Sphingosine 1-phosphate Receptor 2 Mediated Early Stages of Pancreatic and Systemic Inflammatory Response via NF-kappa B Activation in Acute Pancreatitis

doi:10.21203/rs.3.rs-1721396/v2

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Sphingosine 1-phosphate Receptor 2 Mediated Early Stages of Pancreatic and Systemic Inflammatory Response via NF-kappa B Activation in Acute Pancreatitis

https://doi.org/10.21203/rs.3.rs-1721396/v2

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In acute pancreatitis (AP), activation of inflammatory signaling including nuclear factor-kappa B (NF-κB) pathway within acinar cells is known to be an early intracellular event occurring in parallel with pathologic trypsinogen activation. Sphingosine 1-phosphate Receptor 2 (S1PR2) plays a critical role in endothelial inflammation and our previous studies reported that S1PR2 deficiency significantly reduced inflammatory response in liver injury under cholestasis conditions. However, the role of S1PR2 in inflammatory signaling activation within acinar cells and inflammatory responses during acute pancreatitis has not been elucidated. Here we report that S1PR2 was upregulated in whole pancreas during acute pancreatitis conditions. Blockade of S1PR2 by pharmacologic inhibition of S1PR2 by JTE-013 or AAV-mediated knockdown of S1PR2 improved the severity of pancreatic injury as indicated by significant reduction of inflammation and acinar cells death in acute pancreatitis mice. Moreover, S1PR2 is the predominant S1PRs expressed in pancreatic acinar cells and mediates NF-κB activation and early inflammatory response within acinar cell under acute pancreatitis conditions via ROCK signaling pathways, not extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways. In addition, S1PR2 mediated macrophages NF-κB activation, migration and polarization toward M1 phenotype. Therefore, this results demonstrated that S1PR2-mediated early inflammatory response in acinar cell promote the progression of acute pancreatitis, successfully linking the local events to the systematic inflammatory response and leading a novel therapeutic target for acute pancreatitis aimed at halting the progression of inflammatory response.

S1PR2

Acute Pancreatitis

Inflammatory Responses

Acinar Cells

NF- kappa B

Acute Pancreatitis (AP) in both mild and severe forms, with high morbidity and mortality, are critical clinical problems worldwide [1, 2]. Despite recent significant advances, this disease remains the limited understanding of their pathogenesis, heterogeneity of disease presentations, and the lack of effective therapeutic options, which leading fluid resuscitation and supportive care as the only solution for AP patients[3]. More and more studies focus on the sources and pathogenesis of sterile inflammatory response in acute pancreatitis. Acinar cells are the major targets in acute pancreatitis [4]. As a critical role in the pathogenesis of pancreatitis, early activation of inflammatory pathway in the acinar cells lead to local inflammatory reaction and pancreatic injury. Once local inflammatory reaction is un-control, various pro-inflammatory mediators are released into the circulation system, triggering the widespread inflammatory storm, and progressively the systemic inflammatory response syndrome (SIRS) and multi-organ dysfunction (MODS)[5–7].Therefore, therapeutic targeting of early inflammatory events within acinar cells provide the therapeutic choice to prevent disease progression.

Nuclear factor-kappa B (NF-κB) pathway is the most widely studied of inflammatory signaling within the acinar cells, and correlated with greater severity of acute pancreatitis [8]. NF-κB activation and trypsinogen activation are independent early events and play different roles in the development of acute pancreatitis [8–12]. NF-κB is present in the cytoplasm in an inactive from coupled to an inhibitor IκB. Upon activation by infection or cellular stresses, IκB was be phosphorylated, degraded and released NF-κB. Then NF-κB translocate to the nucleus, binds to specific elements located in the promoters of various genes, such as inflammatory factors, adhesion molecules [13, 14].

Recent prominent research efforts have advanced that sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) signaling is a crucial inflammatory mediator and involved in pathogenesis of various inflammatory-related diseases[15]. In endothelial cells, upregulation of S1PR2 activates the ROCK/NF-κB pathway and the p38 MAPK which are critical for the induction of vascular inflammation [16]. In human cholangiocarcinoma cells, activation of S1PR2 further promoted activation of NF-κB and subsequent COX-2 expression and prostaglandin E2 synthesis via ERK1/2 and Akt signaling pathway [17]. Our previous study further indicated that, in cholestasis-induced liver injury, S1PR2 promotes an inflammatory response and participates disease progression [18]. Accumulating evidence showed that S1PR2/3 activation recruits bone marrow-derived monocyte/macrophage to the damaged liver, promotes inflammatory M1 polarization and primes NLRP3 inflammasome and pro-inflammatory cytokines (IL-1β and IL-18) secretion, contributes to various liver injury progression [15, 19, 20]. Except bone marrow-derived monocyte/macrophage, different from neutrophils, the migration and cytoskeletal remodeling were mediated by S1PR2. S1PR2 knockdown significantly lessened neutrophil infiltration and inhibited neutrophil extracellular rap (NET) formation, which ameliorated liver inflammation and fibrosis [21, 22]. Based on the previous studies, S1PR2 is an interesting target for pancreatic local and systemic inflammatory response during acute pancreatitis and is worth to further study.

Here we focus on explored the influence of S1PR2 on early inflammatory response within acinar cell under acute pancreatitis conditions. We found that S1PR2 is overexpression in pancreas, acinar cell and macrophages, and inhibition of S1PR2 activation or knockdown of S1PR2 expression significantly inhibited acinar cells NF-κB activation, inflammatory cytokines release, macrophages recruitment and polarization toward M1 phenotype. Finally, blockade of S1PR2 by JTE-013 also showed protective effect against acute pancreatitis in vivo. In conclusion, these findings opens new perspectives for S1PR2 as a target for therapeutic of acute pancreatitis.

In the current study, we report that activation of S1PR2 plays an essential role in NF-κB activation in acinar cells and macrophages under acute pancreatitis conditions. Inhibition of S1PR2 activation using a specific chemical antagonist or knock down of S1PR2 expression using a gene-specific shRNA significantly inhibited acinar cells NF-κB activation, macrophages migration and polarization toward M1 phenotype. The chemical antagonist of S1PR2, JTE-013, and AAV-mediated knockdown of S1PR2 also showed protective effect against acute pancreatitis in vivo. Our study suggests that targeting of S1PR2-mediated signaling pathways may have therapeutic potential for a subset of acute pancreatitis that currently lack effective therapies.

Materials

Caerulein was purchased from Nanjing peptide (China). LPS was purchased from Aladdin (China). Taurocholate acids (TCA) and sodium taurocholate were from Sigma-Aldrich (USA). JTE-013 was purchased from Cayman Chemicals (USA). The adeno-associated virus carrying the S1PR2-targeting shRNA or scramble control shRNA were obtained from KeyGEN Biotech Company (Obio Technology, China). The β-actin, S1PR2, p-ERK and T-ERK antibodies were purchased from Santa Cruz Biotechnology (USA). NF-κB(p65), RIP3 and Cleaved-Caspase 3 were purchased from Cell Signaling Technology (USA).

Animals and Treatment

All animal experiments were approved by the animal research committee of Jiangnan University and were performed in accordance with the principles of care and use of laboratory animals. ICR mice (male, 8 weeks) were purchased from Shanghai Slac Laboratory Animal CO.LTD (China). For caerulein pancreatitis model, caerulein (200µg/kg) was intraperitoneally administered hourly for ten times and LPS was intraperitoneal injected after the last caerulein injection, while the controls received the identical PBS injections. For taurocholate pancreatitis model, taurocholate acids was infused retrogradely into the pancreatic duct of mice and the controls received the identical PBS injections. For JTE-013 intervention groups, mice were administered intraperitoneally JTE-013 (10mg/kg) one hour before first caerulein injection. After 12 hour, serum and pancreas tissue was harvested for further analysis (n = 5 per group). For S1PR2 knockdown mouse model, mice were constructed by intraperitoneal injection of adeno-associated virus carrying the S1PR2-targeting shRNA or scramble control shRNA. After three weeks, caerulein pancreatitis were induced in accordance with our previous study.

Isolation of Primary Pancreatic Acinar Cells and Primary Peritoneal Macrophage, Cell Culture and Treatment

Pancreatic acinar cells and primary peritoneal macrophage were acquired from mice aged 8 weeks as previously described, respectively [23] [24]. Mouse pancreatic acinar carcinoma 266-6 cells were obtained from Cobioer Biosciences Co, Ltd. and mouse macrophages RAW264.7 were obtained from American Type Culture Collection. All cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin.

Western Blot Analysis

Pancreas and cell sample were homogenized by RIPA lysis buffer and total proteins were isolated and were analyzed by western blot using specific primary and secondary antibodies as previously described [25]. The ECL chemiluminescent kit was used for visualized immunoreactive bands, and Image J software was used for analyzed the density of immunoreactive images.

Immunofluorescence Staining

For tissues, fresh pancreas were fixed, sliced, dewaxed, rehydration, antigen retrieval, blocking with 5% BSA and stained with S1PR2 staining.

For cell sample, cells were plated on coverslips, cultured overnight and treat with TCA (100µM), in the presence or absence of JTE-013 (10µM) for 4 hours. Cells were fixed with 4% paraformaldehyde, penetrated with 0.2% Triton X-100, blocked with 5% BSA for 1 hour, then incubated with NF-κB (p65) antibody (1:200 dilution) overnight at 4°C and Alexa Fluor 488 secondary antibody for 1 hour, and finally filmed with anti-fluorescence quencher including DAPI. Images were recorded using a Carl Zeiss LSM880 microscope.

Real-Time PCR

Extraction of total RNA from cells and real-time RT-PCR were performed as described previously [25]. The mRNA levels of S1PRs, IL-6, TNF-α, CD86, Arginase, CD206, and CD163 were determined. All data were normalized to β-actin as an internal control.

Macrophage Migration Assay

RAW264.7 cells were seeded in the upper chamber and treat with TCA (100µM), in the presence or absence of JTE-013 (10µM) for 24 hours, while the chemoattractant (CCL2, 100 ng/mL) were added to the lower compartments. The migrated cells in lower surface of the chamber were stained by crystal violet solution and detected by a light microscopy as described previously [26].

RNA Interference

Cells were cultured overnight to 50% confluence and transiently transfected with control shRNA or S1PR2 shRNA. After 48 hours, cells were used to perform for further experiments.

Measurement of Serum Lipase and α-Amylase Activity

The blood from mice was collected and centrifuged (4000 rpm, 10 minutes, 4℃) to get the supernatant for further detection. Serum activity of α-amylase and lipase were detected using assay kits (Jiancheng Biotech, China). All kits were used according to the manufacturer’s instructions.

HE Staining

Fresh pancreas were fixed by 4% paraformaldehyde and embedded with paraffin for H&E staining and examined by light microscopy.

Immunohistochemistry staining (IHC)

Fresh pancreas were fixed, sliced, dewaxed, rehydration, antigen retrieval, blocking with 5% BSA and stained with Ly6G and CD68 staining. Finally, images were captured with a light microscope.

Enzyme-linked Immunosorbent Assays (ELISA)

Serum TNF-α and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (Proteintech, China) according to the manufacturer’s protocols.

Statistical analysis

The results were expressed as mean ± SEM. Statistical significance among multiple groups was determined using one-way variance followed by Bonferroni's Multiple Comparison Test. Statistical analyses were performed using GraphPad Prism 8 software and P < 0.05 was considered statistically significant.

Over-expression of S1PR2 in the Pancreas of Acute Pancreatitis Mice

Caerulein-induced acute pancreatitis (Caerulein pancreatitis) and retrograde pancreatic duct injection of taurocholate induced severe necrotizing pancreatitis (Taurocholate pancreatitis) are two widely used experimental acute pancreatitis models in vivo[27]. We examined the expression of S1PR2 in two experimental acute pancreatitis models. In caerulein pancreatitis model, after 6, 12 and 24 hours of repetitive caerulein treatment, serum lipase and amylase levels is upregulated in gradual manner, which revealed that

exposure to caerulein resulted in pancreatic damage (Fig. 1A and B). Caerulein-induced injury stimulated S1PR2 expressions at protein levels significantly after 12 h (Fig. 1C). Similar to the findings in taurocholate pancreatitis, the western blot analysis showed that S1PR2 markedly increased at 12h in pancreas of model mice compared to the control (Fig. 1D). In addition, the immunofluorescence results indicated that positive S1PR2 stain was been obviously up-regulated in acute pancreatitis mice by either caerulein or taurocholate, and most of the anti-S1PR2 staining is localized at the acinar cells (Fig. 1E). The above results indicated that S1PR2 is pancreatic overexpression during acute pancreatitis conditions.

Pharmacologic inhibition of S1PR2 by JTE-013 Alleviates the Severity of Pancreatic injury in Acute Pancreatitis Mice

The significant increase of S1PR2 expression in acute pancreatitis model prompted us to elucidate the function of S1PR2 in acute pancreatitis. At first, we examined whether blockade of S1PR2 expression could affect the initiation and progression of acute pancreatitis. The specific antagonist of S1PR2, JTE-013, was injected to mice one hour before acute pancreatitis induction (Fig. 2A). And 12 hours after the Caerulein challenge, the pancreas injury index including serum lipase, α-amylase (Fig. 2B and C) and inflammatory cytokines concentrations including IL-6, TNF-α (Fig. 2D and E) were significantly upregulated in mice induced by caerulein but decreased significantly after the JTE-013 intervention. Consistent with the changes in pancreatic function, HE staining of pancreas sections revealed that exposure to caerulein resulted in extensive pancreatic edema, inflammatory infiltration and local necrosis, and JTE-013 treatment significantly attenuated histological features of pancreatic injury (Fig. 2F).

In addition, the TUNEL-positive acinar cells significantly increased in response to caerulein supramaximal stimulation as expected, while markedly reduced in JTE-013 intervention group (Fig. 2G). Furthermore, we verified the influence of JTE-013 on cell apoptosis and necroptosis markers, cleaved-caspase 3 and receptor-interacting protein kinase 3(RIP3) in caerulein pancreatitis. After caerulein stimulation, cleaved-caspase3 and RIP3 protein expressions in pancreas tissue were dramatically increased (Fig. 2H). Meanwhile, JTE-013 administration successfully abrogated the expression of these death markers compared with that in caerulein pancreatitis mice (Fig. 2H).

Immunohistochemistry results revealed that the expressions of Ly6G + neutrophils and CD68 + macrophages were been abundantly recruited in caerulein pancreatitis group as compared to normal control group. After JTE-013 administration, the results exhibited a significant reduction the number of Ly6G + neutrophils and CD68 + macrophages in pancreas (Fig. 2I and J). Western blotting also hinted that JTE-013 administration recovered the increase in NF-κB p65 protein expression in acute pancreatitis (Fig. 2H). These above results indicated blockade of S1PR2 by JTE-103 efficiently inhibited inflammatory cells recruitment, infiltration and abated pancreatic inflammatory response, which confirming that S1PR2 inhibition was protective for acute pancreatitis.

S1PR2 Knockdown Obviously Alleviates Pancreatic Damage in Acute Pancreatitis Mice

To determine whether S1PR2 mediated sustaining pathological inflammation in vivo, we subjected mice to intraperitoneal injection of S1PR2-shRNA (Fig. 3A) S1PR2-shRNA markedly and specially downregulated S1PR2 expression in the pancreas of acute pancreatitis mice (Fig. 3B). S1PR2 blockage significantly decreased the levels of lipase and α-amylase (Fig. 3C and D) in the serum and the concentrations of inflammatory cytokines including IL-6, TNF-α (Fig. 3E and F) in the injured mice. Simultaneously, histologic analysis exhibited that the interlobular space, acinar necrosis and inflammatory cell infiltration were predominantly attenuated under S1PR2-shRNA administration (Fig. 3G and H). Moreover, acinar cell death hallmarks (RIP3, PARP, and Caspase3) and NF-κB activation were obviously reduced in the injured pancreas (Fig. 3I). Taken together, these results prove that S1PR2 knockdown may be an excellent therapeutic for acute pancreatitis via alleviated pancreatic injury.

S1PR2 is the Predominant S1PRs Expressed in Pancreatic Acinar Cells

Primary pancreatic acinar cells and acinar carcinoma cell lines 266-6 (mouse) are often used in vitro experimental AP models. PCR analysis demonstrated S1PR2 was the predominant S1PRs expressed in pancreatic acinar cells and 266-6 cells (Fig. 4A and B). Figure 4C and D illustrated that S1RP2 in primary pancreatic acinar cells up-regulated after stimulated with caerulein, cholecystokinin, sodium taurocholate or taurocholate acids (TCA). In 266-6 cells, TCA can induced S1PR2 highly expression in time- and dose- dependent manner (Fig. 4E and F).

NF-κB Activation and Inflammatory Factors Expression in Pancreatic Acinar Cells induced by TCA

Cytokines and inflammatory signaling play a critical role in the progession of acute pancreatitis, was not only released by infiltrating inflammatory cells, but also released by the acinar cells [5]. Due to a short in vitro viability of primary pancreatic acinar cells, we used 266-6 cells to assess the activation of NF-κB. These results showed that TCA can up-regulated protein levels of NF-κB p65 in both time- dependent and dose- dependent manner (Fig. 5A). Moreover, TCA significantly increased NF-κB p65 nuclear translocation (Fig. 5B). Consistent with NF-κB activation, there was a marked elevation in the expression of the NF-κB target gene IL-6 and TNF-α by TCA stimulation (Fig. 5C). In addition, we used CCK-8 method to evaluate the cytotoxic effect of TCA at 266-6 cells, and the dose we used in cells (100 nM) did not cause the cytotoxic effect (Fig. 5D), which hinted that TCA induced activation of NF-κB independent its cytotoxic effect.

S1PR2 Mediates NF-κB Activation induced by TCA in Pancreatic Acinar Cells

To examine whether S1PR2 be responsible for inflammatory response within acinar cells during the early phase of pancreatitis, we performed JTE-013 (antagonist of S1PR2) or S1PR2-shRNA to block these effects. As shown in Fig. 5E and F, either inhibition of S1PR2 activation by JTE-013 or knockdown of S1PR2 expression by S1PR2-shRNA reversed TCA-induced NF-κB activation, and further suggest that TCA activates NF- κB via activation of S1PR2 in pancreatic acinar cells.

S1PR2 is a G protein-coupled receptor and has various biological function by activated downstream multiple signaling pathways, such as the Rho kinase (ROCK), ERK, and p38 signaling pathways [28].Next, we used pharmacological inhibitors of signal transduction, including Y27632 (ROCK inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor) to dissect the responsible signal pathways for NF-κB activation of acinar cells regulated by TCA. The TCA-induced increase of NF-κB protein level were only lessened by Y27632 in mouse 266-6 cells whereas U0126 and SB203580 had no effect (Fig. 5G), indicating that ROCK signal, not ERK and p38 signal, might be necessary for TCA-induced NF-κB activation in pancreatic acinar cells.

S1PR2 is the Predominant S1PRs Expressed in Macrophages and mediated NF-κB Activation in Macrophages

Macrophages were the prominent infiltrating cells in early pancreatitis and is critical in widespread systemic inflammatory response [29]. Studies showed that S1PR2 is highly expression in macrophages and macrophage S1PR2 retarded liver inflammation and fibrogenesis [19]. We analyzed the expression of S1PR2 in isolated primary peritoneal macrophage and RAW264.7 cells in acute pancreatitis models. PCR results demonstrated S1PR2 was the predominant S1PRs expressed in primary peritoneal macrophage and RAW264.7 cells (Fig. 6A and B). Figure 6C and D revealed that TCA can induced S1PR2 highly expression in RAW264.7 via the dose- and time- dependent manner. Figure 6C and D revealed that TCA up-regulated protein levels of NF-κB p65 in both dose- dependent and time- dependent manner. TCA also significantly increased nuclear translocation of NF-κB p65 (Fig. 6E). To identify whether S1PR2 induced NF-κB activation in macrophages, JTE-013 or S1PR2 shRNA will be used in subsequent experiments. Figure 6F and G indicated that TCA significantly increased the protein levels of NF-κB p65, which were reversed by JTE-013 or S1PR2 shRNA. Next, the increase of NF-κB protein level were lessened by Y27632, U0126 and SB203580 in mouse 266-6 cells (Fig. 6H), indicating that ROCK, ERK and p38 signal might be necessary for NF-κB activation in macrophages.

Activation of S1PR2 Promotes Macrophages Migration and Macrophages Polarization toward M1 Phenotype

To verify the effects of S1PR2 activation on macrophage migration, RAW264.7 cells were seeded in the upper chamber of transwell and treated with TCA (100 uM), or vehicle control, in the presence or absence of JTE-013. The migration cells of TCA group were markedly increased compared to control group, which were obviously blocked by JTE-013 (Fig. 6I).

Also, we showed that TCA treatment upregulated the expression of M1 macrophage-marker genes, including IL-6, TNF-α, and CD86, while downregulated the expression of M2 macrophage-marker genes, including Arginase, CD206, and CD163 (Fig. 6J). Moreover, JTE-013 treatment significantly downregulated the expression of M1 marker genes, and particularly promoted the expression of M2 marker genes, which further suggested that activation of S1PR2 promotes macrophages polarization toward M1 phenotype.

We previously reveal that S1PR2 promotes an inflammatory response and participates the progression of cholestasis-induced liver injury. In the present study, we identified a critical role of S1PR2 in acute pancreatitis. Pharmacologic inhibition of S1PR2 signaling by JTE-013 or knock down of S1PR2 expression using a gene-specific shRNA alleviated pancreatic injury and resulted in lower expression of inflammatory response in mouse models of caerulein pancreatitis. Importantly, the protective mechanisms depend on the inhibition of S1PR2 on acinar cells and macrophages, which altered early activation of inflammatory pathways including NF-κB activation in the acinar cells, decreased peritoneal macrophages recruitment and regulated M1 phenotype polarization of macrophages.

Despite significant advances in the last 25 years, the mechanisms responsible for the initiation of acute pancreatitis remains to be elusive. It is widely held that acute pancreatitis was attributed to pre-mature intracellular trypsinogen activation initiate pancreatic acinar cell injury, release various DAMP or cytokines, which lead to inflammatory cells recruited into the damaged pancreas [30, 31]. But in-vitro expression of active trypsin cannot activate NF-κB, which suggest that trypsinogen activation and NF-κB activation are independent events during the early phase of acute pancreatitis. Actually, under conditions of stress, including alcohol abuse, cigarette smoking, or hyperlipidemia, the acinar cells show such unique inflammatory property[32]. Thus far, studies using transgenic animals in acute pancreatitis, such as T7-/- and Ctsb-/- mice, have successfully identified that NF-κB pathway within acinar cells is a key early event and may have a hand in progression of pancreatic and systemic inflammatory response [30]. In current study, TCA induce NF-κB up-expression and translocate to the nucleus in pancreatic acinar cells in both dose- dependent and time- dependent manner, and these effects were blocked by JTE-013 (antagonist of S1PR2) or S1PR2-shRNA, which further S1PR2 activation be responsible for inflammation within acinar cells during the early phase of pancreatitis. Consistent with our reports, upregulation of S1PR2 activates the NF-κB signaling pathway and are critical for the vascular inflammation induction.

It is noteworthy that monocytes/macrophages, but not NK, NKT, and TCRγδ + T cells, participate in the early stage of the pathogenesis of caerulein pancreatitis and emerged at the highest ratio among the immune cells in the damaged pancreas of cerulein-injected mice [33, 34]. In this study, one striking finding was that TCA promoted macrophages migration and regulated macrophages polarization toward M1 phenotype via S1PR2 in vitro. As expected, administration of S1PR2 antagonist JTE-013 results in lower the infiltration number of macrophages and the levels of inflammatory factor during acute pancreatitis in vivo. Emerging evidence indicates that S1PR2 participate in neutrophil recruitment and NETosis, another form of activated neutrophil death, during BDL-induced liver injury [22]. We also demonstrated that blockade of S1PR2 alleviated Ly6G + neutrophils infiltration in acute pancreatitis mice.

S1PR2 is a G protein-coupled receptor and couples with several different G-alpha subunits, such as Gα_(i/o), Gα_q, and Gα_(12/13)[28, 35]. These subunits activation exert different influence in various different pathways, due to the different stimulus and different cell type[36]. Here, we further evaluate which downstream second messenger molecules of S1PR2, such as the Rho kinase (ROCK), ERK and p38 MAPK, is responsible for NF-κB signaling pathway activation in pancreatic acinar cell. These results showed that TCA-induced NF-κB activation and inflammatory factors secretion were impaired by ROCK inhibitors in pancreatic acinar cells, while ERK and p38 inhibitors had no such effect. Nevertheless, in macrophages, ROCK, ERK, and p38 inhibitors impaired TCA-induced NF-κB activation. Consistent with our results, S1PR2 couples dominantly to the Rho/ROCK pathway and leads to NF-κB activation in macrophages. Subsequently, blockade of S1PR2 activates the Rho/Rho kinase/NF-κB signal pathway and reduced cytokine secretion and oxidized LDL uptake [16, 37]. Finally, homology modeling of the S1PR2 docking to TCA predicts that TCA, a low affinity agonist, hydrogen bonds only to Leu 173 of the S1PR2 [38]. All in all, this study highlighted S1PR2 is the critical role in acute pancreatitis. In pancreatic acinar cells, activation S1PR2 by TCA activates NF-κB tran-activation through ROCK signal pathway, leads to the secretion of inflammatory factors, further recruits immune cell, especially macrophage, and promotes M1 phenotype polarization during acute pancreatitis (Fig. 7). Furthermore, our results supported that S1PR2 blockade can alleviate early inflammatory response and provided new compelling information on therapeutic of acute pancreatitis

Author contributions

JY and JS conceived the study and designed the project. JY, XT and BL carried out the experiments and interpreted the data. JY wrote the paper. All authors contributed to draft revisions and approved the final version of the manuscript.

Declaration of competing interest

The authors declare no conflicts of interest related to this work.

Funding

This work was supported by Grants from the National Natural Science Foundation of China (Grants nos.: 81900560), the Natural Science Foundation of Jiangsu Province (Grants nos.: BK20180294), and the fellowship of China Postdoctoral Science Foundation (Grants nos.:2020M671348).

Data Availability Statement

The data that support the findings of this study are available in the methods of this article.

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No competing interests reported.

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Sphingosine 1-phosphate Receptor 2 Mediated Early Stages of Pancreatic and Systemic Inflammatory Response via NF-kappa B Activation in Acute Pancreatitis

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Version 2

Abstract

Figures

Introduction

Materials And Methods

Materials

Animals and Treatment

Isolation of Primary Pancreatic Acinar Cells and Primary Peritoneal Macrophage, Cell Culture and Treatment

Western Blot Analysis

Immunofluorescence Staining

Real-Time PCR

Macrophage Migration Assay

RNA Interference

Measurement of Serum Lipase and α-Amylase Activity

HE Staining

Immunohistochemistry staining (IHC)

Enzyme-linked Immunosorbent Assays (ELISA)

Statistical analysis

Results

Over-expression of S1PR2 in the Pancreas of Acute Pancreatitis Mice

Pharmacologic inhibition of S1PR2 by JTE-013 Alleviates the Severity of Pancreatic injury in Acute Pancreatitis Mice

S1PR2 Knockdown Obviously Alleviates Pancreatic Damage in Acute Pancreatitis Mice

S1PR2 is the Predominant S1PRs Expressed in Pancreatic Acinar Cells

NF-κB Activation and Inflammatory Factors Expression in Pancreatic Acinar Cells induced by TCA

S1PR2 Mediates NF-κB Activation induced by TCA in Pancreatic Acinar Cells

S1PR2 is the Predominant S1PRs Expressed in Macrophages and mediated NF-κB Activation in Macrophages

Activation of S1PR2 Promotes Macrophages Migration and Macrophages Polarization toward M1 Phenotype

Discussion

Declarations

References

Additional Declarations

Status:

Version 2