microRNA-1-3p and T-synthase mRNA have high diagnostic efficacy on intestinal mucosal barrier dysfunction in patients with severe acute pancreatitis.

The clinical data of SAP&IBD patients and SAP patients were compared and summarized in Table  2 . There were no significant differences between the two groups in terms of gender, age, BMI, Comorbidities, Etiology, APACHE II, CT severity index, Ranson score, levels of amylase, blood urea nitrogen, and serum albumin (all p  > 0.05). However, elevated levels of CRP, procalcitonin, IL‐6, and IBD indicators DAO and D‐lactate levels were observed in SAP&IBD patients compared to the SAP group (all p  < 0.001). Comparative analysis of clinical baseline data of subjects. Abbreviations: ALB, serum albumin; BUN, blood urea nitrogen; CRP, C‐reactive protein; DAO, diamine oxidase; IBD, intestinal mucosal barrier dysfunction; PCT, procalcitonin; SAP, severe acute pancreatitis. Expression patterns of miR‐1‐3p and T‐synthase mRNA in peripheral blood of all patients were assessed by means of RT‐qPCR. Compared to the SAP group, serum miR‐1‐3p expression was higher in the SAP&IBD group ( p  < 0.001) (Figure  1A ), while the expression of T‐synthase mRNA in peripheral blood B lymphocytes was lower ( p  < 0.001) (Figure  1B ). To further understand the correlation between miR‐1‐3p and T‐synthase mRNA in peripheral blood of SAP&IBD patients, a Pearson method analysis was performed. The results showed that serum miR‐1‐3p expression in the SAP&IBD patients was negatively correlated with T‐synthase mRNA expression in peripheral blood B lymphocytes ( r  = −0.624, p  < 0.001) (Figure  1C ). miR‐1‐3p and T‐synthase expression levels in peripheral blood of SAP&IBD patients and their correlations. (A) Serum miR‐1‐3p expression and (B) T‐synthase mRNA expression in peripheral blood B lymphocytes in SAP and SAP&IBD patients were determined by RT‐qPCR; (C) The correlation between miR‐1‐3p and T‐synthase mRNA in peripheral blood of SAP&IBD patients was analyzed using the Pearson method. Data were expressed as mean ± standard deviation. Independent sample unpaired t ‐test was used for comparisons between two groups. *** p  < 0.001. The correlation between miR‐1‐3p/T‐synthase mRNA and clinical data of SAP&IBD patients was further analyzed using the Pearson method (Table  3 ). The results showed that serum miR‐1‐3p levels in SAP&IBD patients were positively correlated with their Ranson score, CRP, IL‐6, and IBD indicators DAO and D‐Lactate (all p   0.05). Meanwhile, T‐synthase mRNA levels in peripheral blood B lymphocytes in SAP&IBD patients were negatively correlated with their IL‐6 levels and IBD indicators DAO and D‐Lactate levels (all p   0.05). These results indicate that there is a relationship between the expression levels of miR‐1‐3p and T‐synthase mRNA with the severity of SAP and IBD in patients. Correlation between miR‐1‐3p/T‐synthase in peripheral blood and clinical data of SAP&IBD patients. Abbreviations: ALB, serum albumin; BUN, blood urea nitrogen; CRP, C‐reactive protein; DAO, diamine oxidase; IBD, intestinal mucosal barrier dysfunction; PCT, procalcitonin; SAP, severe acute pancreatitis. The diagnostic efficacy of miR‐1‐3p and T‐synthase in SAP&IBD patients were analyzed with a ROC curve to further evaluate the potential clinical applications of miR‐1‐3p and T‐synthase levels in predicting the risk of IBD in SAP patients (Table  4 , Figure  2 ). The AUC of serum miR‐1‐3p level for predicting IBD in SAP patients was 0.871, the cut‐off value was 1.245 (0.800 sensitivity and 0.960 specificity); Meanwhile, the AUC of T‐synthase mRNA level for predicting IBD in SAP patients was 0.827, the cut‐off value was 0.935 (0.814 sensitivity and 0.800 specificity); whereas the AUC of miR‐1‐3p and T‐synthase mRNA combination for predicting IBD in SAP patients was 0.864, the cut‐off value was 0.555 (0.814 sensitivity and 0.860 specificity). The comparative analysis of MedCalc‐comparison of ROC curves manifested no significant differences in AUC among the three groups (all p  > 0.05). Together, these findings suggest that both miR‐1‐3p and T‐synthase levels possess high diagnostic efficacy for IBD in SAP patients. Diagnostic efficacy of miR‐1‐3p, T‐synthase and their combination on IBD in SAP patients. ROC curves of miR‐1‐3p, T‐synthase and their combination for assessing the risk of IBD in SAP patients. A logistic multivariate regression analysis was performed to further explore whether miR‐1‐3p and T‐synthase were independent risk factors for IBD in SAP patients. The presence of IBD in SAP patients was used as the dependent variable, while CRP, PCT, IL‐6, DAO, D‐Lactate with p  < 0.05, as well as serum miR‐1‐3p, and T‐synthase mRNA levels in peripheral blood B lymphocytes were included as the independent variables in logistic multivariate regression analysis (Table  5 ). After adjusting for CRP, IL‐6, and DAO, the results showed that both serum miR‐1‐3p ( p  = 0.039, OR = 20.881, 95%CI = 1.168–373.285) and T‐synthase mRNA levels in peripheral blood B lymphocytes ( p  = 0.012, OR = 0.004, 95%CI = 0.000–0.303) were independently associated with IBD in SAP patients. Logistic regression analysis of IBD in SAP patients.

AP is a highly‐prevalent gastrointestinal disorder that requires hospital admission and has an annual incidence of 34 per 100,000 person‐years in high‐income countries. 22 Previous research has shown that various miRNAs are dysregulated in several cell types involved in AP such as macrophages, lymphocytes, and acinar cells, and these miRNAs modulate both systemic and local inflammatory responses, thus playing a role in the initiation and progression of AP. 8 Moreover, evidence has come to light indicating that exosomal miR‐1‐3p mediates cell permeability and membrane injury of endothelial cells via SERP1, leading to vascular barrier dysfunction. 23 Elevated miR‐124‐3p in aged distal colonic mucosa is further known to disrupt the mucus barrier and increase the susceptibility to colitis by targeting T‐synthase. 24 In the current study, we performed a series of experiments to further elucidate the clinical roles of miR‐1‐3p and T‐synthase in IBD diagnosis in SAP patients. Our findings revealed that miR‐1‐3p and T‐synthase were independent risk factors for IBD in SAP patients and could assist in IBD diagnosis in these patients. Accumulating evidence has shown that multiple organ dysfunction syndromes and systemic inflammatory response syndromes can result from massive inflammatory cytokine release in the early stage of SAP, followed by pancreatic necrosis and intestinal dysfunction in the later stage. 25 IBD plays a crucial role in the pathophysiology of various diseases, including SAP. 26 The activity of DAO, Endotoxin, and D‐Lactate levels have long been adopted as indicators of small intestinal mucosal mass and integrity. 27 Herein, our findings manifested that in contrast to SAP patients, the levels of CRP, procalcitonin, interleukin‐6 (IL‐6), and IBD indicators (DAO, D‐lactate, and endotoxin) were significantly higher in SAP patients with IBD. Similarly, Ouyang et al. documented apparent IBD in AP rats, indicated by elevated levels of plasma endotoxin and DAO. 28 Moreover, increased serum IL‐6 and CRP levels in SAP patients may reflect not only a systemic inflammatory response in SAP but also a potential involvement in the pathogenesis of the disease. 29 Furthermore, we recorded the APACHE II score, Ranson score, and CT severity index reflecting the severity of SAP. Our findings revealed that there were no significant differences in regard to the APACHE II score, Ranson score, and CT severity index of 100 SAP patients and 70 SAP&IBD patients, suggesting that the presence of intestinal mucosal barrier dysfunction in SAP patients might not be related to the severity of SAP. However, this does not rule out the possibility that the severity of SAP could be a risk factor for intestinal mucosal barrier dysfunction. In addition, our conclusion is based on limited sample size and the possibility of certain data biases in the study, which may impact the generalizability of our findings and thus requires further exploration. Recent studies have further indicated the importance of miRNAs and their roles in multiple immune response‐related disorders, especially in IBD. 30 The increased expression of T‐synthase is thought to help improve the abnormal protein folding and O‐glycosylation of Muc2, which is a protein found in the mucus barrier of the intestine. This improvement in the mucus barrier helps prevent damage and maintain its integrity. 31 Herein, we found elevated levels of serum miR‐1‐3p and reduced mRNA expression of T‐synthase in peripheral blood B lymphocytes of SAP&IBD patients. In addition, our findings showed a negative correlation between serum expression of miR‐1‐3p and T‐synthase mRNA expression in peripheral blood B lymphocytes in SAP&IBD patients. This is consistent with previous research that reported that elevated miR‐1‐3p levels in the colons of older individuals can disrupt the mucus barrier by directly targeting T‐synthase, potentially contributing to the breakdown of intestinal homeostasis. 32 T‐synthase, the rate‐limiting enzyme in O‐glycosylation, has been shown to have decreased enzymatic activity and protein levels in aged colons, thereby affecting its role in maintaining the physiochemical functions of intestinal homeostasis and colonic mucus stability. 24 Herein, our analyses revealed that there were significant correlations between miR‐1‐3p/T‐synthase mRNA and clinical parameters in SAP&IBD patients. The serum level of miR‐1‐3p was positively correlated with the levels of CRP, Ranson score, IL‐6, DAO, and D‐Lactate, whereas the mRNA level of T‐synthase was negatively correlated with the levels of D‐Lactate, IL‐6, and DAO in SAP&IBD patients. These findings provide novel insight into the involvement of miR‐1‐3p and T‐synthase in the pathogenesis of SAP&IBD. Furthermore, our study also highlights the potential diagnostic value of miR‐1‐3p and T‐synthase in SAP patients with IBD. Previously, raised levels of serum miRNAs such as miR‐9, miR‐7, miR‐141, and miR‐122 have been identified as noninvasive biomarkers of AP. 33 However, the diagnostic significance of miR‐1‐3p and T‐synthase on IBD in SAP patients had not been previously explored. We generated ROC curves and discovered that serum miR‐1‐3p level >1.245, T‐synthase mRNA level 0.555 could assist in the diagnosis of IBD in SAP patients. In addition, our logistic multivariate regression analysis also revealed that the levels of serum miR‐1‐3p and T‐synthase mRNA in peripheral blood B lymphocytes were independently associated with IBD in SAP patients. It has been previously reported that miR‐1‐3p plays a role in regulating the endothelial system to help balance the nitric oxide and endothelin systems, correct endothelial dysfunction, protect endothelial function, and ameliorate chronic heart failure. 34 Elevated expression of miR‐1‐3p during incident ischemia has also been identified as a possible risk factor for secondary cardiovascular events. 35 On the other hand, research has shown that mice lacking the gene for intestinal C1galt1, which is involved in T‐synthase activity, developed spontaneous colitis primarily in the distal colon and mucus layer impairment. 36 Together, these findings provide evidence that miR‐1‐3p and T‐synthase are independent risk factors for IBD in SAP patients. In conclusion, our findings provide evidence for the involvement of miR‐1‐3p and T‐synthase in the development of IBD in SAP patients. Our findings suggest that serum levels of miR‐1‐3p and T‐synthase in peripheral blood B lymphocytes are independent risk factors for IBD diagnosis in SAP patients. Nevertheless, further large‐scale and multicenter studies are necessary to confirm these findings and to fully understand the clinical application of miR‐1‐3p and T‐synthase in predicting the risk of IBD in SAP patients. In addition, in this study, we only studied the value of miR‐1‐3p and T‐synthase levels in peripheral blood in assessing the risk of intestinal mucosal barrier dysfunction in SAP patients, without focusing on the treatment plan and clinical results of patients. Successful interventions, such as stone removal and/or stent insertion, may alleviate symptoms and affect clinical results. Additionally, the prognostic value of miR‐1‐3p and T‐synthase levels in peripheral blood in SAP patients deserves further investigation.

Acute pancreatitis (AP) is a global leading cause of hospital admission for gastrointestinal disorders, characterized by elevated pancreatic enzymes, pancreatic changes in abdominal imaging, and severe abdominal pain. 1 Moreover, its incidence continues to exhibit a rising trend all over the world. 2 AP can range from mild, self‐limiting cases that require only supportive care to severe AP (SAP), which can lead to life‐threatening complications. 3 Approximately 20% of AP patients develop moderate or SAP, with necrosis of organ failure or peripancreatic or pancreatic tissue, or both, with a substantial mortality rate of 20%–40%. 4 In the past 10 years, the treatment method of AP has undergone considerable improvements. 5 Unfortunately, despite advancements in treatment methods and access to care, AP continues to result in significant mortality and morbidity. 3 On the other hand, recent evidence has indicated that gastrointestinal failure, primarily mediated by intestinal mucosal barrier dysfunction (IBD), plays a crucial role in the poor outcome of AP and is an independent predictor of SAP, such that SAP‐related IBD management represents a valid strategy to reducing SAP severity. 6 Therefore, it is crucial to advance the search for early and accurate predictors of IBD risk in SAP patients to improve treatment outcomes and patient prognoses. MicroRNAs (miRs) are single‐stranded noncoding RNA molecules that possess the ability to regulate gene expression through translational repression or cleavage, and the discovery of miRs has opened new avenues for the diagnosis and treatment of AP. 7 Due to their stability and ease of detection, miRs have the potential as biomarkers for AP diagnosis and assessment. 8 Unsurprisingly, a plethora of miRNAs are known to participate in the process of IBD in SAP. 9 , 10 , 11 Recent studies have further indicated that miR‐1 can protect against endothelial permeability and alleviate colitis symptoms by restoring impaired barrier function in rat colitis. 12 , 13 However, the role of serum miR‐1‐3p in SAP patients with IBD and its potential as an early predictor remains unclear. T‐synthase (also known as core 1 β1,3‐galactosyltransferase, C1galt1) represents a key enzyme in the process of T antigen generation, whereas deletion of C1galt1 in intestinal epithelial cells is associated with the development of spontaneous colitis and impaired mucus barrier integrity in mice. 14 Moreover, the regulation of T‐synthase was previously correlated with the inability to synthesize T antigen and Tn antigen deposition, leading to a variety of autoimmune diseases. 15 However, domestic and foreign investigations are scarce on the expression levels of T‐synthase in peripheral blood mononuclear cells (PBMCs) of SAP&IBD patients and the correlations between miR‐1‐3p/T‐synthase and IBD in SAP to the best of our knowledge. In lieu of the same, the current study set out to investigate the clinical application value of miR‐1‐3p combined with T‐synthase in predicting the risk of IBD in SAP patients, in an effort to provide novel references for guiding SAP treatment and improving the prognosis of the patients.

All authors declare no conflict of interest.

The current study was authorized by the academic ethics committee of our hospital. All experimental procedures were performed in strict accordance with the Declaration of Helsinki . All the subjects involved were fully informed of the study objective and signed informed consents were obtained prior to sampling. A total of 170 patients with SAP who received treatment at our hospital from June 2020 to June 2022 were s enrolled in this study. Inclusion criteria were as follows: 1. meeting the diagnostic criteria for SAP according to the Guidelines of Diagnosis and Treatment of Severe AP and the Guidelines of Diagnosis and Treatment of AP in China (2013, Shanghai); 2. meeting the diagnostic criteria of AP, which includes the presence of two of the following three characteristics: severe and persistent epigastric pain consistent with AP, radiating to the back; serum amylase activity (or lipase activity) at least three times higher than the upper limit of normal; and characteristic findings of AP in contrast‐enhanced computed tomography, less common magnetic resonance imaging or transabdominal ultrasound 16 ; 3. having clinical manifestations and biochemical changes of AP, along with persistent organ failure (a respiratory, cardiovascular or renal failure that lasts for more than 48 h and cannot recover by itself, involving one or more organs). Exclusion criteria were as follows: 1. patients with concurrent immune diseases; 2. patients with major organ dysfunction or related diseases; 3. patients with psychiatric disorders that impair the ability to participate in the study; 4. patients taking medications that might affect the evaluation of intestinal mucosal barrier function; 5. patients with malignant tumors. Patients were included in the occurrence group (SAP&IBD, n  = 70) upon meeting the following conditions: 1. with critical diseases that might lead to IBD; 2. with symptoms such as abdominal pain, abdominal distension, diarrhea, constipation, or gastrointestinal bleeding, intolerance to food, and signs such as weakening or disappearance of intestinal sounds (excluding changes in intestinal sounds caused by anesthetic drugs) based on the primary disease; 3. plasma endotoxin level > 55.34 EU/L; 4. increased intestinal permeability; 5. with positive bacteria in blood and ascites culture and without other specific infection focus, and with IBD identified by intestinal mucosal pathology. The remaining patients were included in the nonoccurrence group (SAP, n  = 100). During admission, a wide array of baseline data were recorded for the enrolled subjects, including age, gender, body mass index (BMI), diabetes/hypertension/chronic kidney disease complication, etiology (Biliary/Alcoholic/Idiopathic/Hypertriglyceridemia/Post‐ERCP), the Acute Physiology and Chronic Health Evaluation II (APACHE II) score (71 points in total, a higher score represented a severer condition), 17 Ranson score (11 items in total, with 0 and 1 scoring methods for each item. The highest score was 11 points, and a higher score represented a more severe condition), 18 and CT severity index (10 points in total, by the assessment of peripancreatic and pancreatic inflammation degree [0–2 points]), the number and presence of peripancreatic fluid collections (0–2 points), and the degree and presence of pancreatic parenchymal non‐enhancement or necrosis (0–6 points). 19 In addition, on the morning of the first day after admission, 5 mL of fasting blood samples were collected from the elbow vein, and centrifuged at 4°C at 2000 g for 10 min, and the supernatant was transferred to Eppendorf tubes at −80°C for further analysis. The levels of C‐reactive protein (CRP) and interleukin‐6 (IL‐6) levels were determined using Enzyme‐linked immunosorbent assay (ELISA), and procalcitonin (PCT) level was measured by immunoluminescence method, and the kits were purchased from Solarbio (Beijing, China). Blood amylase activity was also detected using the enzyme rate method, and the levels of blood urea nitrogen and serum albumin were determined using an automatic biochemical analyzer (SL800, Hongkang Century Science and Technology Development, Wuhan, Hubei, China). To assess the serum intestinal mucosal barrier‐related indicators, endotoxin levels were determined with the help of Limulus amebocyte lysate turbidimetry, and the kit was purchased from Yes Service Biotech (Shanghai, China); diamine oxidase (DAO) levels were determined by ELISA, and the kit was provided by ADANTI (Wuhan, Hubei, China); D‐lactic acid levels were determined by ELISA, and the kit was provided by SenBeiJia Biological Technology. PBMCs were separated from blood samples using the Ficoll density gradient centrifugation method (Ficoll 1.077 density, Sangon, Shanghai, China). The PBMCs were rinsed thrice with phosphate‐buffered saline (PBS) and resuspended in a PBS solution containing 1% bovine serum albumin. Subsequently, peripheral B lymphocytes were isolated by Dynabeads™ CD19 Pan B (Thermo Fisher Scientific, Shanghai, China) according to the instructions of the manufacturer. 20 Total RNA content was extracted from subject serum/B lymphocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Next, the extracted RNA was converted into the first strand of cDNA with the help of Mirvana miRNA extraction kit (Applied Biosystems, Foster City, CA, USA). RT‐PCR was performed on the cDNA using the ABI7500 system (Applied Biosystems). The reaction conditions were: pre‐denaturation at 95°C for 10 min, denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 34 s, for a total of 40 cycles. The 2 −ΔΔ Ct method was utilized for data analysis, with U6 or GAPDH serving as the internal parameter. 21 Primers were synthesized by Sangon Biotech, and their sequences are shown in Table  1 . Primer sequence. GraphPad Prism 8.01 (GraphPad Software Inc.) and SPSS 21.0 (IBM Corp.) were applied for data mapping and analysis. The normality of the data was tested using the Shapiro–Wilk (W test), and variable data were expressed as mean ± standard deviation , or in count. Non‐paired t ‐test or χ2 test was utilized for comparisons between two groups. The Pearson method was applied for correlation analysis between miR‐1‐3p/T‐synthase mRNA and clinical data of SAP patients with IBD. The diagnostic efficacy of miR‐1‐3p and T‐synthase mRNA in SAP patients with IBD was analyzed using the receiver operating characteristic (ROC) curve. MedCalc‐comparison of ROC curves was used to analyze the difference of area under the curve (AUC). Whether miR‐1‐3p and T‐synthase were independent risk factors for IBD in SAP patients was analyzed by logistic multivariate regression analysis. A value of p  < 0.05 was considered statistically significant.