探索膽道閉鎖初期之疾病標記及腸道微生態失調

Abstract

膽道閉鎖是嬰兒早期發生的進行性膽道發炎及纖維化，造成肝外膽道阻塞。因膽汁無法順利從肝臟排出到腸道，造成肝內細胞傷害。膽道閉鎖有可能在產前或周產期就已經發生。及時的診斷讓膽道閉鎖嬰兒能夠儘早接受葛西氏肝門空腸吻合術，重建膽汁流通，改善嬰兒長期預後。研究顯示膽酸、蛋白質、細胞激素和miRNA等生物標記物在疾病診斷、治療、與致病機轉中有不同的角色。此研究旨在探索膽道閉鎖疾病早期生物標記物，以發現具有早期診斷及預測預後的指標。 本研究一個重要的里程碑為首次在診斷為膽道閉鎖的新生兒中發現了在出生後三天內基質金屬蛋白酶7（MMP-7）升高的情況。MMP-7可作用於廣泛的基質並透過多重路徑來調節發炎反應、表皮細胞通透性及表皮細胞再生。本研究的主要研究結果展示了血片MMP-7水平作為膽道閉鎖篩檢生物標記的證據。與非膽道閉鎖患者相比， MMP-7數值在膽道閉鎖患者中顯著升高，並且不受黃疸照光治療、血片存儲時間及早產狀況等因素影響。重要的是， MMP-7在區分膽道閉鎖和其他先天性或周產期疾病以及健康對照組方面表現出很高的敏感性和特異性，最佳曲線下面積為93.7%。這些發現突顯了MMP-7於膽道閉鎖疾病初期作為檢測工具的潛力，為臨床醫師提供了一種寶貴的方式，加速診斷並改善患者臨床結果與預後。 腸道微生物特徵可做為許多疾病的生物標記，為了解疾病之病理生理和進展提供了啟示。透過研究膽道閉鎖腸道中的微生物特徵有助於早期診斷、改善預後與治療反應。在此研究中，進一步使用16S rRNA次世代定序分析膽道閉鎖患者腸道菌叢，結果顯示健康對照組和膽道閉鎖患者的糞便微生物組成，在葛西氏肝門空腸吻合術前後均有顯著差異。長雙歧桿菌（B. longum）在膽道閉鎖患者中明顯減少，與葛西氏肝門空腸吻合術後γ-谷氨酰轉肽酶升高相關。在膽道閉鎖疾病早期檢測到B. longum的患者與未檢出患者比較，腸道中具B. longum之膽道閉鎖患者葛西氏肝門空腸吻合術後三個月膽紅素有較顯著的下降，且有較低的肝臟移植比例。此研究結果顯示，顯示B. longum在膽道閉鎖疾病初期的重要性，且與預後有高度相關性。使用散彈槍總體基因體定序技術進一步確認16S rRNA次世代定序分析之發現，進一步證實了B. longum在膽道閉鎖中的重要性。在膽道閉鎖患者中腸道B. longum的早期定植和其豐富度可作為治療膽道閉鎖可能之治療方向。 此研究強調了兩個關鍵生物標記MMP-7和B. longum在膽道閉鎖疾病早期階段的重要性。這兩個生物標記在過去中研究顯示對維持腸道屏障完整性和調節免疫功能扮演著重要的角色。MMP-7和B. longum對在診斷為膽道閉鎖的新生兒中，出生後三天內MMP-7升高突顯了其作為早期篩檢或診斷指標的重要性。此外，膽道閉鎖患者B. longum存在減少的情況進一步突顯了其作為預後生物標記的潛力。這些發現強調了這些生物標記在促進早期檢測和預後評估方面的關鍵作用。透過理解B. longum、MMP-7、腸道屏障完整性和免疫失衡之間的相互作用，可能有助於深入探索先天性膽道閉鎖的致病機轉。

Biliary atresia (BA) is characterized by bile duct obstruction, neonatal jaundice, and liver fibrosis, often manifesting prenatally or in early life with prenatal anomalies and early bilirubin elevation. Timely diagnosis of BA enables early Kasai portoenterostomy (KPE) intervention that improves patient outcomes. Current management of BA remain suboptimal due to late diagnosis, and progressive liver fibrosis after KPE. Recent research explores biomarkers, including bile acid, cytokines, and miRNA for diagnostic and therapeutic potential. This study aims to explore early-stage biomarkers for screening and prognosis prediction of BA. The first part of the study marks a milestone as the first to identify elevated levels of MMP-7 within the first three days of life in newborns diagnosed with BA. MMP-7 has broad substrate specificity which underscores its involvement in modulating non-ECM molecules and signaling pathways crucial for inflammation, epithelial permeability, and epithelial regeneration. The main findings of the research reveal evidence supporting the use of dried blood spot (DBS) matrix metallopeptidase 7 (MMP-7) levels as a biomarker for BA in early life. DBS MMP-7 levels were significantly elevated in BA patients compared to non-BA patients, irrespective of factors such as phototherapy, storage duration of DBS, or preterm birth status. Importantly, DBS MMP-7 demonstrated high sensitivity and specificity in distinguishing BA from other congenital or perinatal disorders and healthy controls, with an area under the curve of 93.7%. These findings underscore the potential of DBS MMP-7 as a reliable tool for early BA detection, offering clinicians a valuable means to expedite diagnosis and optimize patient management strategies for improved clinical outcomes. The microbiome signature holds immense promise as a biomarker for various diseases, shedding light on their pathophysiology and progression. Investigating the microbiome signature in BA may unveil novel biomarkers for early diagnosis, prognosis, and treatment response, ultimately enhancing clinical management and patient outcomes in this complex pediatric liver disease. In this study, the analysis of fecal microbial composition using 16S rRNA NGS revealed significant differences between healthy controls (HCs) and BA patients, both before and after Kasai portoenterostomy (KPE). Specifically, Bifidobacterium longum (B. longum) was notably diminished in infants with BA, correlating with elevated gamma-glutamyl transferase levels post-KPE. Patients exhibiting early detectable B. longum showed reduced bilirubin levels and a lower rate of liver transplantation, emphasizing its potential as a prognostic indicator. Confirmation of these findings was obtained through shotgun metagenomic sequencing, further highlighting the significance of B. longum in BA management. Early colonization and augmentation of B. longum levels in the gut may serve as a potential therapeutic avenue to enhance the prognosis of individuals affected by BA. In conclusion, this study underscores the importance of two key biomarkers, MMP-7 and B. longum, in the early stages of BA. Both was known to contribute to maintaining gut barrier integrity and regulating immune function. Elevated levels of MMP-7 within the first three days of life in newborns diagnosed with BA highlight its significance as an early diagnostic indicator. Furthermore, the diminished presence of B. longum in BA patients affirm its potential as a prognostic marker. These findings emphasize the critical role of these biomarkers in facilitating early detection and prognostic assessment, thereby enhancing clinical management and patient outcomes in BA. Understanding the interplay between B. longum, MMP-7, gut barrier integrity, and immune imbalance might provide insights into pathogenesis of BA.