MCU promotes oxidative stress by regulating SIRT3 to destroy the pancreatic duct epithelial cytoskeleton: In vivo and in vitro hypertriglyceridema-induced pancreatitis

MCU promotes oxidative stress by regulating SIRT3 to destroy the pancreatic duct epithelial cytoskeleton: In vivo and in vitro hypertriglyceridema-induced pancreatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article MCU promotes oxidative stress by regulating SIRT3 to destroy the pancreatic duct epithelial cytoskeleton: In vivo and in vitro hypertriglyceridema-induced pancreatitis Junbo Hong, Qingzi Fu, Liang Zhu, Zhenzhen Yang, Jianhua Wan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6249694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The pathogenesis of hypertriglyceridema-induced pancreatitis (HTGP) is complex and not fully understood. The purpose of this study was to investigate the molecular mechanism of MCU in HTGP. Methods We observed the expression levels of MCU and SIRT3 in both in vivo and in vitro HTGP models, and after intervention with RR, an active inhibitor of MCU, and 3-TYP, an active inhibitor of SIRT3, changes in mitochondrial calcium ions, oxidative stress-related indices, the microfilament cytoskeleton, and monolayer cell permeability were detected. Results In vivo and in vitro experiments revealed the upregulation of MCU and downregulation of SIRT3 in caerulein-treated HPDE6-C7 cells and mice, along with increased mitochondrial calcium accumulation, increased ROS and MDA, decreased GSH, destruction of the microfilament cytoskeleton, and increased monolayer permeability. During in vitro experiments, intervention with RR, an active inhibitor of MCU, reversed the above changes, whereas intervention with 3-TYP, an active inhibitor of SIRT3, further exacerbated the above changes. Conclusions MCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions. Health sciences/Diseases/Gastrointestinal diseases/Pancreatic disease Biological sciences/Immunology/Inflammation/Acute inflammation pancreatitis acute disease pathogenesis oxidative stress cytoskeleton Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Acute pancreatitis (AP) is a common problem in gastroenterology, with an annual incidence of up to 34 per 100,000 person-years in developed countries 1 . About 20% of all patients with AP have moderate-to-severe AP, for which the mortality rate is 20–40% 2–4 . Hypertriglyceridemia is the third leading cause of AP after gallstones and alcohol abuse 5 . In China, the incidence of hypertriglyceridemia-induced pancreatitis (HTGP) in AP patients is increasing every year 6 . Owing to improvements in medical technology, the overall mortality rate of AP has decreased significantly 7 , but the mortality rate of severe AP is still about 28% 8 . Several studies have shown that patients with HTGP are more likely to develop organ failure, systemic inflammatory response syndrome (SIRS), and even death than patients who develop AP due to other causes 9,10 . Therefore, the pathogenesis of AP, especially HTGP, needs to be elucidated, as it is poorly understood. The toxicity of free fatty acids (FFAs) and the inflammatory response and release of large quantities of calcium ions caused by FFAs play key roles in the pathogenesis of HTGP 5 . In HTGP animal models, the accumulation of high levels of FFAs leads to ischemia, triggering acidosis and converting trypsinogen into active trypsin, leading to digestion of the pancreas 11 . An increase in the level of a vasoconstrictor (thromboxane A2) and a decrease in the level of a vasodilator (prostaglandin 2) induced by hypertriglyceridemia (HTG) can lead to excessive constriction of the capillary bed and deterioration of the pancreatic microcirculation 5 . The concept of the pancreatic ductal mucosal barrier (PDMB) was first proposed in 1969 12 . This barrier, which is composed of a tightly packed layer of ductal epithelial cells and mucus, protects the pancreatic parenchyma from the contents of the pancreatic duct, such as bile and trypsin 12 . Actin filaments, key components of the cytoskeleton, are essential for providing mechanical support and maintaining cell morphology 13 . The F-actin cytoskeleton also regulates intercellular conjunctions through interactions with the cell membrane 14 . F-actin binding is necessary to promote the formation of adherens junctions (AJs) and tight junctions (TJs) 15 . Our previous studies revealed that the synthetic cholecystokinin analog caerulein (CAE) can induce AP in the human pancreatic duct epithelial cell line HPDE6-C7 and lead to increased permeability of monolayer cells 16 ; however, the specific mechanism still needs further investigation. The mitochondrial Ca 2+ uniporter (MCU) is located in the inner mitochondrial membrane and functions mainly to transport calcium ions from the cytoplasm to the mitochondria. It plays a crucial role in the regulation of mitochondrial homeostasis, cell survival, and aerobic metabolism 17 . Under pathological conditions, calcium ion accumulation in mitochondria caused by the overexpression of MCU can impair mitochondrial function, increase the production of reactive oxygen species (ROS), decrease ATP synthesis, and even promote cell apoptosis 18 . Excessive uptake of mitochondrial calcium strongly influences the production of mitochondrial ROS 19,20 . Calcium ions may lead to an increase in ROS production by directly altering the performance of mitochondrial membranes 21 . In a rat model of AP, antioxidants significantly reversed apoptosis, suggesting that ROS can directly cause apoptosis 22 . In addition, the inhibition of MCU can significantly alleviate acute pancreatitis in mice; however, the specific mechanism is unclear 23 . The active form of silent information regulator 3 (SIRT3) is located in mitochondria and functions in the deacetylation of target proteins. SIRT3 modulates mitochondrial protein expression and activation, reduces ROS generation, and is important for mitochondrial adaptability and stress response 24 . Recent studies have shown that the activation of SIRT3 can effectively alleviate acute pancreatitis in rats 25,26 , suggesting that the inhibition of SIRT3 plays an important role in the pathogenesis of acute pancreatitis. Although many studies have demonstrated the accumulation of calcium ions in mitochondria and damage to cells by ROS, the roles of MCU-mediated mitochondrial calcium influx and ROS in pancreatic duct epithelial cells in HTGP have not been reported. Whether MCU-related mitochondrial calcium accumulation affects the function of the pancreatic ductal epithelial mucosal barrier and the cytoskeleton of pancreatic ductal epithelial cells also remains undetermined. Therefore, in this study, we investigated the mechanism by which MCU and ROS affect the pancreatic duct mucosal barrier in HTGP by inhibiting the activity of MCU and interfering with ROS production. 2. Results 2.1 An acute pancreatitis model in mice and HPDE6-C7 cells was established successfully The expression of MCU in the pancreatic tissue of the mice in the AP group and HTGP group was significantly greater than that in the control group, and the increasing trend in the HTGP group was more obvious than that in the AP group. The expression of SIRT3 in the pancreatic tissue of the mice in the AP group and HTGP group was significantly lower than that in the control group, and the decreasing trend in the HTGP group was more obvious than that in the AP group. H&E staining revealed prominent tissue edema, significant widening of the interlobular space, and infiltration of many inflammatory cells in the pancreatic tissues of the mice in the AP and HTGP groups (Fig. 1 A-D). The pancreatic histopathological scores of the AP group and HTGP group were significantly greater than those of the control group and HTG group, respectively (Fig. 1 I; AP vs. control, t = 22.13, P < 0.0001; HTG vs. HTGP, t = 24.65, P < 0.0001). No significant difference was recorded in the serum amylase levels between the AP group and the control group (Fig. 1 K, AP vs. control, t = 0.81, P = 0.44), but the serum amylase level in the HTGP group was significantly greater than that in the HTG group and AP group (Fig. 1 K, HTG vs. HTGP, t = 24.65, P < 0.0001; AP vs. HTGP, t = 6.21, P < 0.001). The serum triglyceride levels in the HTG and HTGP groups were significantly greater than those in the control and AP groups (Fig. 1 L, HTG vs. control, t = 7.01, P < 0.001; HTGP vs. AP, t = 6.56, P < 0.001). Immunohistochemical results revealed that the AOD of MCU in the AP and HTGP groups was significantly greater than that in the control and HTG groups (Fig. 1 E-H, and J; AP vs. control, t = 5.27, P < 0.0001; HTG vs. HTGP, t = 6.84, P < 0.0001). The western blotting results were the same as those described above; the level of MCU expression in the AP group was significantly greater than that in the control group (Fig. 2 A and B; AP vs. control, t = 7.63, P < 0.05). The expression level of MCU in the HTGP group was also significantly greater than that in the HTG and AP groups (Fig. 2 A and B; HTGP vs. HTG, t = 7.99, P < 0.01; HTGP vs. AP, t = 5.76, P < 0.01). Similarly, in vitro studies revealed that the level of expression of MCU in the CAE group was significantly greater than that in the control group (Fig. 2 C and D; CAE vs. control, t = 9.11, P < 0.001), and the level of expression of MCU in the TG + CAE group was significantly greater than that in the TG and CAE groups (Fig. 2 C and D; TG + CAE vs. TG, t = 6.71, P < 0.01; TG + CAE vs. CAE, t = 2.89, P < 0.05). The expression level of SIRT3 in the AP and HTGP groups was significantly lower than that in the control and HTG groups (Fig. 2 E; AP vs. control, t = 3.841, P < 0.05; HTGP vs. HTG, t = 15.74, P < 0.0001). The expression level of SIRT3 in the HTGP group was also significantly lower than that in the AP group (Fig. 2 E; HTGP vs. AP, t = 6.105, P < 0.01). The same results were observed via immunohistochemistry (Fig. 2 F and G; AP vs. control, t = 8.067, P < 0.0001; HTGP vs. HTG, t = 9.816, P < 0.0001). 2.2 In vitro experiments demonstrated that SIRT3 expression was downregulated in both the CAE and TG + CAE groups, accompanied by a significant increase in the mitochondrial calcium ion concentration and increased oxidative stress (evidenced by elevated ROS and MDA levels, as well as decreased GSH levels). Additionally, microfilament cytoskeleton disruption and increased monolayer cell permeability were observed. Notably, RR intervention significantly reversed these changes. Ruthenium red at a concentration of ≤ 10 µM maintained cell viability at about 90%, whereas ruthenium red at a concentration of > 10 µM significantly inhibited cell viability (Fig. 3 C). Therefore, in subsequent in vitro experiments, we used ruthenium red at a concentration of 10 µM. 3-TYP at a concentration of ≤ 0.25 µM maintained cell viability at about 90%, whereas 3-TYP at a concentration of > 0.25 µM significantly inhibited cell viability (Fig. 3 D and E). Therefore, in subsequent in vitro experiments, we used 3-TYP at a concentration of 0.25 µM. The expression level of SIRT3 in the CAE and TG + CAE groups was significantly lower than that in the control and TG groups (Fig. 3 A and B; CAE vs. control, t = 9.903, P < 0.001; TG + CAE vs. TG, t = 8.730, P < 0.001). The expression level of SIRT3 in the TG + CAE group was also significantly lower than that in the AP group (Fig. 3 A and B; HTGP vs. AP, t = 6.105, P 0.05; TG + RR vs. TG, t = 0.857, P > 0.05). The mean fluorescence intensity of mitochondrial calcium in the CAE group was significantly greater than that in the control group (Fig. 4 A and B; CAE vs. control, t = 15.49, P < 0.0001), and the mean fluorescence intensity of mitochondrial calcium in the TG + CAE group was significantly greater than that in the TG and CAE groups (Fig. 4 A and B; TG + CAE vs. TG, t = 15.64, P < 0.0001; TG + CAE vs. CAE, t = 3.47, P < 0.01). However, ruthenium red significantly decreased the increase in the number of mitochondrial calcium ions in the CAE and TG + CAE groups (Fig. 4 A and B; CAE + RR vs. CAE, T = 9.87, P < 0.0001; TG + CAE + RR vs. TG + RR, t = 12.03, P < 0.0001). The mean green fluorescence intensity of the ROS in the CAE and TG + CAE groups was significantly greater than that in control and TG groups (Fig. 4 C and D; CAE vs. control, 66.49 ± 3.88 vs. 41.82 ± 2.52, t = 13.06, P < 0.0001; TG + CAE vs. TG, 74.33 ± 6.22 vs. 37.79 ± 5.37, t = 10.89, P < 0.0001), and this change was reversed by RR intervention (Fig. 4 C and D; CAE + RR vs. CAE, 44.62 ± 8.23 vs. 66.49 ± 3.88, t = 5.86, P < 0.001; TG + CAE + RR vs. TG + CAE, 49.69 ± 4.73 vs. 74.33 ± 6.22, t = 7.73, P < 0.0001). The MDA concentrations in the CAE and TG + CAE groups were significantly greater than those in the control and TG groups (CAE vs. control, 0.236 ± 0.008 vs. 0.117 ± 0.006, t = 20.04, P < 0.0001; TG + CAE vs. TG, 0.267 ± 0.011 vs. 0.123 ± 0.006, t = 20.46, P < 0.0001) (Fig. 5 A), and the MDA concentration increased more significantly in the TG + CAE group than in the CAE group (TG + CAE vs. CAE, 0.267 ± 0.011 vs. 0.236 ± 0.008, t = 4.063, P < 0.05). Ruthenium red intervention significantly decreased the increase in the MDA concentration in the CAE and TG + CAE groups (Fig. 5 A, TG + CAE + RR vs. TG + CAE, 0.152 ± 0.007 vs. 0.267 ± 0.011, t = 15.59, P < 0.0001; CAE + RR vs. CAE, 0.14 ± 0.014 vs. 0.236 ± 0.008, t = 9.886, P < 0.001). Compared with those in the control and TG + CAE groups, the GSH concentrations in the CAE and TG + CAE groups presented the opposite trend in terms of the MDA concentration (Fig. 5 B, CAE vs. control, 0.435 ± 0.041 vs. 0.817 ± 0.04, t = 11.51, P < 0.001; TG + CAE vs. TG, 0.29 ± 0.067 vs. 0.751 ± 0.074, t = 8.01, P < 0.01); however, this trend was weakened by RR intervention (Fig. 5 B, TG + CAE + RR vs. TG + CAE, 0.689 ± 0.018 vs. 0.29 ± 0.067, t = 9.926, P < 0.001; CAE + RR vs. CAE, 0.722 ± 0.055 vs. 0.435 ± 0.041, t = 7.266, P < 0.01). In the control and TG groups, the microfilaments were regularly arranged in bundles. In the CAE and TG + CAE groups, the structural arrangement of the microfilaments was disordered, the bundle arrangement structure disappeared, and the microfilaments were loose and dispersed. After 24 h of RR intervention, the disordered arrangement, release, and dispersion of the microfilament structures in the CAE and TG + CAE groups were partially improved (Fig. 5 D). The permeability of monolayer cells in the CAE and TG + CAE groups was significantly greater than that in the control and TG groups (Fig. 5 C, CAE vs. control, 914.7 ± 114.4 vs. 585 ± 178.9, t = 3.287, P < 0.05; TG + CAE vs. TG, 1239 ± 142.3 vs. 700.01 ± 149.5, t = 3.367, P < 0.05). Additionally, the permeability of monolayer cells increased more significantly in the TG + CAE group than in the CAE group (Fig. 5 C, TG + CAE vs. CAE, 1239 ± 142.3 vs. 914.7 ± 114.4, t = 3.079, P < 0.05). Ruthenium red intervention significantly decreased the increase in monolayer cell permeability in the CAE and TG + CAE groups (Fig. 5 C, TG + CAE + RR vs. TG + CAE, 786.3 ± 237.6 vs. 1239 ± 142.3, t = 2.833, P < 0.05; CAE + RR vs. CAE, 617.2 ± 179 vs. 914.7 ± 114.4, t = 4.085, P < 0.05). 2.3 In vitro experiments revealed that intervention with 3-TYP, a specific inhibitor of SIRT3 activity, significantly exacerbated mitochondrial calcium ion accumulation, enhanced oxidative stress, disrupted the microfilament cytoskeleton, and increased monolayer cell permeability in both the AP and HTGP groups. Intervention with 3-TYP significantly increased the accumulation of mitochondrial calcium ions in the CEA and TG + CAE groups (Fig. 6 A and B; CAE + TYP vs. CAE, T = 8.86, P < 0.0001; TG + CAE + TYP vs. TG + RR, t = 11.02, P < 0.0001). Intervention with 3-TYP significantly increased ROS accumulation (Fig. 6 C and D, TG + CAE + TYP vs. TG + CAE, 0.389 ± 0.012 vs. 0.267 ± 0.011, t = 12.3, P < 0.001; CAE + TYP vs. CAE, 0.338 ± 0.028 vs. 0.236 ± 0.008, t = 6.098, P < 0.01) and the MDA concentration (Fig. 7 A, TG + CAE + TYP vs. TG + CAE, 0.389 ± 0.012 vs. 0.267 ± 0.011, t = 12.3, P < 0.001; CAE + TYP vs. CAE, 0.338 ± 0.028 vs. 0.236 ± 0.008, t = 6.098, P < 0.01) in the CAE and TG + CAE groups but decreased the GSH concentration in the CAE and TG + CAE groups (Fig. 7 B, TG + CAE + TYP vs. TG + CAE, 0.151 ± 0.016 vs. 0.29 ± 0.067, t = 3.509, P < 0.05, CAE + TYP vs. CAE, 0.244 ± 0.022 vs. 0.435 ± 0.041, t = 7.066, P < 0.01). After 24 h of 3-TYP intervention, the disordered arrangement, release, and dispersion of the microfilament structures in the CAE and TG + CAE groups worsened (Fig. 7 D). 3-TYP intervention significantly increased monolayer cell permeability in the CAE and TG + CAE groups (Fig. 7 C, TG + CAE + TYP vs. TG + CAE, 1443 ± 107.2 vs. 1206 ± 87.45, t = 2.972, P < 0.05; CAE + TYP vs. CAE, 1140 ± 74.79 vs. 981.3 ± 52.98, t = 2.992, P < 0.05). 3. Discussion Caerulein, a cholecystokinin analog, is widely used to induce AP in vivo 27 and in vitro 28–30 . In our study, H&E staining revealed obvious tissue edema, significant widening of the interlobular space, and infiltration of many inflammatory cells in the pancreatic tissues of the mice in the AP and HTGP groups, all of which indicated that the CAE-induced AP model mice successfully developed. In animal studies, the level of MCU expression in the AP group was significantly greater than that in the control group. In an in vitro study, the level of MCU expression in the CAE and TG + CAE groups was significantly greater than that in the control and TG groups, which was consistent with the results of the in vivo study. Moreover, significant changes in mitochondrial calcium ions, ROS, MDA, GSH, microfilament morphology, and monocyte permeability were also observed in the CAE and TG + CAE groups compared with those in the control group after 24 h of co-culture with HPDE6-C7 cells. Therefore, we hypothesized that the co-culture of CAE with HPDE6-C7 may constitute a new model of in vitro AP, which was also reported in our previous studies 31,32 . In this study, the serum triglyceride levels were significantly greater in the hypertriglyceridemia group than in the non-hypertriglyceridemia group. Metabolites of triglycerides, i.e., FFAs, are toxic to pancreatic tissue 5 and play important roles in the development of AP 33 . Unsaturated fatty acids can not only lead to hypocalcemia associated with a poorer prognosis by binding to calcium ions 34 but also increase the levels of tumor necrosis factor, interleukin-6, and other cytokines, triggering or aggravating AP 35,36 . These findings may explain why, in our study, the degree of pancreatic inflammation and the pathological score of pancreatic tissue in the HTGP group were significantly greater than those in the AP group. In this study, HPDE6-C7 cells were stimulated with CAE, a CCK analog, which significantly increased the levels of calcium ions in the cytoplasm and mitochondria, along with a significant increase in the expression of MCU. These results were similar to our observations in the CAE-induced mouse AP model. Our experimental results also revealed that stimulating HPDE6C7 cells with CAE ( 10–7 mol/L) for 24 h induced changes in the intracellular calcium ion concentration, mitochondrial calcium ion concentration, and ROS and MCU levels in HPDE6-C7 cells. The levels of MCU expression in the AP and HTGP groups were significantly greater than those in the control and TG groups, whereas no significant difference was found in the expression of MCU between the TG group and control group or between the HTGP group and AP group. The main function of MCU is to transport calcium ions from the cytoplasm to the mitochondria. Our results also revealed that the mitochondrial calcium concentrations in the HTGP and AP groups were greater than those in the TG and control groups, respectively, and the increase in calcium concentration in the HTGP group was more prominent than that in the AP group. In vitro cell experiments revealed that after CAE- and TG + CAE-stimulated HPDE6-C7 cells for 24 h, the intracellular MCU expression, mitochondrial calcium ion concentration, and ROS level increased significantly, and the increase in the TG + CAE group was more prominent than that in the CAE group. Under physiological conditions, a certain level of calcium ions is required for mitochondrial oxidative phosphorylation and ATP synthesis 18 . We found that HPDE6C7 cells with low expression of MCU induced by lentivirus transfection presented abnormal growth, further verifying the importance of the MCU gene for HPDE6C7 cells under physiological conditions. However, under pathological conditions, the increase in calcium ion levels in mitochondria caused by high expression of MCU is the primary driver of ROS production in mitochondria. The excessive production of ROS can directly lead to metabolic disorders and cell death 37 . Interestingly, these changes in the mitochondrial calcium ion concentration and ROS can be reversed by treatment with ruthenium red, an active inhibitor of MCU. Therefore, we speculate that the MCU-induced increase in mitochondrial calcium levels may be an important factor leading to AP. The toxic effects of free fatty acids may also be involved, which may lead to an increase in intracellular calcium ions, mitochondrial calcium ions, and ROS 38 , which may explain the greater increase in intracellular calcium ions, mitochondrial calcium ions, and ROS in the TG + CAE group than in the CAE group. The PDMB consists of a tightly packed pancreatic ductal epithelium and mucus that protects the pancreatic parenchyma from the contents of the pancreatic duct, such as bile and trypsin 12 . When PDMB is stimulated by inflammatory factors, septicemia, and chemicals, the cytoskeleton in pancreatic ductal epithelial cells is destroyed, and the tight connections between cells are damaged, resulting in pancreatic tissue edema and destruction; these changes induce AP 39 . Actin filaments (AFs), also known as microfilaments (MFs), are among the main components of the cytoskeleton and play a key role in providing mechanical support and maintaining cell morphology. Damage to the MF cytoskeleton, which is involved in the regulation of intercellular connectivity through interactions with the cell membrane 15 , can ultimately damage the PDMB 40 . Our results revealed that the permeability of monolayer cells in the CAE and TG + CAE groups was significantly greater than that in the control and TG groups, and the degree of increase in the TG + CAE group was greater than that in the CAE group. Additionally, the microfilament skeleton of HPDE6-C7 cells in the CAE and TG + CAE groups was significantly damaged, and the degree of damage to the microfilament skeleton was greater in the TG + CAE group. These changes were reversed by the MCU inhibitor ruthenium red. Therefore, we speculated that mitochondrial calcium overload and oxidative stress induced by an increase in MCU may be the causes of the destruction of the microfilament cytoskeleton and impairment of PDMB function. In an experimental study on Alzheimer’s disease, the inhibition of MCU activity by ruthenium red improved mitochondrial calcium ion accumulation, mitochondrial function, and bioenergetics 41 . Similar results were also reported in a study on a disease model of subarachnoid hemorrhage 42 . Similarly, ruthenium red has a protective effect on cardiac ischemia and reperfusion injury (CIR) by reducing calcium accumulation in mitochondria 43 . Ruthenium red can also reduce the accumulation of calcium ions in the cytoplasm and mitochondria after traumatic brain injury 44 . These findings suggest that MCUs may be potential therapeutic targets for various diseases, including AP. SIRT3 belongs to the Sirtuin family and protects mitochondria from damage 45 . SIRT3 modulates mitochondrial protein expression and activation, reduces ROS generation, and is important for mitochondrial adaptability and stress response 24 . The expression of SIRT3 in the CAE group and TG + CAE group was significantly lower than that in the control group and TG group, and this change was reversed by RR intervention. After intervention with 3-TYP, the levels of active inhibitors of SIRT3, ROS, and MDA in the CAE group and TG + CAE group further increased, the levels of GSH and monolayer cell permeability further decreased, and the microfilament cytoskeleton was further damaged. Therefore, we speculated that MCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions. However, this study had several limitations. First, our study was based on animal and cellular data, which may be different from the real disease state in humans. Second, the specific mechanism by which MCUs increase oxidative stress and impair the function of PDMB needs to be further investigated. 5. Conclusions MCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions. The inhibition of MCU activity can significantly reverse the above changes. Thus, MCU might be a potential target for treating AP. 4. Methods 4.1 Formation of experimental groups To conduct in vitro studies, HPDE6-C7 human pancreatic duct epithelial cells (Shanghai Jingfeng Biotechnology Co., LTD, GT1730C) were treated with CAE (100 nM/L, Amquar, EYS113, USA), triglycerides (TG, 2.5 mM/L, Solarbio, T9420, China), ruthenium red (RR, 10 mM/L, Amquar, EI2414, USA) and 3-(1H-1,2,3-triazol-4-yl) pyridine (TYP, 0.25 µmol/L, Amquar, EI2418, USA) for 24 h and then divided into twelve groups: the control group, TG group, CAE group, TG + CAE group, RR group, TG + RR group, CAE + RR group, TG + CAE + RR group, TYP group, TG + TYP group, CAE + TYP group, and TG + CAE + TYP group. In vivo, 20 mice were randomly divided into the following four groups: the control group, HTG group, AP group, and HTGP group. 4.2 Animals The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (CDYFY-IACUC-202311QR029 and CDYFY-IACUC-202409GR018) and conducted in accordance with the regulatory guidelines. All methods were reported in accordance with ARRIVE guidelines. Four-week-old wild-type male C57BL6/J mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. All the mice were housed at 22–24°C in a specific pathogen-free environment and were provided free access to food and water for four weeks. The mice in the control group and acute AP group were fed an ordinary diet (11001, Boaigang. China), and the mice in the HTG group and HTGP group were fed a high-fat diet (1160HLP, Boaigang, China). After the mice were fed for four weeks, they were intraperitoneally injected with 50 µg/kg CAE (EYS113, Amquar, USA) seven times at 1 h intervals to induce AP. The mice in the control group and HTG group were intraperitoneally injected with the same volume of normal saline. Blood was collected from the inferior vena cava of each mouse to determine amylase and triglyceride levels 24 h after the last intraperitoneal injection. Pancreatic tissue was removed for H&E staining, immunohistochemistry, and WB analysis. 4.3 Cell culture The HPDE6-C7 human pancreatic duct epithelial cell line was purchased from Guangzhou Jenniobio Biotechnology Co., Ltd., China. The cells were cultured in a sterile environment at 37°C and 5% CO 2 . Each 100 mL of complete medium contained 89 mL of MEM (8120294, Gibco, USA), 10 mL of FBS (A511-001, Lonsera, Uruguay), and 1 mL of penicillin/streptomycin (P1400, Solarbio, China). The cells were inoculated in 60 mm diameter sterile Petri dishes (430166, Corning, USA) at a density of 10 3 /mL, and when the cell confluence reached 70%, the cells in the different groups were treated with different concentrations of intervention agents for 24 h. 4.4 H&E staining Pancreatic tissues were immersed in 4% paraformaldehyde solution for 12 h, embedded in paraffin, and cut into thin sections. Three random fields in each section were selected to calculate the pancreatic histopathological score. The pancreatic histopathological scoring was performed following the criteria described by Van Laethem et al . 46 . 4.5 Immunohistochemistry Dewaxing, hydration, and thermal repair were performed, and then, 5 µg/mL primary antibody (A00685-1, Boster, China) was added and incubated overnight at 4°C. After the tissues were incubated with an HRP-labeled linked polymer (KIT-5009, MXB biotechnologies, China) for 40 min at 26°C, the signal was detected using DAB (P0202, Beyotime, China). Three random fields were selected for each sample, and the average optical density (AOD) was calculated using ImageJ software. 4.6 Serum triglyceride and amylase detection Mouse venous blood was stored at 26°C for 2 h and centrifuged at 4°C at 3000 RPM for 15 min, after which the supernatant was collected for analysis. All steps in the procedure were performed in strict accordance with the instructions of the triglyceride detection kit (c061-a, Changchun Huili, China) and the amylase detection kit (C033, Changchun Huili, China). The levels of triglycerides and amylase were calculated on the basis of the absorbance measured via a microplate reader. 4.7 Protein extraction After the medium was removed, the cells were washed three times with PBS (P1020, Solarbio, China). Next, 1000 µL of RIPA lysis solution (P0013K, Beyotime, China) containing 10 µL of phenyl methane sulfonyl fluoride (P0100, Solarbio, China) was added to each 60 mm Petri dish, and the cells were placed on ice for 30 min before centrifugation (4°C at 12000 RPM for 15 min). Then, 200 µL of the supernatant was collected, 50 µL of protein loading buffer (P1015, Solarbio, China) was added, and the mixture was mixed. Next, the mixture was heated at 100°C for 10 min to denature the protein. The total protein was stored in a refrigerator at − 20°C. 4.8 Western blotting analysis Proteins were resolved by SDS-PAGE and then transferred to a 0.22 µm PVDF membrane. The membrane was blocked with a 5% skim milk powder solution at 26°C for 45 min. After three washes with TBST solution, the membrane and primary antibodies (D2Z3B, CST, UK, 1:1000) were incubated at 4°C for 12 h. After washing again with TBST, the membrane and secondary antibodies (5151P, CST, UK, 1:10000) were incubated at 26°C in the dark for 1 h. The fluorescence signal was detected using a Li-COR Odyssey dual-color infrared fluorescence imaging system. 4.9 Cytotoxicity assay A Cell Counting Kit-8 (CCK-8, C0038) was purchased from Beyotime Biotechnology Co., Ltd. HPDE6-C7 cells were seeded in 96-well plates at a density of 5000 cells/100 µL. Five different final concentrations of ruthenium red (1 µmol/L, 5 µmol/L, 10 µmol/L, 50 µmol/L, and 100 µmol/L) were used. After incubation for 24 h in 5% CO 2 at 37°C, 10 µL of CCK-8 solution was added to each well and incubated in the cell culture box for another 1 h. Finally, the absorbance was measured at 450 nm. 4.10 Mitochondrial calcium ion detection DMSO (D8371, Solarbio, China) was added to 50 µg of Rhod-2 AM (R1245MP, Invitrogen, USA) to produce 100 µL of mother liquor at a concentration of 4 mmol/L, which was stored at − 20°C. Next, 6 µL of mother liquor was added to 5994 µL of HBSS (H1025, Solarbio, China) to prepare a working solution with a concentration of 4 mmol/L. After the medium was removed and the cells were washed three times with HBSS, 1 mL of stock solution was added to each 60 mm petri dish and incubated at 37°C for 30 min. After washing once with PBS, 1 mL of HBSS was added, and the mixture was incubated at 37°C for 30 min. After washing with PBS again two times, 1 mL of mitochondrial green fluorescent probe (Mito-Tracker Green, C1048, Beyotime, China) diluted 5000 times with HBSS was added, and the mixture was incubated at 37°C for 15 min. The red and green fluorescence signals were imaged under an inverted fluorescence microscope after the samples were washed twice with PBS. After three fields of view were randomly selected for each sample, ImageJ software was used to calculate the mean fluorescence intensity value for subsequent statistical analysis. 4.11 ROS detection When the degree of HPDE6-C7 cell fusion in a 60 mm diameter sterile Petri dish reached 70%, the cells were washed with PBS three times, and then 2 mL of serum-free medium supplemented with 10 mM/L DCFH-DA (S0033S, Beyotime, China) was added to each dish. The cells were incubated for 20 min in a cell culture box at 37°C and washed with serum-free cell culture medium three times. Then, the green fluorescence images captured under a fluorescence microscope were analyzed via ImageJ software. Similarly, we randomly selected three random regions to calculate the average fluorescence intensity for subsequent statistical analysis. 4.12 MDA detection The cells in the six-well plate were lysed with RIPA lysis buffer (P0013K, Beyotime, China) for 30 min and then centrifuged. The supernatant was collected for subsequent analysis. An MDA detection kit (S0131S, Beyotime, China) was purchased from Biotime Biotechnology Co., Ltd. The MDA test working fluid and MDA standard were prepared following the manufacturer’s instructions. In total, 100 µL of a standard substance at different concentrations and different groups of samples to be tested was added to 200 µL of the MDA detection working solution and then heated in boiling water for 15 min. After cooling to 26°C and centrifugation (1000 ×g, 26°C, 10 min), 200 µL of the supernatant was collected and added to a 96-well plate. The absorbance was subsequently measured at 532 nm via a microplate reader. The concentration of MDA was calculated on the basis of a standard curve. A BCA protein concentration determination kit (P0010, Beyotime, China) was purchased from Biotime Biotechnology Co., Ltd. The BCA working solution and protein standard were prepared following the manufacturer’s instructions. In each well of the 96-well plate, 20 µL of protein standard or test sample at different concentrations was added successively, followed by the addition of 200 µL of BCA working solution. After the plate was incubated for 30 min at 37°C, the absorbance was measured at 562 nm using a microplate reader. The protein concentration of each group was calculated based on a standard curve. The amount of MDA per milligram of protein was calculated by dividing the concentration of MDA by the corresponding total protein concentration. 4.13 GSH detection After HPDE6-C7 cells were washed with PBS three times, the cells were resuspended and lysed by ultrasonication. The broken cell suspension was centrifuged at 3500 rpm for 10 min, after which 0.1 mL of the supernatant was removed for subsequent analysis. Following the instructions of the GSH and GSSH determination kits (S0053, Beyotime, China), 0.1 mL of reagent 1, 0.1 mL of reagent 2, and 0.025 mL of reagent 3 were added to the wells containing the blank, standard, and test samples, respectively. The plate was left undisturbed for 5 min, after which the absorbance was measured at 405 nm using a microplate reader. 4.14 Immunofluorescence microfilament staining After HPDE6-C7 cells were washed on sterile glass plates twice with PBS at 37°C, 4% paraformaldehyde was added at 26°C for 10 min. The cells were again washed twice with PBS, and then 0.5% Triton X-100 was added for 5 min. Next, 200 µL of phalloidin (200 nmol/L; CA1610, Solarbio, China) was added to the glass plate and incubated at 37°C in the dark for 30 min. After washing twice with PBS, 200 µL of DAPI (100 nmol/L; S2110, Solarbio, China) was added to stain the nuclei. Finally, the morphological characteristics of the microfilaments were observed under a fluorescence microscope. 4.15 Permeability test of monolayer cells First, 100 µL of complete medium containing about 100,000 cells was added to the apical compartment of the Transwell system (3413, Corning, USA), and 600 µL of complete medium was added to the basolateral compartment. After 24 h of culture in an incubator at 37°C, the different groups were treated with the corresponding intervention agent for 24 h. After the culture medium was removed and the cells were washed with PBS three times, 200 µL of FITC-Dextran 4000 (1 mg/mL; 46944, Sigma, USA) was added to the apical compartment, and 600 µL of PBS was added to the basolateral compartment. The cells were then incubated for 2 h. After incubation, 2 µL of liquid from the basolateral compartment was removed from the 96-well plate, and the absorbance was measured via a microplate reader (excitation/emission wavelength: 490/520 nm). 4.16 Statistical methods The measurement data were statistically analyzed via the unpaired sample t-test in GraphPad Prism 8 software, and P < 0.05 was considered statistically significant. Declarations Additional information The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the National Natural Science Foundation of China [grant numbers 82460134]; Natural Science Foundation of Jiangxi Province [20232BAB206021]. Author Contribution Shiyu Zhang: Conceptualization, Methodology, Software. Junbo Hong.: Data curation, Writing- Original draft preparation. Qingzi Fu: Data curation, Writing- Original draft preparation. Liang Zhu: Visualization. Zhenzhen Yang: Supervision. Jianhua Wan: Software, Validation. Qiaofeng Chen, Data curation. Peng Chen: Writing- Reviewing and Editing. Acknowledgements This work was supported by the Key Laboratory Project of Digestive Diseases in Jiangxi Province (2024SSY06101), and Jiangxi Clinical Research Center for Gastroenterology (20223BCG74011). Data Availability Data will be made available on request from the corresponding author. References Xiao, A. Y. et al. Global incidence and mortality of pancreatic diseases: a systematic review, meta-analysis, and meta-regression of population-based cohort studies. Lancet Gastroenterol. Hepatol. 1 , 45-55. https://doi.org/10.1016/s2468-1253(16)30004-8 (2016) Schepers, N. J. et al. Impact of characteristics of organ failure and infected necrosis on mortality in necrotising pancreatitis. Gut 68 , 1044-1051. https://doi.org/10.1136/gutjnl-2017-314657 (2019) Gurusamy, K. S., Belgaumkar, A. P., Haswell, A., Pereira, S. 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Immunopharmacol. 117 , 109981. https://doi.org/10.1016/j.intimp.2023.109981 (2023) Van Laethem, J. L. et al. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology 108 , 1917-1922. https://doi.org/10.1016/0016-5085(95)90158-2 (1995) Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6249694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432150895,"identity":"a0782937-49db-4ad2-ac48-c19b98075780","order_by":0,"name":"Junbo Hong","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Junbo","middleName":"","lastName":"Hong","suffix":""},{"id":432150896,"identity":"72d29123-d8e9-4b78-ade0-533b94066774","order_by":1,"name":"Qingzi Fu","email":"","orcid":"","institution":"Department of Medical Genetics, Jiangxi Maternal and Child Health Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qingzi","middleName":"","lastName":"Fu","suffix":""},{"id":432150898,"identity":"e96a24a9-c336-4e90-b91a-9a947c236541","order_by":2,"name":"Liang Zhu","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zhu","suffix":""},{"id":432150899,"identity":"6d7d66dd-7931-4698-9e1d-850f1e03567b","order_by":3,"name":"Zhenzhen Yang","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Zhenzhen","middleName":"","lastName":"Yang","suffix":""},{"id":432150900,"identity":"8044ed19-2bc9-430c-a5f1-97077817526d","order_by":4,"name":"Jianhua Wan","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Wan","suffix":""},{"id":432150901,"identity":"1bf383e3-b0d8-4280-9553-ec2fb04399db","order_by":5,"name":"Qiaofeng Chen","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Qiaofeng","middleName":"","lastName":"Chen","suffix":""},{"id":432150902,"identity":"61fc15ee-2f2d-4f68-b64b-56a8854d39a6","order_by":6,"name":"Peng Chen","email":"","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Chen","suffix":""},{"id":432150903,"identity":"401b19d2-36a9-4931-af4b-6e508181a304","order_by":7,"name":"Shiyu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYFCCBIYPDAY2cvbHmw9ABA4Q1sI4g6EizZjhzLEEUrScOZzIcMPHgDgt5uw5hs28bYeBGnm+PfjZxiDHdyOB8XMBHi2WPW9AWtLzmKV7txv2tjEYS95IYJaegUeLwY0c88e8bdbFbDJnt0kztjEkbriRwMbMg18LyBbmxB6JnGcgLfXEaeE545w4QyKHDaQlwYCgljPPChvnAAPZgOeYmWTPOQnDmWceNkvj1XI8eWPDG2BUGrA3P5P4UWYjz3c8+eBnfFoYGDgMmJAUSAAxYwNeDQwM7A8YfxBQMgpGwSgYBSMcAAB4+lCqBzWZxwAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Shiyu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-18 05:54:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6249694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6249694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79431034,"identity":"fed03b49-6843-4b7c-8b44-5a3009b311bc","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":677583,"visible":true,"origin":"","legend":"\u003cp\u003eMouse models of AP, HTG and HTGP were successfully constructed. A-D, HE staining of pancreatic tissue from the mice in each group. E-H, Immunohistochemical staining images of MCU in the pancreatic tissue of the mice in each group. The brown region in the figure is the MCU-positive region, and the darker the brown region is, the greater the expression of MCU. I, Pancreatic histopathological scores of the mice in each group. J, AOD values of the MCU immunohistochemistry results of the mice in each group. K, Blood amylase levels in each group of mice; L, Serum triglyceride levels of the mice in each group. AP, acute pancreatitis; HTGP, hypertriglyceridema-induced pancreatitis; average optical density, AOD; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/629badf04c9c8a9e8e1be7a1.jpeg"},{"id":79431038,"identity":"3cebadba-de6f-41a8-84ae-8d832bccd214","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":941704,"visible":true,"origin":"","legend":"\u003cp\u003eA, Expression of MCU in the pancreatic tissues of each group of mice; COX IV was used as a loading control for western blotting. B, Relative expression level of MCU in the pancreatic tissues of each group of mice. The above results were obtained from three independent experiments. C, Expression of MCU in the HPDE6-C7 cells of each group. COX IV was used as a loading control for western blotting. D, Relative expression level of MCU in the HPDE6-C7 cells of each group. The above results were obtained from three independent experiments. E, Expression of SIRT3 in the pancreatic tissues of each group of mice. COX IV was used as a loading control for western blotting. F, AOD level of SIRT3 expression in the pancreatic tissues of each group of mice. G - H, Immunohistochemistry of SIRT3 expression in the pancreatic tissues of each group of mice. AP, acute pancreatitis; HTGP, hypertriglyceridemia-induced pancreatitis; average optical density, AOD. *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/e485ce30db1928a5df6ba146.jpeg"},{"id":79431916,"identity":"71cad4a2-328c-49f7-9fcc-8092c7abaa1a","added_by":"auto","created_at":"2025-03-28 10:56:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253255,"visible":true,"origin":"","legend":"\u003cp\u003eA, Expression of SIRT3 in HPDE6-C7 cells in each group; COX IV was used as a loading control for western blotting. B, Relative expression level of SIRT3 in HPDE6-C7 cells in each group. The above results were obtained from three independent experiments. C, Cytotoxicity assay of HPDE6-C7 cells cultured with different concentrations of ruthenium red. D-E, Cytotoxicity assay of HPDE6-C7 cells cultured with 3-TYP at different concentrations *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/2c6f38bf726f5667ba688ffd.jpeg"},{"id":79431037,"identity":"f76acd42-56ba-4406-92a7-02886dd5d141","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":652842,"visible":true,"origin":"","legend":"\u003cp\u003eA, Immunofluorescence images of mitochondrial calcium ions in HPDE6-C7 cells from each group. The stronger the red fluorescence is, the higher the concentration of mitochondrial calcium ions. B, The average fluorescence intensity of the mitochondrial calcium ions in HPDE6-C7 cells in each group. C, The average fluorescence intensity of the ROS in HPDE6-C7 cells in each group. D, Immunofluorescence images of the ROS in HPDE6-C7 cells in each group; the stronger the green fluorescence is, the higher the concentration of ROS; reactive oxygen species, ROS; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/1c852346c7e8af855f4d6649.jpeg"},{"id":79431035,"identity":"f25f0330-821f-4d92-a833-e0dd6c38cf90","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":385706,"visible":true,"origin":"","legend":"\u003cp\u003eA. Comparison of MDA concentrations in HPDE6-C7 cells in each group; B, Comparison of GSH concentration in HPDE6-C7 cells in each group; C, Monolayer cell permeability of HPDE6-C7 cells in each group. D. Microfilament cytoskeleton staining of HPDE6-C7 cells in each group, in which the red is microfilament cytoskeleton and the blue is nucleus. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/42032cfd7a969f49a13cf53d.jpeg"},{"id":79431043,"identity":"73ffc0ca-fc8d-46b5-84d0-a264ca7a4db4","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":682892,"visible":true,"origin":"","legend":"\u003cp\u003eA. Immunofluorescence images of mitochondrial calcium ions of HPDE6-C7 cells in each group. The stronger the red fluorescence, the higher the concentration of mitochondrial calcium ions. B, The average fluorescence intensity of mitochondrial calcium ion of HPDE6-C7 cells in each group; C, The average fluorescence intensity of ROS of HPDE6-C7 cells in each group D, The immunofluorescence images of ROS of HPDE6-C7 cells in each group, the stronger the green fluorescence, the higher the concentration of ROS; reactive oxygen species, ROS; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/c86c8a8ad68fcf7b6c60250d.jpeg"},{"id":79431044,"identity":"f8118e84-8c65-44d4-a480-06f553dc82a6","added_by":"auto","created_at":"2025-03-28 10:48:38","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":370818,"visible":true,"origin":"","legend":"\u003cp\u003eA. Comparison of MDA concentrations in HPDE6-C7 cells in each group; B, Comparison of GSH concentration in HPDE6-C7 cells in each group; C, Monolayer cell permeability of HPDE6-C7 cells in each group. D. Microfilament cytoskeleton staining of HPDE6-C7 cells in each group, in which the red is microfilament cytoskeleton and the blue is nucleus. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/a014d763a5f4fe57e749b1ad.jpeg"},{"id":82614800,"identity":"aaad6cb9-6d4c-4b3f-bd2a-94e7de073960","added_by":"auto","created_at":"2025-05-13 11:32:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5305298,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/95aaf539-f9c9-40af-a750-8acdbd1d12e3.pdf"},{"id":79432193,"identity":"8b6fb71f-254e-4802-980e-97fc919d4ed6","added_by":"auto","created_at":"2025-03-28 11:04:38","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":883022,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6249694/v1/9dbc569db9057b2632268fb7.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"MCU promotes oxidative stress by regulating SIRT3 to destroy the pancreatic duct epithelial cytoskeleton: In vivo and in vitro hypertriglyceridema-induced pancreatitis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute pancreatitis (AP) is a common problem in gastroenterology, with an annual incidence of up to 34 per 100,000 person-years in developed countries \u003csup\u003e1\u003c/sup\u003e. About 20% of all patients with AP have moderate-to-severe AP, for which the mortality rate is 20\u0026ndash;40% \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Hypertriglyceridemia is the third leading cause of AP after gallstones and alcohol abuse \u003csup\u003e5\u003c/sup\u003e. In China, the incidence of hypertriglyceridemia-induced pancreatitis (HTGP) in AP patients is increasing every year \u003csup\u003e6\u003c/sup\u003e. Owing to improvements in medical technology, the overall mortality rate of AP has decreased significantly \u003csup\u003e7\u003c/sup\u003e, but the mortality rate of severe AP is still about 28% \u003csup\u003e8\u003c/sup\u003e. Several studies have shown that patients with HTGP are more likely to develop organ failure, systemic inflammatory response syndrome (SIRS), and even death than patients who develop AP due to other causes \u003csup\u003e9,10\u003c/sup\u003e. Therefore, the pathogenesis of AP, especially HTGP, needs to be elucidated, as it is poorly understood.\u003c/p\u003e \u003cp\u003eThe toxicity of free fatty acids (FFAs) and the inflammatory response and release of large quantities of calcium ions caused by FFAs play key roles in the pathogenesis of HTGP \u003csup\u003e5\u003c/sup\u003e. In HTGP animal models, the accumulation of high levels of FFAs leads to ischemia, triggering acidosis and converting trypsinogen into active trypsin, leading to digestion of the pancreas \u003csup\u003e11\u003c/sup\u003e. An increase in the level of a vasoconstrictor (thromboxane A2) and a decrease in the level of a vasodilator (prostaglandin 2) induced by hypertriglyceridemia (HTG) can lead to excessive constriction of the capillary bed and deterioration of the pancreatic microcirculation \u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe concept of the pancreatic ductal mucosal barrier (PDMB) was first proposed in 1969 \u003csup\u003e12\u003c/sup\u003e. This barrier, which is composed of a tightly packed layer of ductal epithelial cells and mucus, protects the pancreatic parenchyma from the contents of the pancreatic duct, such as bile and trypsin \u003csup\u003e12\u003c/sup\u003e. Actin filaments, key components of the cytoskeleton, are essential for providing mechanical support and maintaining cell morphology \u003csup\u003e13\u003c/sup\u003e. The F-actin cytoskeleton also regulates intercellular conjunctions through interactions with the cell membrane \u003csup\u003e14\u003c/sup\u003e. F-actin binding is necessary to promote the formation of adherens junctions (AJs) and tight junctions (TJs) \u003csup\u003e15\u003c/sup\u003e. Our previous studies revealed that the synthetic cholecystokinin analog caerulein (CAE) can induce AP in the human pancreatic duct epithelial cell line HPDE6-C7 and lead to increased permeability of monolayer cells\u003csup\u003e16\u003c/sup\u003e; however, the specific mechanism still needs further investigation.\u003c/p\u003e \u003cp\u003eThe mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e uniporter (MCU) is located in the inner mitochondrial membrane and functions mainly to transport calcium ions from the cytoplasm to the mitochondria. It plays a crucial role in the regulation of mitochondrial homeostasis, cell survival, and aerobic metabolism \u003csup\u003e17\u003c/sup\u003e. Under pathological conditions, calcium ion accumulation in mitochondria caused by the overexpression of MCU can impair mitochondrial function, increase the production of reactive oxygen species (ROS), decrease ATP synthesis, and even promote cell apoptosis \u003csup\u003e18\u003c/sup\u003e. Excessive uptake of mitochondrial calcium strongly influences the production of mitochondrial ROS \u003csup\u003e19,20\u003c/sup\u003e. Calcium ions may lead to an increase in ROS production by directly altering the performance of mitochondrial membranes \u003csup\u003e21\u003c/sup\u003e. In a rat model of AP, antioxidants significantly reversed apoptosis, suggesting that ROS can directly cause apoptosis \u003csup\u003e22\u003c/sup\u003e. In addition, the inhibition of MCU can significantly alleviate acute pancreatitis in mice; however, the specific mechanism is unclear\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe active form of silent information regulator 3 (SIRT3) is located in mitochondria and functions in the deacetylation of target proteins. SIRT3 modulates mitochondrial protein expression and activation, reduces ROS generation, and is important for mitochondrial adaptability and stress response \u003csup\u003e24\u003c/sup\u003e. Recent studies have shown that the activation of SIRT3 can effectively alleviate acute pancreatitis in rats\u003csup\u003e25,26\u003c/sup\u003e, suggesting that the inhibition of SIRT3 plays an important role in the pathogenesis of acute pancreatitis.\u003c/p\u003e \u003cp\u003eAlthough many studies have demonstrated the accumulation of calcium ions in mitochondria and damage to cells by ROS, the roles of MCU-mediated mitochondrial calcium influx and ROS in pancreatic duct epithelial cells in HTGP have not been reported. Whether MCU-related mitochondrial calcium accumulation affects the function of the pancreatic ductal epithelial mucosal barrier and the cytoskeleton of pancreatic ductal epithelial cells also remains undetermined. Therefore, in this study, we investigated the mechanism by which MCU and ROS affect the pancreatic duct mucosal barrier in HTGP by inhibiting the activity of MCU and interfering with ROS production.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cb\u003e2.1 An acute pancreatitis model in mice and HPDE6-C7 cells was established successfully The expression of MCU in the pancreatic tissue of the mice in the AP group and HTGP group was significantly greater than that in the control group, and the increasing trend in the HTGP group was more obvious than that in the AP group. The expression of SIRT3 in the pancreatic tissue of the mice in the AP group and HTGP group was significantly lower than that in the control group, and the decreasing trend in the HTGP group was more obvious than that in the AP group.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eH\u0026amp;E staining revealed prominent tissue edema, significant widening of the interlobular space, and infiltration of many inflammatory cells in the pancreatic tissues of the mice in the AP and HTGP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). The pancreatic histopathological scores of the AP group and HTGP group were significantly greater than those of the control group and HTG group, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI; AP vs. control, t\u0026thinsp;=\u0026thinsp;22.13, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; HTG vs. HTGP, t\u0026thinsp;=\u0026thinsp;24.65, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). No significant difference was recorded in the serum amylase levels between the AP group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, AP vs. control, t\u0026thinsp;=\u0026thinsp;0.81, P\u0026thinsp;=\u0026thinsp;0.44), but the serum amylase level in the HTGP group was significantly greater than that in the HTG group and AP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, HTG vs. HTGP, t\u0026thinsp;=\u0026thinsp;24.65, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; AP vs. HTGP, t\u0026thinsp;=\u0026thinsp;6.21, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The serum triglyceride levels in the HTG and HTGP groups were significantly greater than those in the control and AP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, HTG vs. control, t\u0026thinsp;=\u0026thinsp;7.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; HTGP vs. AP, t\u0026thinsp;=\u0026thinsp;6.56, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eImmunohistochemical results revealed that the AOD of MCU in the AP and HTGP groups was significantly greater than that in the control and HTG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H, and J; AP vs. control, t\u0026thinsp;=\u0026thinsp;5.27, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; HTG vs. HTGP, t\u0026thinsp;=\u0026thinsp;6.84, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The western blotting results were the same as those described above; the level of MCU expression in the AP group was significantly greater than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B; AP vs. control, t\u0026thinsp;=\u0026thinsp;7.63, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression level of MCU in the HTGP group was also significantly greater than that in the HTG and AP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B; HTGP vs. HTG, t\u0026thinsp;=\u0026thinsp;7.99, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; HTGP vs. AP, t\u0026thinsp;=\u0026thinsp;5.76, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, in vitro studies revealed that the level of expression of MCU in the CAE group was significantly greater than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D; CAE vs. control, t\u0026thinsp;=\u0026thinsp;9.11, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the level of expression of MCU in the TG\u0026thinsp;+\u0026thinsp;CAE group was significantly greater than that in the TG and CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, t\u0026thinsp;=\u0026thinsp;6.71, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; TG\u0026thinsp;+\u0026thinsp;CAE vs. CAE, t\u0026thinsp;=\u0026thinsp;2.89, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression level of SIRT3 in the AP and HTGP groups was significantly lower than that in the control and HTG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; AP vs. control, t\u0026thinsp;=\u0026thinsp;3.841, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; HTGP vs. HTG, t\u0026thinsp;=\u0026thinsp;15.74, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The expression level of SIRT3 in the HTGP group was also significantly lower than that in the AP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE; HTGP vs. AP, t\u0026thinsp;=\u0026thinsp;6.105, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The same results were observed via immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and G; AP vs. control, t\u0026thinsp;=\u0026thinsp;8.067, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; HTGP vs. HTG, t\u0026thinsp;=\u0026thinsp;9.816, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 In vitro experiments demonstrated that SIRT3 expression was downregulated in both the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups, accompanied by a significant increase in the mitochondrial calcium ion concentration and increased oxidative stress (evidenced by elevated ROS and MDA levels, as well as decreased GSH levels). Additionally, microfilament cytoskeleton disruption and increased monolayer cell permeability were observed. Notably, RR intervention significantly reversed these changes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRuthenium red at a concentration of \u0026le;\u0026thinsp;10 \u0026micro;M maintained cell viability at about 90%, whereas ruthenium red at a concentration of \u0026gt;\u0026thinsp;10 \u0026micro;M significantly inhibited cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Therefore, in subsequent in vitro experiments, we used ruthenium red at a concentration of 10 \u0026micro;M. 3-TYP at a concentration of \u0026le;\u0026thinsp;0.25 \u0026micro;M maintained cell viability at about 90%, whereas 3-TYP at a concentration of \u0026gt;\u0026thinsp;0.25 \u0026micro;M significantly inhibited cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E). Therefore, in subsequent in vitro experiments, we used 3-TYP at a concentration of 0.25 \u0026micro;M.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression level of SIRT3 in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly lower than that in the control and TG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B; CAE vs. control, t\u0026thinsp;=\u0026thinsp;9.903, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, t\u0026thinsp;=\u0026thinsp;8.730, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The expression level of SIRT3 in the TG\u0026thinsp;+\u0026thinsp;CAE group was also significantly lower than that in the AP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B; HTGP vs. AP, t\u0026thinsp;=\u0026thinsp;6.105, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was no significant difference in the expression of SIRT3 between the RR and control groups or between the TG\u0026thinsp;+\u0026thinsp;RR and TG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B; RR vs. control, t\u0026thinsp;=\u0026thinsp;1.531, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05; TG\u0026thinsp;+\u0026thinsp;RR vs. TG, t\u0026thinsp;=\u0026thinsp;0.857, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean fluorescence intensity of mitochondrial calcium in the CAE group was significantly greater than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B; CAE vs. control, t\u0026thinsp;=\u0026thinsp;15.49, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the mean fluorescence intensity of mitochondrial calcium in the TG\u0026thinsp;+\u0026thinsp;CAE group was significantly greater than that in the TG and CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, t\u0026thinsp;=\u0026thinsp;15.64, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; TG\u0026thinsp;+\u0026thinsp;CAE vs. CAE, t\u0026thinsp;=\u0026thinsp;3.47, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, ruthenium red significantly decreased the increase in the number of mitochondrial calcium ions in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B; CAE\u0026thinsp;+\u0026thinsp;RR vs. CAE, T\u0026thinsp;=\u0026thinsp;9.87, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR vs. TG\u0026thinsp;+\u0026thinsp;RR, t\u0026thinsp;=\u0026thinsp;12.03, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mean green fluorescence intensity of the ROS in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly greater than that in control and TG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D; CAE vs. control, 66.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 vs. 41.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52, t\u0026thinsp;=\u0026thinsp;13.06, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, 74.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.22 vs. 37.79\u0026thinsp;\u0026plusmn;\u0026thinsp;5.37, t\u0026thinsp;=\u0026thinsp;10.89, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and this change was reversed by RR intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D; CAE\u0026thinsp;+\u0026thinsp;RR vs. CAE, 44.62\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23 vs. 66.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88, t\u0026thinsp;=\u0026thinsp;5.86, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR vs. TG\u0026thinsp;+\u0026thinsp;CAE, 49.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73 vs. 74.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.22, t\u0026thinsp;=\u0026thinsp;7.73, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The MDA concentrations in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups were significantly greater than those in the control and TG groups (CAE vs. control, 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 vs. 0.117\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006, t\u0026thinsp;=\u0026thinsp;20.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, 0.267\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011 vs. 0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006, t\u0026thinsp;=\u0026thinsp;20.46, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and the MDA concentration increased more significantly in the TG\u0026thinsp;+\u0026thinsp;CAE group than in the CAE group (TG\u0026thinsp;+\u0026thinsp;CAE vs. CAE, 0.267\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011 vs. 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, t\u0026thinsp;=\u0026thinsp;4.063, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Ruthenium red intervention significantly decreased the increase in the MDA concentration in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR vs. TG\u0026thinsp;+\u0026thinsp;CAE, 0.152\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 vs. 0.267\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011, t\u0026thinsp;=\u0026thinsp;15.59, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; CAE\u0026thinsp;+\u0026thinsp;RR vs. CAE, 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 vs. 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, t\u0026thinsp;=\u0026thinsp;9.886, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with those in the control and TG\u0026thinsp;+\u0026thinsp;CAE groups, the GSH concentrations in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups presented the opposite trend in terms of the MDA concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, CAE vs. control, 0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041 vs. 0.817\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, t\u0026thinsp;=\u0026thinsp;11.51, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067 vs. 0.751\u0026thinsp;\u0026plusmn;\u0026thinsp;0.074, t\u0026thinsp;=\u0026thinsp;8.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); however, this trend was weakened by RR intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR vs. TG\u0026thinsp;+\u0026thinsp;CAE, 0.689\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 vs. 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067, t\u0026thinsp;=\u0026thinsp;9.926, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CAE\u0026thinsp;+\u0026thinsp;RR vs. CAE, 0.722\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 vs. 0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041, t\u0026thinsp;=\u0026thinsp;7.266, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the control and TG groups, the microfilaments were regularly arranged in bundles. In the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups, the structural arrangement of the microfilaments was disordered, the bundle arrangement structure disappeared, and the microfilaments were loose and dispersed. After 24 h of RR intervention, the disordered arrangement, release, and dispersion of the microfilament structures in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups were partially improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe permeability of monolayer cells in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly greater than that in the control and TG groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, CAE vs. control, 914.7\u0026thinsp;\u0026plusmn;\u0026thinsp;114.4 vs. 585\u0026thinsp;\u0026plusmn;\u0026thinsp;178.9, t\u0026thinsp;=\u0026thinsp;3.287, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; TG\u0026thinsp;+\u0026thinsp;CAE vs. TG, 1239\u0026thinsp;\u0026plusmn;\u0026thinsp;142.3 vs. 700.01\u0026thinsp;\u0026plusmn;\u0026thinsp;149.5, t\u0026thinsp;=\u0026thinsp;3.367, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the permeability of monolayer cells increased more significantly in the TG\u0026thinsp;+\u0026thinsp;CAE group than in the CAE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, TG\u0026thinsp;+\u0026thinsp;CAE vs. CAE, 1239\u0026thinsp;\u0026plusmn;\u0026thinsp;142.3 vs. 914.7\u0026thinsp;\u0026plusmn;\u0026thinsp;114.4, t\u0026thinsp;=\u0026thinsp;3.079, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Ruthenium red intervention significantly decreased the increase in monolayer cell permeability in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR vs. TG\u0026thinsp;+\u0026thinsp;CAE, 786.3\u0026thinsp;\u0026plusmn;\u0026thinsp;237.6 vs. 1239\u0026thinsp;\u0026plusmn;\u0026thinsp;142.3, t\u0026thinsp;=\u0026thinsp;2.833, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CAE\u0026thinsp;+\u0026thinsp;RR vs. CAE, 617.2\u0026thinsp;\u0026plusmn;\u0026thinsp;179 vs. 914.7\u0026thinsp;\u0026plusmn;\u0026thinsp;114.4, t\u0026thinsp;=\u0026thinsp;4.085, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 In vitro experiments revealed that intervention with 3-TYP, a specific inhibitor of SIRT3 activity, significantly exacerbated mitochondrial calcium ion accumulation, enhanced oxidative stress, disrupted the microfilament cytoskeleton, and increased monolayer cell permeability in both the AP and HTGP groups.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntervention with 3-TYP significantly increased the accumulation of mitochondrial calcium ions in the CEA and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B; CAE\u0026thinsp;+\u0026thinsp;TYP vs. CAE, T\u0026thinsp;=\u0026thinsp;8.86, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP vs. TG\u0026thinsp;+\u0026thinsp;RR, t\u0026thinsp;=\u0026thinsp;11.02, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntervention with 3-TYP significantly increased ROS accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP vs. TG\u0026thinsp;+\u0026thinsp;CAE, 0.389\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 vs. 0.267\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011, t\u0026thinsp;=\u0026thinsp;12.3, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CAE\u0026thinsp;+\u0026thinsp;TYP vs. CAE, 0.338\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028 vs. 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, t\u0026thinsp;=\u0026thinsp;6.098, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the MDA concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP vs. TG\u0026thinsp;+\u0026thinsp;CAE, 0.389\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012 vs. 0.267\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011, t\u0026thinsp;=\u0026thinsp;12.3, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; CAE\u0026thinsp;+\u0026thinsp;TYP vs. CAE, 0.338\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028 vs. 0.236\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008, t\u0026thinsp;=\u0026thinsp;6.098, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups but decreased the GSH concentration in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP vs. TG\u0026thinsp;+\u0026thinsp;CAE, 0.151\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016 vs. 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.067, t\u0026thinsp;=\u0026thinsp;3.509, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, CAE\u0026thinsp;+\u0026thinsp;TYP vs. CAE, 0.244\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022 vs. 0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041, t\u0026thinsp;=\u0026thinsp;7.066, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 24 h of 3-TYP intervention, the disordered arrangement, release, and dispersion of the microfilament structures in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups worsened (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e3-TYP intervention significantly increased monolayer cell permeability in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP vs. TG\u0026thinsp;+\u0026thinsp;CAE, 1443\u0026thinsp;\u0026plusmn;\u0026thinsp;107.2 vs. 1206\u0026thinsp;\u0026plusmn;\u0026thinsp;87.45, t\u0026thinsp;=\u0026thinsp;2.972, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; CAE\u0026thinsp;+\u0026thinsp;TYP vs. CAE, 1140\u0026thinsp;\u0026plusmn;\u0026thinsp;74.79 vs. 981.3\u0026thinsp;\u0026plusmn;\u0026thinsp;52.98, t\u0026thinsp;=\u0026thinsp;2.992, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eCaerulein, a cholecystokinin analog, is widely used to induce AP in vivo \u003csup\u003e27\u003c/sup\u003e and in vitro \u003csup\u003e28\u0026ndash;30\u003c/sup\u003e. In our study, H\u0026amp;E staining revealed obvious tissue edema, significant widening of the interlobular space, and infiltration of many inflammatory cells in the pancreatic tissues of the mice in the AP and HTGP groups, all of which indicated that the CAE-induced AP model mice successfully developed. In animal studies, the level of MCU expression in the AP group was significantly greater than that in the control group. In an in vitro study, the level of MCU expression in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly greater than that in the control and TG groups, which was consistent with the results of the in vivo study. Moreover, significant changes in mitochondrial calcium ions, ROS, MDA, GSH, microfilament morphology, and monocyte permeability were also observed in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups compared with those in the control group after 24 h of co-culture with HPDE6-C7 cells. Therefore, we hypothesized that the co-culture of CAE with HPDE6-C7 may constitute a new model of in vitro AP, which was also reported in our previous studies \u003csup\u003e31,32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the serum triglyceride levels were significantly greater in the hypertriglyceridemia group than in the non-hypertriglyceridemia group. Metabolites of triglycerides, i.e., FFAs, are toxic to pancreatic tissue \u003csup\u003e5\u003c/sup\u003e and play important roles in the development of AP \u003csup\u003e33\u003c/sup\u003e. Unsaturated fatty acids can not only lead to hypocalcemia associated with a poorer prognosis by binding to calcium ions \u003csup\u003e34\u003c/sup\u003e but also increase the levels of tumor necrosis factor, interleukin-6, and other cytokines, triggering or aggravating AP \u003csup\u003e35,36\u003c/sup\u003e. These findings may explain why, in our study, the degree of pancreatic inflammation and the pathological score of pancreatic tissue in the HTGP group were significantly greater than those in the AP group.\u003c/p\u003e \u003cp\u003eIn this study, HPDE6-C7 cells were stimulated with CAE, a CCK analog, which significantly increased the levels of calcium ions in the cytoplasm and mitochondria, along with a significant increase in the expression of MCU. These results were similar to our observations in the CAE-induced mouse AP model. Our experimental results also revealed that stimulating HPDE6C7 cells with CAE (\u003csup\u003e10\u0026ndash;7\u003c/sup\u003e mol/L) for 24 h induced changes in the intracellular calcium ion concentration, mitochondrial calcium ion concentration, and ROS and MCU levels in HPDE6-C7 cells. The levels of MCU expression in the AP and HTGP groups were significantly greater than those in the control and TG groups, whereas no significant difference was found in the expression of MCU between the TG group and control group or between the HTGP group and AP group. The main function of MCU is to transport calcium ions from the cytoplasm to the mitochondria. Our results also revealed that the mitochondrial calcium concentrations in the HTGP and AP groups were greater than those in the TG and control groups, respectively, and the increase in calcium concentration in the HTGP group was more prominent than that in the AP group. In vitro cell experiments revealed that after CAE- and TG\u0026thinsp;+\u0026thinsp;CAE-stimulated HPDE6-C7 cells for 24 h, the intracellular MCU expression, mitochondrial calcium ion concentration, and ROS level increased significantly, and the increase in the TG\u0026thinsp;+\u0026thinsp;CAE group was more prominent than that in the CAE group. Under physiological conditions, a certain level of calcium ions is required for mitochondrial oxidative phosphorylation and ATP synthesis \u003csup\u003e18\u003c/sup\u003e. We found that HPDE6C7 cells with low expression of MCU induced by lentivirus transfection presented abnormal growth, further verifying the importance of the MCU gene for HPDE6C7 cells under physiological conditions. However, under pathological conditions, the increase in calcium ion levels in mitochondria caused by high expression of MCU is the primary driver of ROS production in mitochondria. The excessive production of ROS can directly lead to metabolic disorders and cell death \u003csup\u003e37\u003c/sup\u003e. Interestingly, these changes in the mitochondrial calcium ion concentration and ROS can be reversed by treatment with ruthenium red, an active inhibitor of MCU. Therefore, we speculate that the MCU-induced increase in mitochondrial calcium levels may be an important factor leading to AP. The toxic effects of free fatty acids may also be involved, which may lead to an increase in intracellular calcium ions, mitochondrial calcium ions, and ROS \u003csup\u003e38\u003c/sup\u003e, which may explain the greater increase in intracellular calcium ions, mitochondrial calcium ions, and ROS in the TG\u0026thinsp;+\u0026thinsp;CAE group than in the CAE group.\u003c/p\u003e \u003cp\u003eThe PDMB consists of a tightly packed pancreatic ductal epithelium and mucus that protects the pancreatic parenchyma from the contents of the pancreatic duct, such as bile and trypsin \u003csup\u003e12\u003c/sup\u003e. When PDMB is stimulated by inflammatory factors, septicemia, and chemicals, the cytoskeleton in pancreatic ductal epithelial cells is destroyed, and the tight connections between cells are damaged, resulting in pancreatic tissue edema and destruction; these changes induce AP \u003csup\u003e39\u003c/sup\u003e. Actin filaments (AFs), also known as microfilaments (MFs), are among the main components of the cytoskeleton and play a key role in providing mechanical support and maintaining cell morphology. Damage to the MF cytoskeleton, which is involved in the regulation of intercellular connectivity through interactions with the cell membrane \u003csup\u003e15\u003c/sup\u003e, can ultimately damage the PDMB \u003csup\u003e40\u003c/sup\u003e. Our results revealed that the permeability of monolayer cells in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly greater than that in the control and TG groups, and the degree of increase in the TG\u0026thinsp;+\u0026thinsp;CAE group was greater than that in the CAE group. Additionally, the microfilament skeleton of HPDE6-C7 cells in the CAE and TG\u0026thinsp;+\u0026thinsp;CAE groups was significantly damaged, and the degree of damage to the microfilament skeleton was greater in the TG\u0026thinsp;+\u0026thinsp;CAE group. These changes were reversed by the MCU inhibitor ruthenium red. Therefore, we speculated that mitochondrial calcium overload and oxidative stress induced by an increase in MCU may be the causes of the destruction of the microfilament cytoskeleton and impairment of PDMB function. In an experimental study on Alzheimer\u0026rsquo;s disease, the inhibition of MCU activity by ruthenium red improved mitochondrial calcium ion accumulation, mitochondrial function, and bioenergetics \u003csup\u003e41\u003c/sup\u003e. Similar results were also reported in a study on a disease model of subarachnoid hemorrhage \u003csup\u003e42\u003c/sup\u003e. Similarly, ruthenium red has a protective effect on cardiac ischemia and reperfusion injury (CIR) by reducing calcium accumulation in mitochondria \u003csup\u003e43\u003c/sup\u003e. Ruthenium red can also reduce the accumulation of calcium ions in the cytoplasm and mitochondria after traumatic brain injury \u003csup\u003e44\u003c/sup\u003e. These findings suggest that MCUs may be potential therapeutic targets for various diseases, including AP.\u003c/p\u003e \u003cp\u003eSIRT3 belongs to the Sirtuin family and protects mitochondria from damage \u003csup\u003e45\u003c/sup\u003e. SIRT3 modulates mitochondrial protein expression and activation, reduces ROS generation, and is important for mitochondrial adaptability and stress response \u003csup\u003e24\u003c/sup\u003e. The expression of SIRT3 in the CAE group and TG\u0026thinsp;+\u0026thinsp;CAE group was significantly lower than that in the control group and TG group, and this change was reversed by RR intervention. After intervention with 3-TYP, the levels of active inhibitors of SIRT3, ROS, and MDA in the CAE group and TG\u0026thinsp;+\u0026thinsp;CAE group further increased, the levels of GSH and monolayer cell permeability further decreased, and the microfilament cytoskeleton was further damaged. Therefore, we speculated that MCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions.\u003c/p\u003e \u003cp\u003eHowever, this study had several limitations. First, our study was based on animal and cellular data, which may be different from the real disease state in humans. Second, the specific mechanism by which MCUs increase oxidative stress and impair the function of PDMB needs to be further investigated.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eMCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions. The inhibition of MCU activity can significantly reverse the above changes. Thus, MCU might be a potential target for treating AP.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Formation of experimental groups\u003c/h2\u003e \u003cp\u003eTo conduct in vitro studies, HPDE6-C7 human pancreatic duct epithelial cells (Shanghai Jingfeng Biotechnology Co., LTD, GT1730C) were treated with CAE (100 nM/L, Amquar, EYS113, USA), triglycerides (TG, 2.5 mM/L, Solarbio, T9420, China), ruthenium red (RR, 10 mM/L, Amquar, EI2414, USA) and 3-(1H-1,2,3-triazol-4-yl) pyridine (TYP, 0.25 \u0026micro;mol/L, Amquar, EI2418, USA) for 24 h and then divided into twelve groups: the control group, TG group, CAE group, TG\u0026thinsp;+\u0026thinsp;CAE group, RR group, TG\u0026thinsp;+\u0026thinsp;RR group, CAE\u0026thinsp;+\u0026thinsp;RR group, TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;RR group, TYP group, TG\u0026thinsp;+\u0026thinsp;TYP group, CAE\u0026thinsp;+\u0026thinsp;TYP group, and TG\u0026thinsp;+\u0026thinsp;CAE\u0026thinsp;+\u0026thinsp;TYP group.\u003c/p\u003e \u003cp\u003eIn vivo, 20 mice were randomly divided into the following four groups: the control group, HTG group, AP group, and HTGP group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Animals\u003c/h2\u003e \u003cp\u003e The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (CDYFY-IACUC-202311QR029 and CDYFY-IACUC-202409GR018) and conducted in accordance with the regulatory guidelines. All methods were reported in accordance with ARRIVE guidelines. Four-week-old wild-type male C57BL6/J mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. All the mice were housed at 22\u0026ndash;24\u0026deg;C in a specific pathogen-free environment and were provided free access to food and water for four weeks. The mice in the control group and acute AP group were fed an ordinary diet (11001, Boaigang. China), and the mice in the HTG group and HTGP group were fed a high-fat diet (1160HLP, Boaigang, China). After the mice were fed for four weeks, they were intraperitoneally injected with 50 \u0026micro;g/kg CAE (EYS113, Amquar, USA) seven times at 1 h intervals to induce AP. The mice in the control group and HTG group were intraperitoneally injected with the same volume of normal saline. Blood was collected from the inferior vena cava of each mouse to determine amylase and triglyceride levels 24 h after the last intraperitoneal injection. Pancreatic tissue was removed for H\u0026amp;E staining, immunohistochemistry, and WB analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Cell culture\u003c/h2\u003e \u003cp\u003eThe HPDE6-C7 human pancreatic duct epithelial cell line was purchased from Guangzhou Jenniobio Biotechnology Co., Ltd., China. The cells were cultured in a sterile environment at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Each 100 mL of complete medium contained 89 mL of MEM (8120294, Gibco, USA), 10 mL of FBS (A511-001, Lonsera, Uruguay), and 1 mL of penicillin/streptomycin (P1400, Solarbio, China). The cells were inoculated in 60 mm diameter sterile Petri dishes (430166, Corning, USA) at a density of 10\u003csup\u003e3\u003c/sup\u003e/mL, and when the cell confluence reached 70%, the cells in the different groups were treated with different concentrations of intervention agents for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.4 H\u0026amp;E staining\u003c/h2\u003e \u003cp\u003ePancreatic tissues were immersed in 4% paraformaldehyde solution for 12 h, embedded in paraffin, and cut into thin sections. Three random fields in each section were selected to calculate the pancreatic histopathological score. The pancreatic histopathological scoring was performed following the criteria described by Van Laethem \u003cem\u003eet al\u003c/em\u003e. \u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eDewaxing, hydration, and thermal repair were performed, and then, 5 \u0026micro;g/mL primary antibody (A00685-1, Boster, China) was added and incubated overnight at 4\u0026deg;C. After the tissues were incubated with an HRP-labeled linked polymer (KIT-5009, MXB biotechnologies, China) for 40 min at 26\u0026deg;C, the signal was detected using DAB (P0202, Beyotime, China). Three random fields were selected for each sample, and the average optical density (AOD) was calculated using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Serum triglyceride and amylase detection\u003c/h2\u003e \u003cp\u003eMouse venous blood was stored at 26\u0026deg;C for 2 h and centrifuged at 4\u0026deg;C at 3000 RPM for 15 min, after which the supernatant was collected for analysis. All steps in the procedure were performed in strict accordance with the instructions of the triglyceride detection kit (c061-a, Changchun Huili, China) and the amylase detection kit (C033, Changchun Huili, China). The levels of triglycerides and amylase were calculated on the basis of the absorbance measured via a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Protein extraction\u003c/h2\u003e \u003cp\u003eAfter the medium was removed, the cells were washed three times with PBS (P1020, Solarbio, China). Next, 1000 \u0026micro;L of RIPA lysis solution (P0013K, Beyotime, China) containing 10 \u0026micro;L of phenyl methane sulfonyl fluoride (P0100, Solarbio, China) was added to each 60 mm Petri dish, and the cells were placed on ice for 30 min before centrifugation (4\u0026deg;C at 12000 RPM for 15 min). Then, 200 \u0026micro;L of the supernatant was collected, 50 \u0026micro;L of protein loading buffer (P1015, Solarbio, China) was added, and the mixture was mixed. Next, the mixture was heated at 100\u0026deg;C for 10 min to denature the protein. The total protein was stored in a refrigerator at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Western blotting analysis\u003c/h2\u003e \u003cp\u003eProteins were resolved by SDS-PAGE and then transferred to a 0.22 \u0026micro;m PVDF membrane. The membrane was blocked with a 5% skim milk powder solution at 26\u0026deg;C for 45 min. After three washes with TBST solution, the membrane and primary antibodies (D2Z3B, CST, UK, 1:1000) were incubated at 4\u0026deg;C for 12 h. After washing again with TBST, the membrane and secondary antibodies (5151P, CST, UK, 1:10000) were incubated at 26\u0026deg;C in the dark for 1 h. The fluorescence signal was detected using a Li-COR Odyssey dual-color infrared fluorescence imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eA Cell Counting Kit-8 (CCK-8, C0038) was purchased from Beyotime Biotechnology Co., Ltd. HPDE6-C7 cells were seeded in 96-well plates at a density of 5000 cells/100 \u0026micro;L. Five different final concentrations of ruthenium red (1 \u0026micro;mol/L, 5 \u0026micro;mol/L, 10 \u0026micro;mol/L, 50 \u0026micro;mol/L, and 100 \u0026micro;mol/L) were used. After incubation for 24 h in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C, 10 \u0026micro;L of CCK-8 solution was added to each well and incubated in the cell culture box for another 1 h. Finally, the absorbance was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.10 Mitochondrial calcium ion detection\u003c/h2\u003e \u003cp\u003eDMSO (D8371, Solarbio, China) was added to 50 \u0026micro;g of Rhod-2 AM (R1245MP, Invitrogen, USA) to produce 100 \u0026micro;L of mother liquor at a concentration of 4 mmol/L, which was stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Next, 6 \u0026micro;L of mother liquor was added to 5994 \u0026micro;L of HBSS (H1025, Solarbio, China) to prepare a working solution with a concentration of 4 mmol/L. After the medium was removed and the cells were washed three times with HBSS, 1 mL of stock solution was added to each 60 mm petri dish and incubated at 37\u0026deg;C for 30 min. After washing once with PBS, 1 mL of HBSS was added, and the mixture was incubated at 37\u0026deg;C for 30 min. After washing with PBS again two times, 1 mL of mitochondrial green fluorescent probe (Mito-Tracker Green, C1048, Beyotime, China) diluted 5000 times with HBSS was added, and the mixture was incubated at 37\u0026deg;C for 15 min. The red and green fluorescence signals were imaged under an inverted fluorescence microscope after the samples were washed twice with PBS. After three fields of view were randomly selected for each sample, ImageJ software was used to calculate the mean fluorescence intensity value for subsequent statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.11 ROS detection\u003c/h2\u003e \u003cp\u003eWhen the degree of HPDE6-C7 cell fusion in a 60 mm diameter sterile Petri dish reached 70%, the cells were washed with PBS three times, and then 2 mL of serum-free medium supplemented with 10 mM/L DCFH-DA (S0033S, Beyotime, China) was added to each dish. The cells were incubated for 20 min in a cell culture box at 37\u0026deg;C and washed with serum-free cell culture medium three times. Then, the green fluorescence images captured under a fluorescence microscope were analyzed via ImageJ software. Similarly, we randomly selected three random regions to calculate the average fluorescence intensity for subsequent statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.12 MDA detection\u003c/h2\u003e \u003cp\u003eThe cells in the six-well plate were lysed with RIPA lysis buffer (P0013K, Beyotime, China) for 30 min and then centrifuged. The supernatant was collected for subsequent analysis.\u003c/p\u003e \u003cp\u003eAn MDA detection kit (S0131S, Beyotime, China) was purchased from Biotime Biotechnology Co., Ltd. The MDA test working fluid and MDA standard were prepared following the manufacturer\u0026rsquo;s instructions. In total, 100 \u0026micro;L of a standard substance at different concentrations and different groups of samples to be tested was added to 200 \u0026micro;L of the MDA detection working solution and then heated in boiling water for 15 min. After cooling to 26\u0026deg;C and centrifugation (1000 \u0026times;g, 26\u0026deg;C, 10 min), 200 \u0026micro;L of the supernatant was collected and added to a 96-well plate. The absorbance was subsequently measured at 532 nm via a microplate reader. The concentration of MDA was calculated on the basis of a standard curve.\u003c/p\u003e \u003cp\u003eA BCA protein concentration determination kit (P0010, Beyotime, China) was purchased from Biotime Biotechnology Co., Ltd. The BCA working solution and protein standard were prepared following the manufacturer\u0026rsquo;s instructions. In each well of the 96-well plate, 20 \u0026micro;L of protein standard or test sample at different concentrations was added successively, followed by the addition of 200 \u0026micro;L of BCA working solution. After the plate was incubated for 30 min at 37\u0026deg;C, the absorbance was measured at 562 nm using a microplate reader. The protein concentration of each group was calculated based on a standard curve. The amount of MDA per milligram of protein was calculated by dividing the concentration of MDA by the corresponding total protein concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.13 GSH detection\u003c/h2\u003e \u003cp\u003eAfter HPDE6-C7 cells were washed with PBS three times, the cells were resuspended and lysed by ultrasonication. The broken cell suspension was centrifuged at 3500 rpm for 10 min, after which 0.1 mL of the supernatant was removed for subsequent analysis. Following the instructions of the GSH and GSSH determination kits (S0053, Beyotime, China), 0.1 mL of reagent 1, 0.1 mL of reagent 2, and 0.025 mL of reagent 3 were added to the wells containing the blank, standard, and test samples, respectively. The plate was left undisturbed for 5 min, after which the absorbance was measured at 405 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.14 Immunofluorescence microfilament staining\u003c/h2\u003e \u003cp\u003eAfter HPDE6-C7 cells were washed on sterile glass plates twice with PBS at 37\u0026deg;C, 4% paraformaldehyde was added at 26\u0026deg;C for 10 min. The cells were again washed twice with PBS, and then 0.5% Triton X-100 was added for 5 min. Next, 200 \u0026micro;L of phalloidin (200 nmol/L; CA1610, Solarbio, China) was added to the glass plate and incubated at 37\u0026deg;C in the dark for 30 min. After washing twice with PBS, 200 \u0026micro;L of DAPI (100 nmol/L; S2110, Solarbio, China) was added to stain the nuclei. Finally, the morphological characteristics of the microfilaments were observed under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.15 Permeability test of monolayer cells\u003c/h2\u003e \u003cp\u003eFirst, 100 \u0026micro;L of complete medium containing about 100,000 cells was added to the apical compartment of the Transwell system (3413, Corning, USA), and 600 \u0026micro;L of complete medium was added to the basolateral compartment. After 24 h of culture in an incubator at 37\u0026deg;C, the different groups were treated with the corresponding intervention agent for 24 h. After the culture medium was removed and the cells were washed with PBS three times, 200 \u0026micro;L of FITC-Dextran 4000 (1 mg/mL; 46944, Sigma, USA) was added to the apical compartment, and 600 \u0026micro;L of PBS was added to the basolateral compartment. The cells were then incubated for 2 h. After incubation, 2 \u0026micro;L of liquid from the basolateral compartment was removed from the 96-well plate, and the absorbance was measured via a microplate reader (excitation/emission wavelength: 490/520 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.16 Statistical methods\u003c/h2\u003e \u003cp\u003eThe measurement data were statistically analyzed via the unpaired sample t-test in GraphPad Prism 8 software, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAdditional information\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China [grant numbers 82460134]; Natural Science Foundation of Jiangxi Province [20232BAB206021].\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShiyu Zhang: Conceptualization, Methodology, Software. Junbo Hong.: Data curation, Writing- Original draft preparation. Qingzi Fu: Data curation, Writing- Original draft preparation. Liang Zhu: Visualization. Zhenzhen Yang: Supervision. Jianhua Wan: Software, Validation. Qiaofeng Chen, Data curation. Peng Chen: Writing- Reviewing and Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the Key Laboratory Project of Digestive Diseases in Jiangxi Province (2024SSY06101), and Jiangxi Clinical Research Center for Gastroenterology (20223BCG74011).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXiao, A. 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L.\u003cem\u003e et al.\u003c/em\u003e Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. \u003cem\u003eGastroenterology\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 1917-1922. https://doi.org/10.1016/0016-5085(95)90158-2 (1995)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pancreatitis, acute disease, pathogenesis, oxidative stress, cytoskeleton","lastPublishedDoi":"10.21203/rs.3.rs-6249694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6249694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe pathogenesis of hypertriglyceridema-induced pancreatitis (HTGP) is complex and not fully understood. The purpose of this study was to investigate the molecular mechanism of MCU in HTGP.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe observed the expression levels of MCU and SIRT3 in both in vivo and in vitro HTGP models, and after intervention with RR, an active inhibitor of MCU, and 3-TYP, an active inhibitor of SIRT3, changes in mitochondrial calcium ions, oxidative stress-related indices, the microfilament cytoskeleton, and monolayer cell permeability were detected.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn vivo and in vitro experiments revealed the upregulation of MCU and downregulation of SIRT3 in caerulein-treated HPDE6-C7 cells and mice, along with increased mitochondrial calcium accumulation, increased ROS and MDA, decreased GSH, destruction of the microfilament cytoskeleton, and increased monolayer permeability. During in vitro experiments, intervention with RR, an active inhibitor of MCU, reversed the above changes, whereas intervention with 3-TYP, an active inhibitor of SIRT3, further exacerbated the above changes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMCU may be involved in the pathogenesis of AP by inhibiting the expression of SIRT3, resulting in increased oxidative stress and destruction of the microfilament cytoskeleton and PDMB functions.\u003c/p\u003e","manuscriptTitle":"MCU promotes oxidative stress by regulating SIRT3 to destroy the pancreatic duct epithelial cytoskeleton: In vivo and in vitro hypertriglyceridema-induced pancreatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-28 10:48:33","doi":"10.21203/rs.3.rs-6249694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"55158d8a-0505-49c9-be67-121b2ee2bb00","owner":[],"postedDate":"March 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46031396,"name":"Health sciences/Diseases/Gastrointestinal diseases/Pancreatic disease"},{"id":46031397,"name":"Biological sciences/Immunology/Inflammation/Acute inflammation"}],"tags":[],"updatedAt":"2025-05-13T11:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-28 10:48:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6249694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6249694","identity":"rs-6249694","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}