Rn-Ji Chng ,Hui-Lin Wng ,Mng-Bin Qin ,Zhi-Hi Ling ,Ji-Ping H ,Yu-L Wi ,Hong-Zong Fu ,Guo-Du Tng ,*

a Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China

b Department of Gastroenterology, The First People’s Hospital of Nanning City, The Fifth Affiliated Hospital of Guangxi Medical University, Nanning 530022,China

c Department of Chemotherapy, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China

d Department of Gastroenterology, Second Affiliated Hospital of Guangxi Medical University, Nanning 530 0 07, China

e Department of Gastroenterology, Liuzhou General Hospital, Liuzhou 545006, China

f Department of Gastroenterology, Guangxi International Zhuang Medical Hospital, Nanning 530 0 01, China

Keywords:Acute pancreatitis Ghrelin Inflammatory cytokine Acinar cells

ABSTRACT Background: Previous studies have provided conflicting results regarding whether the serum ghrelin concentration can reflect the severity of acute pancreatitis (AP).The present study examined the correlation between the serum ghrelin concentration and AP severity in animal models and investigated whether altered ghrelin expression in pancreatic acinar cells influences IKK β/NF-κB signaling and pro-inflammatory cytokine production.Methods: Mild or severe AP was induced in rats by intraperitoneal injection of cerulein or retrograde cholangiopancreatic duct injection of sodium taurocholate,respectively.After successful model induction,serum ghrelin,tumor necrosis factor-α (TNF-α),and interleukin-6 (IL-6) concentrations were determined by enzyme-linked immunosorbent assay,and IKK β/NF-κB activation was assessed by immunohistochemistry.Subsequently,stable overexpression or knockdown of ghrelin in AR42J cells was achieved by lentiviral transfection.After transfected cells and control cells were treated with cerulein for 24 h,the TNF-α and IL-1 β levels in the supernatants were determined by enzyme-linked immunosorbent assay,and the expression levels of p-p65,IKK β,and p-IKK β were detected by Western blotting.Results: In rat AP models,AP severity was correlated with increased IKK β/NF-κB activation,proinflammatory cytokine production,and ghrelin secretion.The levels of pro-inflammatory cytokines TNF-α and IL-1 β as well as IKK β/NF-κB signaling activity were increased upon knockdown of ghrelin in the AP acinar cell model and decreased with ghrelin overexpression.Conclusions: Serum ghrelin is related to the severity of AP.Ghrelin may play a protective role in the pathogenesis of AP by inhibiting the pro-inflammatory cytokines and the activation of the IKK β/NF-κB signaling pathway.

Introduction

Acute pancreatitis (AP) is a common and suddenly occurring pancreatic inflammatory disease that can develop via multiple etiologies.Although the majority of AP cases (about 80%) are mild and can be successfully treated using conservative measures,severe cases (about 20%) require intensive care unit admission and even surgical intervention to control the severe complications associated with the complex disease course,such as multiple organ dysfunction syndrome [1] .Despite the diversity of therapeutic strategies currently available,severe AP (SAP) continues to be associated with a high mortality rate (10% -30%) [2] .

The pathogenesis of AP is complex.In the early stages of AP,inflammatory reactions can lead to endothelial cell dysfunction,which in turn activates coagulation,leading to microcirculation disturbance [3] .Laboratory tests in AP patients revealed elevated levels of inflammatory markers in the blood as well as increased Ddimer,soluble fms-like tyrosine kinase 1 (sFlt-1),and angiopoietin-2 levels [3] .The severity of AP largely depends on the severity of inflammation,which is limited by endogenous defense mechanisms [4] .Thus,the identification of endogenous protective factors against AP progression and elucidation of the underlying protective mechanisms will provide important insights for the development of new preventive or therapeutic strategies for this life-threatening disease.

Ghrelin,an endogenous peptide hormone first identified in the stomach [5] and later found to be extensively expressed in a variety of tissues and organs,including the pancreas [6,7],has been shown to protect the pancreas against inflammatory injury [4] .Ghrelin was shown to inhibit both the exocrine and endocrine functions of the pancreas by directly acting on isletβ-cells [8] .While some studies have demonstrated that serum ghrelin levels can reflect the severity of AP [9-13],others did not find this correlation [14,15] .In animal models of AP,ghrelin treatment prior to AP induction was shown to significantly attenuate the inflammatory response and pancreatic tissue damage [16,17] .Several mechanisms,such as innate immune system activation and inflammatory response inhibition,have been proposed to explain the protective effect of ghrelin against AP;however,the detailed molecular mechanisms of ghrelin’s activity in AP remain largely unknown.

The intra-pancreatic activation of digestive enzymes (mainly trypsin) and subsequent pancreatic autodigestion,which initiates local or systemic inflammation by inducing the release of proinflammatory cytokines such as tumor necrosis factor-α(TNF-α)and interleukin-6 (IL-6),are regarded as central mechanisms in the early pathogenesis of AP [18] .Activation of nuclear factor-κB(NF-κB),an early event in AP,has been suggested to be involved in the activation of trypsin and the subsequent inflammatory response [4] .Intriguingly,a previous study implicated ghrelin in stimulating pancreatic enzyme secretion [19],and in an animal model of AP,the protective effect of exogenous ghrelin against AP was attributed to the suppression of NF-κB activation and the blockade of the inflammatory response [17] .However,the protective and therapeutic effects of ghrelin against AP are traditionally believed to be indirect and dependent on the induction of other factors,such as growth hormone and insulin-like growth factor-1 [3],and it remains unclear whether endogenous ghrelin (especially pancreatic ghrelin) can act upon NF-κB signaling in pancreatic acinar cells.

Our recent study found that endogenous inhibition of the ghrelin gene in pancreatic acinar cells promotes the expression of intracellular inflammatory factors,suggesting that pancreatic ghrelin may have a protective effect against AP [20] .Accordingly,we hypothesized that pancreatic ghrelin may inhibit NF-κB activation and pro-inflammatory cytokine production in pancreatic acinar cells.To test this hypothesis,we utilized a pancreatic acinar cell (AR42J) model of cerulein-induced experimental AP to examine the impact of ghrelin overexpression or knockdown on the activation of IKKβ/NF-κB signaling and the production of the proinflammatory cytokines TNF-αand IL-6.Given the currently conflicting data on the correlation between ghrelin and the severity of AP,we also used two animal models of AP to investigate whether the serum ghrelin concentration is associated with the severity of inflammation and IKKβ/NF-κB activation.For these rat models of AP,we applied the commonly used cerulein intraperitoneal injection method to induce moderate AP (MAP) and retrograde injection of sodium taurocholate to induce SAP.

Materials and methods

Animals

Adult male Sprague-Dawley rats,weighing 250-300 g,were provided by the Laboratory Animal Center of Guangxi Medical University (Nanning,China) and maintained in accordance with the Laboratory Animal Care and Use Regulations of Guangxi Medical University.The rats received regular rat chow for 1 week to acclimatize to the new environment.Prior to model induction,the rats were fasted for 12 h with access to water.All animal experiments were approved by the Ethics Committee of our hospital(201,701,007).

Animal model preparation and sample collection

A total of 32 rats were randomly divided into four groups(n=8/group):MAP group,MAP control group (C-M group),SAP group,and SAP control group (C-S group).MAP was induced by intraperitoneal injections of cerulein (50μg/kg,dissolved in saline;Sigma-Aldrich,St Louis,MO,USA) at 1-h intervals for a total of seven times.Rats in the C-M group were injected with saline instead of cerulein.SAP was induced by retrograde cholangiopancreatic duct injection of 4% sodium taurocholate (1 mL/kg;TCI Shanghai,China) at a rate of 0.1 mL/min.The bile duct was temporarily occluded at the liver hilum with a micro-vein clamp to prevent reflux of the infused material to the liver.The clamp was removed after 2 min of injection.The rats in the C-S group underwent a sham-operation.The pancreas was flipped several times without duct puncture.After surgery,the duodenum and pancreas were carefully returned to the peritoneal cavity,and the skin incision was closed with continuous sutures.The animals were left to recover for 1 h and then placed individually into squirrel cages.

Twenty-four hours after the induction of AP,the animals were anesthetized by intraperitoneal injection of 10% chloral hydrate(3 mL/kg body weight).Blood samples were collected via abdominal aorta puncture under anesthesia,allowed to clot,and centrifuged to collect the serum,which was aliquoted and stored at-20 °C for subsequent use.The animals were then sacrificed,and pancreatic tissue samples were collected for histopathological examination.

Histopathology

Pancreatic tissue was fixed in 10% formalin,embedded in paraffin,and cut into slices for morphological evaluation by hematoxylin and eosin (HE) staining.After microscopic images were taken,the improved Schmidt method [21] was adopted to semi-quantitatively assess parenchymal damage with regard to interstitial edema,acinar necrosis,hemorrhage and fat necrosis,and inflammatory infiltrates.

Immunohistochemistry

Immunohistochemical staining for phosphorylated (p)-p65 and p-IKKβwas performed using an immunohistochemical SP-9000 kit(Zhongshan Goldenbridge,Beijing,China).The prepared sections were boiled in 0.01 mmol/L citrate buffer for 2 min in a pressure cooker for antigen retrieval.After incubation with hydrogen peroxide (3%) for 15 min to inactivate endogenous peroxidase activity,5% goat serum was applied as the blocking agent for 10 min at room temperature.The sections were then incubated with rabbit anti-rat anti-p-p65 antibody (1:400 dilution;Bioworld Technology,St Louis Park,MN,USA) or anti-p-IKKβantibody (1:150 dilution;Cell Signaling Technology,Danvers,MA,USA) overnight at4 °C.After washing with phosphate-buffered saline (PBS),the sections were incubated with a goat anti-rabbit secondary antibody and visualized using a 3,3-diaminobenzidine kit (Zhongshan Goldenbridge).The stained sections were observed under a microscope(CX41,Olympus,Tokyo,Japan).

To evaluate p-p65 and p-IKKβstaining,a semi-quantitative scoring system based on the staining intensity and percentage of positive cells was used.Staining intensity was graded as 0 (negative),1 (weak),2 (moderate),or 3 (intense),and the percentage of positive cells was scored on a 4-point scale as follows:0,＜5%positive cells;1,5% -25%;2,26% -50%;and 3,＞50%.The sum of the scores for staining intensity and the percentage of positive cells was used as the final score:0 (-);1-2 (+);3-4 (++);and＞4(+++).

Biochemical determination

Serum amylase activity was measured using a HITACHI-7170 auto-biochemical analyzer (HITACHI,Tokyo,Japan).The levels of TNF-αand IL-1βin the serum or cell supernatant were measured using rat TNF-αand IL-1βenzyme-linked immunosorbent assay (ELISA) kits (Neobioscience,Shenzhen,China) according to the manufacturer’s instructions.Serum ghrelin was measured using a rat ghrelin enzyme immunoassay kit (RayBiotech Technologies,Norcross,GA,USA) following the manufacturer’s protocol.

Cell culture, transfection, and treatment

AR42J cell lines with stable overexpression or silencing of ghrelin were obtained as previously reported [22] .Different groups of cells (AR42J cells [AR42J],AR42J cells transfected with the empty vector [normal control,NC],AR42J cells overexpressing ghrelin [AR42J+ghrelin],and AR42J cells with ghrelin knockdown [AR42J+ghrelin shRNA]) were treated with cerulein(10-7mmol/L) for 24 h,and untreated cells were used as controls.After treatments,the cells were used for Western blotting analysis,and cell supernatants were collected for ELISA.

qRT-PCR

Total RNA was extracted from different groups of cells using a Total RNA Miniprep Kit (Axygen,Union City,CA,USA) according to the manufacturer’s instructions.After reverse transcription using a reverse transcription kit (TaKaRa,Kusatsu,Japan),qPCR was carried out on an ABI 7500 Real-Time PCR system (Bio-Rad Laboratories,Hercules,CA,USA) using SYBR Green Mix (TaKaRa).Quantification of ghrelin mRNA expression was performed using glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal reference.Relative expression of ghrelin mRNA was quantified using the 2-ΔΔCtmethod.The sequences of primers used for PCR amplification were:ghrelin forward,5 ′ -AGA AAG GAA TCC AAG CCA CC-3 ′ and reverse,5 ′ -TGC CAA CAT CGA AGG GAG C-3 ′ ;GAPDH forward,5 ′ -TGG CAT GGA CTG TGG TCA TG-3 ′ and reverse,5 ′ -TGG GTG TGA ACC ACG AGA AA-3 ′ .Eleven oligonucleotide fragments were designed and synthesized on the CDS region sequence of ghrelin gene,and BamH I restriction site was added at the 5 ′end of the gene,and Nhe I restriction site was added at the 3 ′ end(Table 1).

Table 1 The sequences of primers used to amplify the full-length cDNA of ghrelin.

Western blotting analysis

Cells were subjected to lysis using radioimmunoprecipitation assay buffer (Beyotime,Shanghai,China).The protein concentration in cell lysates was measured using a bicinchoninic acid protein assay (Beyotime).Equal amounts of proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore,Burlington,MA,USA).The membrane was then blocked with 5% nonfat milk in Tris-buffered saline containing 0.05%Tween 20 and incubated with a primary antibody against p-p65(1:1000;Cell Signaling Technology),IKKβ(1:1000;Cell Signaling Technology),p-IKKβ(1:1000;Cell Signaling Technology),ghrelin(1:600;Proteintech,Rosemont,IL,USA),or GAPDH (1:3000;Proteintech) overnight at 4 °C,followed by incubation with an IRDyeconjugated secondary antibody (Licor,Lincoln,NE,USA) at room temperature for 1 h.Subsequently,the protein bands were detected using a Licor Odyssey infrared fluorescent imaging system(Licor),and band intensity was quantified with ImageJ software and normalized to that for GAPDH.

Statistical analysis

SPSS 16.0 software (IBM Corp.,Armonk,NY,USA) was used for statistical analyses.Data were expressed as the mean ± standard deviation (SD).The differences between the mean values of each group were determined by one-way analysis of variance (ANOVA)after testing for data normality.APvalue less than 0.05 was considered statistically significant.

Results

Successful development of rat models of MAP and SAP

Fig.1.Successful creation of rat models of MAP and SAP.A-D:Histopathological images of pancreas specimens from rats in the C-M group (A,HE staining,original magnification × 400),C-S group (B,HE staining,original magnification × 200),MAP group (C,HE staining,original magnification × 400),and SAP group (D,HE staining,original magnification × 400).E:Scoring of pancreatic histopathological changes in different groups;F:serum levels of amylase in the different groups.*P ＜ 0.05,vs.the SAP group;# P ＜ 0.05,vs.the MAP group.

Histopathological analysis showed that the rats in the two control groups exhibited a normal pancreatic architecture (Fig.1 A and B),the rats in the SAP group showed typical SAP manifestations,such as severe hemorrhage,interstitial edema,massive inflammatory cell infiltration,and extensive acinar cell necrosis (Fig.1 D).In contrast,the pancreatic histopathological changes in the MAP group were milder (Fig.1 C).The pathological score was significantly higher in the SAP group than that in the C-S group and MAP groups (bothP＜0.05);this score was higher in MAP group than that in the C-M group (P＜0.05;Fig.1 E).In addition,the serum level of amylase was significantly higher in the SAP group than that in the C-S group and MAP groups (bothP＜0.05);this level was higher in the MAP group than that in the C-M group (P＜0.05;Fig.1 F).Collectively,these data indicate that MAP and SAP models were successfully created.

Fig.2.Immunohistochemical staining of p-p65 in pancreas specimens from the different model and control groups (original magnification × 400).A:C-M group;B:C-S group;C:MAP group;D:SAP group.E:The semi-quantitative analysis of p-p65 immunohistochemical staining in the different groups.*P ＜ 0.05,vs.the SAP group;# P ＜ 0.05,vs.the MAP group.

AP severity is associated with IKK β/NF- κB activity and pro-inflammatory cytokine production

p-p65 expression was significantly higher in the SAP group than that in the C-S and MAP groups (bothP＜0.05);p-p65 was also higher in the MAP group than that in the C-M group(P＜0.05) (Fig.2).Similar results were obtained for p-IKKβexpression (Fig.3).In addition,the serum levels of TNF-αand IL-1βwere significantly higher in the SAP group (TNF-α:55.08 ±14.12 pg/mL;IL-1β:277.33 ± 39.52 pg/mL) than that in the C-S group (TNF-α:15.32 ± 4.85 pg/mL;IL-1β:103.90 ± 50.57 pg/mL;P＜0.05) and the MAP group (TNF-α:28.95 ± 10.20 pg/mL;IL-1β:94.73 ± 48.82 pg/mL;P＜0.05);these cytokines were also higher in the MAP group than that in the C-M group (TNF-α:9.68 ± 2.75 pg/mL;IL-1β:43.27 ± 24.27 pg/mL;P＜0.05;Fig.4 A).Altogether,these findings indicate that AP severity is associated with increases in IKKβ/NF-κB activity and pro-inflammatory cytokine production.

AP severity is related to ghrelin secretion

As shown in Fig.4 B,the serum level of ghrelin was significantly higher in the SAP group (187.13 ± 56.15 pg/mL) than that in the CS group (78.67 ± 14.62 pg/mL;P＜0.05) and the MAP group (28.15± 7.94 pg/mL;P＜0.05);no significant difference was observed between the MAP group and C-M group (22.85 ± 4.92 pg/mL;P＞0.05).These findings suggest that ghrelin secretion correlates with the severity of AP.

Fig.3.Immunohistochemical staining of p-IKK β in pancreas specimens from the different model and control groups.A:C-M group (original magnification × 400);B:C-S group (original magnification × 400);C:MAP group (original magnification × 200);D:SAP group (original magnification × 400).E:The semi-quantitative analysis of p-IKK β immunohistochemical staining in the different groups.*P ＜ 0.05,vs.the SAP group;# P ＜ 0.05,vs.the MAP group.

Ghrelin overexpression inhibits IKKβ /NF-κ B activation and pro-inflammatory cytokine production in cerulein-treated pancreatic acinar cells

Transfection of AR42J cells with lentiviral vector expressing ghrelin significantly up-regulated the expression of ghrelin mRNA and protein compared with the expression levels in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05;Fig.5 A,B and G).Overexpression of ghrelin in AR42J cells significantly reduced the expression of p-p65,IKKβ,and p-IKKβcompared with the corresponding levels in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05;Fig.5 C-G).Treatment of AR42J cells with cerulein significantly up-regulated the expression of p-p65,IKKβ,and p-IKKβ,whereas overexpression of ghrelin significantly reduced the cerulein-induced upregulation of these proteins.

As shown in Fig.6 A,overexpression of ghrelin in AR42J cells significantly reduced the levels of TNF-αand IL-1βcompared with those in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05).Overexpression of ghrelin significantly reduced the cerulein-induced upregulation of these proinflammatory cytokines in AR42J cells.

Fig.4.Pro-inflammatory cytokine production and ghrelin secretion in model and control groups.A:serum pro-inflammatory cytokine production in different groups;B:Serum ghrelin levels in different groups.*P ＜ 0.05,vs.the SAP group;# P ＜ 0.05,vs.the MAP group;.

Ghrelin knockdown activated IKKβ /NF-κ B and increased pro-inflammatory cytokine production in cerulein-treated pancreatic acinar cells

The transfection of AR42J cells with lentiviral vector expressing ghrelin shRNA significantly down-regulated the expression of ghrelin mRNA and protein compared with the levels in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05;Fig.7 A,B and G).Knockdown of ghrelin in AR42J cells also significantly up-regulated the expression of p-p65,IKKβ,and p-IKKβcompared with these levels in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05;Fig.7 C-G).While treatment of AR42J cells with cerulein significantly up-regulated the expression of p-p65,IKKβ,and p-IKKβ,knockdown of ghrelin further significantly enhanced the cerulein-induced upregulation of these proteins.

Regarding the impact of ghrelin knockdown on proinflammatory cytokine production in cerulein-treated pancreatic acinar cells,knockdown of ghrelin in AR42J cells significantly increased the levels of TNF-αand IL-1βcompared with those in untreated AR42J cells or AR42J cells transfected with the empty vector (allP＜0.05;Fig.6 B).While treatment of AR42J cells with cerulein significantly up-regulated the secretion of TNF-αand IL-1β,knockdown of ghrelin further significantly enhanced the cerulein-induced upregulation of these pro-inflammatory cytokines.

Fig.5.Ghrelin overexpression inhibits IKK β/NF-κB activation in cerulein-treated pancreatic acinar cells.A:Relative expression of the ghrelin gene in each group of AR42J cells.* P ＜ 0.05,vs.NC (normal control) cells.B:Relative expression of the ghrelin protein in each group of AR42J cells.* P ＜ 0.05,vs.NC cells.C:Western blotting analysis of p-p65,IKK β,p-IKK β,ghrelin,and GADPH expression in different groups of AR42J cells.D-G:Relative gene expression of p-p65 (D),IKK β (E),p-IKK β (F),and ghrelin (G) in relation to GAPDH gene expression in different groups of AR42J cells.* P ＜ 0.05,vs.NC;# P ＜ 0.05,vs.AR42J;§ P ＜ 0.05,vs.AR42J+ghrelin;ζ P ＜ 0.05,vs.NC+cerulein.

Discussion

The present study investigated the association between the severity of AP and serum levels of ghrelin and IKKβ/NF-κB activation in rat models of AP,as well as the impact of ghrelin overexpression or knockdown on IKKβ/NF-κB activation and proinflammatory cytokine production in pancreatic acinar AR42J cells treated with cerulein.In rat models of AP,endogenous ghrelin expression was found to be correlated with the severity of AP and IKKβ/NF-κB activity.In addition,ghrelin silencing increased the secretion of the proinflammatory factors TNF-αand IL-1βand the activity of IKKβ/NF-κB in pancreatic acinar AR42J cells treated with cerulein,with ghrelin overexpression having the opposite effects.

Fig.6.Effect of ghrelin overexpression or knockdown on pro-inflammatory cytokine production by cerulein-treated pancreatic acinar cells.A:Effect of ghrelin overexpression on pro-inflammatory cytokine production.* P ＜ 0.05,vs.NC;# P ＜ 0.05,vs.AR42J;§ P ＜ 0.05,vs.AR42J+ghrelin;ζ P ＜ 0.05,vs.NC+cerulein.B:Effect of ghrelin knockdown on pro-inflammatory cytokine production.* P ＜ 0.05,vs.NC;# P ＜ 0.05,vs.AR42J; § P ＜ 0.05,vs.AR42J+ghrelin shRNA;ζ P ＜ 0.05,vs.NC+cerulein.

Previous studies demonstrated that increased serum ghrelin levels are associated with a more severe course of AP in both patients [11,12] and animal models [9,10],suggesting that serum ghrelin levels can be used to predict the severity of AP.In conflict with these observations,Kanat et al.[14] and Türko ˘glu et al.[15] found no correlation between this hormone and the severity of AP.Our previous study [13] revealed that decreased serum ghrelin is a potential predictor of more SAP in the early course of the disease.The present study showed that serum levels of ghrelin were significantly higher in rats with SAP than in those with MAP,which is consistent with most previous studies [9-12] .We speculate that the discrepancy regarding this association has two explanations.First,the criteria used for evaluating AP severity,the statistical methods used,and the characteristics of study subjects (e.g.,type of pancreatitis) are different across studies.Additionally,serum ghrelin originates from various tissues (including pancreatic tissue),and thus,its levels may be affected by many factors.Our finding that the ghrelin level was significantly increased in AR42J cells treated with cerulein implies that ghrelin produced in the pancreas may directly correlate with AP severity,although this speculation remains to be confirmed in animal models and AP patients.

Unexpectedly,although the serum levels of ghrelin were significantly higher in both animal models of AP than in the respective control groups,the rats in the MAP group had lower serum levels of ghrelin than those in the C-S group.Because rats in the C-S group only underwent a sham-operation,we surmise that surgical stress may lead to an increase in the serum level of ghrelin,given that ghrelin can be produced in a variety of tissues [4] .In agreement with this speculation,stress-induced ghrelin secretion has been reported in previous studies [23,24] .These observations further suggest that confounding factors may exist when assessing the association between serum ghrelin levels and AP severity and that pancreatic ghrelin,rather than circulating ghrelin,may better reflect the severity of AP.

A key finding of this study is that ghrelin can act upon the noncanonical NF-κB pathway,which depends on the phosphorylation of IκB by IKKβ[25] in pancreatic acinar cells.A previous study discovered that exogenous ghrelin can suppress the development of AP possibly by reducing NF-κB overactivation and inflammatory cytokine production in a rat model of SAP [17] .However,whether ghrelin produced in pancreatic acinar cells acts upon NFκB activation remains unclear.Notably,ghrelin expression has been demonstrated in both exocrine and endocrine pancreas [6,26,27] .We speculate that,similar to EGF receptor [28],a ghrelin receptor is expressed in the AR42J cell membrane,and ghrelin exerts the above effects via binding to this ghrelin receptor.

Fig.7.Ghrelin knockdown promotes IKK β/NF-κB activation in cerulein-treated pancreatic acinar cells.A:Relative expression of the ghrelin gene in each group of AR42J cells.* P ＜ 0.05,vs.NC.B:Relative expression of the ghrelin protein in each group of AR42J cells.* P ＜ 0.05,vs.NC.C:Western blotting analysis of p-p65,IKK β,p-IKK β,ghrelin,and GADPH expression in different groups of AR42J cells.D-G:Relative gene expression of p-p65 (D),IKK β (E),p-IKK β (F),and ghrelin (G) in relation to GAPDH gene expression in different groups.* P ＜ 0.05,vs.NC;# P ＜ 0.05,vs.AR42J;§ P ＜ 0.05,vs.AR42J+ghrelin shRNA;ζ P ＜ 0.05,vs.NC+cerulein.

Our findings that the overexpression/silencing of ghrelin in pancreatic acinar cells suppressed/increased the secretion of proinflammatory factors TNF-αand IL-1βand the activation of IKKβ/NF-κB in cerulein-treated pancreatic acinar cells provide direct evidence that endogenous pancreatic ghrelin can inhibit both NF-κB activation and proinflammatory cytokine production in pancreatic acinar cells.However,the mechanisms by which endogenous pancreatic ghrelin inhibits NF-κB activation remain unclear.IKKβcan be rapidly activated by TNF-αand IL-1 and is necessary for NF-κB activation [25] .Due to the extensive anti-inflammatory effects of ghrelin [4],it may decrease NF-κB activation by suppressing TNF-αand IL-1 production and thereby decreasing IKKβactivation,as was observed in the present study.However,NFκB signaling may also act upstream of proinflammatory cytokine production [4] .Thus,other known mechanisms may mediate the ghrelin-induced inhibition of NF-κB activation.Consistent with this notion,in addition to IKKβ/NF-κB activation,unphosphorylated IKKβexpression was also down-regulated by ghrelin treatment in pancreatic acinar cells.Further research is needed to clarify these mechanisms.

Acinar cell destruction is an early event of AP [29] in which NF-κB activation may play a critical role possibly by participating in trypsin activation and the subsequent inflammatory reaction [4],thereby contributing to the development of AP [30,31] .In this regard,NF-κB appears to be an ideal therapeutic target for AP.However,because NF-κB has many important physiological functions,direct blockade of NF-κB may lead to physiological dysfunction.Our findings that ghrelin can inhibit NF-κB activation and proinflammatory cytokine production in pancreatic acinar cells suggest that local administration of ghrelin inhibiting pancreatic NF-κB activity may be a less detrimental strategy for AP prevention and treatment.Notably,no clinical research on ghrelin in the treatment of AP was found in the current literature.

Several limitations of this study should be noted.First,two animal models were used as MAP and SAP models,and to what extent they reflect differing severities of the human condition remains unclear.Second,cerulein-treated AR42J cells were used as a cellular model of AP.Although this cell line retains many characteristics of normal pancreatic acinar cells,is widely used as an in vitro model to study pancreatic diseases [32],and expresses ghrelin [33],this line was derived from a transplantable tumor of a rat exocrine pancreas.Therefore,extrapolation of animal findings to the human condition requires caution.Third,the concentrations of ghrelin in cell cultures before and after the experiments were not detected in this study,and experiments to investigate the existence of ghrelin receptors on AR42J cells were not conducted.Finally,we only examined the association between serum ghrelin and AP severity,although pancreatic ghrelin may better reflect the severity of AP.Future studies are needed to resolve these problems.

In conclusion,our study showed that serum levels of ghrelin correlated with AP severity in the employed animal models.Ghrelin in pancreatic acinar cells was found to inhibit IKKβ/NF-κB activation and reduce pro-inflammatory cytokine production.Thus,the administration of ghrelin may be a potential strategy for the prevention and treatment of AP.However,more studies,especially in humans,are needed to confirm our findings.

Acknowledgments

None.

CRediTauthorshipcontributionstatement

Ren-JieChang:Conceptualization,Formal analysis,Methodology,Software,Validation,Visualization,Writing -original draft,Writing -review &editing.Hui-LinWang:Conceptualization,Formal analysis,Methodology,Software,Validation,Visualization,Writing -original draft,Writing -review &editing.Meng-Bin Qin:Conceptualization,Investigation,Software,Validation,Visualization,Writing -review &editing.Zhi-HaiLiang:Conceptualization,Investigation,Software,Validation,Visualization,Writing -review &editing.Jia-PingHe:Conceptualization,Data curation,Formal analysis,Resources,Software,Validation,Visualization,Writing -review &editing.Yu-LeWei:Conceptualization,Data curation,Formal analysis,Resources,Software,Validation,Visualization,Writing -review &editing.Hong-ZongFu:Conceptualization,Data curation,Formal analysis,Resources,Software,Validation,Visualization,Writing -review &editing.Guo-DuTang:Conceptualization,Funding acquisition,Investigation,Project administration,Software,Supervision,Validation,Visualization,Writing -review &editing.

Funding

This study was supported by grants from the National Natural Science Foundation of China (81260087 and 81560111),and Guangxi Natural Science Foundation (2017GXNSFAA198068).

Ethicsapproval

This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (201,701,007).All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Competinginterest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.