Introduction

Acute pancreatitis (AP) is characterized by inflammation of the pancreas, which can be either localized or systemic. Its development involves a complex interplay of biological mechanisms, including self-digestion, activation of inflammatory agents, and cell death1,2,3. Pathologically, AP is classified into acute edematous pancreatitis and acute hemorrhagic necrotizing pancreatitis4. Notably, acute edematous pancreatitis, which is quite common, can progress to acute hemorrhagic necrotizing pancreatitis. The lesions in acute edematous pancreatitis can affect part or all of the pancreas, with the tail being the most frequently involved region. Pathological examination typically reveals pancreatic enlargement, congestion, edema, infiltration of inflammatory cells, and minor local necrosis5.

Central to the pathological progression of AP are the apoptosis and swelling of pancreatic cells, both of which significantly influence the extent of pancreatic tissue damage. These factors can also trigger systemic inflammation, thereby exacerbating the severity of the condition and increasing mortality risk. The concept of pan-apoptosis, encompassing various cell death pathways such as apoptosis, necrosis, and autophagy, contributes to pancreatic cell damage during AP. This damage is precipitated by the activation of pan-apoptosis and the presence of inflammatory cytokines during oxidative stress. Cellular swelling, often linked to dysfunctional ion channels and the secretion of inflammatory agents, not only impairs cellular functionality but also raises the risk of cell death and tissue damage6,7.

PLD2, a crucial enzyme in the phosphatidylinositol signaling pathway, significantly influences various cellular processes, including cell growth, movement, specialization, and programmed cell death. Although prior research has elucidated the biological functions of PLD2 in various cell types, its precise role in AP and the underlying mechanisms remain incompletely understood. NRF2 and NF-κB are pivotal transcription factors that regulate antioxidant and inflammatory responses, respectively8. Activation of Nrf2 enhances cellular antioxidant capacity, whereas activation of NF-κB stimulates the production of inflammatory agents. Thus, maintaining a balance between these two pathways is crucial for managing the progression of inflammatory conditions. Our previous study revealed that PLD2 plays a key role in regulating pan-apoptotic processes (apoptosis, pyroptosis, and necrosis) by activating the Nrf2 antioxidant pathway and inhibiting the NF-κB inflammatory pathway9. However, the interactions between PLD2 and the Nrf2/NF-κB pathways, and their roles in AP, have not been fully clarified in the existing literature.

This study aims to investigate the molecular mechanisms through which PLD2 regulates both pan-apoptosis and edema in pancreatic cells via the Nrf2 and NF-κB pathways. By simulating the conditions of acute pancreatitis, we examined the impact of PLD2 overexpression on the expression of pan-apoptosis-associated proteins in pancreatic cells, as well as changes in cellular edema. Through this research, our objective is to elucidate the function of PLD2 in AP and establish a theoretical foundation for developing novel therapeutic approaches.

Materials and methods

Cell culture and model establishment

AR42J cells, derived from the rat pancreas and obtained from ATCC, were maintained in a 37 °C incubator with 5% CO2. The cells were cultured in F-12 K medium supplemented with 10% FBS. To establish an in vitro pancreatitis model, 10 nM mitomycin was introduced to the model group. A control group consisted of AR42J cells cultured with an equivalent amount of phosphate-buffered saline.

Cell transfection

A PLD2 overexpression plasmid (PLD2), based on PcDNA and sourced from GeneChem, was utilized. AR42J cells were transfected with a 20 nM oligonucleotide or 10 ng plasmid using Lipofectamine 3000 (Thermo Fisher Scientific Shier Technology) following the manufacturer’s instructions. Transfection efficiency was assessed 48 h post-transfection using RT-qPCR.

Experimental grouping

According to the research plan, the cultured cells were divided into three groups: the control group (AR42J cells cultured under regular conditions), the model group (AR42J cells treated with 10 nM mitomycin to induce an in vitro pancreatitis model), and the PLD2 overexpression group (AR42J cells transfected with the PLD2 overexpression plasmid post-model cell treatment).

Western blot analysis

Cells were lysed using RIPA buffer, and protein concentration was determined using the BCA method. To detect the levels of PLD2, Nrf2, NFκB proteins, and pan-apoptotic related proteins, nuclear and cytoplasmic proteins were isolated using extraction kits (Beyotime). Samples were processed under the same conditions to reduce experimental errors, using consistent biological materials and processing procedures. Proteins were separated by polyacrylamide gel electrophoresis based on their molecular weight and charge. Subsequently, the proteins were transferred onto a solid carrier such as a nitrocellulose or polytetrafluoroethylene membrane.

A 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 50 µg of protein, which was then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 0.1-20% and 5% bovine serum albumin (Sigma Aldrich) for 1 h, followed by an overnight incubation at 4 °C. The membrane was rinsed with Tris-buffered saline containing 0.05% Tween and incubated at room temperature for 2 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Ab6721). An enhanced chemiluminescence detection system (Pierce Biotech) was used to detect immune response bands, and a Gel Pro analyzer was employed for analysis.

RT-qPCR analysis

The mRNA expression levels of cytokines TNF-α, IL-6, and IL-10 were analyzed using RT-qPCR to assess cellular inflammatory levels. Total RNA was extracted using the RNeasy Mini Kit as per the manufacturer’s guidelines. The PrimeScript first-strand cDNA synthesis kit (Takara) was used for reverse transcription of total RNA, and the miRcute miRNA first-strand cDNA synthesis kit was utilized to synthesize miRNA into cDNA. RT-qPCR analysis was conducted on a Roder-Gene Q device using either the SYBR Green PCR Master Mix or the miScript SYBR Green PCR Kit. Marker and miR-5132-5p levels were normalized using GAPDH or U6, and mRNA levels were determined using the 2-ΔΔCt method.

Analysis of apoptosis and necrosis

Apoptosis was detected by flow cytometry using the Annexin V-FITC/PI double staining technique. Cells grown to 80% confluence were collected post-treatment and gently suspended in 1X binding buffer to adjust the cell concentration to 1 × 106 cells/mL. For each sample, 100 µL of the cell mixture was extracted, and 5 µL of Annexin V-FITC and 10 µL of acrylamide (PI) were added for labeling. After a 15-minute incubation at room temperature in the dark, an additional 400 µL of 1X binding buffer was added, and samples were analyzed for apoptosis using flow cytometry. Cell necrosis was assessed through the lactate dehydrogenase (LDH) release test. Cells were evenly distributed across a 96-well plate to ensure consistent cell numbers for standardized data. Following treatment, the culture supernatant containing free-floating cells was removed. LDH activity was measured using an LDH release kit to obtain quantitative results.

Detection of cell Edema

Cell volume was measured using flow cytometry after inducing temporary porosity in the cells. The flow cytometer sensor gauged cell volume as cells passed through, estimating the volume based on the intensity of scattered light from the laser beam. Atomic absorption spectrometry was used to measure sodium (Na+) and potassium (K+) levels in cells. Following cell lysis, the sample was gasified, and absorption values were compared with a standard curve of recognized concentration to determine Na+ and K+ concentrations. Cell membrane integrity was assessed through the LDH release test. LDH was released into the culture medium upon cell membrane damage. After cell staining, the culture supernatant was collected and mixed with the LDH reaction solution. Following a 30-minute incubation, changes in the reaction solution were measured by a spectrometer at a specific wavelength. Higher absorbance indicated more LDH release, reflecting more severe cell membrane damage.

Statistical analysis

All data were processed using SPSS 20.0 statistical analysis software (IBM, USA). Measurement data are represented by “mean ± standard deviation” (± s). Inter-group comparisons were performed using one-way ANOVA or repeated measurement ANOVA, and inter-group pairwise comparisons were performed using the LSD-t-test. Counting data were expressed as a percentage (%), and inter-group comparisons were made using χ2 analysis. A p-value of < 0.05 was considered statistically significant.

Results

Western blot analysis

The PLD2 protein expression in the model group was significantly lower compared to the control group, whereas the overexpression group exhibited a marked increase in PLD2 protein expression compared to the model group (P < 0.05) (Fig. 1).

Fig. 1
Fig. 1
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Western Blot Analysis of PLD2 Protein Expression Note: The PLD2 protein expression is significantly lower in the model group compared to the control group (P < 0.05). The PLD2 overexpression group exhibits significantly higher PLD2 protein expression than the model group (P < 0.05). (* indicates P < 0.05)

Effect of PLD2 on cytokines

RT-qPCR analysis revealed that the mRNA expression levels of inflammatory cytokines TNF-α, IL-6, and IL-10 were significantly higher in the model group compared to the control group (P < 0.05). In contrast, the PLD2 overexpression group showed significantly lower mRNA expression levels of TNF-α, IL-6, and IL-10 compared to the model group (P < 0.05) (Fig. 2).

Fig. 2
Fig. 2
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mRNA Expression Levels of Inflammatory Cytokines Note: The mRNA levels of TNF-α, IL-6, and IL-10 are significantly elevated in the model group compared to the control group (P < 0.05). The PLD2 overexpression group shows significantly lower levels of TNF-α, IL-6, and IL-10 mRNA compared to the model group (P < 0.05). (* indicates P < 0.05)

Effect of PLD2 on the expression of Nrf2/NF-κB

Nrf2 protein expression was significantly reduced in the model group compared to the control group (P < 0.05), while it was significantly elevated in the PLD2 overexpression group compared to the model group (P < 0.05). Additionally, NF-κB protein expression was significantly higher in the model group than in the control group (P < 0.05), whereas it was significantly lower in the PLD2 overexpression group compared to the model group (P < 0.05) (Fig. 3).

Fig. 3
Fig. 3
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Expression of Nrf2 and NF-κB Proteins Note: Nrf2 protein expression is significantly reduced in the model group compared to the control group (P < 0.05) and significantly elevated in the PLD2 overexpression group compared to the model group (P < 0.05). NF-κB protein expression is significantly higher in the model group than in the control group (P < 0.05) and significantly lower in the PLD2 overexpression group compared to the model group (P < 0.05). (* indicates P < 0.05)

PLD2 affected pan-apoptosis through Nrf2/NF-κB

The rate of programmed cell death and cell death due to injury was significantly higher in the model group compared to the control group (P < 0.05). Conversely, the rate of programmed cell death and cell death due to injury was significantly lower in the PLD2 overexpression group compared to the model group (P < 0.05) (Fig. 4).

Fig. 4
Fig. 4
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Rates of Programmed Cell Death and Cell Death Due to Injury Note: The rates of programmed cell death and cell death due to injury are significantly higher in the model group compared to the control group (P < 0.05). These rates are significantly lower in the PLD2 overexpression group compared to the model group (P < 0.05). (* indicates P < 0.05; ** indicates P < 0.01)

PLD2 affected cell Edema through Nrf2/NF-κB

In comparison to the control group, the model group exhibited a significant increase in cell volume, Na + content, and LDH release (P < 0.05). Conversely, the PLD2 overexpression group demonstrated a significant decrease in cell volume, Na + content, and LDH release when compared to the model group (P < 0.05) (Fig. 5).

Fig. 5
Fig. 5
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Measures of Cell Edema Note: Compared to the control group, the model group shows a significant increase in cell volume, Na + content, and LDH release (P < 0.05). The PLD2 overexpression group demonstrates a significant decrease in cell volume, Na + content, and LDH release compared to the model group (P < 0.05). (* indicates P < 0.05)

Expression of Pan-apoptosis related proteins

The model group exhibited a significant decrease in the expression of RIPK1 protein compared to the control group (P < 0.05). Conversely, the PLD2 overexpression group demonstrated a significant increase in the expression of RIPK1 protein compared to the model group (P < 0.05). The protein expression levels of CASP8, FADD, and ZBP1 were significantly higher in the model group compared to the control group (P < 0.05). However, in the PLD2 overexpression group, the protein expression levels of CASP8, FADD, and ZBP1 were significantly lower compared to the model group (P < 0.05) (Fig. 6).

Fig. 6
Fig. 6
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Expression Levels of Pan-Apoptosis Related Proteins Note: RIPK1 protein expression is significantly decreased in the model group compared to the control group (P < 0.05) and significantly increased in the PLD2 overexpression group compared to the model group (P < 0.05). The protein expression levels of CASP8, FADD, and ZBP1 are significantly higher in the model group compared to the control group (P < 0.05) and significantly lower in the PLD2 overexpression group compared to the model group (P < 0.05). (* indicates P < 0.05)

Discussion

Acute pancreatitis can extend its impact beyond the local pancreatic injury, affecting the entire body and giving rise to systemic inflammatory response syndrome, potentially leading to multiple organ dysfunction syndrome10,11. The initiation of apoptosis triggers a cascade of intricate signal transduction processes, encompassing diverse intracellular signal molecules and pathways, such as protein kinases, nuclear factors, and cell death receptors12,13,14. Disruption of these signaling pathways in AP may result in cellular dysfunction and cytoskeleton damage, subsequently causing cell content leakage and the excessive release of inflammatory factors. These factors further exacerbate both local and systemic inflammatory reactions.

Cell edema plays a dual negative role in AP. Firstly, pancreatic cell edema contributes to microcirculation disorders, reducing tissue blood flow, subsequently lowering tissue oxygen partial pressure, resulting in hypoxia and disruptions in energy metabolism15. Furthermore, alterations in the electrolyte permeability of the cell membrane disrupt the ion equilibrium in the internal and external cellular environment, particularly causing an imbalance in sodium and potassium ions16. This imbalance has the potential to modify osmotic pressure within and outside the cells, ultimately exacerbating cellular edema.

Changes in cell signal transduction play a pivotal role in the development of AP17,18. The expression level of PLD2 is strongly correlated with the degree of inflammation in the pancreas. The study revealed a significant decrease in PLD2 expression in the AP model, correlating with pancreatic cell damage and increased inflammatory response. This downregulation could impact the equilibrium of signals in cells, particularly in the regulation of cell viability and programmed cell death. Significantly, the upregulation of PLD2 led to a reduction in the levels of pro-inflammatory agents such as TNF-α, IL-6, and IL-10 mRNA. TNF-α and IL-6, primary contributors to inflammation, are typically associated with tissue damage and the escalation of the inflammatory reaction. IL-10, recognized as a controller of the inflammatory response, possesses anti-inflammatory properties. The excessive expression of PLD2 was found to suppress these factors, suggesting its potential to alleviate the inflammatory condition of cells by exerting a negative regulatory effect on crucial inflammatory mediators. This regulation may be achieved by modifying the cellular signal transduction network, such as suppressing the activation of inflammatory pathways, thereby mitigating the harm caused by inflammation.

The progression of AP is facilitated by the combined effects of oxidative stress and the inflammatory response19,20,21. Nrf2, a crucial factor in cellular antioxidant defense, is typically triggered during periods of oxidative stress, initiating the production of various antioxidant enzymes to combat oxidative harm. In contrast, NF-κB, serving as the central transcription factor of the inflammatory response, gets activated upon cellular infection or inflammatory triggers, stimulating the synthesis of inflammatory molecules and initiating inflammatory responses. In the AP model, the inhibition of Nrf2 downregulation implies a decrease in cellular antioxidant capability, potentially exacerbating oxidative stress and causing harm to cellular structure and function, including lipid peroxidation and protein oxidation. Simultaneously, the upregulation of NF-κB leads to heightened production and release of inflammatory agents, exacerbating cellular and tissue harm. These changes form a vicious circle in the context of AP, damaging cell structure and promoting the expansion of inflammation.

Nevertheless, this research discovered that the excessive expression of PLD2 disrupts this harmful cycle. PLD2 overexpression significantly amplifies Nrf2 expression, potentially boosting the cells’ antioxidant defense capability, mitigating harm caused by reactive oxygen species (ROS), and decelerating the production of lipid peroxidation products. Moreover, through suppressing NF-κB activity, PLD2 overexpression hinders the generation of inflammatory agents, ultimately relieving cellular harm and tissue lesions induced by inflammation. This suggests that PLD2 may protect pancreatic cells from AP-induced damage by regulating the Nrf2/NF-κB pathway. The results of this research propose that PLD2 not only has a defensive function in AP but achieves this protective impact by controlling the Nrf2/NF-κB pathway. By regulating PLD2, it is possible to achieve a balance between antioxidant stress and inflammatory stress, offering a fresh approach to treating AP.

Pan-apoptosis, including classical apoptosis, necrosis, and focal death22,23, represents a comprehensive mode of cell death where multiple signaling pathways interconnect to facilitate cellular demise. Acute pancreatitis, as an acute inflammatory reaction, involves not only apoptosis but also necrosis and focal death driven by excessive production of inflammatory mediators24. Our research highlights the significant role of RIPK1 (receptor-interacting protein kinase 1) as a regulatory molecule in the pan-apoptosis process. The reduction in its expression implies that cells may favor alternative death pathways, such as necrosis or focal death, over apoptosis. This inference is supported by elevated expression levels of CASP8 (Caspase-8), FADD (Fas-associated protein with death domain), and ZBP1 (Z-DNA binding protein 1). Apoptosis initiation involves Caspase-8, a crucial cysteine protease, and FADD, an adaptable protein capable of enlisting and activating Caspase-8. Additionally, ZBP1 serves as a regulator of cellular demise and inflammation associated with viral infections. Overexpression of PLD2 (phosphatidylcholine-specific phospholipase D2) appears to counteract these changes in the pan-apoptotic pathway. PLD2 potentially enhances the stability or recovery of specific cell survival signals by augmenting the expression of RIPK1. Simultaneously, it diminishes the expressions of CASP8, FADD, and ZBP1, thereby minimizing death signals transmitted through these pathways. Hence, PLD2 serves as a safeguard in the pan-apoptosis procedure, restoring a balance vital in preventing excessive demise of pancreatic cells. The function of RIPK1 is intricate, as it can facilitate or impede cell demise, contingent upon its interaction with other proteins. Suppression of RIPK1 expression can impair the ability of cells to block the necrosis pathway, resulting in non-apoptotic demise of pancreatic cells. The restoration of RIPK1 expression resulting from the excessive expression of PLD2 could potentially mitigate the impairment of cellular function and the disruption of cellular structure induced by inflammation.

During AP, cellular swelling is a prevalent phenomenon, characterized by an increase in cell size and an upsurge in Na + concentration within cells, indicating potential disruption in the equilibrium of ion channels and pumps. The presence of cell edema not only impacts cellular function but also poses a risk to cell structure, consequently elevating the likelihood of cell demise25,26. Furthermore, cellular swelling can exacerbate the inflammatory response by inducing elevated cell membrane stress, making the cell membrane more susceptible to damage. LDH, a cellular enzyme, serves as a significant biochemical indicator of cell membrane integrity damage. LDH release into the external environment occurs when there is a disruption in the cell membrane27,28. The rise in LDH secretion in the model of pancreatic inflammation indicates the presence of cellular membrane harm, aligning with cellular harm and swelling caused by inflammation. Notably, our research discovered that the regulation of cellular size and Na + concentration was evident with PLD2 overexpression, suggesting a noteworthy protective effect against cellular swelling induced by pancreatic inflammation. This protective impact potentially involves the control of ion channels and pumps by means of PLD2’s influence on intracellular signal transmission, thereby upholding the equilibrium of ion levels within and outside the cell and diminishing the likelihood of cellular swelling. Further support for this protective effect is observed in the reduced LDH release caused by PLD2 overexpression, reflecting improved cell membrane integrity. Cell survival is heavily dependent on the integrity of the cell membrane, acting as a protective barrier between cells and the external environment29. The excessive expression of PLD2 may enhance the stability of the cellular membrane, mitigating damage caused by an inflammatory response. This effect could be linked to the role of PLD2 in regulating phospholipid metabolism in the cell membrane.

Strengths and limitations

Despite the promising findings of our study, several limitations must be acknowledged. The study’s sample size was relatively small, which may limit the generalizability of the results. Additionally, the study population may lack regional representativeness, and results might differ in diverse demographic or geographic settings. Our findings are primarily based on animal models of acute pancreatitis, which, while valuable, may not fully replicate the complexity of human disease. Differences in physiology and immune response between animals and humans could influence the applicability of the results to clinical settings. The study focused on short-term responses to acute pancreatitis and PLD2 overexpression. Long-term effects and potential chronic implications of PLD2 regulation were not explored, which could be critical for understanding the full therapeutic potential and safety of targeting PLD2. While we identified key signaling pathways influenced by PLD2, the precise molecular mechanisms through which PLD2 exerts its protective effects remain incompletely understood. Further studies are required to elucidate these mechanisms in detail. The study did not include clinical data from human patients with acute pancreatitis. Future research should aim to validate these findings in clinical settings to confirm the translational potential of PLD2 as a therapeutic target. The study did not thoroughly investigate the potential side effects or unintended consequences of PLD2 overexpression. Understanding these aspects is crucial for developing safe therapeutic interventions.

Despite these limitations, the study has several notable strengths. This study provides novel insights into the role of PLD2 in regulating inflammatory responses and cell survival during acute pancreatitis, offering new avenues for therapeutic intervention. The research incorporated a comprehensive analysis of multiple aspects of cell signaling, inflammation, and cell death, providing a holistic understanding of the molecular dynamics involved in acute pancreatitis. By identifying PLD2 as a potential therapeutic target, the study opens up possibilities for the development of targeted therapies aimed at modulating PLD2 activity to treat or mitigate acute pancreatitis. The study employed a robust experimental design with well-controlled conditions, enhancing the reliability and validity of the findings. The research elucidated the involvement of key signaling pathways, such as Nrf2 and NF-κB, in the protective effects of PLD2, which could inform future studies focused on these pathways. The findings lay a solid foundation for future research, including larger multi-center studies, long-term investigations, and clinical trials, to further explore and validate the therapeutic potential of PLD2.

Conclusion

Our study sheds light on the protective role of PLD2 in pancreatic cells during acute pancreatitis. This protective function is attributed to its potential to regulate the expression of proteins associated with widespread cell death by activating Nrf2 and inhibiting the NF-κB signaling pathway. Consequently, this regulatory mechanism contributes to the alleviation of cell swelling and mitigates damage to the cell membrane. The identification of this protective role offers a novel perspective on the molecular processes underlying pancreatic inflammation, presenting a promising avenue for future therapeutic interventions.