The role of antimicrobial peptides in pancreatic diseases and related research progress

The innate immune system is the body's first barrier against the invasion of foreign pathogens, and antimicrobial peptides are an important part of
the innate immune system.
Antimicrobial peptides in the human intestine are closely related to digestive diseases, and in inflammatory bowel disease and gastrointestinal inflammation, antimicrobial peptides help the recruitment and activation of immune cells, the production of cytokines, and the regulation
of inflammatory responses.
Recent studies have found that antimicrobial peptides also play a pivotal role in the occurrence and development of pancreatic diseases, and may become a breakthrough in the treatment of pancreatic diseases
.
This article reviews the role of common antimicrobial peptides in pancreatic diseases and the research progress of related treatments
.

I.
Overview

Antimicrobial peptide, also known as host defense peptide, contains 12~50 amino acids, molecular mass of 10 000, and has cationic
properties because of arginine and lysine residues.
Antimicrobial peptides are an important part of the innate immune system, have a broad-spectrum antibacterial effect, can also inhibit the activity of fungi, parasites, viruses and tumor cells, and are effective against a variety of drug-resistant bacteria, not easy to develop drug resistance
.
Antimicrobial peptides are typically stored in large cytoplasmic granules, then released into the crypt lumen of the small intestine, and exert immunomodulatory effects through various signaling pathways such as NF-κB, VDR, and MAPK to support
host defense functions.
Antimicrobial peptides in the human body can be divided into 10 types according to their different structures, the most common of which are defensins and cathelicidin-related antimicrobial peptides (CRAMP).

1
.
Defensin: Defensin was first isolated, purified and determined by Ganz in 1980.
At present, more than 20 structural analogues have been found in animals of different species, which are rich in the disulfide bond formed between cysteine and cysteine, collectively known as defensins, which are divided into α, β and θ3 categories according to their molecular topology, and only α-defensins and β-defensins
are expressed in the human body.
Among them α defensins 1, 2 and 3 are mainly secreted by neutrophils, so they are also called human neutrophil peptides, and α-defensins 5 and 6 are mainly secreted
by small intestinal Pan cells.
The content of α-defensin 5 mRNA in the human gut is about 4 times
that of α-defensin 6.
Other studies have found that α-defensin 5 mRNA expression levels tend to increase gradually from the jejunum to the ileum, reaching their highest
at the end of the ileum.
The antibacterial activity of α-defensin is mainly achieved
by penetrating the cell membrane of bacteria and single-celled pathogens to cause viral agglutination and promote its phagocytosis.
β-defensins are mainly produced by epithelial cells and keratinocytes, and the antibacterial effect is similar to that of α-defensins, inhibiting bacterial cell wall synthesis, and additionally exerting antiviral activity
through direct interaction with viruses and indirect interaction with target cells.

2.
CRAMP: CRAMP is another major member of the antimicrobial peptide family, initially found in neutrophils and later found to be expressed
in a variety of cells such as epithelial cells and keratinocytes.
The protein encoded by the Cathelicidin gene is hCAP-18, LL-37, the 37 amino acid fragments at the C-terminus of the protein, and is the mature form
of hCAP-18 treated with protease-3.
The antimicrobial profile of CRAMP is similar to that of defensins, and it also exerts a certain regulatory effect
on a variety of host cells, especially epithelial cells and immune cells.

3.
Other antibacterial peptides: In addition to defensin and CRAMP, antibacterial peptides in the human body include hepcidin, heparin-binding protein, silkwormin, frogeetin, etc
.
Silva-Vaz et al.
found that hepcidin was a better prognostic indicator
than CRP 48 hours after the onset of AP symptoms.
The prospective study by Sjobeck et al.
found that heparin-binding protein is expected to be an early predictor
of AP progression to severe sepsis.
It can be seen that although these antimicrobial peptides are expressed in less amount in the human body, they still have high clinical value
.

Second, the expression of antimicrobial peptides in pancreatic tissue

The expression of antimicrobial peptides in human pancreatic tissues is not fully understood
.
Stenwall et al.
found that in normal pancreatic tissues β-defensin 1 and 4 expression was negative, α-defensin 1, 4, β-defensin 2, 3, glycoprotein 2, cathelicidin, regenerating islet-derived protein 3α (Reg3α) expression was positive, in which α-defensin 1 and glycoprotein 2 were localized in pancreatic exocrine tissues.
α-defensin 4 and β-defensin 3 are expressed only in islet tissues, and the rest are expressed
in both islet and exocrine tissues.
At the same time, the researchers found that the expression of antimicrobial peptides in pancreatic inflammatory tissues was significantly lower than that in normal pancreatic tissues.
β-defensin 1 is negatively expressed in normal pancreatic tissue but positive in the inflammatory cell-infiltrated parenchyma of the pancreate
.
Another scholar conducted a comprehensive detection of the pancreatic proteome of pancreatic cancer patients through proteomics technology, found that the antimicrobial peptide Reg3α in the patient's pancreatic juice was overexpressed, and found another new protein with a degree of up to 85% of Reg3α, which was named pancreatitis-associated protein 2
.

Third, the role of antimicrobial peptides in pancreatic diseases

Huang et al.
found that the intestinal microbiota of rats with acute necrotising pancreatitis (ANP) was disordered, the expression levels of lysozyme and α-defensin mRNA decreased significantly, while the expression of TNF-α, IL-1β and IL-17A in plasma and distal ileum increased, and the intestinal barrier function was impaired.
It suggests that disturbance of the intestinal microbiota and decrease in α-defensins during ANP may be one of
the causes of intestinal barrier dysfunction.
Some scholars have also found that the acquired defect of α-defensin 5 may be an important cause
of intestinal bacterial translocation during ANP.
Deng et al.
found that CRAMP gene-deficient cnlp-/-ANP mouse models had a more severe pancreatic inflammatory response
than wild-type mice.
The above results show that antimicrobial peptides have a protective effect
in the initiation and development of ANP, whether it is defensin or CRAMP.
Cunha et al.
found that the expression of α-defensin 5 and α-defensin 7 in the intestine of ANP rats was increased, and the expression level of older rats was higher in younger rats, which may be related to
the defect of intestinal barrier functional integrity in old rats, resulting in further bacterial translocation 。 It was also found that even though systemic inflammation responses were similar between older and younger rats, local inflammation of the terminal ileum was more severe and the prognosis was worse in older rats, suggesting that α-defensin may play a key role in the development of the course of ANP, and high expression of α-defensin may mediate the development of more severe local inflammation
in older rats 。 Tiszlavicz et al.
found that gene variants in human β-defensin 1 and β-defensin 2 may be risk factors for SAP, and that high copy numbers of β-defensin 4 variants may predict a more severe prognosis, which is consistent with
the above findings.
Obviously, antimicrobial peptides have a very effective protective effect in the course of SAP disease, but when antimicrobial peptides encode gene mutations, they may aggravate local intestinal damage and lead to poor prognosis, so the multiple effects of antimicrobial peptides on the inflammatory process of AP still need to be confirmed by further research
.

Antimicrobial peptides also have a role
in CP.
Pausch et al.
found that compared with normal pancreatic tissue, α-defensin 1 was elevated in CP, and the increase in α-defensin 1 was associated with the expression of local pro-inflammatory cytokines in tissues by CD15 immunohistochemical staining, suggesting that α-defensin 1 may be related to
the course and development of CP.

Some studies have shown that antimicrobial peptides have antitumor effects
.
α-defensins 1, 2, and 3 can be used as tumor markers, small doses of α-defensin can promote tumor growth, while large doses of α-defensin can lyse and kill tumor cells
.
However, different studies have found that different types of antimicrobial peptides encoded by the same gene show different effects
on pancreatic cancer.
Wang Xue et al.
studied the activity of cathelicidin gene encoded peptide BF-30 in the in vitro anti-tumor effect of mouse pancreatic cancer cell Panc02, and found that BF-30 can penetrate the cell membrane, bind genomic DNA, and inhibit Panc02 cell migration and proliferation
。 However, Sainz et al.
found that hCAP-18/LL-37 encoded by cathelicidin gene can promote the occurrence and development of pancreatic tumors, hCAP-18/LL-37 is strongly expressed in the tumor matrix of advanced primary and secondary pancreatic ductal adenocarcinoma, and recombinant LL-37 is passed through formyl peptide receptor 2 (FPR2) and P2X purine receptor 7 receptor (P2X purinoceptor 7 recombinant protein, P2X7R) dependently enhances the expression, self-renewal, invasion and tumorigenicity
of genes associated with pancreatic cancer stem cells.
Tumorigenesis can be inhibited by knocking out the cathelicidin gene in mice or by pharmacological inhibition of FPR2 and P2X7R receptors

Fourth, the application of antibacterial peptides in the treatment of pancreatic diseases

Further research has been done on the related pathways related to the regulation of intestinal antimicrobial peptides, and it was found that the expression level of α-defensin 7 in rats in the intervention group using MAPK inhibitors was higher than that in the ANP group, and the histopathological score of pancreas in the rats in the intervention group was significantly lower than that in the ANP group, indicating that the reduction in the expression of α-defensin 7 regulated by the p38MAPK signaling pathway may be related to
the progression of ANP 。 Decreased expression of α-defensin 7 can lead to tissue inflammatory damage, which in turn destroys the intestinal immune barrier, triggers intestinal infection, and may even lead to pancreatic necrosis and systemic inflammatory response syndrome, resulting in adverse outcomes
such as organ failure.
It can be seen that blocking the p38MAPK signaling pathway to regulate the expression level of α-defensin 7 may become a breakthrough in SAP therapy
.

With the increase in the incidence of AP, convenient and feasible preventive measures have attracted more and more attention from patients
.
Xiong et al.
found that supplementation with okra pectin (OP) or crude okra pectin (COP) inhibited the production of pro-inflammatory cytokines in the pancreatic and upregulated β-defensin 1 and CRAMP, and colonic histology also confirmed that OP or COP can effectively maintain intestinal barrier function
.
Another study has found that low-methoxyl pectin (LMP) can play a similar role
.
These studies have shown that dietary OP and LMP supplementation can upregulate intestinal antimicrobial peptides, effectively improve intestinal damage and reduce inflammatory response triggered by AP, and both can be used as effective nutritional interventions
for AP and related intestinal injury 。 Other scholars have found that dietary supplementation of bacitracidase can effectively prevent CP, and the bacitracidase secreted by Bacillus subtilis can upregulate antimicrobial peptides in the intestine, and can reduce inflammatory cytokines (interferon γ, IL-2, 4, 5, 10, 13) and immunoglobulins in the pancreas, protect the pancreas and intestinal area, and help them restore function, so the regulation of intestinal antimicrobial peptides and inflammatory cytokines through dietary bacitracidase may become an effective way
to maintain pancreatic health and prevent CP.

K-ras mutant pancreatic cancer cells have been found to exhibit a strong macropinocytosis effect, a process that can be used in
the design of anti-cancer targeted therapies.
The study of Du et al.
found that recombinant defensin custom-made containing β-defensin 2 and human serum albumin was more cytotoxic to K-ras mutant cells than to wild-type cells, and that the recombinant defensin could inhibit cancer cell proliferation and induce apoptosis
of mitochondrial pathway 。 The defensin fusion protein Ec-LDP-D5 prepared by Liu Wenjuan et al.
containing α-defensin 5, lidamycin apoprotein (LDP) and EGFR-directing ligand peptide (Ec) can bind to PNAC1 and ASPC pancreatic cancer cells with high EGFR expression, thereby producing strong killing activity and inducing apoptosis on pancreatic cancer cells
。 It suggests that the above recombinant defensins may play a positive role in targeted therapy for pancreatic cancer, which also provides new ideas
for the treatment of pancreatic cancer in the future.

In summary, antimicrobial peptides are polypeptides widely present in the human body and are an important part of the innate immune system, with broad-spectrum bactericidal effects and inhibition of various biological activities
such as fungi, parasites, viruses and tumor cells.
Changes in the expression of antimicrobial peptides in pancreatic diseases suggest that they play a role in the progression of pancreatic diseases, but the multiple effects are still unclear and require more basic and clinical studies
.