Ameliorative effect of Atractylodes macrocephala essential oil combined with Panax ginseng total saponins on 5-fluorouracil induced diarrhea is associated with gut microbial modulation

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Abstract

Ethnopharmacological relevance

Traditional Chinese medicine (TCM) holds that deficiency of spleen-Qi is the major pathogenesis of chemotherapy-induced diarrhea (CID). Herb pair of Atractylodes macrocephala Koidz. (AM) and Panax ginseng C. A. Mey. (PG) has good effects of supplementing Qi and strengthening spleen.

Aim of the study

To investigate therapeutic effects and mechanism of Atractylodes macrocephala essential oil (AMO) and Panax ginseng total saponins (PGS) alone and in combination (AP) on 5-fluorouracil (5-FU) chemotherapy induced diarrhea in mice.

Materials and methods

The mice were administered with AMO, PGS and AP respectively for 11 days, and intraperitoneally injected with 5-FU for 6 days since the 3rd day of the experiment. During the experiment, the body weights and diarrhea scores of mice were recorded daily. Thymus and spleen indexes were calculated after sacrifice of the mice. Pathological changes in ileum and colonic tissues were examined by hematoxylin-eosin (HE) staining. And the content levels of intestinal inflammatory cytokines were measured by enzyme-linked immmunosorbent assays (ELISA). 16S rDNA Amplicon Sequencing was used to analyze and interpret the gut microbiota of fecal samples.

Results

AP significantly inhibited body weights loss, diarrhea, reductions of thymus and spleen indexes, and pathological changes of ileums and colons induced by 5-FU. Neither AMO nor PGS alone significantly improved above-mentioned abnormalities. Besides, AP could significantly suppressed the 5-FU-mediated increases of the intestinal inflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-1β and IL-17), while AMO or PGS only inhibited some of them after 5-FU chemotherapy. Gut microbiota analysis indicated that 5-FU induced overall structural changes of gut microbiota were reversed after AP treatment. Additionally, AP significantly modulated the abundances of different phyla similar to normal values, and restored the ratios of Firmicutes/Bacteroidetes (F/B). At genus level, AP treatment dramatically decreased potential pathogens like Bacteroides, Ruminococcus, Anaerotruncus and Desulfovibrio. AP also antagonized the abnormal effects of AMO and PGS alone on certain genera like Blautia, Parabacteroides and Lactobacillus. Neither AMO nor PGS alone inhibited changes of gut microbial structure caused by 5-FU.

Conclusions

AP, combination of AMO and PGS, not AMO or PGS alone, significantly ameliorated diarrhea, inhibited intestinal pathology, and modulated gut microbial structure in 5-FU induced mice. AP also antagonized abnormal effects of AMO or PGS on certain genera. The results illustrated that gut microbiota was involved in the combined effects of AP on 5-FU induced diarrhea.

Introduction

As an efficient chemotherapeutic agent, 5-fluorouracil (5-FU) is widely applied for treatment of malignant tumors in the gastrointestinal tract, head, neck, chest, and ovary. And 5-FU is the first-line drug for colorectal cancer in clinic. The action mechanism of 5-FU is to block the transformation of uracil nucleic acid into thymine nucleic acid in the tumor cells, then affect the synthesis and repairing of DNA and RNA to achieve its cytotoxic effect (Afzal et al., 2009; Ducreux et al., 2015; Longley et al., 2003). However, 5-FU also produces chemotherapy-induced diarrhea (CID), one of the most common adverse reactions that plagues many patients (Filho et al., 2016). The incidence of diarrhea in patients treated with 5-FU was up to 50%–80%, which seriously affected the progress and efficacy of chemotherapy (Iacovelli et al., 2014; Rosenoff et al., 2006). Consequently, it is of significant importance to find effective therapy for 5-FU induced CID.

At present, non-drug interventions and drug interventions have been imported into the clinical treatment of CID. Non-drug interventions include reasonable diet, and supplement with salt, sugar and other nutrients. Drugs such as loperamide and octreotide are commonly used in anti-diarrhea therapy of CID (Benson et al., 2004). In addition, ethnomedicines are also adopted to treat CID with their own unique therapy in various countries. Traditional Chinese medicine (TCM) is one typical ethnomedicine that has been practiced for more than 2000 years in East Asian countries including China, Japan and Korea (Qi et al., 2010). TCM holds that chemotherapeutic drugs would trigger Qi consumption, spleen deficiency, stomach disharmony and endophytic dampness, resulting in conductive dysfunction of intestines. In TCM theory, the treatment strategy of CID should be mainly depended on supplementing Qi and strengthening spleen (Wang et al., 1994).

The dried roots of Atractylodes macrocephala Koidz. (AM) and Panax ginseng C. A. Mey. (PG) are the typical herbal medicines in TCM with same effects of supplementing Qi and strengthening spleen (Li et al., 2014). AM and PG are usually used as herb pair (the simplest form of Chinese herbal compatibility) with the effects of supplementing Qi and strengthening spleen to treat diarrhea. For example, AM and PG were documented in classical anti-diarrheal formulae such as Shen Ling Bai Zhu San, Si Jun Zi Tang from Taiping Huimin Heji Ju Fang (Song dynasty, China) and Bu Zhong Yi Qi Tang from Pi Wei Lun (Yuan dynasty, China) (Fig. 1). Several previous studies had reported that all the three formulae possess the ability of alleviating CID (Bai et al., 2017; Chen et al., 2019; Gou et al., 2016). In addition, our previous study showed that Shenzhu Capsule which only contains AM and PG have potential effects on the treatments of diarrhea, colitis (xiexie syndrome), and other gastrointestinal diseases (Feng et al., 2018). However, no study has discussed the effect and mechanism of AM and PG in treating CID, whether in combination or alone.

Now gut microbiota is deemed to be a potential factor in understanding the therapeutic mechanism of TCM (Feng et al., 2019). Modern studies indicate that gut microbiota plays a crucial role in maintaining intestinal homeostasis. Healthy gut microbiota contributes to the intestinal mucosal protection, metabolism, immune homeostasis and response, and pathogen suppression (Thursby and Juge, 2017; Pickard et al., 2017). Disordered gut microbiota impairs the physiological and immune functions of the human body directly or indirectly, inducing side reactions like diarrhea (Patel et al., 2016; Zhao and Shen, 2010). Researches had shown that 5-FU remarkably shifted the structure of gut microbiota in diarrheic mice (Li et al., 2017). Therefore, the effects of AM and PM on 5-FU induced diarrhea may be mediated by gut microbiota. However, whether AM and PG alone and in combination could prevent 5-FU induced diarrhea by modulating gut microbiota is still unknown.

In order to investigate anti-diarrhea effects and underlying mechanism of AM and PG, we used 5-FU to simulate a diarrheal model in mice. Here, we focused on the potential effects of the single and combined administration (AP) of Atractylodes macrocephala essential oil (AMO) and Panax ginseng total saponins (PGS), the active components respectively extracted from AM and PG, on diarrhea, intestinal pathology and microbial structure after 5-FU chemotherapy.

Section snippets

Chemicals and reagents

5-FU injection was provided by Tianjin JinYao Pharmaceutical Co., Ltd (Tianjin, China). Sodium carboxymethyl cellulose, n-hexane and formaldehyde were purchased from Chengdu Kelong Chemical Reagent Plant (Chengdu, China). 95% ethanol was provided by Chengdu Cologne Chemical Co., Ltd (Chengdu, China). Ginsenosides standards of Rg1 (purity ≥ 98%), Rd (purity = 99.23%), Rc (purity = 99.11%), Rb1 (purity = 98.58%), Rb2 (purity = 98.68%), Re (purity = 99.50%) and Rf (purity = 99.62%) were provided

Chemical compositions of AMO

As shown in Fig. 3, atractylone (60.45%), γ-elemene (8.16%), eudesma-3,7(11)-diene (7.68%), 1-methoxy-2-(1-methyl-2-methylenecyclopentyl) benzene (3.77%), isopetasan (3.26%), β-eudesmene (2.46%), germacrene B (1.83%), 3,5,11-eudesmatriene (1.70%), juniper camphor (1.42%) and 2-acetyl-1,8-dihydroxy-3,6-dimethylnaphthalene (1.01%) were identified in AMO. Atractylone was considered to be the representative compound of AMO.

Ginsenosides contents in PGS

Ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2 and Rd in PGS were isolated and

Discussion

5-FU is an essential chemotherapeutic drug for the treatment of gastrointestinal cancers with a strong inhibitory function on tumor cells, but it often produces side effects such as CID (Tsuyuki. et al., 2012). According to TCM theory, treatment of CID should be based on supplementing Qi and strengthening spleen (Shao et al., 2016). In our study, AM and PG, the typical herbs with effects of supplementing Qi and strengthening spleen, were used alone and in combination to treat 5-FU induced

Conclusion

Our results indicated that AP, combination of AMO and PGS, not AMO or PGS alone, effectively alleviated 5-FU induced diarrhea and intestinal pathology. Moreover, the results illustrated that gut microbiota was involved in the ameliorative effect of AP on 5-FU induced diarrhea. And the combined effects of AP included inhibiting 5-FU induced structural changes of gut microbiota and antagonizing the abnormal effects of AMO and PGS alone on some genera after 5-FU chemotherapy. Our study revealed

Author's contributions

JW and WWF designed the experiment process, obtained data, analyzed data, and wrote the manuscript. SYZ, LC, YCS, FT, JLH and XX collected data, analyzed and explained data, and revised the manuscript. HA and CP obtained funding, conceived and designed experiment, supervised process, wrote and revised the manuscript. All the authors have read and confirmed the final manuscript.

Conflicts of interest

All the authors claimed that there was no conflict of interest.

Acknowledgements

All the authors in this study thanked National Natural Science Foundation of China (grant number 81503272) and Department of Science and Technology of Sichuan Province (grant number 2017JY0187) for strong supports.

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