Background
Pancreatitis (both acute and chronic) is among the three most common noncancerousGastrointestinal diagnoses and has accounted for a 12% increase in emergency room visits since 2006 [
1]. Acute pancreatitis (AP) is the first stage of an inflammatory disease continuum in the pancreas that can progress to recurrent AP (RAP), with 36% of patients with RAP further developing chronic pancreatitis (CP) [
2,
3]. Despite its high incidence, early diagnosis and treatment remain intractable.
To date, diagnostic radiologic imaging techniques including abdominal CT scanning and MRI in addition to endoscopic procedures including endoscopic retrograde cholangiopancreatography have been used to diagnose CP [
4,
5]. However, clinicians need a diagnostic test that can accurately identify patients early in the disease process, but no such test currently exists due to a lack of sensitive blood, imaging and functional biomarkers [
6,
7]. In this study, micro-PET/CT with two radiotracers has been applied to evaluate CP. And to the best of our knowledge, it is the first time that this approach is anticipated to serve as a valuable method for the early diagnosis of CP.
Most studies on CP have focused mainly on the mechanism of pancreatic fibrosis [
8,
9]. However, inhibiting pancreatic fibrosis as the main clinical therapeutic direction is no longer progressing. The manifestation of fatty degeneration of acinar cells in patients with CP, especially in patients with alcoholic chronic pancreatitis (ACP), has been ignored. In patients with hereditary pancreatitis, lipomatous atrophy of the pancreas leading to pancreatic insufficiency is a frequent occurrence and increases with age [
10]. In most cases, pancreatic steatosis is associated with metabolic syndrome and alcohol abuse. Chronic alcohol abuse increases pancreatic lipid accumulation, inducing pancreatic steatosis, and is usually seen in people who consume more than 30 g of ethanol per day [
11]. These findings suggest a strong correlation between disordered lipid metabolism and CP.
An important pathway in lipid metabolism [
12], the arachidonic acid (AA) pathway and derivatives of AA, such as prostaglandins, thromboxanes, and leukotrienes, are also important mediators of inflammation and involved in the inflammatory process [
13]. Prostaglandin E2 (PGE
2), generally recognized as an important mediator of inflammation, regulates many pathways of inflammation [
14,
15]. St13, also called HIP (Hsc70-interacting protein), has been reported to be involved in lipid metabolism as a member of the chaperone family. For instance, a study showed that fraxin reduced lipid peroxidation, and the subsequent increase in internal reactive oxygen species accompanying with upregulated St13 [
16]. Furthermore, HIP overexpression could reverse the functional inhibition of glucocorticoid receptor (GR), which is strongly associated with lipid metabolism, and enhance its functional maturation [
17‐
19]. GR controls the secretion of glucocorticoids with a regular daily rhythm by time-dependent chromatin binding and target gene transcription to control daily cycles of glucose and triglyceride metabolism [
20], moreover, GR plays a pivotal role in activating metabolic genes, including those mediating hyperglycaemia, hyperlipidaemia and obesity [
21]. Nonetheless, the relationship between St13 and lipid metabolism is not well understood.
The cleavage of trypsinogen contributes to the initiation of AP, and increased trypsin activity sensitizes mice to the development of pancreatitis [
22]. In view of this, we established a humanized
PRSS1 mouse model to simulate the pathogenesis of AP and CP [
23‐
25]. We sought to use this model to identify diagnostic targets and therapeutic methods for CP in this study. We first confirmed the occurrence of lipid metabolic disorder in CP by both mouse and human tissues; specifically, dysregulation of the AA pathway was common to both ACP and nACP. Next, we showed that
PRSS1Tg mice in which St13 had been deleted displayed elevated pancreatic acinar injury and were prone to steatosis. Stromal cell-derived factor 2-like 1 (Sdf2l1) was identified as a regulatory binding partner of St13 that protects against CP pathogenesis.
68Ga-FAPI-04 PET/CT may be very hopeful in early diagnosis in CP.
Methods and materials
Human pancreatic tissues and study approval
Human pancreatic tissues were collected from 6 nACP patients (mean age = 44.33), 7 ACP patients (mean age = 49.43) and 5 patients with benign pancreatic tumours or peritumoural normal pancreatic tissues (mean age = 41.60) as controls. All patients were hospitalized in Nanfang Hospital, Southern Medical University between 2015 and 2020 (Additional file
7: Table S1). The study was approved by the Ethics Committee of the Southern Medical University and written consent was obtained from each patient.
Mouse models and adSt13- and shSdf2l1-expressing adenovirus
The preclinical PRSS1 (GenBank Accession Number: NM_002769.4) transgenic (PRSS1Tg) mice were used for CP models. We used caerulein and alcohol on PRSS1Tg mice to mimic human nACP and ACP development. St13 knock mice were constructed.
The pancreases of
PRSS1Tg mice were infected with adenoviral vectors harboring full-length St13 for St13 overexpression and shSdf2l1 fragments for Sdf2l1 silencing. Blank adenovirus was used as negative control. All adeno-associated virus (PackGene Biotech, China) used are listed in Additional file
4: Fig. S4. Please see the Supplementary materials and methods for more details.
Proteomics
Proteomic analysis has been described previously [
23]. The UniProt database was used to screen and identify the differential expression proteins induced by ISO in mice using 2 times as the change threshold and
p < 0.05 as the standard.
Transmission electron microscopy (TEM) and immunoelectron microscopy (IEM)
As previously described [
26], ultrastructural examination was performed using a transmission electron microscope to observe lipid droplets in pancreatic acinar cells. The sub-cellular localization of St13 and Sdf2l1 were analyzed by immunogold staining, of which the experimental methods has been adjusted, referring to the previous literatures [
27]. Please refer to the Supplementary materials and methods for more details.
Coimmunoprecipitation and mass spectrometry (MS)
The coimmunoprecipitation was performed to detect the proteins binding to St13 directly. The fusion protein bound to St13 were separated by SDS-PAGE and stained with a Silver Staining Kit (Beyotime Biotechnology, Shanghai, China). After digesting the peptides with trypsin, the specific proteins were analyzed on a mass spectrometer (Thermo Fisher, Waltham, USA) and identified using Protein Pilot 5.0 (AB Sciex, USA). Experimental details can be received in the Supplementary materials and methods.
Plasmid synthesis and co-immunoprecipitation
To map the specific region of St13 required for its interaction with Sdf2l1, four fragments of St13 harbouring amino acids with Flag tags were constructed: #1, (aa 1–371), #2, (aa 1–112); #3, (aa 113–214) and #4, (aa 215–317). 293 cells over-expressing each fragment of St13 were immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-Flag and anti-Sdf2l1 antibodies. Empty plasmid transfected 293 cells served as the normal control (NC).
Micro-PET/CT
PET/CT imaging was performed using the SIEMENS Inveon micro-PET/CT scanner at Nanfang PET Center, Nanfang Hospital, Southern Medical University. 68Ga-DOTA-TATE and 68Ga-FAPI-04 were used as the radiotracer biodistributions for identification of mouse pancreas. Please refer to the Supplementary materials and methods for more experimental details.
Gas chromatography-mass spectrometry (GC–MS; Agilent 6890 GC coupled to an Agilent 5973 MS System, Waldbronn, Germany) and liquid chromatography-MS/MS (LC–MS/MS; Agilent 1100 HPLC-System, Darmstadt, Germany) were applied for the determination of fatty acids, triglycerides, cholesterol, phospholipids and eicosanoid levels.
Fatty acids, triglycerides, cholesterol, phospholipids and eicosanoid were extracted from pancreatic tissues respectively by liquid/liquid extraction and solid/liquid extraction. For further details, please see the Supplementary materials and methods.
Cell viability assay
Pancreatic acinar cells were cultured with 10% FBS and 0.25 mg/ml of trypsin inhibitor in Waymouth’s medium for 24 h. After culturing, cell viability was measured using a Cell viability assay Kit (BBI, USA). The percentage of growth was determined on Flow cytometer. Experiments were repeated at least three times with triplicate samples.
Details of methods for human tissues, mice strains and materials used, immunohistochemistry, Oil Red O staining, immunofluorescence assay and the quantification of proteins by quantitative real-time PCR, western blot and ELISA are described in the Supplementary materials and methods section.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0, SPSS 24.0 and MATLAB R2018a software. The continuous variables are expressed as mean ± standard error. Significant differences between two groups were analyzed by Student’s t-test, and one-way analysis of variance was performed to investigate the differences among more than two groups. P < 0.05 was considered statistically significant.
Discussion
In order to better simulate clinical (alcoholic) and experimental (non-alcoholic) pancreatitis, two mice CP models were used in the present study. Our data identified St13 as a critical protein in acinar lipid metabolic disorder in CP. The molecular co-chaperone St13 was overexpressed in the pancreatic tissues of CP model mice and patients and found to protect against acinar steatosis and injury. Experiments conducted to screen binding partners of St13 identified Sdf2l1, which was confirmed to bind St13 and found to promote AA pathway homeostasis in acinar cells by regulating the IRE1α-XBP1s pathway in CP. Parecoxib inhibited the AA pathway to protect against acinar injury in CP (Fig.
6). These results provide the first indication that St13 regulates AA pathway in CP by binding Sdf2l1. Theoretically, St13, Sdf2l1 and parecoxib could be clinically translated into valuable therapeutic targets to overcome the limitations of CP therapies. Meanwhile,
68Ga-FAPI-04 PET/CT is a promising imaging method for the early diagnosis of CP.
Alcohol abuse and smoking are the 2 most critical modifiable risk factors that affect the transition from AP to CP [
3]. The combination of alcohol and cigarette smoke promotes the development of CP by inducing dysregulation of the unfolded protein response (UPR) and proteostatic mechanisms in acinar cells [
32]. Compared to nACP, more severe lipid metabolism disorders existed in ACP, which hints that the pancreas is more sensitive to alcohol damage. In
l-arginine-induced AP, pancreatic lipid metabolism is profoundly disrupted. Specifically, in L-arginine-induced AP, the levels of saturated free fatty acids (FFAs) are markedly decreased, and the levels of long-chain polyunsaturated fatty acids and AA metabolites are markedly increased [
33]. However, to the best of our knowledge, no research on lipid metabolic disorder in CP in detail has been reported, and this idea is worthy of in-depth study.
The action of chaperone systems is crucial for client protein folding, translocation, and unfolding. Breakdown of the chaperone system can lead to protein misfolding and aggregation, which can ultimately cause cell death [
34]. Although St13 plays a crucial role in the chaperone system, unfortunately, its function, especially in specific diseases, has not received attention and has not been further studied. The ER is critical for the proper folding, maturation and secretion of transmembrane and secreted proteins. ER stress and activation of the UPR (unfolded protein response) help to determine cell fate and function [
35]. Indeed, we discovered that ER stress plays a critical role in acinar cell apoptosis in both AP and CP [
23,
24]. The function of molecular chaperones is closely related to ER-associated proteins. Thus, we sought to further study Sdf2l1 as a research target, which was identified by the screen.
Sdf2l1, a component of a chaperone complex with the ER-resident protein ERdj3 (DNAJB11), prevents protein aggregation during the BIP chaperone cycle, which acts as a “valve” for the 3 ER stress pathways [
36]. Therefore, interference with Sdf2l1 causes this “valve” open and activates ER stress. Chronic ER stress is thought to cause metabolic disorders in various tissues [
37]. Recently, Takayoshi Sasako et al. reported that suppression of Sdf2l1 caused sustained ER stress, leading to insulin resistance and hepatic steatosis in obesity and diabetes, which indicated that the induction of Sdf2l1 as an ER stress response molecule mediates lipid metabolism [
38]. Our data revealed that Sdf2l1 within the St13-Sdf2l1 complex plays a protective role in acinar injury and steatosis through regulation of the AA pathway. These results changed our previous view that St13 was only a chaperone of HSC70. In CP, St13 plays a protective role in acinar steatosis by binding Sdf2l1.
Importantly, the IRE1α-XBP1s pathway, the most ancient branch of the UPR, is closely related to lipid metabolism during ER stress [
39‐
41]. In addition, the production of COX-2 and mPGES-1 in leukocytes can be modulated by the IRE1α-XBP1s signalling pathway, which then promotes the production of PGE
2 in the AA metabolic pathway; this link suggests a new approach to exploit anti-inflammatory drugs and analgesics [
29]. Interestingly, these findings were consistent with those of our previous clinical study indicating that a selective COX-2 inhibitor, parecoxib, reduced the morbidity due to complications and abdominal infection among patients with mild and moderately severe AP [
42]. In this study, we found that the disruption of Sdf2l1 triggered the IRE1α-XBP1s pathway and promoted COX-2 and PGE
2 production. Based on above results, St13 and Sdf2l1 may be translated into therapeutic target of CP including COX-2 inhibitors in future.
Another major aim of the study was to identify a new diagnostic method for CP to address limitations in currently available diagnostic methods. This is the first report showing the use of micro-PET/CT to evaluate pancreatic morphology and fibrosis in CP models, and the results indicated that this technique accurately reflects dynamic changes in pancreatic morphology. Since advanced CP is progressive and irreversible, early diagnosis and medical interventions are essential to improve the long-term outcomes of CP patients [
43]. Early diagnosis of CP has long been a clinical challenge.
68Ga-DOTA-TATE offers high sensitivity and specificity for imaging of the pancreas and has especially important diagnostic value for pancreatic neuroendocrine tumours (NETs) [
44]. However, in this study, as CP developed, the decrease in functional acinar cells led to the absence of
68Ga-DOTA-TATE uptake in the pancreas, indicating that
68Ga-DOTA-TATE cannot be used for CP diagnosis.
6FAP is a membrane serine coenzyme that belongs to the type II serine protease family. It displays both dipeptidase and collagenase activity and mediates degradation of the extracellular matrix. Generally, FAP is expressed mainly on the surface of activated fibroblast cell membranes and is rarely expressed in healthy tissues under normal conditions. FAP was shown to be expressed in solid tumours, rheumatoid arthritis tissues, atherosclerotic plaques, and fibrotic tissues [
45]. However,
68Ga-FAPI-04 has not been applied to evaluate fibrosis in CP. In this study, we used two radiotracers to evaluate the imaging of CP at 2 different time points (2 and 4 weeks) in 2 CP models. Unlike the tracer
68Ga-DOTA-TATE,
68Ga-FAPI-04 identified morphological and fibrotic changes in the early stage (2 weeks) in CP mouse models. However, in the late stage,
68Ga-FAPI-04 could not be used to image the fibrotic pancreas, because activated fibroblast cells had transformed into fibrocytes and the expression of FAP gradually decreased. Based on the detection of characteristic FAP expression,
68Ga-FAPI-04 PET-CT could play a role in the early diagnosis of CP, which should be validated in future clinical work.
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