Protective effect of andrographolide against ulcerative colitis by activating Nrf2/HO-1 mediated antioxidant response

Abstract

Ulcerative colitis (UC) is a recurring inflammatory bowel disease, in which oxidative stress plays a role in its progression, and regulation of the oxidative/antioxidative balance has been suggested as a potential target for the treatment of UC. The aim of this study was to evaluate the protective effect of andrographolide against UC and its potential antioxidant properties by modulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Dextran sulfate sodium (DSS) -induced UC mice and the LPS-induced HT29 inflammatory cell model were established to uncover the potential mechanisms of andrographolide. ML385, a Nrf2 inhibitor, was used in both models to assess whether andrographolide exerts a protective effect against UC through the Nrf2/HO-1 pathway. The in vivo experiment showed that andrographolide ameliorated the symptoms and histopathology of DSS-induced mice and restored the expressions of ZO-1, Occludin-1 and Claudin-1. Meanwhile, DSS-induced oxidative stress and inflammation were suppressed by andrographolide treatment, along with the upregulation of key proteins in the Nrf2/HO-1 pathway. In vitro experiments showed that andrographolide attenuated LPS-induced excessive generation of ROS in HT29 cells, reduced inflammatory factors, and upregulated the expression of proteins related to tight junctions and Nrf2/HO-1 pathway. In addition, ML385 abolished the beneficial effect of andrographolide. In conclusion, the protective effect of andrographolide against UC may involve the suppression of oxidative stress and inflammation via the Nrf2/HO-1 pathway.

Keywords: Nrf2/HO-1 pathway; andrographolide; inflammation; oxidative stress; ulcerative colitis.

FIGURE 1

Andrographolide ameliorated DSS-induced colitis in mice. (A) Representative images of the colon. (B) Colon length. (C) Pictures of H&E staining (amplification 200, bar = 100 μM). The data are presented as mean ± SD (n = 6). ***p < 0.001, compared with control group; ##P < 0.01, ###P < 0.001, compared with the DSS group.

FIGURE 2

Andrographolide exerted protective effect on intestinal barrier of UC. (A) The protein expressions of Occludin-1, Claudin-1, and ZO-1 in mice colon tissues. (B) Quantitation of the expression of Occludin-1, Claudin-1, and ZO-1 protein. The results are presented as mean ± SD (n = 3). ^* P < 0.05, ^** P < 0.01, compared with control group; ^# P < 0.05, ^## P < 0.01, compared with the DSS group. (C) The protein expressions of Occludin-1 and Claudin-1 in HT29 cells. (D) Quantitation of the expression of Occludin-1 and Claudin-1 protein. The results are presented as mean ± SD (n = 3). ^** P < 0.01, compared with control group; ^# P< 0.05, ^## P < 0.01, compared with the LPS group.

FIGURE 3

Andrographolide modulated the levels of inflammatory cytokines. (A) The expression of MPO in mice colon tissues (amplification 100, bar = 100 μM). (B) The levels of serum LPS. (C) The protein expressions of IL-17 and IL-23 in mice colon tissues. (D) Relative levels of IL-17 and IL-23 expressions. (E) The mRNA expressions of IL-17 and IL-23 in mice colon tissues. (F) The mRNA expressions of IL-1β, IL-6 and TNFα. The results are presented as mean ± SD (n = 3–5). ^* P < 0.05, ^** P < 0.01, ^*** p < 0.001, compared with control group; ^# P < 0.05, ^## P < 0.01, compared with the DSS group. (G) The protein expressions of IL-17, IL-23, IL-1β and p-p65 in LPS primed HT-29 cells. (H) Relative levels of IL-17, IL-23, IL-1β and p-p65. The results are presented as mean ± SD (n = 3). ^** P < 0.01, compared with control group; ^# P < 0.05, ^## P < 0.01, compared with the LPS group.

FIGURE 4

Andrographolide inhibited oxidative stress. Effect of andrographolide on the content of MDA (A), the activity of GSH (B) and SOD (C) level in the colonic tissues. The data are presented as mean ± SD (n = 5). ^*** P < 0.001, compared with control group; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001, compared with the DSS group. (D) Representative images of ROS level in HT29 cells.

FIGURE 5

Andrographolide activated the Nrf2/HO-1 signaling pathway. (A) The protein expressions of Nrf2 and HO-1 in mice colon tissues. (B) Relative levels of Nrf2 and HO-1 expressions. The results are presented as mean ± SD (n = 3). ^** P < 0.01, compared with control group; ^## P < 0.01, compared with the DSS group. (C) The protein expressions of Nrf2 and HO-1 in LPS primed HT-29 cells. (D) Relative levels of Nrf2 and HO-1 expressions. The results are presented as mean ± SD (n = 3). ^** P < 0.01, compared with control group; ^# P < 0.05, ^## P < 0.01, compared with the LPS group.

FIGURE 6

Inhibition of Nrf2 abolished the protective effects of andrographolide in DSS-induced colitis mice. (A) body weight. (B) Representative images of the colon. (C) Colon length. (D) Pictures of H&E staining (amplification 100, bar = 100 μM). (E–G) The mRNA levels of Occludin-1, Claudin-1, and ZO-1 in mice colon tissues. (H) The protein expressions of Occludin-1, Claudin-1, and ZO-1 in mice colon tissues. (I) Quantitation of the expression of Occludin-1, Claudin-1, and ZO-1 protein. The data are presented as mean ± SD (n = 3–10). ^* P < 0.05, ^** P < 0.01.

FIGURE 7

Inhibition of Nrf2 attenuated the protective effects of andrographolide on inflammatory factors and oxidative stress through Nrf2/HO-1 pathway in DSS-induced colitis mice. (A, B) The level of MDA and SOD. (C, D) The mRNA levels of IL-17 and IL-23 in mice colon tissues. (E) The protein expressions of IL-17, IL-23, Nrf2 and HO-1 in mice colon tissues. (F) Quantitation of the expression of IL-17, IL-23, Nrf2, and HO-1 protein. The data are presented as mean ± SD (n = 3–5). ^* P < 0.05, ^** P < 0.01.

FIGURE 8

Inhibition of Nrf2 attenuated the protective effects of andrographolide on inflammatory factors. (A) The protein expressions of IL-1β, IL-17, IL-23, and P-p65 in HT29 cells. (B) Quantitation of the expression of IL-1β, IL-17, IL-23, and P-p65 protein. The data are presented as mean ± SD (n = 3). ^* P < 0.05, ^** P < 0.01.

FIGURE 9

The protective effect of Andrographolide against ulcerative colitis by suppressing oxidative stress and inflammation through activation of the Nrf2/HO-1 pathway.