1. Introduction
Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic disorder of the gastrointestinal tract characterized by inflammation. Individuals with UC typically suffer with recurrent inflammation of the colon whereas in CD patients the inflammatory responses can occur in the terminal ileum and colon [
1]. Although the precise aetiology of IBD still remains unknown, and there are some cases reporting genetic susceptibility of some individuals, a dysbiosis of the gut microbiota remains a consistent underlying feature [
2]. Environmental factors, diet and lifestyle have also been widely recognized as potential triggers for IBD. Recent rapid increases in the incidence of IBD in developing countries have occurred along with considerable shifts toward Western style dietary habits [
3]. Dietary intervention is also being considered as a means of IBD prevention or treatment, due to the significant influence it has on the metabolism and diversity of gut microbiota populations [
4,
5].
Recent studies suggest there are beneficial effects of polyphenol-rich foods on human health, and may provide promising alternative approaches to preventing or treating chronic diseases, including IBD [
6]. Polyphenolic constituents which possess potent antioxidant activities function not only as free-radical scavengers, but also modulators of cellular redox signalling pathways during different physiological/pathological stages. Their anti-inflammatory activities are also widely acknowledged since they inhibit the production and the release of several inflammatory mediators like interleukins, tumour necrosis factor, nitric oxide, etc. [
6]. More importantly, recent evidence suggests that dietary polyphenols modulate human intestinal microbial populations due to their bacteriostatic or bactericidal actions [
7], which could be of particular value for treating IBD given the apparent role of gut microbes as a contributor to the condition.
Propolis is a resinous substance collected by honeybees,
Apis mellifera, from various plant sources, which contains abundant polyphenolic compounds [
8]. Our previous studies using different animal models showed that polyphenol-rich propolis extracts exhibited significant anti-inflammatory effects, probably via modulating the production of key inflammatory mediators and by blocking the activation of NF-κB [
9,
10]. Propolis’ chemical properties vary slightly depending on the geographic location from which it is derived, but a broad spectrum of bioactivities including anti-inflammatory effects are consistently present, suggesting it could be used to treat diseases, such as IBD.
Here, we describe a study to determine whether the addition of a Chinese propolis extract to a Western style diet can reduce the severity of colitis induced by dextran sulphate sodium (DSS) in rats and using this model to examine the contribution that the gut microbiota may play in this protection. The major shifts in population of microbiota in DSS-induced colitis in rats are similar to those in human UC [
11,
12] and we will examine the contribution that the gut microbiota may play in any protection observed with dietary propolis in this model.
4. Discussion
IBD comprises a group of chronic intestinal inflammatory disorders that are now very common across the world. Current treatments for UC using anti-inflammatory or immunosuppressive drugs are often unable to sustain long-term clinical remission, which highlights the need for novel treatment therapies. The potential for diet to prevent or improve outcomes of the disease is still relatively poorly explored. Here, the ability of dietary intake of Chinese propolis in preventing DSS-induced colitis in rats was studied, including the role that modulation of the gut microbiota might play.
An increasing number of dietary components, such as bioactive dietary peptides, amino acids, prebiotic fibres, and polyunsaturated fatty acids, are recognized as having gut health benefits [
18]. Many plant-derived phytochemicals, including polyphenolic compounds, seem especially beneficial and are often linked to anti-inflammatory and anti-oxidant activities. For instance, UC patients receiving a daily standardized anthocyanin-rich bilberry preparation for six weeks showed 63% remission [
19]. A polyphenol-rich cranberry extract has also been found to help protect against diet-induced obesity, insulin resistance, and intestinal inflammation, which is associated with gut microbiota population changes [
20]. Here, we used DSS to induce colitis in rodents, which is widely recognized as a model resembling human ulcerative colitis [
12]. The group of untreated control animals was included in the first phase of the study solely as a reference point to demonstrate that treatment with DSS actually generated the symptoms of colitis. As anticipated, overt colitis was generated as shown by the DAI results and by the histology sections we included (as showed in
Figure 1). We observed a lower weight gain and a higher colon length/weight value in the untreated control rats compared with the 0% propolis group (
Supplementary Table S1). Interestingly, the present study showed that addition of 0.3% Chinese propolis extract to the diet was able to reduce the severity of DSS-induced colitis. This extract has been used previously in our studies and shown to have anti-inflammatory activities. Recently, we found that propolis may strengthen intestinal barrier function in Caco-2 cell monolayers by activating AMPK and ERK signalling. Moreover, rats fed with propolis (same batch and amount as used here) exhibited increased expression of the tight junction protein ZO-1 in the colonic epithelium [
21]. Chinese propolis contains an abundance of polyphenolic compounds which may be responsible for these effects, including chrysin [
22], kaempferol [
23], apigenin [
24], and caffeic acid [
25], which have been demonstrated to prevent DSS-induced colitis. This also complements dietary supplementation studies carried out using Brazilian green propolis, which has been shown to alleviate colitis induced by acetic acid or trinitrobenzene sulfonic acid in rodents [
26,
27]. The Brazilian green propolis, with a local botanic origin of
Baccharis dracunculifolia DC (Asteraceae), also has abundant phenolic acids, including caffeic acid and prenylated
p-coumaric acids (Artepillin C and Baccarin). In contrast, the botanic origins of the Chinese propolis used in the present study is poplar trees (
Populus sp.) and flavonoids and flavonoid esters are the main polyphenols in the propolis derived from this source [
13]. Therefore, our findings presented here suggest there is a therapeutic potential of propolis for IBD irrespective of its geographical and botanical origins.
A disruption of gut microbiota populations is associated with many diseases, including IBD [
28,
29]. Such disruptions are also often linked to intestinal permeability changes which will facilitate the uptake of harmful agents [
30]. In UC patients, microbial dysbiosis is generally characterized by reduced bacterial diversity and an increase in the ratio of Bacteroidetes/Firmicutes [
31,
32]. Shifts in microbiota are also observed in experimentally-induced colitis in animals. In our study, DSS-induced colitis was accompanied by shifts in gut microbiota populations, including the bacterial families Ruminococcaceae and Bacteroidaceae, which has also been found in previous studies [
33]. In addition, when compared to normal (non-colitis) rats, there was a lower abundance of bacteria belonging to the order Lactobacillales, which comprises the lactic acid bacteria with well-known probiotic properties. A previous study which used the DSS-induced colitis rat model noted a similar change in lactic acid bacteria, finding that colonic injury was negatively correlated with Firmicutes, Actinobacteria, Lactobacillales, and
Lactobacillus [
2]. In addition, we also noticed that the SCFA values from the untreated control were lower than the DSS-challenged group, which is in contrast to a previously published result [
34]. Nevertheless, some studies suggest that DSS challenge will boost the SCFA production [
35,
36].
Dietary propolis has shown potential to modulate gut microbial populations, but the evidence is limited. Propolis can inhibit the growth of some bacteria, especially pathogens, and has been examined for use in farm animals such as chickens and cows [
37,
38]. In a previous study in mice fed a high-fat diet, supplementation with Brazilian green propolis was found to have beneficial effects, stabilizing the gut microbiota profile. Longer-term treatment with propolis (0.2% in the diet) promoted increases in numbers of Bacteroides and
Helicobacter, and reductions in numbers of
Oscillopira and
Blautia [
39]. Our study, where rats were on a Western-style diet background, has now shown that supplementation with Chinese propolis (0.3%) leads to greater bacterial diversity and increases in Proteobacteria and Acidobacteria when compared to rats in the 0% propolis and non-DSS controls. Interestingly, increases in bacteria of the phyla Proteobacteria, such as
E. coli, are regularly associated with IBD [
40]. Given this, our observation of protection against colitis with a propolis treatment which also increases Proteobacteria seems counter-intuitive. One explanation is that some members of the phylum increased in response to propolis out-compete other more pathogenic members that could contribute to the inflammatory processes of the disease. Acidobacteria are widely detected by 16S rRNA sequencing in different environments; nevertheless, their roles or physiological activities during colitis remain elusive [
41]. Our finding that propolis supplementation during DSS-induced colitis increases Acidobacteria, suggesting that Acidobacteria might have an important and beneficial role in gut health which should be explored further. Using LEfSe analysis to examine differences in the relative abundances of microbial taxa, the 0.3% propolis treatment resulted in a trend of a return to non-DSS treatment abundances for the Bacteroides and Bacteroidaceae. The lowering of the abundances of these microbes could also potentially explain the reduced colitis severity seen with the 0.3% propolis as these bacteria have been linked to poor gut health outcomes and often highly abundant in colitis patients [
42].