Urolithin A's Role in Alleviating Severe Acute Pancreatitis via Endoplasmic Reticulum-Mitochondrial Calcium Channel Modulation.

Severe acute pancreatitis (SAP), characterized by pancreatic acinar cell death, currently lacks effective targeted therapies. Ellagic acid (EA), rich in pomegranate, shows promising anti-inflammatory and antioxidant effects in SAP treatment. However, the roles of other forms of EA, such as plant extracellular vesicles (EVs) extracted from pomegranate, and Urolithin A (UA), converted from EA through gut microbiota metabolism in vivo, have not been definitively elucidated. Our research aimed to compare the effects of pomegranate-derived EVs (P-EVs) and UA in the treatment of SAP to screen an effective formulation and to explore its mechanisms in protecting acinar cells in SAP. By comparing the protective effects of P-EVs and UA on injured acinar cells, UA showed superior therapeutic effects than P-EVs. Subsequently, we further discussed the mechanism of UA in alleviating SAP inflammation. In vivo animal experiments found that UA could not only improve the inflammatory environment of pancreatic tissue and peripheral blood circulation in SAP mice but also revealed that the mechanism of UA in improving SAP might be related to mitochondria and endoplasmic reticulum (ER) through the results including pancreatic tissue transcriptomics and transmission electron microscopy. Further research found that UA could regulate ER-mitochondrial calcium channels and reduce pancreatic tissue necroptosis. In vitro experiments of mouse pancreatic organoids and acinar cells also confirmed that UA could improve pancreatic inflammation by regulating the ER-mitochondrial calcium channel and necroptosis pathway proteins. This study not only explored the therapeutic effect of plant EVs on SAP but also revealed that UA could alleviate SAP by regulating ER-mitochondrial calcium channel and reducing acinar cell necroptosis, providing insights into the pathogenesis and potential treatment of SAP.

Inflammation and Acinar Cell Dual-Targeting Nanomedicines for Synergistic Treatment of Acute Pancreatitis via Ca2+ Homeostasis Regulation and Pancreas Autodigestion Inhibition.

Severe acute pancreatitis (AP) is a life-threatening pancreatic inflammatory disease with a high mortality rate (∼40%). Existing pharmaceutical therapies in development or in clinical trials showed insufficient treatment efficacy due to their single molecular therapeutic target, poor water solubility, short half-life, limited pancreas-targeting specificity, etc. Herein, acid-responsive hollow mesoporous Prussian blue nanoparticles wrapped with neutrophil membranes and surface modified with the N,N-dimethyl-1,3-propanediamine moiety were developed for codelivering membrane-permeable calcium chelator BAPTA-AM (BA) and trypsin activity inhibitor gabexate mesylate (Ga). In the AP mouse model, the formulation exhibited efficient recruitment at the inflammatory endothelium, trans-endothelial migration, and precise acinar cell targeting, resulting in rapid pancreatic localization and higher accumulation. A single low dose of the formulation (BA: 200 µg kg-1, Ga: 0.75 mg kg-1) significantly reduced pancreas function indicators to close to normal levels at 24 h, effectively restored the cell redox status, reduced apoptotic cell proportion, and blocked the systemic inflammatory amplified cascade, resulting in a dramatic increase in the survival rate from 58.3 to even 100%. Mechanistically, the formulation inhibited endoplasmic reticulum stress (IRE1/XBP1 and ATF4/CHOP axis) and restored impaired autophagy (Beclin-1/p62/LC3 axis), thereby preserving dying acinar cells and restoring the cellular "health status". This formulation provides an upstream therapeutic strategy with clinical translation prospects for AP management through synergistic ion homeostasis regulation and pancreatic autodigestion inhibition.

Chronic exposure to BDE-47 aggravates acute pancreatitis and chronic pancreatitis by promoting acinar cell apoptosis and inflammation.

The effect of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a persistent environmental pollutant commonly used as a flame retardant in various consumer products, on pancreatitis has not been clearly elucidated, although it has been reported to be toxic to the liver, nervous system, and reproductive system. Acute pancreatitis (AP) and chronic pancreatitis (CP) models were induced in this study by intraperitoneal injection of caerulein. The aim was to investigate the impact of BDE-47 on pancreatitis by exposing the animals to acute (1 week) or chronic (8 weeks) doses of BDE-47 (30 mg/kg in the low-concentration group and 100 mg/kg in the high-concentration group). Additionally, BDE-47 was utilized to stimulate mouse bone marrow-derived macrophages, pancreatic primary stellate cells, and acinar cells in order to investigate the impact of BDE-47 on pancreatitis. In vivo experiments conducted on mice revealed that chronic exposure to BDE-47, rather than acute exposure, exacerbated the histopathological damage of AP and CP, leading to elevated fibrosis in pancreatic tissue and increased infiltration of inflammatory cells in the pancreas. In vitro experiments showed that BDE-47 can promote the expression of the inflammatory cytokines Tnf-α and Il-6 in M1 macrophages, as well as promote acinar cell apoptosis through the activation of the PERK and JNK pathways via endoplasmic reticulum stress. The findings of this study imply chronic exposure to BDE-47 may exacerbate the progression of both AP and CP by inducing acinar cell apoptosis and dysregulating inflammatory responses.

CaMK II Inhibition Attenuates ROS Dependent Necroptosis in Acinar Cells and Protects against Acute Pancreatitis in Mice.

As a calcium-regulated protein, CaMK II is closely related to cell death, and it participates in the development of pathological processes such as reperfusion injury, myocardial infarction, and oligodendrocyte death. The function of CaMK II activation in acute pancreatitis (AP) remains unclear. In our study, we confirmed that the expression of p-CaMK II was increased significantly and consistently in injured pancreatic tissues after caerulein-induced AP. Then, we found that KN93, an inhibitor of CaMK II, could mitigate the histopathological manifestations in pancreatic tissues, reduce serum levels of enzymology, and decrease oxidative stress products. Accordingly, we elucidated the effect of KN93 in vitro and found that KN93 had a protective effect on the pancreatic acinar cell necroptosis pathway by inhibiting the production of ROS and decreasing the expression of RIP3 and p-MLKL. In addition, we identified the protective effect of KN93 on AP through another mouse model induced by pancreatic duct ligation (PDL). Together, these data demonstrated that CaMK II participates in the development of AP and that inhibiting CaMK II activation could protect against AP by reducing acinar cell necroptosis, which may provide a new idea target for the prevention and treatment of AP in the clinic.

Effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate the systemic inflammatory response in acute pancreatitis.

This research was carried out to investigate the effect and mechanism of Angelic Shaoyaosan mediated AMPK/SIRT1 positive feedback loop to promote autophagy and regulate systemic inflammatory response in acute pancreatitis. In this study, the rat pancreatic acini AR42J cells were chosen as the research object, the application of hyla induced pancreatic acinar cells made model for acute pancreatitis, application of different concentrations of angelica peony spread effect on building cells, thus divided into control group, built in the module, the low concentration group, concentration and high concentration groups, determined by MTT method was applied to explore the above categories in cell proliferation, cell apoptosis was measured by flow cytometry, the expression of inflammatory factors in cell supernatant was determined by enzyme-linked immunoassay, and the expression of autophagy marker proteins LC3- ? and P62 was determined by Western-Bolt method. In order to explore the relationship between AMPK and SIRT1, immunoco-precipitation method was used to determine the interaction between AMPK and SIRT1, and dual luciferase experiment was used to explore the effect of AMPK on SIRT1. The AICAR group, BLM-275 group and negative control group were established. To explore the effect of SIRT1 on AMPK, we established SRT 1720 group, EX-527 group and control group. Direct binding between AMPK and SIRT1 should be determined by chromatin co-precipitation assay. In order to further explore the effect of AMPK/SIRT1 positive feedback loop on the systemic inflammatory response of acute pancreatitis, this study selected the medium-concentration Danggui Shaoyajiao SAN group as the control group (group C), and applied AMPK inhibitor BLM-275 and SIRT1 inhibitor EX 527 to the effect of medium-concentration Danggui Shaoyajiao SAN cells, respectively. The expression of autophagy marker proteins LC3- ? and P62 in groups A and B were determined by the Western-Bolt method. Results showed that compared with the control group, the cell survival rate, the expression of AMPK, SIRT1 and LC3-II in the model group were decreased, and the apoptosis rate of iNOS, IL-2, TNF-?, P62 and apoptosis were increased in the model group (P<0.05). the levels of iNOS, IL-2, TNF-?, P62 and cell survival rate in low, medium and high concentration groups decreased gradually, while the expressions of AMPK, SIRT1, LC3-II and cell apoptosis rate increased (P<0.05). The levels of iNOS, IL-2 and TNF-? in the three groups were gradually decreased with the increase of the concentration (P<0.05). Immunoprecipitation showed that AMPK and SIRT1 could bind to each other in cells. The double luciferase experiment indicated that the reporter gene containing the SIRT1 binding site was constructed. The luciferase activity was increased in THE AICAR group and decreased in the BLM-275 group (P<0.05). The reporter gene containing the AMPK promoter binding site was constructed. The luciferase activity in SRT1720 group was increased, while that in EX-527 group was decreased. SIRT1 could directly bind to the AMPK promoter. SIRT1 and LC3- ? protein expressions in group A were down-regulated, and P62 protein was increased (P<0.05). The protein expressions of AMPK and LC3- ? in group B were down-regulated, and the protein expression of P62 was increased (P<0.05). It concluded that AMPK can directly bind to activate SIRT1 expression, and SIRT1 expression can also activate AMPK, forming a positive feedback loop between the two. Therefore, Angelic Shaoyaodong decoction can mediate AMPK/SIRT1 positive feedback pathway to promote autophagy and regulate systemic inflammatory response in acute pancreatitis.

Pyruvate and Glutamine Define the Effects of Cholecystokinin and Ethanol on Mitochondrial Oxidation, Necrosis, and Morphology of Rat Pancreatic Acini.

OBJECTIVES: The objective of this study was to test whether pyruvate and glutamine affect the ethanol and cholecystokinin (CCK) effects on the mitochondrial function, viability, and morphology of rat pancreatic acini. METHODS: Respiration was measured with Clark oxygen electrode. Mitochondrial membrane potential, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), cell morphology, and viability were studied with fluorescence microscopy. RESULTS: In vitro, CCK (0.1 nM) caused pyruvate-dependent stimulation of basal and uncoupled respiration, and the effects were abolished by ethanol (20 mM). The combination of ethanol with CCK (2 hours) caused necrosis of approximately 40% acinar cells in medium with glucose, but not with pyruvate and/or glutamine. Cholecystokinin (10 nM) or ethanol with 0.1 nM CCK caused plasma membrane blebbing not related to apoptosis only when both glutamine and pyruvate were present. Glutamine, but not pyruvate, decreased NAD(P)H level and prevented the effects of ethanol with CCK on mitochondrial membrane potential and NAD(P)H, but, in combination with CCK and ethanol, decreased the uncoupled respiration. In vivo, the combination of ethanol (4 g/kg) and CCK (20 pmol/kg) suppressed basal and uncoupled respiration and caused acinar cell blebbing, but not necrosis. CONCLUSIONS: The lack of sufficient substrate supply in vitro makes pancreatic acinar cells susceptible to necrosis caused by ethanol and CCK in clinically relevant concentrations.

Single-cell analyses of human pancreas: characteristics of two populations of acinar cells in chronic pancreatitis.

Chronic pancreatitis (CP) is a complex inflammatory disorder with numerous associated genetic and environmental risk factors. The most distressing characteristic of CP is recalcitrant pain, often requiring surgical resection including total pancreatectomy with islet autotransplantation (TPIAT). We studied five consented subjects undergoing pancreatic resection and processed isolated cells for single-cell RNA sequencing (scRNA-Seq). Using high-dimensional transcriptomic cluster analysis, we identified 11 unique cell clusters in the pancreas tissue. These cell clusters include a cluster of undifferentiated/dedifferentiated cells and two unique clusters of acinar cells, one of which appears to be in a transitional stage. To determine the cellular response to protease inhibitor and stimulation, we treated aliquots of cells from one subject with a protease inhibitor cocktail with and without bethanechol (a muscarinic receptor agonist) at 100 and 400 µM and compared gene expression profiles. The protease inhibitors appeared to reduce cell stress. Pancreatic digestive enzymes and islet hormones were upregulated in both doses of bethanechol-treated cells compared with naïve cells. High-dose bethanechol appeared to be toxic and consistent with hyperstimulation. These studies demonstrate the feasibility of investigating human acinar cell physiology at the single-cell level and initial evidence that these cells retain responsiveness to agonist stimulation with predicted second messenger and transcriptomic responses.NEW & NOTEWORTHY We conducted single cell RNA sequencing on pancreas tissue from five individuals. We identified eleven unique cell clusters including a large population of dedifferentiated cells as well as two unique clusters of acinar cells, one of which appears to exist in a transitional state. We also examined the cellular response of pancreas tissue to stimulation and identified affected genes and pathways, including pancreatic digestive enzymes.

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway.

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

Emodin inhibits the progression of acute pancreatitis via regulation of lncRNA TUG1 and exosomal lncRNA TUG1.

Acute pancreatitis (AP) is one of the most frequent gastrointestinal diseases and has no specific treatment. It has been shown that dysfunction of pancreatic acinar cells can lead to AP progression. Emodin is a natural product, which can alleviate the symptoms of AP. However, the mechanism by which emodin regulates the function of pancreatic acinar cells remains unclear. Thus, the present study aimed to investigate the mechanism by which emodin modulates the function of pancreatic acinar cells. To mimic AP in vitro, pancreatic acinar cells were cotreated with caerulein and lipopolysaccharide (LPS). Exosomes were isolated using the ExoQuick precipitation kit. Western blot analysis, Nanosight Tracking analysis and transmission electron microscopy were performed to detect the efficiency of exosome separation. Gene expression was detected by reverse transcription­quantitative PCR. The levels of IL­1ß and TNF­α were detected by ELISA. The data indicated that emodin significantly decreased the levels of IL­1ß and TNF­α in the supernatant samples derived from AR42J cells cotreated with caerulein and LPS. In addition, emodin significantly promoted the proliferation of AR42J cells cotreated with caerulein and LPS, and inhibited apoptosis, while the effect of emodin was reversed by long non­coding (lnc)RNA taurine upregulated 1 (TUG1) overexpression. The expression level of TUG1 in AR42J cells or exosomes derived from AR42J cells was significantly increased following treatment of the cells with LPS and caerulein, while this effect was notably reversed by emodin treatment. In addition, exosomes derived from caerulein and LPS cotreated AR42J cells inhibited the differentiation and anti­inflammatory function of regulatory T cells, while treatment of the cells with emodin significantly decreased this effect. In conclusion, the data indicated that emodin inhibited the induction of inflammation in AR42J cells by regulating the expression of cellular and exosomal lncRNA. Therefore, emodin may be used as a potential agent for the treatment of AP.

Inhibition of hypoxia-inducible factor-1α alleviates acinar cell necrosis in a mouse model of acute pancreatitis.

Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.

Escin Sodium Improves the Prognosis of Acute Pancreatitis via Promoting Cell Apoptosis by Suppression of the ERK/STAT3 Signaling Pathway.

Acute pancreatitis (AP), an inflammatory disorder of the pancreas, can cause systemic inflammatory responses. Escin Sodium (ES), a natural mixture of triterpene saponins extracted from the dry ripe fruit of Fructus Aesculi or horse chestnut crude, has been demonstrated to have antiedematous, anti-inflammatory, and antiexudative effects. We here aim to investigate the effects of ES pretreatment on AP in vivo and in vitro and explore its potential molecular mechanism. In the present study, we demonstrated that ES pretreatment could apparently decrease amylase and lipase, downregulate inflammatory cytokines, and attenuate pancreatic damage. Additionally, the increased expression of apoptotic-related proteins and the results of flow cytometry demonstrated the effects of ES on promoting apoptosis in acinar cells. Moreover, ES could enhance mitochondrial membrane potential (MMP, ΔΨm) and reactive oxygen species (ROS) level and reduce intracellular calcium concentration, which are closely related to mitochondrial-mediated death. The effect of ES pretreatment on acinar cell apoptosis was furtherly confirmed by the regulatory pathway of the ERK/STAT3 axis. These results suggest that ES attenuates the severity of AP by enhancing cell apoptosis via suppressing the ERK/STAT3 signaling pathway. These findings provide evidence for ES which is treated as a novel and potent therapeutic for the treatment of AP.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Insulin protects acinar cells during pancreatitis by preserving glycolytic ATP supply to calcium pumps.

Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.

Circ_ZFP644 attenuates caerulein-induced inflammatory injury in rat pancreatic acinar cells by modulating miR-106b/Pias3 axis.

(AP) is a kind of inflammatory misorder existing in pancreas. Non-coding RNAs (ncRNAs) have been reported to play important roles in development of AP. The current study was designed to explore the role of circular RNA zinc finger protein 644 (circRNA circ_ZFP644) in caerulein-induced AR42J cells. AP model in vitro was established by exposure of rat pancreatic acinar AR42J cells to caerulein. Amylase activity was measured using a kit. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the levels of several inflammatory factors. The expression of circ_ZFP644, microRNA (miR)-106b and protein inhibitor of activated STAT 3 (Pias3) was detected by quantitative real-time PCR (qRT-PCR) or western blot assay. And flow cytometry was employed to monitor cell apoptosis. Western blot assay was also conducted to analyze the expression of apoptosis-related proteins. The association among circ_ZFP644, miR-106b and Pias3 was validated by dual-luciferase reporter assay. Caerulein treatment activated amylase activity and promoted the secretion of inflammatory cytokines in AR42J cells. Circ_ZFP644 and Pias3 were downregulated, but miR-106b was upregulated in caerulein-induced AR42J cells. Enforced expression of circ_ZFP644 or miR-106b inhibition could reduce amylase activity and inflammatory cytokine secretion, while promote apoptosis in caerulein-induced AR42J cells, which was almost reversed by Pias3 knockdown. Circ_ZFP644 targeted miR-106b to upregulate Pias3 expression. Circ_ZFP644 might exert its anti-inflammation and pro-apoptosis roles in caerulein-induced AR42J cells by regulating miR-106b/Pias3 axis.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury.

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

Pancreatic ductal deletion of S100A9 alleviates acute pancreatitis by targeting VNN1-mediated ROS release to inhibit NLRP3 activation.

Recent studies have proven that the overall pathophysiology of pancreatitis involves not only the pancreatic acinar cells but also duct cells, however, pancreatic duct contribution in acinar cells homeostasis is poorly known and the molecular mechanisms leading to acinar insult and acute pancreatitis (AP) are unclear. Our previous work also showed that S100A9 protein level was notably increased in AP rat pancreas through iTRAQ-based quantitative proteomic analysis. Therefore, we investigated the actions of injured duct cells on acinar cells and the S100A9-related effects and mechanisms underlying AP pathology in the present paper. Methods: In this study, we constructed S100A9 knockout (s100a9-/-) mice and an in vitro coculture system for pancreatic duct cells and acinar cells. Moreover, a variety of small molecular inhibitors of S100A9 were screened from ChemDiv through molecular docking and virtual screening methods. Results: We found that the upregulation of S100A9 induces cell injury and inflammatory response via NLRP3 activation by targeting VNN1-mediated ROS release; and loss of S100A9 decreases AP injury in vitro and in vivo. Moreover, molecular docking and mutant plasmid experiments proved that S100A9 has a direct interaction with VNN1 through the salt bridges formation of Lys57 and Glu92 residues in S100A9 protein. We further found that compounds C42H60N4O6 and C28H29F3N4O5S can significantly improve AP injury in vitro and in vivo through inhibiting S100A9-VNN1 interaction. Conclusions: Our study showed the important regulatory effect of S100A9 on pancreatic duct injury during AP and revealed that inhibition of the S100A9-VNN1 interaction may be a key therapeutic target for this disease.

Chaiqin chengqi decoction ameliorates acute pancreatitis in mice via inhibition of neuron activation-mediated acinar cell SP/NK1R signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaiqin chengqi decoction (CQCQD) and its derivatives have been widely used in China for the early management of patients with acute pancreatitis (AP). Numerous studies demonstrate the anti-inflammatory and anti-oxidative effects of CQCQD and derivatives, but whether these effects can be attributed to suppressing neurogenic inflammation, has never been studied. AIM OF THE STUDY: To investigate the effects of CQCQD on substance P (SP)-neurokinin 1 receptor (NK1R) based neurogenic inflammation in an experimental AP model. MATERIAL AND METHODS: For AP patients on admission, pain score was accessed by visual analog scale (VAS); the levels of serum SP and expressions of pancreatic SP and NK1R were also determined. For in vivo study, mice received 7 intraperitoneal injections of cerulein (50 µg/kg) at hourly intervals to induce AP, whilst controls received normal saline injections. In the treatment groups, CQCQD (10 g/kg, 200 µl) was intragastrically given at the third, fifth, and seventh of the cerulein injection or the NK1R antagonist CP96345 (5 mg/kg) was intraperitoneally injected 30 min before the first cerulein administration. The von Frey test was performed to evaluate pain behavior. Animals were sacrificed at 12 h from the first cerulein/saline injection for severity assessment. Pharmacology network analysis was used to identify active ingredients of CQCQD for AP and pain. In vitro, freshly isolated pancreatic acinar cells were pre-treated with CQCQD (5 mg/ml), CP96345 (1 µM), or selected active compounds of CQCQD (12.5, 25, and 50 µM) for 30 min, followed by SP incubation for another 30 min. RESULTS: The VAS score as well as the levels of serum SP and expressions of pancreatic SP-NK1R were up-regulated in moderately severe and severe patients compared with those with mild disease. CQCQD, but not CP96345, consistently and significantly ameliorated pain, pancreatic necrosis, and systemic inflammation in cerulein-induced AP as well as inhibited NK1R internalization of pancreatic acinar cells. These effects of CQCQD were associated with reduction of pancreatic SP-NK1R and neuron activity in pancreas, dorsal root ganglia, and spinal cord. Baicalin, emodin, and magnolol, the top 3 active components of CQCQD identified via pharmacology network analysis, suppressed NK1R internalization and NF-κB signal pathway activation in isolated pancreatic acinar cells. CONCLUSIONS: CQCQD ameliorated cerulein-induced AP and its associated pain via inhibiting neuron activation-mediated pancreatic acinar cell SP-NK1R signaling pathways and its active compounds baicalin, emodin, and magnolol contributed to this effect.

A multi-strategy platform for quality control and Q-markers screen of Chaiqin chengqi decoction.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory disorder of the pancreas that is associated with substantial morbidity and mortality. Chaiqin chengqi decoction (CQCQD) has been proven clinically to be an effective treatment for AP for decades in West China Hospital. Quality control for CQCQD containing many hundreds of characteristic phytochemicals poses a challenge for developing robust quality assessment metrics. PURPOSE: To evaluate quality consistency of CQCQD with a multi-strategy based analytical method, identify potential quality-markers (Q-markers) based on drug properties and effect characteristics, and endeavor to establish CQCQD as a globally-accepted medicine. METHODS: A typical analysis of constitutive medicinal plant materials was performed following the Chinese Pharmacopoeia. The extraction process was optimized through an orthogonal array (L9(34)) to evaluate three levels of liquid to solid ratio, soaking time, duration of extraction, and the number of extractions. An ultra-high-performance liquid chromatography (UHPLC) fingerprinting combined with absolute quantitation of multi chemical marker compounds, coupled with similarity, hierarchical clustering analysis (HCA), and principal component analyses (PCA) were performed to evaluate 10 batches of CQCQD. On the basis of systematic analysis of fundamental features of CQCQD in treating AP, the potential Q-marker screen was proposed through detection of quality transfer and efficacy for chemical markers. UHPLC coupled with quadrupole orbitrap mass spectrometry were used to determine compounds in medicinal materials, decoctions and plasma. Network pharmacology and taurolithocholic acid 3-sulfate induced pancreatic acinar cell death were used to evaluate the correlation between chemical markers and anti-pancreatitis activity. A cerulein induced AP murine model was used to validate quality assessed CQCQD batches at clinically-equivalent dose. The effective content of chemical markers was predicted using linear regression analysis on quantitative information between validated batches and the other batches. RESULTS: The chemical markers and other physical and chemical indices in the original materials met Chinese Pharmacopoeia standards. A total of 22 co-existing fingerprint peaks were selected and the similarity varied between 0.946 and 0.990. Batch D10 possessed the highest similarity index. HCA classified the 10 batches into 2 main groups: 7 batches represented by D10 and 3 batches represented by D1. During the initial Q-marker screen stage, 22 compounds were detected in both plant materials and decoctions, while 13 compounds were identified in plasma. Network pharmacology predicted the potential targets and pathway of AP related to the 22 compounds. All 10 batches showed reduced necrosis below 60% with the best effect achieved by D10 (~40%). The spectrum-efficacy relationship analyzed by Pearson correlation analysis indicated that emodin, rhein, aloe emodin, geniposide, hesperridin, chrysin, syringin, synephrine, geniposidic acid, magnolol, physcion, sinensetin, and baicalein showed positive correlation with pancreatic acinar cell death protection. Similar to the in vitro evaluation, batch D10 significantly reduced total histopathological scores and biochemical severity indices at a clinically-equivalent dose but batch D1 did not. The content of naringin, narirutin and baicalin in batches D1, D5 and D9 consistently exceeds the upper limit of the predicted value. Eight markers whose lower limit is predicted to be close to 0 contributed less to the material basis for AP protection. CONCLUSION: Despite qualified materials used for CQCQD preparation, the clinical effect depends on appropriate content range of Q-markers. Emodin, rhein, aloe emodin, magnolol, hesperidin, synephrine, baicalein, and geniposide are considered as vital Q-markers in the primary screen. This study proposed a feasible platform for producing highly consistent batches of CQCQD in future study.

Development of a functional salivary gland tissue chip with potential for high-content drug screening.

Radiation therapy for head and neck cancers causes salivary gland dysfunction leading to permanent xerostomia. Limited progress in the discovery of new therapeutic strategies is attributed to the lack of in vitro models that mimic salivary gland function and allow high-throughput drug screening. We address this limitation by combining engineered extracellular matrices with microbubble (MB) array technology to develop functional tissue mimetics for mouse and human salivary glands. We demonstrate that mouse and human salivary tissues encapsulated within matrix metalloproteinase-degradable poly(ethylene glycol) hydrogels formed in MB arrays are viable, express key salivary gland markers, and exhibit polarized localization of functional proteins. The salivary gland mimetics (SGm) respond to calcium signaling agonists and secrete salivary proteins. SGm were then used to evaluate radiosensitivity and mitigation of radiation damage using a radioprotective compound. Altogether, SGm exhibit phenotypic and functional parameters of salivary glands, and provide an enabling technology for high-content/throughput drug testing.

Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.