Crystal structure of 6‐guanidinohexanoyl trypsin near the optimum pH reveals the acyl‐enzyme intermediate to be deacylated

The force driving the conversion from the acyl intermediate to the tetrahedral intermediate in the deacylation reaction of serine proteases remains unclear. The crystal structure of 6‐guanidinohexanoyl trypsin was determined at pH 7.0, near the optimum reaction pH, at 1.94 Å resolution. In this structure, three water molecules are observed around the catalytic site. One acts as a nucleophile to attack the acyl carbonyl carbon while the other two waters fix the position of the catalytic water through a hydrogen bond. When the acyl carbonyl oxygen oscillates thermally, the water assumes an appropriate angle to catalyze the deacylation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Chronic pancreatitis: role of oxidative stress and antioxidants

Abstract

Chronic pancreatitis (CP) is characterized by pain, and exocrine and endocrine insufficiency of pancreas. Several hypotheses have been put forward to explain the hitherto partially understood pathophysiology of CP. In the past decade, animal and clinical studies have suggested that an increased chronic oxidative stress (OS) plays a key role in pathophysiology of CP and perpetuates its clinical and histological symptoms (pain and fibrosis–necrosis, respectively). Mounting OS in pancreatic acinar cells is a result of overproduction of free radicals (FR) during xenobiotic metabolism. It has been shown that Phase I cytochrome P450 enzymes of xenobiotic pathway are induced when exposed to a xenobiotic overload including alcohol, tobacco, smoke and other dietary toxins, which exceeds the capacity of Phase II conjugation due to limited glutathione availability. Consequently, there is an overload of toxic metabolites as well as FR. Additionally, bioactivation of subsequently entering compounds may occur increasing their toxicity. Such an imbalance overwhelms the antioxidant capacity of the body resulting in undefended chronic OS that derails the normal physiology of pancreatic acinar cells since FR act as second messengers controlling the cellular signaling. OS hypothesis is further supported by the studies that demonstrated that antioxidant supplementation ameliorated pain. Moreover, animal studies have demonstrated a cessation of fibrotic cascade with antioxidant supplementation. In a recent large randomized controlled trial, it was demonstrated that antioxidant supplementation led to a significant reduction in pain, and also lowered the OS in patients with alcoholic or idiopathic CP.     

急性胰腺炎患者血清PAB、HMGB1、SIL-2R、ACE2水平变化及意义

摘要 目的：探讨急性胰腺炎患者血清前白蛋白（PAB）、高迁移率族蛋白 B1（HMGB1）、可溶性白介素2受体(SIL-2R)、紧张素转化酶2（ACE2）水平变化及意义。方法：选择2018年7月至2020年7月于我院进行治疗的急性胰腺炎患者83例患者进行研究,设为病例组,并选择我院同期体检健康者70例作为对照组,分析患者血清PAB、HMGB1、SIL-2R、ACE2及急性生理功能和慢性健康状况评分系统Ⅱ（APACHEII）评分水平变化情况及之间的相关性,采用受试者工作特征曲线分析血清PAB、HMGB1、SIL-2R、ACE2的诊断价值。结果：病例组患者血清PAB、ACE2水平显著低于对照组,HMGB1、SIL-2R、APACHEII评分水平显著高于对照组,差异显著（P＜0.05）;轻症组血清PAB、ACE2水平显著高于重症组,HMGB1、SIL-2R、APACHEII评分水平显著低于重症组,差异显著（P＜0.05）;Pearson相关分析结果显示,血清PAB、ACE2与APACHEⅡ之间呈负相关（P<0.05）,血清HMGB1、SIL-2R与APACHEⅡ之间呈正相关（P<0.05）;血清PAB预测急性胰腺炎的临界值为147.09 mg/L,灵敏度为80.54%,特异度为85.68%,AUC为0. 845,血清HMGB1预测急性胰腺炎的临界值为85.34 ng/L,灵敏度为81.65%,特异度为86.69%,AUC为0. 998,血清SIL-2R预测急性胰腺炎的临界值为25.47 pg/mL,灵敏度为81.59%,特异度为87.57%,AUC为0. 978,血清ACE2预测急性胰腺炎的临界值为298.57 pg/mL,灵敏度为80.19%,特异度为83.58%,AUC为0. 867。结论：血清PAB、HMGB1、SIL-2R、ACE2的表达与APACHEII评分之间密切相关,对急性胰腺炎具有较高的预测价值。     

大柴胡汤对轻症急性胰腺炎患者Th17/Treg免疫平衡和血清水通道蛋白的影响

摘要 目的：观察大柴胡汤对轻症急性胰腺炎（MAP）患者辅助性T细胞17 /调节性T细胞（Th17/Treg）免疫平衡和血清水通道蛋白（AQP）的影响。方法：按照随机数字表法，将天津中医药大学第一附属医院2020年4月~2022年4月期间收治的95例MAP患者分为对照组（西医基础治疗，47例）和研究组（对照组基础上接受大柴胡汤治疗，48例）。对比两组中医证候积分、Th17/Treg免疫平衡、AQP1、AQP5、AQP6和实验室指标。结果：两组治疗7 d后两胁胀痛，矢气则舒，右中上腹痛，恶心呕吐，抑郁易怒，嗳气呃逆，大便不畅评分均下降，且研究组低于对照组（P<0.05）。两组治疗7 d后Treg细胞比例升高，Th17细胞比例、Th17/Treg下降，且研究组的改善幅度大于对照组（P<0.05）。两组治疗7 d后AQP1、AQP5、AQP6升高，且研究组高于对照组（P<0.05）。两组治疗7 d后淀粉酶（AMY）、脂肪酶（LIP）、C反应蛋白（CRP）下降，且研究组低于对照组（P<0.05）。结论：大柴胡汤治疗MAP疗效确切，可以缓解患者的临床症状，有效改善MAP患者Th17/Treg免疫平衡和血清AQP1、AQP5、AQP6水平。     

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.     

白藜芦醇联合乌司他丁治疗急性胰腺炎的疗效分析

目的：观察白藜芦醇联合乌司他丁对大鼠急性胰腺炎（AP）的治疗效果。方法：将250只雄性Wister大鼠随机分为5组,分别为假手术（A）组、急性胰腺炎组（B组）、白藜芦醇组（C组）、乌司他丁组（D组）和白藜芦醇联合乌司他丁治疗组（C＋D组）。制备各组AS大鼠模型,观察各组腹水量及血淀粉酶、脂肪酶、TNF-α、NF—κB、丙二醛（MDA）及超氧化物歧化酶（SOD）水平,并观察胰腺病理改变。结果：B组与A组相比,SOD水平明显降低（P〈0．05）,MDA水平明显升高（P〈0．05）;C、D及C＋D组与B组相比,血清SOD水平明显升高（P〈0．05）,MDA水平明显降低（P〈0．05）;C＋D组与C组、D组相比,血清SOD水平明显升高（P〈O．05）,血清MDA水平明显降低（P〈0．05）。与B组比较,C和D组血淀粉酶、脂肪酶、TNF-α、病理学评分均降低,细胞超微结构损伤减轻,有显著性差异（P〈0．05或P〈0．01）。C＋D组上述指标改善较C组和D组更明显,差异有显著性（P〈0．05）。结论：乌司他丁、白藜芦醇通过升高SOD保护性因子、降低MDA损伤性因子,对急性胰腺炎有治疗作用,尤其在乌司他丁与白藜芦醇合用时效果更显著。     

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 μM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control (P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group (P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.     

104 例重症急性胰腺炎的手术治疗与非手术治疗对比分析

目的:探讨重症急性胰腺炎的有效治疗方法以及施行外科手术治疗的时机与指征.方法:回顾性分析104例重症胰腺炎患者的临床治疗资料,其中63例患者行非手术治疗,41例患者行手术治疗,观察比较患者治愈率、死亡率、并发症发生率、住院时间以及治疗前后血尿淀粉酶的变化.结果:①非手术组与手术组间治愈率、死亡率和平均住院时间无明显差异(P＞0.05),但手术组并发症发生率显著高于非手术组(P＜0.05).②早期手术组与延期手术组间治愈率比较无明显差异(P＞0.05),但延期手术可有效降低患者死亡率与并发症发生率(P＜0.05).③经过治疗,早期与延期手术组患者血尿淀粉酶水平均明显降低(P＜0.01),且早期手术组下降幅度大于延期手术组(P＜0.05).结论:非手术治疗与手术治疗的综合治疗可有效提高SAP的治疗效果,同时手术治疗中应尽量避免早期手术.     

Age-dependent vulnerability to experimental acute pancreatitis is associated with increased systemic inflammation and thrombosis

The severity and mortality rates of acute pancreatitis (AP) are significantly elevated in the elderly population. However, due to a lack of appropriate animal models, the underlying mechanisms for this age‐dependent vulnerability remain largely unknown. The purpose of this study was to characterize a murine model of AP, which displays age‐associated severity, and to use this model to identify pathophysiologies that are distinctive of the aged with AP. AP was induced in young (4–5 months), middle‐aged (12–13 months), and aged (23–25 months) C57BL/6 mice by repeated injection of caerulein, a homologue of the gastrointestinal hormone cholecystokinin. Approximately 10% of aged mice died during AP, while young and middle‐aged mice showed no mortality. Although both young and aged mice exhibited early signs of edema and inflammation in the pancreas, kidney, and lung, young mice showed signs of recovery within 24 h, while aged mice exhibited increasingly severe tissue damage and cell death. There was a significant age‐dependent increase in pancreatic neutrophil activation and systemic inflammation as assessed by pancreatic myeloperoxidase and plasma interleukin‐6 (IL‐6) concentration, respectively. Importantly, aged but not young mice with AP showed significantly elevated thrombosis in the lung and kidney as well as a marked increase in plasma concentration of plasminogen activator inhibitor‐1 (PAI‐1), a primary inhibitor of the fibrinolytic system. These results demonstrate that aging is associated with increased severity of AP characterized by augmented and prolonged pancreatic inflammation and the presence of multiple extra‐pancreatic sequelae including thrombosis.     

Interactions between enteroviruses and autophagy in vivo

Autophagy plays a protective role during many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway. However, one family of viruses, the picornaviruses, has gone one step further, by actively exploiting autophagy. Using mice in which Atg5 has been conditionally deleted in pancreatic acinar cells, we have studied the outcome of infection by coxsackievirus B3 (CVB3), a member of the enterovirus genus and picornavirus family. Two key findings emerged: disruption of autophagy (1) dramatically compromised virus replication in vivo, and (2) significantly limited pancreatic disease.