Pulsatilla Decoction Can Treat the Dampness-Heat Diarrhea Rat Model by Regulating Glycerinphospholipid Metabolism Based Lipidomics Approach

Abstract

Ethnopharmacological relevance: Diarrhea is a major medical problem in clinical practice. According to the theory of traditional Chinese medicine (TCM), different types of diarrhea should be treated with different TCM formulations based on the targeted medical condition. Dampness-heat diarrhea (DHD) is a serious diarrheal disease and Pulsatilla decoction (PD), a TCM, has been found effective against DHD.

Objective: The aim of this study was to clarify the mechanism of action of PD in DHD using an untargeted lipidomics strategy.

Materials and methods: Wistar rats were randomized to four groups, including the control group, model group, PD groups and self-healing group. The PD groups were given a daily intragastric gavage of PD at doses of 3.76 g/kg. The rat model of DHD established by such complex factors as high-sugar and high-fat diet, improper diet, high temperature and humidity environment, drinking and intraperitoneal injection of Escherichia coli., which imitated the inducing conditions of DHD. Then the clinical symptoms and signs, blood routine, serum inflammatory cytokines levels and the histopathological changes of main organs were detected and observed to evaluate DHD model and therapeutic effect of PD. Lipid biomarkers of DHD were selected by comparing the control and model groups with the colon lipidomics technology and an ultra-high performance liquid chromatography (UHPLC) coupled with Q Exactive plus mass analyzer. Multivariate statistical analysis and pattern recognition were employed to examine different lipids within the colon of PD-treated rats.

Results: The clinical symptoms and signs of the model rats were consistent with the diagnostic criteria of DHD. After treatment with PD, the clinical symptoms and signs of the rats with DHD were improved; the indexes of blood routine and inflammatory cytokines levels tended to be normal. The lipidomics profile of the model group were evidently disordered when compared to the control group. A total of 42 significantly altered lipids between the model-control groups were identified by multivariate statistical analysis. DHD may result from such lipid disorders which are related to glycerophospholipid metabolism, arachidonic acid (AA) metabolism, and sphingolipid metabolism. After PD treatment, the lipidomic profiles of the disorders tended to recover when compared to the model group. Twenty lipid molecules were identified and some glycerophospholipids and AA levels returned close to the normal level.

Conclusion: Glycerophospholipid metabolism may play an important role in the treatment of dampness-heat induced diarrhea using PD.

Keywords: arachidonic acid metabolism; colon lipidomics; dampness-heat diarrhea; glycerophospholipid metabolism; pulsatilla decoction.

FIGURE 1

High performance liquid chromatography chromatogram of the standard mixture (A) and 3 PD samples (B–D), Peaks 1: aesculin; 2: aesculetin; 3: jateorhizine hydrochloride; 4: palmatine chloride; and 5: berberine hydrochloride.

FIGURE 2

Symptoms of rats with DHD. Rats in the control group (A,B). Rats treated in a high temperature and humidity environment, high-sugar and high-fat diet (C). DHD symptoms such as sloppy diarrhea, anus redness, and swelling (D–F), respectively.

FIGURE 3

Body weight changes for rats in the control group, model group, self-healing group, and pulsatilla decoction treatment group during the experiment.

FIGURE 4

The contents of inflammatory cytokines in serum. (A) IL-1β, (B) IL-2, (C) IL-6, (D) TNF-α; control group; model group; Self-healing group; Pulsatilla decoction treatment group. *P < 0.05 vs control group; ^#P < 0.05 vs model group.

FIGURE 5

Histopathological changes of the ileum. (A–A1) Control group, the mucosal epithelium was integral, morphologies of the villi were normal, and venous congestion was not observed in the lamina propria (LP). (B–B1) Model group, infiltration of eosinophils was observed in the LP and around the crypt; integrity of the mucosal epithelium was destroyed and some epithelia were exfoliated; villi epithelium showed degeneration and necrosis. (C–C1) Self-healing group, eosinophils were reduced in the LP and around the crypt; integrity of the mucosal epithelium was destroyed, and parts of the epithelium were exfoliated; villi epithelium showed amelioration. (D–D1) PD group, epithelium was regenerated, integrity was recovered, and eosinophil infiltration decreased. Triangular arrows indicate eosinophil infiltration into LP. Pins indicate injuries of mucosal epithelium. Original magnification, × 400. Scale bar represents 20 μm.

FIGURE 6

Histopathological changes of the colon. (A–A1) Control group, epithelium was integral, brush border was clearly visible, and morphologies of the veins in LP were normal although red cells could be seen within them. (B–B1) Model group, epithelium showed necrosis and exfoliation, and integrity was destroyed; few eosinophil infiltrations were present in the LP around the intestinal glands; many red cells were observed in the veins and arteries of LP. (C–C1) Self-healing group, epithelium showed necrosis and exfoliation and the integrity was destroyed; there was no eosinophil infiltration in the LP around the intestinal glands. (D–D1) PD group, mucosal epithelium was regenerated, integrity was recovered, neutrophil infiltration decreased in the LP, and congestion was effectively alleviated. Arrows in B and B1 indicate the injuries in the mucosal epithelium, few eosinophil infiltration and severe congestion, respectively. Original magnification, ×400. The scale bar represents 20 μm.

FIGURE 7

Typical base peak intensity chromatograms (BPC) for the colon of rats from the control group in positive ion mode (A) and negative ion mode (B), and those from the model group in positive ion mode (C) and negative ion mode (D).

FIGURE 8

Typical base peak intensity chromatograms (BPC) for the colon of rats from the PD group in positive ion mode (A) and negative ion mode (B), and those from the self-healing group in positive ion mode (C) and negative ion mode (D).

FIGURE 9

Lipid subgroup and lipid molecule count according to the International Lipid Classification and Nomenclature Committee and based on LipidSearchsoftware version 4.1.

FIGURE 10

Multivariate data analysis of metabolites in rat colon based on UHPLC-Orbitrap MS. (A) PCA score plot of all samples and QC samples; (B) PCA score plot of Model-Control; (C) PLS-DA score plot of Model-Control; (D) OPLS-DA score plot of Model-Control; (E) PLS-DA displacement test of Model-Control; (F) OPLS-DA displacement test of Model-Control; (G) volcano plot of Model-Control, red point represents the different lipids (FC > 2.0, P-value < 0.05); (H) Fold-change analysis of the different lipids between Model and Control (C-Control, M-model, P-PD, S-self-healing).

FIGURE 11

Heatmap visualization and hierarchical clustering analysis (A) and the correlation analysis results of the Control group (B) based on significant difference in lipids.

FIGURE 12

Differential expression levels (mean) of 20 differential lipids in different groups. A comparison of the relative intensities of the potential biomarkers in the control, model, PD, and self-healing groups. *P < 0.05 vs control group; ^#P < 0.05 vs model group.

FIGURE 13

Possible metabolic pathway maps associated with the lipid biomarkers in dampness-heat induced diarrhea and dampness-heat induced diarrhea with PD treatment. Biomarkers in blue are those increased in the control-model. Biomarkers in red are those decreased biomarkers in the control-model. The up arrows represent biomarkers which were significantly increased after PD treatment. The down arrows represent the biomarkers which were significantly decreased after PD treatment. The circles represent biomarkers which were not significantly changed after PD treatment.