Objective.The lack of certain trace elements such as selenium, molybdenum, magnesium or related nutrients in the soil, water quality and food in the disease area, which caused disturbance of myocardium metabolism and resulted in injury and necrosis. The aim of the study was to explore the mechanism of ibuprofen alleviating myocardial injury caused by acute pancreatitis (AP). Method. We have established AP cell model and rat model. HE staining is used for histological examination. ELISA is used to determine the levels of proinflammatory cytokines (TNF-α and IL-6) and markers of myocardial injury (LDH and CK-MB). qRT-PCR and Western blot are used to analyze the mRNA and protein levels of related genes. Results. The expression level of AIM2 was significantly increased in AP cells; downregulation of AIM2 alleviated inflammation and myocardial injury induced by AP cells; ibuprofen could inhibit the expression of AIM2 and alleviate inflammation and myocardial injury induced by AP cells. In vivo experiments have found that ibuprofen can inhibit the expression of AIM2 to alleviate myocardial injury in AP rat. Conclusion. Ibuprofen can alleviate myocardial injury caused by acute pancreatitis by inhibiting the expression of AIM2.

1. Introduction

Patients with infectious conditions may exacerbate hidden or well-controlled primary arrhythmias due to several factors, such as fever, electrolyte disturbances, drug interactions, adrenergic stress, and, eventually, the septic patient’s myocardial injury [1]. Acute pancreatitis (AP) is one of the most frequent gastrointestinal diseases and has no specific treatment. It has been shown that dysfunction of pancreatic acinar cells can lead to AP progression [2]. In AP, gut barrier injury resulting in increased mucosal permeability may lead to translocation of intestinal bacteria, necrosis of pancreatic and peripancreatic tissue, and infection, often accompanied by multiple organ dysfunction syndromes. Preserving gut microbial homeostasis may reduce the systemic effects of AP. A growing body of evidence suggests the possible involvement of the gut microbiome in various pancreatic diseases, including AP [3]. Therefore, the mitigation of myocardial injury due to acute pancreatitis has become a clinical problem that needs to be addressed urgently. Under this prudent study, we chose ibuprofen.

Ibuprofen (IBP) is one ubiquitous drug prescribed as an anti-inflammatory, analgesic, and antipyretic [4]. Inflammatory injury caused by acute pancreatitis (AP) is also a symptom of inflammation. It is also important to pay attention to the ibuprofen positive effects to defend against acute pancreatitis. In clinical practice, non-opioid (e.g., ibuprofen/naproxen) and opioid (e.g., oxycodone) forms of analgesia are widely used in pediatric patients with AP or CP, whereas pancreatic enzyme replacement therapy may be beneficial for patients with abdominal pain, steatorrhea, and malnutrition. Despite the disparity in the age of onset, pediatric CP patients display some similarities to adults in terms of disease progress. To reduce the risk of developing pancreatic exocrine inefficiency, diabetes, and pancreatic cancer in the future, clinicians need to be aware of the current diagnostic approach and treatment methods for ARP and CP and refer them to a pediatric gastroenterologist in a timely manner [5]. Thus, high doses of corticosteroids or nonsteroidal anti-inflammatory agents such as ibuprofen share the ability to prevent aggregation and embolization of stimulated granulocytes to patent vessel downstream and inhibit their production of toxic oxygen radicals. These properties suggest the use of these agents in myocardial infarction and shock states, particularly shock lung [6].

Therefore, based on the above effective effects of ibuprofen, we tried to guess whether ibuprofen could alleviate acute pancreatitis (AP) by modulating AIM2, and we treated acute pancreatitis (AP) cells with ibuprofen and performed real experiments to verify it. We are providing a roadmap of human microbial contributions to AP with potential clinical benefits. If we can confirm that ibuprofen alleviates myocardial injury in AP rat by inhibiting AIM2, the contribution to AP provides a roadmap with potential clinical benefit.

2. Materials and Methods

2.1. Ethics Statement

The study was conducted in line with the Guidelines for the care and use of laboratory animals issued by the National Research Council of the United States. The study obtained the approval of the Ethics Committee of our hospital (Ethic approval no.JHU0023017).

2.2. Types of Cells and Reagents

The AR42J acinar cell line was purchased from ATCC (#CRL-1492, Manassas, VA, USA). DMEM solution, fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Mediatech (Herndon, VA, USA) and Gibco (Grand Island, NY, USA); cerulein (bombesin, sigma, C9026), ibuprofen (sigma, 14883), and ELISA kit were purchased from Excell (Shanghai, China); Trizol reagent and ThermoScript RT-PCR system were purchased from Invitrogen (Grand Island, NY, USA) [7].

2.3. Establishment of the Rat Model of Acute Pancreatitis (AP)

15 C57BL/6 male rats aged 8-12 weeks purchased from the animal experiment center of our hospital, weighing between 25 and 30g. The rats were raised under the standard laboratory conditions at 28 °C and 50% humidity for one week. Before constructing the model, all rats were forbidden to drink for 12 hours. The working solution was prepared by dissolving cerulein powder in physiological saline at different concentrations. Cerulein is an analog of cholecystokinin, which can cause pancreatic exocrine and pancreatic cell inflammation. Intraperitoneal injection of cerulein 6 times can induce acute pancreatitis (AP), which is induced by continuous injection. Rats were divided into 3 groups, 5 in each group, AP group: intraperitoneal injection of cerulein on the 1st and 2nd day, each injection of 50μg/kg, once every 1h for a total of 6 injections; AP+ibuprofen group: Inject cerulein intraperitoneally in the same way. Starting from day 3, intraperitoneally inject 25 mg/kg ibuprofen twice a day, with an interval of 2 hours each time, for 5 consecutive days. In the control group, inject equal volume of physiology brine. Seven days after the first injection of cerulein or saline, the rats were euthanized, blood was taken from the inferior vena cava, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at – 80 °C for later use. At the same time, the pancreatic tissue was separated and used 4% paraformaldehyde. Fix overnight and then embed in paraffin. The study obtained the approval of the Ethics Committee of our hospital [8]

2.4. Cell Culture and Treatment

AR42J acinar cells were cultured in DMEM solution containing 10% FBS and 1% penicillin/streptomycin solution and placed in an incubator containing 5% CO2 at 37 °C. The cell culture solution was changed every 2 days before stimulation with cerulein. Pancreatic acinar cell inflammation induced by 10 nmol/L cerulein was used as an in vitro model of acute pancreatitis (AP), and then transfection was performed in cells grouped as si-NC and si-AIM2. 1 × 105 cells were inoculated in each well of a six-well plate. When the cell confluence rate reaches 60%-70%, add 750 μl OptiMEM solution to each well, taking 125 μl OptiMEM and 5 μl Lipo3000 (L3000001, Invitrogen, Carlsbad, CA, USA) to mix, and let it stand for 5 min; then, take 125 μl OptiMEM and plasmid/siRNA, and let it stand for 5 min; then, mix the two, and let it stand for 20 min, add 250 μl of solution into the well, and shake it gently and evenly. Finally, we put it in the incubator; after 16 hours, we change the solution, and cellular RNA can be extracted after 48 hours; also, cellular protein can be extracted after 72 hours for subsequent experiments.

AP cells were treated with ibuprofen (the concentration and time of ibuprofen treatment could not be determined, no relevant literature reference). The cells were divided into 2 groups: AP group and AP + ibuprofen treatment group [7]. The rats were euthanised by overdosed pentobarbital sodium (150 mg/kg).

2.5. qRT-PCR

According to the manufacturer’s instructions, miRNA was isolated from EVs, tissues, and cells by using the mirVanaTM PARISTM RNA kit (AM1556, Invitrogen, CA, USA). For mRNA analysis, cDNA was synthesized randomly from 1 μg of total RNA using the First Strand cDNA Synthesis Kit (K1622, Fermentas, USA) was used to synthesize cDNA randomly from 1 μg of total RNA; for miRNA analysis, the TaqManTM MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems, USA) was used to synthesize cDNA for miRNA. Finally, the Fast SYBR Green PCR kit (Applied biosystems) and the ABI PRISM 7300 RT-PCR system (Applied biosystems) were used to analyze RNA quantitatively, and each sample was repeated for three replicate wells. miR-30c was used with U6 as the internal reference, and the remaining genes were used with GAPDH. The relative gene expression was analyzed by the 2-ΔΔCt method, calculated as △Ct=CT (target gene)−CT (internal reference) and △△Ct=△Ct (Experimental group)−△Ct (control group), repeated the experiment 3 times, and took the average value. All primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. and the primer sequences were as follows Table 1.

2.6. Western Blotting

Western Blot methods were used to detect related indicators [9]. First, we took out the cells, and washed the cell surface twice with PBS, added 60ul RI + PA cloud biological solution +1% and use the performance +1% cloud phosphorylase technology (Beyotime Biotech Inc., Shanghai, China), after scraping the cells off, on the sky ice) Buffer for 45 minutes. After centrifugation, the protein supernatant was separated, and the protein concentration was detected using the BCA kit (Beyotime Biotech Inc., Shanghai, China). Second, use 1/4 volume of 5× loading buffer in protein solution (Beyotime Biotech Inc., Shanghai, China), boil at 100 °C for 10 minutes to spoil the protein, and then take 20 ug of each protein solution and separate it by electrophoresis in 10% SDS-PAGE gel, and transfer the separated protein gel to PVDF membrane for membrane transfer; after the membrane transfer was completed, put the PVDF membrane into the closure solution containing 5% BSA for 2 hours (Table 2). After incubation, the strips were washed 3 times with TBST solution for 5 min each time, and then the strips were placed in the HRP-labeled secondary antibody and incubated at room temperature for 1 h. After incubation, the strips were washed 3 times with TBST solution again, and the ECL working solution (BM101, Biomiga, USA) was added dropwise to the strips for 1 min. After 1 min, the strips were developed using a BioSpectrum 600 imaging system (Ultra-Violet Products, UK). Lastly, protein strip gray values were calculated using Image J software, and the target protein gray value/GAPDH gray value was used as the relative protein expression. The experiment was repeated three times.

2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

According to the manufacturer’s protocol, the cell culture supernatant and rat serum were collected using an ELISA kit to determine the secretion of TNF-α (#900-M54, PeproTech, USA), IL-6 (#900-K86, PeproTech, USA), LDH (#900-K44, PeproTech, USA), and CK-MB (#900-M87, PeproTech, USA). Each sample was assayed at least three times by using an enzyme marker (Bio-Rad) [7].

2.8. HE Staining

Murine tumor tissues were collected and fixed in 4% paraformaldehyde solution, dewaxed with xylene for 15 minutes, the sections were immersed in a mixture of xylene and alcohol (1 : 1) for 3 minutes, and then immerse them in 100%, 95%, 90%, 80%, 70%, and 50% ethanol for 2 minutes, stain with hematoxylin for 5 minutes, rinse with tap water for 3 minutes, 1% hydrochloric acid for 2 seconds, rinse with tap water for 2 minutes, then immerse in 50%, 70%, and 80% ethanol for 2 minutes, and eosin soak for 5 seconds, stained with 90%, 95%, 100% ethanol each for 2minutes, and finally two consecutive times of xylene for 15 minutes each, mount the slides. The sections were sealed, then fixed, and observed under a bright field microscope. In the observation index, the nucleus was stained blue, and the cytoplasm was stained red [10].

2.9. Statistical Analysis

All data were statistically analyzed using Graphpad 7 software, and the data were expressed as mean ± standard deviation, with tests for normality and homogeneity. Data were compared between two groups using an unpaired -test and between multiple groups using a one-way analysis of variance (ANOVA). When the data did not show a normal distribution or equal variance, a rank sum test was performed. was considered statistically significant.

3. Results

3.1. Establishment of Acute Pancreatitis (AP) Cell Model

We first used cerulein to treat AR42J acinar cells to construct an AP cell model and extract the cell culture supernatant and used an ELISA kit to detect pro-inflammatory cytokines (TNF-α and IL-6) and myocardial injury markers (LDH and CK-MB); the results showed that the contents of TNF-α, IL-6, CK-MB, and LDH in AP cells increased significantly, indicating that the AP cell model was successfully constructed. Besides, we found more apoptotic cells in AP group (Figures 1(a) and 1(b)).

3.2. AIM2 Can Exacerbate AP-Induced Myocardial Injury

Checking the literature found that the activation of AIM2 inflammasome can aggravate AP systemic inflammation and organ failure [11]. Therefore, we detected the expression level of AIM2 in AP cells by qRT-PCR ,and Western blot found that AIM2 was overexpressed in AP cells (Figures 2(a) and 2(b)). In order to understand whether the high expression of AIM2 was associated with the inflammatory injury caused by AP cells, we downregulated AIM2 in AP cells, and qRT-PCR test showed that the downregulation was successful (Figure 2(c)). The ELISA kit test found that compared with AP cells, the contents of TNF-α, IL-6, CK-MB, and LDH in the AP + si-AIM2 group were significantly reduced (Figure 2(d)). Based on the above results, it can be inferred that AIM2 is highly expressed in AP cells and can exacerbate the myocardial inflammatory injury induced by AP.

3.3. Ibuprofen Can Alleviate AP-Induced Myocardial Injury by Inhibiting AIM2

One study has shown that ibuprofen and diclofenac treatments reduce the proliferation of pancreatic acinar cells upon inflammatory injury and mitogenic stimulation [8]. Therefore, we guessed whether ibuprofen could alleviate AP by regulating AIM2. We treated AP cells with ibuprofen, and the results of qRT-PCR and Western blot showed that the expression of AIM2 in the ibuprofen-treated group was significantly reduced in the ibuprofen-treated group compared with the PBS group (Figures 3(a) and 3(b)). ELISA detected the content of inflammatory factors and myocardial injury-related factors, and the results showed that compared with the PBS group, the levels of TNF-α, IL-6, CK-MB, and LDH in the ibuprofen-treated group were significantly reduced (Figure 3(c)). In summary of the above results, we can conclude that ibuprofen can alleviate AP-induced myocardial injury by inhibiting AIM2.

3.4. Ibuprofen Relieves Myocardial Injury in AP Rat by Inhibiting AIM2

Finally, we verified the alleviating effect of ibuprofen on AP by in vivo experiments. We constructed an AP mouse model by injecting cerulein and treated it with ibuprofen. HE staining observed the pathological conditions of the pancreatic tissue of each group of rats and found that it was similar to the control group. In comparison, the pancreatic tissue of AP rats showed prominent edema and was infiltrated by inflammatory cells. After treatment with ibuprofen, the pancreatic inflammation was significantly improved (Figure 4(a)). The expression of AIM2 in pancreatic tissue was detected by qRT-PCR and Western blot. It was found that compared with the control group, the expression of AIM2 in the pancreatic tissue of AP rats increased, while it was decreased after treatment with ibuprofen (Figures 4(b) and 4(c)). ELISA detected the expression levels of related factors in the serum of rats. The results showed that compared with the control group, the levels of TNF-α, IL-6, CK-MB, and LDH in the serum of AP rats were significantly increased, while the levels of TNF-α, IL-6, CK-MB, and LDH were decreased after treatment with ibuprofen (Figure 4(d)). The above results indicated that ibuprofen can alleviate myocardial injury in AP rats by inhibiting AIM2.

4. Discussion

Acute pancreatitis (AP), an inflammatory condition of the pancreas, destroys the exocrine cells by releasing various pro-inflammatory cytokines that activate the stellate cells. However, the underlying molecular mechanism remains unclear [12]. AR42J are immortalized pancreatic adenocarcinoma cells that share similarities with pancreatic acinar cells. AR42J is often used as a cell-culture model of cerulein- (CN-) induced acute pancreatitis (AP) [13]. Ibuprofen is a widely used and well-tolerated analgesic and antipyretic. It is desirable to have a formulation with a rapid rate of absorption because it is required for rapid pain relief and temperature reduction [14]. Based on the above literature arguments, we purchased these materials to do experiments and to justify our topic that ibuprofen can alleviate acute pancreatitis– (AP–) induced myocardial injury by inhibiting AIM2.

The present study aimed to examine the positive effect of ibuprofen. In the present study, a rat model of AP was established. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with and the possibility of working with genetically modified strains of mice) that may be relevant to the study [15].

To determine the function of AIM2, we detected the expression level of AIM2 in AP cells by qRT-PCR and Western blot and found that AIM2 was overexpressed in AP cells. The luciferase assay verified whether miR-219a targeted AIM2, and RT-qPCR and Western blot detected the miR-219a and AIM2 expression in myocardial tissues. miR-219a was significantly increased in myocardial tissues from mice treated with Sev, and the area of MI and cardiomyocyte apoptosis were decreased. The miR-219a inhibitor reversed the action of Sev. Moreover, overexpression of AIM2 or induction of the TLR4 pathway aggravated myocardial I/R injury alleviated by miR-219a [16].

Then, the study aims to analyze how we exert functions of ibuprofen; our results demonstrated that ibuprofen can alleviate myocardial injury caused by acute pancreatitis by inhibiting the expression of AIM2.

5. Conclusion

The expression level of AIM2 was significantly increased in AP cells; downregulation of AIM2 alleviated inflammation and myocardial injury induced by AP cells; ibuprofen could inhibit the expression of AIM2 and alleviate inflammation and myocardial injury induced by AP cells. In vivo experiments have found that ibuprofen can inhibit the expression of AIM2 to alleviate myocardial injury in AP rat.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

Chen Ke guaranteed of integrity of the entire study, and Shaohua Wang confirmed the study concepts.