Department of General Surgery, Children's Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai, China.
Pathology Department, Children's Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai, China.
Front Immunol. 2022 Aug 25;13:875593. doi: 10.3389/fimmu.2022.875593. eCollection 2022.
Biliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine.
To study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values.
Low expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts).
Bioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFβ signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFβ signaling pathways. The TGFβ signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFβ pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR.
The downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.
先天性胆道闭锁（BA）是一种儿童期肝脏疾病，其特征为纤维性阻塞和肝外胆道系统阻塞，是婴儿中最常见和最严重的胆道疾病之一。无论 BA 的初始损伤如何，肝脏和胆道的显著炎症和纤维化都是最突出的特征。在 BA 的人类患者和小鼠模型中发现了先天或适应性免疫的异常。我们之前报道过，BA 患儿存在游离血清肉碱等脂代谢异常。
研究肝过氧化物酶体增殖物激活受体-α（PPARα）信号通路和法尼醇 X 受体（FXR）在 BA 和 BA 纤维化中的基因和蛋白表达水平，并评估其临床价值。
在基因表达综合数据库中验证了 BA 中 PPARα 和 NR1H4（FXR）的低表达。基于 PPARα 和 NR1H4 表达的基因集富集分析确定了功能差异。根据 PPARα、NR1H4 和 SMAD3 的表达水平，使用共识聚类将来自 GSE46960 的 BA 患者分为两个簇，并进行免疫浸润分析。最后，使用我院 58 例病例进行实验验证。（IHC：10 例胆道闭锁，10 例胆总管囊肿；PCR：10 例胆道闭锁，14 例胆总管囊肿；WB：10 例胆道闭锁，4 例胆总管囊肿）。
生物信息学分析表明，胆道闭锁组 PPARα、CYP7A1 和 NR1H4（FXR）的表达明显低于对照组。在 BA 患者中，包括 TGFβ 信号通路、P53 信号通路、PI3K-AKT-mTOR 信号通路等在内的更多 BA 特异性通路被富集。此外，低 NR1H4 表达与炎症反应、IL6/STAT3 信号通路、早期雌激素反应、IL2 STAT5 信号通路和 TGFβ 信号通路等有关。TGFβ 信号通路在两组中均有显著差异。根据 PPARα、NR1H4 和 SMAD3 的表达情况，使用共识聚类将 BA 患者分为两个簇。在簇 2 中，SMAD3 表达较高，而 PPARα 和 NR1H4 表达较低。与簇 1 相比，簇 2 中免疫细胞浸润更高，免疫组化证实了这一点。免疫组化、Western blot 分析和实时 PCR 结果显示，BA 患者的 PPARα 和 NR1H4 基因和蛋白表达水平均低于对照组。
PPARα 和 NR1H4（FXR）信号通路的下调可能与胆道闭锁密切相关。
