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高甘油三酯血症延缓急性胰腺炎修复的临床与基础研究
中文摘要

急性胰腺炎(acute pancreatitis,AP)是常见的消化系统疾病之一,全球年发病率约13-45/10万,且呈逐渐上升趋势。AP通常由胆石症、酒精摄入以及高甘油三酯血症(hypertriglyceridemia,HTG)等因素诱发,表现为胰腺外分泌系统的急性炎症性损伤,胰腺组织病理改变包括水肿、渗出、症细胞浸润以及腺泡细胞坏死。胰腺经过炎症消褪、细胞间质生成再降解以及腺泡细胞增殖等一系列损伤修复过程后,基本恢复正常形态功能,但一些药物如吗啡或者代谢性疾病如糖尿病可延缓上述修复过程。HTG是一种常见的代谢性疾病,临床表现为血清甘油三酯(triglyceride,TG)水平升高。目前HTG已成为我国AP的第二大病因,高甘油三酯血症性胰腺炎(hypertriglyceridemic acute pancreatitis,HTG-AP)占AP比例高达13-25.6%。HTG可加重AP严重程度,AP患者预后包括多脏器损伤、炎症反应等与血清TG水平呈正相关。此前有研究表明HTG能够影响心肌以及肝细胞再生,但HTG是否影响AP后胰腺修复尚不明确。本研究旨在探讨HTG对AP胰腺修复的影响,加深对HTG-AP病理生理的认知。 首先,在临床层面我们探讨了血清TG水平与重症急性胰腺炎(severe acute pancreatitis,SAP)患者胰腺修复程度的关系,对2016年1月至2017年1月入住重症胰腺炎中心的SAP患者进行长达1年的随访,调查患者出院1年后的胰腺修复状况。根据患者发病72 h内最高TG水平,将入组患者分为4组:正常血清甘油三酯组(normal triglyceridemia,NTG)(TG<1.7mmol/L),轻度HTG(1.7-5.7 mmol/L),中度HTG(5.7-11.3 mmol/L),重度HTG(≥11.3 mmol/L)。由于患者胰腺组织很难获取,考虑到AP发病后与胰腺组织损伤修复相并行的外分泌功能的动态变化,我们采用粪弹力蛋白酶(fecal elastase-1,FE-1)——一种胰腺外分泌功能的经典标志物来间接评价胰腺修复的状况。根据纳入标准与排除标准,本研究最终共纳入93名SAP患者,20名患者未能参加随访,最终NTG组、轻、中、重度HTG组分别有11、37、11、14名患者检查了FE-1水平。除血清TG水平外,四组患者之间的其他基线特征无显著差异。分析四组的FE-1变化趋势后发现,TG水平越高FE-1越低(P〓<0.001),进行post-hoc分析后发现中度和重度HTG患者FE-1水平显著低于NTG组患者,提示血清TG水平与SAP患者胰腺修复程度呈负相关。 进而,我们在动物模型中观察HTG对AP胰腺修复的影响。本部分采用了两种HTG动物模型,泊洛沙姆(poloxamer407,P407)诱导的HTG小鼠和糖基磷脂酰肌醇高密度脂蛋白结合蛋白1(glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1,GPIHBP1)基因缺陷小鼠。腹腔注射雨蛙素建立AP动物模型,分别于造模后第1、3、5、7天麻醉动物处死取材,动态观察胰腺组织病理变化,同时评估胰腺修复相关的纤维炎性反应以及腺泡细胞增生反应。实验结果显示,AP造模1天后NTG及HTG组小鼠胰腺均呈现显著的急性期病理损伤。NTG组小鼠造模3天后胰腺组织开始显著修复,第7天胰腺基本恢复正常形态结构;而HTG组小鼠胰腺修复显著延迟,TG水平越高修复越慢,重度HTG小鼠AP造模后第7天胰腺形态结构仍未恢复正常。相比于NTG组小鼠, HTG组小鼠与AP修复相关的纤维炎性反应以及腺泡细胞增生均显著延迟。 最后,为明确降脂治疗能否改善HTG-AP修复进程,我们采用临床最常用的降脂药物——非诺贝特,于AP造模后6h处理NTG小鼠和HTG小鼠,动态观察不同剂量非诺贝特(100㎎/㎏/d、100㎎/㎏/bid、100㎎/㎏/tid)处理小鼠1、 3、5天后胰腺病理改变,并通过免疫组化评估降脂治疗后腺泡细胞增生的变化。结果发现,相比于HTG-AP组,非诺贝特处理组中100㎎/㎏/tid剂量组造模后第3天TG水平显著降低、腺泡细胞增生反应加强,第5天胰腺组织坏死面积显著下降。此外,非诺贝特的干预对于NTG组小鼠AP后的修复无显著影响。 综上所述,我们的临床研究及动物实验结果首次表明HTG延迟AP后的损伤修复过程,且修复程度与血清TG水平呈负相关,使用非诺贝特强化降脂可促进HTG小鼠胰腺修复。研究结果丰富了对AP损伤修复的临床认知,为HTGAP的治疗提供新的思路和理论基础。 关键词:高甘油三酯血症,急性胰腺炎,修复,纤维炎性反应,增生反应,非诺贝特

英文摘要

Acute pancreatitis (AP) is a common digestive system disease. The annual incidence in the world is about 13-45 per 100,000, and it is gradually increasing. AP is usually induced by cholelithiasis, alcohol intake, and hypertriglyceridemia (HTG). It is characterised as an acute inflammatory injury of the pancreatic exocrine system. Pathological changes of the pancreas include edema, exudation, acinar cell necrosis and infiltration of inflammatory cells. After the pancreas undergoes a series of repair processes including inflammation fading, cell mesenchyme re-degradation, and acinar cell proliferation, the normal architecture is restored. However, some drugs such as morphine or metabolic diseases such as diabetes can delay the above repair process. HTG is a common metabolic disease, with clinical manifestations of elevated serum triglyceride (TG) levels. At present, HTG has become the second leading cause of AP in China, and the proportion of hypertriglyceridemic acute pancreatitis (HTG-AP) accounted for 13-25.6% of AP. HTG can increase the severity of AP, and the higher the serum TG level, the worse the patient's prognosis. Previous studies have shown that HTG can affect myocardium and hepatocyte regeneration, but whether HTG affects AP after pancreatic repair is not yet clear. Therefore, the purpose of this study was to investigate the effect of HTG on the repair of pancreatic tissue after the onset of AP, and to deepen the understanding of the pathophysiology of HTG-AP. We first observed the relationship between serum TG levels and the degree of pancreatic repair in patients with severe acute pancreatitis (SAP). From January 2015 to December 2016, we performed long-term follow-up in patients with severe pancreatitis at the General Hospital of the Nanjing Military Region. After a 1-year follow-up, the patient was investigated for pancreatic repair after 1 year of discharge. Based on the highest TG level within 72 hours of disease onset, the patients were divided into four groups: normal triglyceridemia (NTG) (TG <1.7 mmol/L) and mild HTG (1.7-5.7 mmol/L), moderate HTG (5.7-11.3 mmol/L), and severe HTG (≥T1.3 mmol/L). Due to the difficulty in obtaining pancreatic tissue in patients, taking into account the dynamic changes of exocrine function parallel to the repair of pancreatic tissue after the onset of AP, we used fecal elastase-1 (FE-1), an classical markers of pancreatic exocrine function, indirectly assess the condition of pancreatic repair. According to the inclusion criteria and exclusion criteria, 93 SAP patients were included in this study, and 20 patients failed to participate in the follow-up. Finally, 11, 37, 11 and 14 patients in the NTG, mild, moderate, and severe HTG groups were examined for FE-1 level. There were no significant differences in other baseline characteristics between the four groups except serum TG levels. After analyzing the trend of FE-1 in the four groups, it was found that the higher the TG level, the lower the FE-1 (P〓<0.001). The post-hoc analysis showed that the levels of FE-1 in patients with moderate and severe HTG were significantly lower than those in the NTG group, suggesting a negative correlation between serum TG levels and pancreatic repair in SAP patients. We further observed the effect of HTG on AP repair in animal models. Two animal models of HTG were used in animal experiments. One was poloxamer 407 (P407)-induced HTG mice, and the other was glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) gene-deficient mice. The animal model of HTG-AP was established by intraperitoneal injection of cerulein. The animals were killed after anesthesia on days 1, 3, 5, and 7 after the model establishment. The pathological changes of pancreatic tissue were dynamically observed. At the same time, through WB and immunohistochemical methods, changes in fibroblast inflammation related to pancreatic repair and acinar cell proliferation were dynamically observed. The experimental results showed that after 1 day of AP modeling, the pancreas of NTG and HTG mice showed significant acute pathological lesions. In the NTG group, the pancreatic tissue began to repair after 3 days of modeling, and the pancreas returned to its normal morphological structure on the 7th day. In the HTG group, the pancreas repair was significantly delayed, and the higher the level of serum TG, the slower the repair was. The morphological structure of the pancreas did not return to normal on the 7th day in the severe HTG group. Compared with mice in the NTG group, the HTG group mice were significantly delayed in the inflammatory reaction and acinar cell proliferation associated with AP repair. Finally, in order to determine whether lipid-lowering therapy can improve the course of HTG-AP repair, we applied the most commonly used TG-lowering drug, fenofibrate, to treat NTG mice and HTG mice 6 hours after AP modeling. The pathological changes of pancreas were observed in mice treated with different doses of fenofibrate (100㎎/㎏/d, 200㎎/㎏/d, 300㎎/㎏/d) for 1, 3, and 5 days, and the effect on proliferation were observed by immunohistochemistry. The results showed that compared with the HTG-AP group, the TG levels were significantly decreased and acinar cell proliferation was enhanced on the third day after the 300 ㎎/㎏/d dose in the fenofibrate treatment group. The area of pancreatic tissue necrosis on the 5th day was significant decreased, while 100 ㎎/㎏/d and 200 ㎎/㎏/d did not have this effect. In addition, the treatment with fenofibrate had no significant effect on the repair of AP after NTG mice. Our clinical studies and animal experiments have shown that HTG delays the repair process after AP, and the degree of repair is negatively correlated with serum TG levels. The use of fenofibrate for lipid-lowering therapy can promote pancreatic repair in HTG mice. The findings enriched the clinical understanding of AP injury repair and provided new ideas and theoretical basis for the treatment of HTG-AP. Keywords: Hypertriglyceridemia, Acute pancreatitis, Regeneration, Fibroinflammatory response, Acinar cell proliferation, Fenofibrate

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