Protective effect of Andrographolide on 5-Fu induced intestinal mucositis by regulating p38 MAPK signaling pathway

Abstract

Aims: Intestinal mucositis is the most common side effect of 5-fluorouracil (5-Fu) treatment in cancer patients. Previous research suggested that andrographolide (Andro) attenuated the intestinal injury in colitis or diarrhea in mice. The present study was aimed at investigating the protective effect of Andro against 5-Fu induced intestinal mucositis and the underlying mechanism.

Main methods: BALB/C mice were injected 5-Fu at a dose of 100 mg/kg for 5 days to induce intestinal mucositis. Andro at different doses (25, 50, 100 mg/kg/day) was administered. Weight loss, diarrhea score, cellular apoptosis and proliferation were evaluated. Apoptosis related proteins were detected by Western blotting. Then, NCM460 cells were used to explore the possible mechanism in vitro. The effect of Andro on the anti-tumor efficacy of 5-Fu was investigated in H22 tumor-bearing mice.

Key findings: Andro significantly ameliorated the 5-Fu induced weight loss and diarrhea. The apoptosis of intestinal cells was also attenuated by Andro treatment both in vivo and in vitro. Besides, Andro markedly down-regulated the 5-Fu-induced protein expression of caspase8/3, Bax and the phosphorylation of p38. Moreover, 5-Fu significantly reduced the viability of NCM460 cells, which was restored by the Andro pretreatment. Furthermore, asiatic acid, an agonist of p38 MAPK, reversed the anti-apoptotic effect of Andro in NCM460 cells. Andro did not weaken the anti-H22 tumor effect of 5-Fu in vivo.

Significance: We have demonstrated that p38 MAPK inhibition mediates anti-apoptotic effects of Andro against 5-Fu induced intestinal mucositis, suggesting that Andro may benefit the patients undergoing 5-Fu based chemotherapy.

Introduction

Generally, 5-Fluorouracil (5-Fu), a pyrimidine analog, is used for chemotherapy of solid tumors, including colorectal and breast cancers [1]. Approximately, 2 million patients are annually treated with 5-Fu worldwide [2]. As an anti-metabolite drug, 5-Fu can inhibit the cell-proliferation by 85% by selectively killing tumor cells and rapidly proliferating cells (e.g. intestinal cells) [3,4]. >50% of patients, who received 5-Fu treatment, suffered from intestinal mucositis. About 15% of patients, who received a chemotherapy regimen including 5-Fu developed grade 3 or worse diarrhea and required hospitalization [5,6]. Even 1–5% of patients died due to 5-Fu-induced diarrhea [6].

However, the optimum approach to treat the side effect is still sparse. Supplements, such as anti-ulcer drugs, probiotics, dietary supplements, and antibiotics can ameliorate the symptoms of 5-Fu induced intestinal mucositis, but the effects of these drugs are still limited in clinical applications [[7], [8], [9], [10], [11], [12], [13]]. Besides, the pathogenesis of 5-Fu related mucositis is still undefined and may include cellular apoptosis, inflammation, gut barrier disruption, oxidative stress, dysbiosis, and diarrhea related nutrition deficiency [7,[9], [10], [11],14,15]. Many studies suggested that apoptosis might be involved in the intestinal cell injury, and the activation of p38 mitogen-activated protein kinases (p38 MAPK) could contribute to the 5-Fu-induced p53-dependent intestinal apoptosis [16,17]. The p38 pathway is essentially integrated with apoptosis, and the upregulation of p53 and caspase8 is accompanied with cell death too [18]. In addition, Bcl-2/Bax (anti-apoptotic/pro-apoptotic members) mediated the mitochondrial membrane permeability during apoptosis [19]. Consequently, adjuvant therapy of cellular apoptosis might effectively improve the quality of life and enhance the safety of patients undergoing 5-Fu based chemotherapy.

Andrographolide (Andro) is a labdane diterpenoid lactone from Andrographis paniculata (Burm. f) Nees, a traditional herbal medicine in China [20]. In recent decades, Andro has been widely used in Southeast Asian and Scandinavian countries for its anti-viral and anti-inflammatory effects [21,22]. Many studies have shown that Andro and its analogs can alleviate diarrhea in colitis mice [[23], [24], [25]], and senna and castor oil-induced diarrhea [26]. However, no studies have reported whether Andro can alleviate 5-Fu induced intestinal mucositis.

Our previous research explored the dose-effect relationship to intestinal mucosa injury induced by 5-Fu and found that 50–100 mg/kg 5-Fu could induce intestinal injury in mice [27]. In this research, we administered 100 mg/kg 5-Fu to mice for establishing the intestinal mucositis model, and then, the protective effect of Andro against 5-Fu-induced intestinal injury and its possible mechanism were investigated both in vivo and in vitro. Also, we further evaluated whether Andro could affect the anti-tumor effect of 5-Fu when they were co-administered.