Fibulin-1 Improves Caerulein-Induced Acute Pancreatitis In Vitro by Inhibiting Ferroptosis

Xiaowei Mai: conceptualization, investigation, writing – original draft, writing – review and editing, methodology, funding acquisition, visualization, software, formal analysis, project administration, resources, data curation, supervision, validation. ShuLiu He: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, software, formal analysis, project administration, resources, supervision, data curation. YangYang Li: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, methodology, validation, visualization, project administration, formal analysis, software, data curation, supervision, resources. Lei Zhang: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, software, formal analysis, project administration, resources, supervision, data curation. QiZhong Xu: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, software, formal analysis, project administration, resources, supervision, data curation. YinSheng Wen: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, software, formal analysis, project administration, resources, supervision, data curation.

This article does not contain any studies with human participants or animals performed by any of the authors.

FBLN1 protein expression was decreased after Caerulein treatment in AR42J cells (Figure  1A ). Consistently, the fluorescence intensity of FBLN1 also reduced following Caerulein treatment (Figure  1B ), suggesting that FBLN1 exhibited lower expression in the in vitro model of acute pancreatitis. FBLN1 exhibited lower expression in acute pancreatitis. The groups were separated into the control and Caerulein group. (A) FBLN1 protein expression was examined through western blot, and (B) fluorescence intensity through IF assay. ^^^ p  < 0.001. Next, FBLN1 protein expression was found to be reduced after Caerulein treatment, and this could be reversed after FBLN1 overexpression (Figure  2A ). Furthermore, cell viability was decreased after Caerulein treatment, while this change was restored after FBLN1 amplification (Figure  2B ). Cell apoptosis was increased after Caerulein treatment, and this effect could be attenuated following FBLN1 overexpression (Figure  2C,D ). The levels of TNF‐α and IL‐1β were found to be upregulated after Caerulein treatment, but could be offset after FBLN1 overexpression (Figure  2E ). Collectively, FBLN1 could inhibit caerulein‐triggered inflammatory injury in AR42J cells. FBLN1 inhibited caerulein‐triggered inflammatory injury in AR42J cells. The groups were separated into the control, Caerulein, Caerulein + NC and Caerulein + FBLN1 group. (A) FBLN1 protein expression was determined through western blot, (B) cell viability was confirmed through CCK‐8 assay, (C, D) cell apoptosis was measured through flow cytometry, and (E) the levels of TNF‐α and IL‐1β were examined through ELISA. ^^^ p  < 0.001 versus the control group; # p  < 0.05, ## p  < 0.01 versus the Caerulein + NC group. Fe 2+ levels were increased after Caerulein treatment, and this effect could be counteracted following FBLN1 overexpression (Figure  3A ). ROS levels were also elevated after Caerulein treatment, and this effect could be alleviated after FBLN1 overexpression (Figure  3B ). Besides, the protein expression of GPX4 was reduced, and ACSL4 was increased after Caerulein treatment, and these changes could be similarly reversed after FBLN1 overexpression (Figure  3C ). The increased levels of TNF‐α and IL‐1β mediated by Caerulein treatment were reduced after FBLN1 overexpression, and these were weakened following treatment with erastin, a ferroptosis inducer (Figure  3D ). These data indicate that FBLN1 can suppress Caerulein‐evoked ferroptosis in AR42J cells. FBLN1 suppressed caerulein‐evoked ferroptosis in AR42J cells. (A) The Fe 2+ level was examined using the Fe kit. The groups were separated into a control, Caerulein, Caerulein + NC, and Caerulein + FBLN1 group. ^^^ p  < 0.001 versus the control group; # p  < 0.05 versus the Caerulein + NC group. (B) ROS levels were assessed using the ROS kit. The groups were separated into the control, Caerulein, Caerulein + NC, and Caerulein + FBLN1 groups. (C) The protein expressions of GPX4 and ACSL4 were determined through western blot. For this, the groups were separated into the control, Caerulein, Caerulein + NC, and Caerulein + FBLN1 group. ^^^ p  < 0.001 versus the control group; # p  < 0.05, ### p  < 0.001 versus the Caerulein+NC group. (D) The levels of TNF‐α and IL‐1β were assessed through ELISA. The groups were separated into the control, Caerulein, Caerulein + NC, Caerulein + FBLN1 and Caerulein + FBLN1 + Erastin group. ^^^ p  < 0.001 versus the control group; ## p  < 0.01 versus the Caerulein + NC group; & p  < 0.05 versus the Caerulein + FBLN1 group. Moreover, NRF2 and HO‐1 protein expression levels were decreased after Caerulein treatment, and these changes could be reversed following FBLN1 amplification (Figure  4 ), indicating that FBLN1 can activate the NRF2/HO‐1 pathway. FBLN1 activated the NRF2/HO‐1 pathway. The groups were separated into the control, Caerulein, Caerulein + NC, and Caerulein + FBLN1 groups, and the protein expressions of NRF2 and HO‐1 were evaluated through Western blot. ^^^ p  < 0.001 versus the control group; ## p  < 0.01, ### p  < 0.001 versus the Caerulein + NC group.

Although FBLN1 has been implicated in numerous diseases [ 6 , 7 , 8 , 9 , 10 ], its significance in acute pancreatitis remained unclear. This study showed that FBLN1 exhibited lower expression in acute pancreatitis. Inflammation is known to be a pivotal process in acute pancreatitis, and many studies have focused on strategies aimed at reducing inflammatory injury. For instance, Eriodictyol was shown to modulate the ZBP1‐dependent signaling to reduce inflammation and necrosis in acute pancreatitis [ 20 ]. In addition, sulfiredoxin‐1 has been demonstrated to influence the ROS/Cathepsin B axis to relieve injury and inflammation [ 21 ]. Moreover, Gastrodin was found to inhibit the p38/NF‐κB pathway to alleviate the macrophage inflammation cascade, improving acute pancreatitis [ 22 ]. In l ‐arginine‐evoked acute pancreatitis, Genistein has also been shown to reduce inflammation and apoptosis [ 23 ]. Consistent with the importance of inflammatory injury in acute pancreatitis, the present study showed that FBLN1 inhibited Caerulein‐triggered inflammatory injury in AR42J cells. Ferroptosis is one form of oxidative stress‐related cell death characterized by the accumulation of lipid ROS [ 24 , 25 ]. Increasing evidence has verified that ferroptosis participates in the progression of acute pancreatitis. For example, Hspb1 interacted with Anxa2 to relieve cell apoptosis and ferroptosis, improving severe acute pancreatitis [ 26 ]. In addition, S100A11 strengthened acinar cell ferroptosis to aggravate acute pancreatitis [ 27 ]. In severe acute pancreatitis‐evoked acute lung injury, matrine triggered the UCP2/SIRT3/PGC1α pathway to improve oxidative stress and ferroptosis [ 28 ]. Furthermore, recent studies have emphasized the significance of FBLN1 in regulating ferroptosis by demonstrating that FBLN1 may suppress the TGF‐β/Smad pathway to weaken free iron levels and ferroptosis in acute respiratory distress syndrome [ 29 ], and that the upregulation of FBLN1 in aggravating cell viability and migration in endometriosis for suppressing EFEMP1‐dependent ferroptosis [ 30 ]. Herein, we further found that FBLN1 suppressed Caerulein‐evoked ferroptosis in AR42J cells, indicating that ferroptosis inhibition may be one important mechanism through which FBLN1 protects against Caerulein‐induced cellular injury. The NRF2/HO‐1 pathway is an important node regulating inflammation, oxidative stress, and ferroptosis [ 31 , 32 ]. Investigations on the NRF2/HO‐1 pathway revealed that Angiotensin‐(1‐7) can stimulate the Keap1/NRF2/HO‐1 pathway to mitigate intestinal barrier dysfunction in acute pancreatitis [ 33 ], AKBA modulates the pathway to restrain oxidative stress in macrophages and relieve experimental pancreatitis [ 34 ], and KAN0438757 (a PFKFB3 inhibitor) also affecting the NRF2/HO‐1 pathway to reduce the progression of severe acute pancreatitis [ 35 ]. Additionally, chlorogenic acid regulates the NRF2/HO‐1 pathway to suppress NLPR3 inflammasome activation in severe acute pancreatitis [ 36 ]. However, whether FBLN1 regulates the NRF2/HO‐1 pathway in acute pancreatitis remains unclear. In this study, FBLN1 was found to activate the NRF2/HO‐1 pathway, suggesting that this pathway may participate in the protective effect of FBLN1 against Caerulein‐induced injury. In conclusion, our findings suggest that FBLN1 can alleviate Caerulein‐induced acute pancreatitis in vitro by inhibiting ferroptosis. Nevertheless, several limitations remain in this study, including the lack of animal experiments, human samples, and clinical investigations, and that FBLN1 expression was not examined in an independent cohort with a larger number of clinical samples. Thus, further studies are needed to validate the role of FBLN1 in acute pancreatitis progression and to clarify its regulatory mechanisms in vivo and in clinical settings.

Acute pancreatitis is an inflammatory disease of the pancreas [ 1 ], with approximately 20% of patients developing severe acute pancreatitis and complications such as hypovolemic shock, secondary infection, pancreatic necrosis, or multiple organ dysfunction [ 2 ]. Currently, there is no effective treatment for acute pancreatitis, making its treatment challenging [ 3 ]. Therefore, clarifying the complex pathophysiology of acute pancreatitis and identifying effective bio‐targets are crucial for identifying promising therapeutic approaches for acute pancreatitis. Fibulin‐1 (FBLN1) is an extracellular matrix (ECM) protein that has been found to be involved in cell migration, adhesion, and tissue repair [ 4 , 5 ]. FBLN1 plays a pivotal role in maintaining the structural integrity and functionality of diverse tissues, and increasing evidence has shown that it participates in the development of multiple diseases. For example, in colorectal cancer, FBLN1 has been found to be downregulated through promoter hypermethylation [ 6 ]. In non‐small cell lung cancer, it was reported to be a promising diagnostic biomarker for brain metastasis [ 7 ]. In lung cancer, cell‐derived matrix FBLN1 was shown to suppress epidermal growth factor receptor [ 8 ]. In addition, FBLN1 has been reported to be a biomarker for early cervical cancer detection [ 9 ]. FBLN1 has also been shown to enhance Smad2 phosphorylation and promote chondrocyte proliferation [ 10 ]. Importantly, a previous study used data‐independent acquisition (DIA) technology combined with mass spectrometry to quantitatively analyze plasma protein profiles of severe acute pancreatitis patients and healthy volunteers, demonstrating lower FBLN1 expression in severe acute pancreatitis patients [ 11 ]. However, the regulatory roles of FBLN1 in acute pancreatitis remain to be fully investigated. Ferroptosis is a form of programmed cell death characterized by the accumulation of iron‐dependent lipid peroxidation [ 12 , 13 ]. Recent studies have indicated that ferroptosis is closely associated with the pathogenesis of acute pancreatitis [ 14 , 15 ], but the influences of FBLN1 on ferroptosis in acute pancreatitis remain unclear. In this study, we aimed to investigate the regulatory effects of FBLN1 in acute pancreatitis progression.

The authors declare no conflicts of interest.

Human pancreatic acinar AR42J cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI‐1640 (Gibco, Waltham, MA, USA) with 10% FBS at 37°C/5% CO 2 . Then, the cells were treated with 10 nmol/L Caerulein (MB2573, Meilunbio, Dalian, China) for 24 h to mimic acute pancreatitis [ 16 , 17 ], establishing the acute pancreatitis cell model. The pcDNA3.1 plasmids target FBLN1 (FBLN1) and the negative control (NC) were purchased from GenePharma (Shanghai, China). Transfections (transient transfection) for these plasmids into AR42J cells were performed using Lipofectamine 2000 (Invitrogen, USA). AR42J cells were lysed using the RIPA buffer (Beyotime, Shanghai, China), and the concentration of proteins was assessed using the BCA method. Proteins (25 μg per lane) were separated by Sodium Dodecyl Sulfate—Polyacrylamide Gel Electrophoresis (SDS‐PAGE, 10%), and moved onto PVDF membranes (Merck Millipore, Burlington, MA, USA). After blocking with 5% (w/v) non‐fat dry milk, primary antibodies were added to the membranes and incubated for 12 h at 4°C, and then with appropriate secondary antibody (1:1000; ab7090, Abcam, Shanghai, China) for 2 h at room temperature. At last, protein blots were examined using an enhanced chemiluminescence (ECL) kit, and the images were analyzed using ImageJ (National Institutes of Health, USA) [ 18 , 19 ]. The primary antibodies used in this experiment were: FBLN1 (1:2000; ab211536, Abcam, Shanghai, China), GPX4 (1:5000; ab180824), ACSL4 (1:5000; ab269613), ULK1 (1:1000; ab125656), NRF2 (1:5000; ab76026), HO‐1 (1:1000; ab229429), and Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH, 1:1000; ab181602). AR42J cells grown on glass coverslips were fixed with 4% PFA and permeabilized with 0.3% Triton X‐10. After blocking with 5% goat serum/TBS, the cells were incubated with the primary antibody against FBLN1 (Abcam, Shanghai, China) overnight at 4°C, followed by incubation with the secondary antibody (1:1000; A‐21202, Invitrogen, Carlsbad, CA, USA; 1 h, 37°C, in the dark). Cell nuclei were counterstained with DAPI, and images were captured using an Olympus BX63 microscope (Olympus, Tokyo, Japan). The CCK‐8 assay kit (CK04‐05; Dojindo, Kumamoto, Japan) was used for this assay. Briefly, AR42J cells (1 × 10 3 cells/well) were seeded onto 96‐well plates, 10 μL of CCK‐8 reagent was added to each well, and they were allowed to react. Then, optical density (OD) at 450 nm was assessed using a spectrophotometer (ND‐ONE‐W, Thermo Fisher Scientific, Waltham, MA, USA). For cell apoptosis, AR42J cells were stained with annexin V‐fluorescein isothiocyanate (BD Biosciences, San Jose, CA, USA) and PI (50 μg/mL; Beyotime, Shanghai, China) in the dark. After washing, cell apoptosis was determined through flow cytometry (BD Biosciences, San Diego, CA, USA). The TNF‐α ELISA kit (ab181421, Abcam, Shanghai, China) and the IL‐1β ELISA kit (ab46052, Abcam, Shanghai, China) were utilized for determining the levels of TNF‐α and IL‐1β in the cellular supernatant, respectively. Briefly, the prepared sample homogenates and standard solution were added to the 96‐well plates, and cultured at 37°C for 1.5 h. After the colorimetric reaction, the optical absorbance was determined at 450 nm using a spectrophotometer (ND‐ONE‐W, Thermo Fisher Scientific, Waltham, MA, USA). A series of standard dilutions was used to construct a calibration curve. Fe 2+ levels were examined using the Fe kit (ab83366, Abcam, Shanghai, China). Cells were washed with PBS, digested with trypsin, and centrifuged at 15,000 g for 5 min at 4°C. The supernatant was collected and added to the reagent containing iron reductase. After 0.5 h, the OD was measured by a microplate reader at 593 nm. The ROS kit (E004‐1‐1, Nanjing Jiancheng Technology Co. Ltd. Nanjing, China) was used for this assay. Briefly, AR42J cells were mixed with Dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) and allowed to react for staining up to 30 min. After washing, images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan). Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, USA). The assumption of normality was assessed using the Shapiro−Wilk test. Comparisons were made through the Student's t ‐test or one‐way Analysis of Variance (ANOVA). p value < 0.05 was considered statistically significant.