Pulsatilla decoction alleviates colitis by enhancing autophagy and regulating PI3K‑Akt‑mTORC1 signaling pathway

Abstract

The aim of the present study was to investigate the therapeutic effect of Pulsatilla decoction (PD) on ulcerative colitis (UC) and to elucidate its potential molecular mechanisms. C57BL/6 mice expressing natural killer (NK)1.1 were used as experimental animals in the present study and a model of oxazolone‑induced colitis was established. Mice were randomly divided into the following five groups: i) PD group; ii) oxazolone‑induced colitis group; iii) IL‑13 intervention group; iv) 5‑aminosalicylic acid positive control group; and v) negative control group (equal volume saline gavage). A total of 10 animals were used in each group. The effects of PD on UC and the association between this regimen and the PI3K‑Akt‑mTORC1 signaling pathway were evaluated by disease activity index (DAI), hematoxylin and eosin staining, reverse transcription‑quantitative PCR (RT‑qPCR), immunofluorescence assay, ELISA and western blotting. The UC models were successfully established by injecting oxazolone gavage solution. Clinical colitis evaluation and histological examination revealed that PD reduced the DAI values in oxazolone‑induced colitis in mice and the degree of infiltration in NK1.1 cells. PD significantly reduced the secretion of IL‑13, as determined using an ELISA. In addition, western blotting and RT‑qPCR analyses demonstrated that Beclin1 and LC3II/I expression levels were downregulated following treatment of the mice with PD. In addition, PD not only partially restored alterations in the expression of tight junction proteins in the colon tissues, but also suppressed the activation of the PI3K‑Akt‑mTORC1 signaling pathway. The data indicated that this regimen could alleviate oxazolone‑induced UC in mice, which could significantly reduce tissue inflammation and autophagy. The mechanism of action was associated with the PI3K‑Akt‑mTORC1 signaling pathway.

Keywords: PI3K‑Akt‑mTORC1; Pulsatilla decoction; autophagy; ulcerative colitis.

Figure 1.

PD alleviates the symptoms of ulcerative colitis in mice. (A) Representative images from H&E-stained sections of the colon derived from different groups of mice. Magnification, ×200. (B) H&E and (C) DAI scores of oxazolone-induced colitis mice were measured following oral administration of PD, anti-IL-13 intervention or 5-ASA for 7 days. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. normal control group; ^##P<0.01 vs. model control group. PD, Pulsatilla decoction; H&E, hematoxylin and eosin; DAI, disease activity index; 5-ASA, 5-aminosalicylic acid.

Figure 2.

PD reduces colon weight and length. (A) The length and weight were evaluated from the different groups of mice. Statistical analysis of colon (B) weight and (C) length. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. normal control group; ^#P<0.05, ^##P<0.01 vs. model control group. PD, Pulsatilla decoction; 5-ASA, 5-aminosalicylic acid.

Figure 3.

PD inhibits ulcerative colitis inflammatory response. Expression levels of the inflammatory cytokines (A) MPO and (B) IL-13 were determined in colon tissues by ELISA. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. normal control group; ^##P<0.01 vs. model control group. PD, Pulsatilla decoction; MPO, myeloperoxidase; 5-ASA, 5-aminosalicylic acid.

Figure 4.

Effect of PD on autophagy in oxazolone-induced colitis tissue. (A and B) Expression levels of LC3 in the colon tissues of oxazolone-induced colitis mice treated with different drugs were detected using immunofluorescence analysis. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. normal control group; ^##P<0.01 vs. model control group. PD, Pulsatilla decoction; 5-ASA, 5-aminosalicylic acid.

Figure 5.

PD downregulates the expression levels of autophagy-associated genes and proteins. Detection of autophagy-associated expression via (A-C) western blotting and (D and E) reverse transcription-quantitative PCR. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. *P<0.05 **P<0.01 vs. normal control group; ^#P<0.05, ^##P<0.01 vs. model control group. PD, Pulsatilla decoction; 5-ASA, 5-aminosalicylic acid.

Figure 6.

Protective effects of PD against oxazolone-induced colitis are exerted via regulation of the PI3K-Akt-mTORC1 signaling pathway. (A-D) Assessment of the mRNA expression levels of the PI3K-Akt-mTORC1 signaling pathway members. (E and F) Protein expression of the PI3K-Akt-mTORC1 signaling pathway was detected by western blot analysis. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. **P<0.01 vs. normal control group; ^#P<0.05 vs. model control group. PD, Pulsatilla decoction; 5-ASA, 5-aminosalicylic acid; p-, phosphorylated; ATG13, autophagy-related protein 13.

Figure 7.

PD protects the colon epithelial barrier through alteration of tight junction proteins. (A) Reverse transcription-quantitative PCR analysis of occludin, ZO-1 and claudin-2 in oxazolone-induced colitis tissues. (B and C) Occludin, ZO-1 and claudin-2 expression levels were detected by western blot analysis. The data are representative of three independent experiments and are expressed as the mean ± standard deviation. **P<0.01 vs. normal control group; ^#P<0.05 vs. model (anti IL-13) group. PD, Pulsatilla decoction; ZO-1, zona occludens protein 1.