Temporal metabolic trajectory analyzed by LC-MS/MS based targeted metabolomics in acute pancreatitis pathogenesis and Chaiqin Chengqi decoction therapy.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory disorder of pancreas that lacks effective specific drugs as well as gold standard laboratory tests for diagnosis and severity assessment. Chaiqin chengqi decoction (CQCQD) has been proven to alleviate the severity and mortality of AP, but its underlying mechanisms remain incompletely understood. PURPOSE: To investigate the correlation between metabolic trajectories of the serum and pancreas, the metabolic pathways with respect to the onset and progression of AP, and investigate the effect of CQCQD in modulating the dysregulated pancreatic metabolism of AP. METHODS: Serum and pancreas samples from cerulein-induced AP mice were collected for pathology, biochemical index assessment, LC-MS/MS based metabolomics and functional validation over the course of 1 - 24 h. The temporal trends of pancreatic and serum metabolites in AP were analyzed using Mfuzz clustering algorithm, and their associations were revealed by Pearson correlation analysis. The metabolic trajectories and pathways across multi-timepoints were analyzed by univariate and multivariate statistical analyses, and the AP-related metabolic pathways were further screened by metabolite correlation and network interaction analyses. Finally, the changes in metabolite levels and metabolic trajectory after CQCQD therapy were identified, and the altered expression of related metabolic enzymes was verified by RT-qPCR, western blotting, and immunohistochemistry. RESULTS: Amino acid metabolism was significantly altered in the pancreas and serum of AP, but with different trends. The unsynchronized "open" and "closed" metabolic trajectories in pancreas and serumrevealed that metabolic processes occur earlier in peripheral rather than local tissue, with the most obvious changes occuring at 12 h in the pancreas which were also consistent with the inflammation score results. Several amino acid intermediates showed strong positive correlation between serum and pancreas, and therein serum cystathionine was positively correlated to 33 pancreatic metabolites. In particular, the correlations between the levels of pancreatic cystathionine and methionine, serine, and glutathione (GSH) emphasized the importance of trans-sulfuration to GSH metabolism for AP progression. CQCQD treatment reversed the metabolic trajectory of the pancreas, and also restored the levels of cystathionine and glutathione synthase. CONCLUSION: Our results have defined a unique time-course metabolic trajectory for AP progression in both the serum and pancreas; it has also revealed a key role of CQCQD in reversing AP-associated metabolic alterations, thus providing new metabolic targets for the treatment and prognosis of AP.

Targeting Macrophage Migration Inhibitory Factor in Acute Pancreatitis and Pancreatic Cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in the pathogenesis of inflammation and cancer. It is produced by various cells and circulating MIF has been identified as a biomarker for a range of diseases. Extracellular MIF mainly binds to the cluster of differentiation 74 (CD74)/CD44 to activate downstream signaling pathways. These in turn activate immune responses, enhance inflammation and can promote cancer cell proliferation and invasion. Extracellular MIF also binds to the C-X-C chemokine receptors cooperating with or without CD74 to activate chemokine response. Intracellular MIF is involved in Toll-like receptor and inflammasome-mediated inflammatory response. Pharmacological inhibition of MIF has been shown to hold great promise in treating inflammatory diseases and cancer, including small molecule MIF inhibitors targeting the tautomerase active site of MIF and antibodies that neutralize MIF. In the current review, we discuss the role of MIF signaling pathways in inflammation and cancer and summarize the recent advances of the role of MIF in experimental and clinical exocrine pancreatic diseases. We expect to provide insights into clinical translation of MIF antagonism as a strategy for treating acute pancreatitis and pancreatic cancer.

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.

Dynamic analysis of pulmonary computed tomography (CT) characteristics in cured coronavirus disease 2019 (COVID-19) patients.

BACKGROUND: To retrospectively analyze the pulmonary computed tomography (CT) characteristics and dynamic changes in the lungs of cured coronavirus disease 2019 (COVID-19) patients at discharge and reexamination. METHODS: A total of 155 cured COVID-19 patients admitted to designated hospitals in Yunnan Province, China, from February 1, 2020, to March 20, 2020, were included. All patients underwent pulmonary CT at discharge and at 2 weeks after discharge (during reexamination at hospital). A retrospective analysis was performed using these two pulmonary CT scans of the cured patients to observe changes in the number, distribution, morphology, and density of lesions. RESULTS: At discharge, the lung CT images of 15 cured patients showed no obvious lesions, while those of the remaining 140 patients showed different degrees of residual lesions. Patients with moderate disease mostly had multiple pulmonary lesions, mainly in the lower lobes of both lungs. At reexamination, the lung lesions in the patients with moderate disease had significantly improved (P<0.05), and the lung lesions in the patients with severe disease had partially improved, especially in patients with multi-lobe involvement (χ 2 =3.956, P<0.05). At reexamination, the lung lesions of patients with severe disease did not show significant changes (P>0.05). CONCLUSIONS: The pulmonary CT manifestations of cured COVID-19 patients had certain characteristics and variation patterns, providing a reference for the clinical evaluation of treatment efficacy and prognosis of patients.

Pain Management in Acute Pancreatitis: A Systematic Review and Meta-Analysis of Randomised Controlled Trials.

Background: Pain management is an important priority in the treatment of acute pancreatitis (AP). Current evidence and guideline recommendations are inconsistent on the most effective analgesic protocol. This systematic review and meta-analysis of randomised controlled trials (RCTs) aimed to compare the safety and efficacy of analgesics for pain relief in AP. Methods: A literature search was performed to identify all RCTs assessing analgesics in patients with AP. The primary outcome was the number of participants who needed rescue analgesia. Study quality was assessed using Jadad score. Pooled odds ratios (ORs) or weighted mean differences (WMDs) with 95% confidence intervals (CI) were analysed using a random-effects model. Results: Twelve studies comprising 699 patients with AP (83% mild AP) were analysed. The tested analgesics significantly decreased the need for rescue analgesia (3 studies, OR.36, 95% CI 0.21 to 0.60) vs. placebo or conventional treatment. The analgesics also improved the pain score [Visual Analogue Scale (Δ-VAS)] at 24 h (WMD 18.46, 0.84 to 36.07) and by the 3rd to 7th days (WMD 11.57, 0.87 to 22.28). Opioids vs. non-opioids were associated with a decrease in the need for rescue analgesia (6 studies, OR 0.25, 95% CI 0.07 to 0.86, p = 0.03) but without significance in pain score. In subgroup analyses, opioids were similar to non-steroidal anti-inflammatory drugs (NSAIDs) regarding the primary outcome (4 studies, OR 0.56, 95% CI 0.24 to 1.32, p = 0.18). There were no significant differences in other clinical outcomes and rate of adverse events. Other studies, comparing epidural anaesthesia vs. patient-controlled analgesia and opioid (buprenorphine) vs. opioid (pethidine) did not show significant difference in primary outcome. Study quality issues significantly contributed to overall study heterogeneity. Conclusions: NSAIDs and opioids are equally effective in decreasing the need for rescue analgesia in patients with mild AP. The relative paucity of trials and high-quality data in this setting is notable and the optimal analgesic strategy for patients with moderately severe and severe AP still requires to be determined.

Chaiqin chengqi decoction alleviates severity of acute pancreatitis via inhibition of TLR4 and NLRP3 inflammasome: Identification of bioactive ingredients via pharmacological sub-network analysis and experimental validation.

BACKGROUND: Chaiqin chengqi decoction (CQCQD) is a Chinese herbal formula derived from dachengqi decoction. CQCQD has been used for the management of acute pancreatitis (AP) in the West China Hospital for more than 30 years. Although CQCQD has a well-established clinical efficacy, little is known about its bioactive ingredients, how they interact with different therapeutic targets and the pathways to produce anti-inflammatory effects. PURPOSE: Toll-like receptor 4 (TLR4) and the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pro-inflammatory signaling pathways, play a central role in AP in determining the extent of pancreatic injury and systemic inflammation. In this study, we screened the bioactive ingredients using a pharmacological sub-network analysis based on the TLR4/NLRP3 signaling pathways followed by experimental validation. METHODS: The main CQCQD bioactive compounds were identified by UPLC-QTOF/MS. The TLR4/NLRP3 targets in AP for CQCQD active ingredients were confirmed through a pharmacological sub-network analysis. Mice received 7 intraperitoneal injections of cerulein (50 µg/kg; hourly) to induce AP (CER-AP), while oral gavage of CQCQD (5, 10, 15 and 20 g/kg; 3 doses, 2 hourly) was commenced at the 3rd injection of cerulein. Histopathology and biochemical indices were used for assessing AP severity, while polymerase chain reaction, Western blot and immunohistochemistry analyses were used to study the mechanisms. Identified active CQCQD compounds were further validated in freshly isolated mouse pancreatic acinar cells and cultured RAW264.7 macrophages. RESULTS: The main compounds from CQCQD belonged to flavonoids, iridoids, phenols, lignans, anthraquinones and corresponding glycosides. The sub-network analysis revealed that emodin, rhein, baicalin and chrysin were the compounds most relevant for directly regulating the TLR4/NLRP3-related proteins TLR4, RelA, NF-κB and TNF-α. In vivo, CQCQD attenuated the pancreatic injury and systemic inflammation of CER-AP and was associated with reduced expression of TLR4/NLRP3-related mRNAs and proteins. Emodin, rhein, baicalin and chrysin significantly diminished pancreatic acinar cell necrosis with varied effects on suppressing the expression of TLR4/NLRP3-related mRNAs. Emodin, rhein and chrysin also decreased nitric oxide production in macrophages and their combination had synergistic effects on alleviating cell death as well as expression of TLR4/NLRP3-related proteins. CONCLUSIONS: CQCQD attenuated the severity of AP at least in part by inhibiting the TLR4/NLRP3 pro-inflammatory pathways. Its active ingredients, emodin, baicalin, rhein and chrysin contributed to these beneficial effects.

Aqueous extraction from dachengqi formula granules reduces the severity of mouse acute pancreatitis via inhibition of pancreatic pro-inflammatory signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachengqi decoction (DCQD) belongs to a family of purgative herbal formulas widely used in China for the treatment of acute pancreatitis (AP). AP is a prevalent digestive disease currently without an effective pharmacological intervention. Formula granules have become the preferred method for delivery of herbal formulation in China given its benefit of potency retention, dosing precision and ease of use. The efficacy of DCQD formula granules (DFGs) in experimental AP models has not been investigated. AIM OF THE STUDY: To analyse and compare the differences in chemical composition of DFGs, with their aqueous extraction (AE) and chloroform extraction (CE) derivatives. To assess their efficacy on severity and targeted pancreatic pro-inflammatory signalling pathways in freshly isolated acinar cells and two models of experimental AP. MATERIAL AND METHODS: UPLC-Q-TOF-MS was used to analyse chemical components of DFGs and their extractions. Freshly isolated mouse pancreatic acinar cells were treated with taurolithocholic acid 3-sulphate disodium salt (TLCS, 500 µM) with or without DFGs, AE and CE. Apoptotic and necrotic cell death pathway activation was measured by caspase 3/7 (10 µl/mL) and propidium iodide (PI, 1 µM), respectively, using a fluorescent plate reader. Necrotic acinar cells were also counted by epifluorescence microscopy. Mice received either 7 intraperitoneal injections of caerulein (50 µg/kg) at hourly intervals or retrograde infusion of TLCS (3 mM, 50 µl) to induce AP (CER-AP and TLCS-AP, respectively). In CER-AP, mice received oral gavage of DFGs (2.1, 4.2 and 5.2 g/kg), AE (0.6, 1.2, and 2.4 g/kg) and CE (4, 9 and 17 mg/kg), or matched DFGs (1.8 g/kg) and AE (1 g/kg) for 3 times at 2-hourly intervals, or a single intraperitoneal injection of DCQD-related monomers rhein (20 mg/kg), narigeinine (25 mg/kg), and honokiol (5 mg/kg) begun at the 3rd injection of caerulein. In TLCS-AP, DFGs (4.2 g/kg) were given orally at 1, 3 and 5 h post-surgery. Disease severity and pancreatic pro-inflammatory markers were determined. RESULTS: The main effective anthraquinones and their glycosides, flavonoids and their glycosides, polyphenols and lignans were found in the DFGs. A higher proportion of polar components including glycosides attached to anthraquinones, phenols and flavonoids was found in AE. Conversely, lower polar components containing methoxy substituted flavonoids and anthraquinones were more abundant in CE. DFGs were given at 4.2 g/kg, a consistent reduction in the pancreatic histopathology score and severity indices was observed in both CER-AP and TLCS-AP. In vitro, AE significantly reduced both apoptotic and necrotic cell death pathway activation, while CE increased TLCS-induced acinar cell necrosis. In vivo, AE at dose of 1.2 g/kg consistently reduced pancreatic histopathological scores and myeloperoxidase in the CER-AP that were associated with suppressed expression of pro-inflammatory meditator mRNAs and proteins. CE increased lung myeloperoxidase and failed to protect against CER-AP in all dosages. AE was demonstrated to be more effective than DFGs in reducing pancreatic histopathological scores and myeloperoxidase. CONCLUSIONS: AE from DFGs alleviated the severity of mouse AP models via an inhibition of pancreatic pro-inflammatory signalling pathways. Efficacy of AE on experimental AP was more potent than its original DFGs and DCQD monomers.

Improving Small Intestinal Motility in Experimental Acute Necrotising Pancreatitis by Modulating the CPI-17/MLCP Pathway Using Chaiqin Chengqi Decoction.

Protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), a specific inhibitor of myosin light-chain phosphatase (MLCP) regulated by proinflammatory cytokines, is central for calcium sensitisation. We investigated the effects of chaiqin chengqi decoction (CQCQD) on the CPI-17/MLCP pathway in the small intestinal smooth muscle cells (SMCs) and strips (SMS) in an AP model. Necrotising AP was induced in rats by intraperitoneal injections (IPI) of L-ornithine (3.0 g/kg, pH 7.0; hourly × 2) at 1 hour apart; controls received saline. In treatment groups, carbachol (CCh; 60 µg/kg, IPI) or CQCQD (20 g/kg; 2-hourly × 3, intragastric) was administered. The necrotising AP model was associated with systemic inflammation (serum IL-1ß and TNF-α) and worsened jejunum histopathology and motility (serum vasoactive intestinal peptide and intestinal fatty acid-binding protein) as the disease progressed. There was decreased intracellular calcium concentration ([Ca2+]i) SMCs. Contractile function of isolated SMCs was reduced and associated with down-regulated expression of key mRNAs and proteins of the CPI-17/MLCP pathway as well as increased IL-1ß and TNF-α. CQCQD and CCh significantly reversed these changes and the disease severity. These data suggest that CQCQD can improve intestinal motility by modulating the CPI-17/MLCP pathway in small intestinal smooth muscle during AP.