Bile acid metabolomics identifies chenodeoxycholic acid as a therapeutic agent for pancreatic necrosis

Abstract

Bile acids are altered and associated with prognosis in patients with acute pancreatitis (AP). Here, we conduct targeted metabolomic analyses to detect bile acids changes in patients during the acute (n = 326) and the recovery (n = 133) phases of AP, as well as in healthy controls (n = 60). Chenodeoxycholic acid (CDCA) decreases in the acute phase, increases in the recovery phase, and is associated with pancreatic necrosis. CDCA and its derivative obeticholic acid exhibit a protective effect against acinar cell injury in vitro and pancreatic necrosis in murine models, and RNA sequencing reveals that the oxidative phosphorylation pathway is mainly involved. Moreover, we find that overexpression of farnesoid X receptor (FXR, CDCA receptor) inhibits pancreatic necrosis, and interfering expression of FXR exhibits an opposite phenotype in mice. Our results possibly suggest that targeting CDCA is a potential strategy for the treatment of acinar cell necrosis in AP, but further verification is needed.

Keywords: acinar cells; acute pancreatitis; bile acids metabolomics; chenodeoxycholic acid; farnesoid X receptor; obeticholic acid; oxidative phosphorylation; pancreatic necrosis.

Graphical abstract

Figure 1

Flow chart of the study design for the cohort

Figure 2

Changes in the metabolic components of serum bile acids in patients during the acute phase of AP (A) Principal component analysis (PCA) of targeted bile acids metabonomic. (B) Volcano plot representing the levels of 17 significantly changed bile acids up- or downregulated in the acute phase of AP patients (n = 326) with respect to healthy controls (HCs, n = 60). Components of serum bile acids with significant changes are presented in red (significant increase) or in green (significant decrease). (C) Heatmap of mean normalized bile acids metabolite concentrations derived from targeted bile acids metabonomic profiling in the acute phase of AP patients (n = 326) and healthy controls (HCs, n = 60). (D) Cleveland dot plot showing a ranked log2 transformation of fold changes of serum bile acids. Green, blue, and red dots represent the fold changes between patients with MAP (n = 99), MASP (n = 144), and SAP (n = 83), with respect to HCs (n = 60), respectively. (E) Scatterplots showing log2 transformation of the normalized concentrations of serum CDCA and CA in patients with MAP (n = 99), MASP (n = 144), SAP (n = 83), and healthy controls (n = 60). (F) Scatterplots showing the changes in log2 transformation of the normalized concentrations of serum CDCA and CA in the acute phase of AP patients grouped according to the different intervals between onset and hospital admission (≤48 h, n = 118; 48–96 h, n = 146; >96 n = 62) and healthy controls (HCs, n = 60). Numbers indicate the mean and standard error of the mean (SEM). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05. NS, no significance.

Figure 3

Changes in the metabolic components of serum bile acids in patients during the recovery phase of AP (A) Principal component analysis (PCA) of targeted bile acids metabonomic. (B) Volcano plot showing the levels of changed bile acids up- or downregulated in patients during the recovery phase of AP concerning the acute phase of AP. The levels of CDCA and CA with significant increases are presented in red. (C) Heatmap of mean normalized bile acids metabolite concentrations derived from targeted bile acids metabonomic profiling in patients during the acute phase of AP (acute, n = 133) and the recovery phase of AP (post, n = 133) with MAP (n = 31), MASP (n = 44), and SAP (n = 58). (D) Cleveland dot plot showing a ranked log2 transformation of fold changes of serum bile acids. Green, blue, and red dots represent the fold changes between the recovery phase of AP with respect to the acute phase of AP with MAP (n = 31), MASP (n = 44), and SAP (n = 58), respectively. (E) Scatterplots displaying the changes of the normalized concentrations of serum CDCA and CA in patients during the acute phase of AP (acute, n = 133) and the recovery phase of AP (post, n = 133) with MAP (n = 31), MASP (n = 44), and SAP (n = 58). Numbers indicate the mean and standard error of the mean (SEM). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 vs. acute group.

Figure 4

Serum CDCA and CA were significantly associated with pancreatic necrosis (A and B) Association analysis between the normalized concentrations of serum CDCA and CA and acute necrotic collection (ANC [n = 186] < NO-ANC [n = 140]), organ failure (OF [n = 108], NO-OF [n = 218]), acute respiratory distress syndrome (ARDS [n = 92], NO-ARDS [n = 234]), acute kidney injury (AKI [n = 53], NO-AKI [n = 273]), and pancreatic necrosis infection (IPN [n = 19], NO-IPN [n = 307]). Numbers indicate the mean and standard error of the mean (SEM). ∗∗p < 0.01 and ∗p < 0.05. NS, no significance. (C) Forest plot of risk factors for pancreatic necrosis, IPN, ARDS, AKI, and shock in AP patients that were analyzed using univariate logistic regression and multivariate logistic regression of corrected age, gender, body mass index, etiology, and disease parameter.

Figure 5

CDCA and its derivative OCA regulate the mitochondrial oxidative phosphorylation pathway to promote cellular ATP production attenuated acinar cell necrosis in vitro and in vivo (A) Scatterplots showing changes in serum CDCA and CA levels in mice with AP (n = 5). (B) qPCR detected the mRNA levels of key enzymes of hepatic bile acids synthesis (CYP7A1, CYP27A1,CYP8B1,CYP7B1) (n = 5). (C) CCK-induced primary acinar cell damage was detected by LDH release after concentration gradients CDCA (0.1, 0.25, 0.5, 1, 25 μM) or OCA (1, 2.5, 5 μM) were treated. (D and E) Immunofluorescent imaging (200x, scale bar represents 50 μM) of calcein-AM/PI stained acinar cells and phosphatidylinositol fluorescence intensity of injured acinar cells quantified (n = 6–8). (F) Diagram of caerulein-induced experimental AP model and OCA intervention in mice. (G) Change in serum activity of amylase and lipase. (H) H＆E staining images (100x and 400x, scale bars represent 100 μM and 25 μM) and histological scores (edema, inflammation, necrosis) of pancreatic tissues in AP mice (n = 7). (I) Differential genes enrichment analysis by KEGG in RNA-seq of pancreatic acinar cells with or without the addition of OCA. (J) GSEA enrichment analysis of differential genes. (K) The levels of ATP production, mitochondrial respiratory chain complex I and V activities, and NADP enzyme activity in the pancreatic acinar cells (n = 4–6). Numbers indicate the mean and standard error of the mean (SEM). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. NS, no significance.

Figure 6

Expression of FXR by acinar cells regulates pancreatic necrosis in mice with AP (A) Expression and relative quantification of FXR in pancreatic tissue of caerulein-induced AP mice by western blotting. (B) IHC staining of FXR in tissue homogenates obtained from caerulein-induced AP mice confirmed that FXR expression was significantly elevated (n = 4). (C) Western blots showing FXR expression in pancreatic tissue of mice was significantly increased after 3 weeks injection of AAV-FXR in mouse pancreatic tissue (n = 3). (D and E) H&E staining images (400x, scale bar represents 25 μM), histological scores (edema, inflammation, and necrosis) of pancreatic tissues, and changes in serum activity of amylase and lipase after AP induced by caerulein in AAV-FXR overexpressing mice (n = 7). (F) Western blots showing FXR expression in pancreatic tissue of mice was significantly decreased after 3 weeks injection of AAV-shFXR in mouse pancreatic tissue (n = 3). (G and H) H&E staining images (400x, scale bar represents 25 μM), histological scores (edema, inflammation, and necrosis) of pancreatic tissues and changes in serum activity of amylase and lipase after AP induced by caerulein in AAV-shFXR interfering mice (n = 7). Numbers indicate the mean and standard error of the mean (SEM). ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05.