating
   the levels of PTX3 or the activity of its receptors, it may help
   alleviate inflammatory responses and inhibit excessive immune
   reactions, thereby providing new strategies for the treatment of
   related diseases. PTX3 is considered a potential biomarker for early
   diagnosis and monitoring disease progression. By tracking changes in
   PTX3 levels, it can help assess disease activity and severity.

   In conclusion, this research demonstrated that PTX3 may exacerbate
   acinar cell damage in AP by modulating the oxidative phosphorylation
   pathway. In the future, the research and development of PTX3 inhibitors
   may be expected to provide a new direction for clinical treatment.

Author contributions

   Wenyue Wang: conceptualization, methodology, investigation, data
   curation, visualization, writing-original draft. Yaning Wang:
   investigation, data curation, visualization, writing-original draft.
   Chenchen Yuan: conceptualization, methodology, investigation, data
   curation, visualization supervision, project administration. Fei Cao:
   methodology, investigation. Weisong Tang: methodology, validation.
   Qingtian Zhu: methodology, validation. Xiaowu Dong: supervision,
   project administration. Lihui Deng: investigation. Guotao Lu:
   conceptualization, resources. Qing Xia: supervision. Weiwei Chen:
   conceptualization, writing – review & editing, supervision, project
   administration, funding acquisition.

Funding

   This study was supported by the National Natural Science Foundation
   (No.82004291); 333 High-Level Talents Training Project of Jiangsu
   Province and Six Talent Peaks Project of Jiangsu Province
   (No.WSN-11325).

Data availability

   The original RNA-Seq transcriptome data has been deposited to National
   Center for Biotechnology Information (NCBI) under the BioProject number
   PRJNA 1219994. The mass spectrometry proteomics data have been
   deposited to the ProteomeXchange Consortium
   (https://proteomecentral.proteomexchange.org) via the iProX partner
   repository with the dataset identifier PXD060566.

Declarations

Ethics approval and consent to participate

   All animal experiments were conducted in accordance with the Principles
   of Laboratory Animal Care (NIH publication 85Y23, revised 1996), and
   were approved by Science and Technology Commission of the Medical
   College of Yangzhou University (YXYLL-2024-074).

Competing interests

   The authors declare no competing interests.

Disclosure

   The authors report there are no competing interests to declare.

Footnotes

   Publisher’s note

   Springer Nature remains neutral with regard to jurisdictional claims in
   published maps and institutional affiliations.

   Wenyue Wang and Yaning Wang contributed equally.

References