Background
Obesity is characterized by an excessive fat storage in adipose tissue. It contributes to oxidative stress and chronic inflammation which lead to major disorders such as type 2 diabetes and cardiovascular diseases [
1-
3]. Several mediators causing obesity-related oxidative stress and inflammation have been reported. First, the over-regulated metabolic activity occurring in the adipose tissue during obesity constitutes a main source of reactive oxygen species (ROS) like H
2O
2. Continuously generated by the mitochondria, ROS are kept in check by endogenous cellular antioxidant mechanisms such as superoxide dismutase (SOD) and catalase. An imbalance between ROS production and the cellular antioxidant defence system causes oxidative stress [
4]. In adipose cells, ROS overproduction alters endoplasmic reticulum and mitochondrial functions as well as cell signalling which induce an inflammatory state [
1,
5]. The molecular mechanism involved could be partly based on ROS-induced production of the inflammatory cytokine Tumor Necrosis Factor alpha (TNFα). TNFα plays a crucial role in insulin resistance through the down-regulation of insulin-stimulated glucose uptake, insulin receptor auto-phosphorylation and insulin receptor substrate-1 [
6]. These effects contribute to the decrease in lipid accumulation within the adipose tissue and to obesity-associated cardiovascular disorders. Thus, additionally to ROS, TNFα represents a second important mediator involved in adipose tissue inflammation during obesity. Its protein levels are significantly increased in adipose tissue of obese humans and its production could result from adipose tissue remodelling characterized by macrophage accumulation [
7]. This macrophage infiltration has been reported in adipose tissue of obese patients. It could be responsible for the major part of the locally-produced TNFα, and mainly contributes to the production of the pro-inflammatory cytokine Interleukin-6 (IL-6) through the activation of Nuclear Factor-κB (NF-κB) signalling pathway [
5-
7]. Much attention has been paid on the mechanisms responsible for macrophage infiltration, and it has been suggested that macrophages present within white adipose tissue could derive from preadipocytes [
8,
9]. Moreover, adipose cells are able to secrete the chemokine Monocyte Chemoattractant Protein-1 (MCP-1), which is a recruiting factor for circulating monocytes reported to be over-produced in obesity [
10]. Similarly to TNFα, both IL-6 and MCP-1 are critically involved in insulin resistance and chronic inflammation [
11,
12]. More and more studies indicate that such adipokines could also result from the immune response of adipose tissue to an increased level of the lipopolysaccharide (LPS) endotoxin from Gram-negative bacteria during obesity [
13]. According to Cani et al. [
14], obesity-related excessive dietary lipid intake facilitates the absorption of endotoxins, leading to a higher plasma LPS level. This metabolic endotoxaemia from the gut microbiota could act as a triggering factor in the development of insulin resistance and type 2 diabetes [
15-
17]. Finally, as ROS and TNFα, LPS may also act as a major obesity-related inflammatory mediator.
No effective pharmacological treatments against obesity-associated oxidative stress and inflammation have been found yet. Thus, the identification of natural compounds able to increase the antioxidant and anti-inflammatory capacity of the body is of high interest. Plant polyphenols constitute the most abundant antioxidants provided by the human diet. More than 5,000 molecules have been identified and classified into main groups, namely phenolic acids, flavonoids, stilbenes, lignans and curcuminoids [
18]. A large array of biological properties has been attributed to polyphenols including antioxidant, antibacterial, antiviral and anti-inflammatory activities [
19-
21]. Recently, we demonstrated that polyphenols protected preadipocytes against mitochondrial alterations and inflammation caused by H
2O
2-mediated oxidative stress. Our data also showed that antioxidant and anti-inflammatory effects of polyphenols depended on their chemical nature, dose and time of exposure [
22,
23]. There is more and more evidence that the consumption of medicinal plants could contribute to increase the daily intake of polyphenols [
18]. Three medicinal plants from the Indian Ocean area are traditionally used in order to reduce the incidence of obesity and diabetes, namely
Antirhea borbonica J.F. Gmelin (Rubiaceae),
Doratoxylon apetalum (Poir.) Radlk. (Sapindaceae) and
Gouania mauritiana Lam. subsp.
Mauritiana (Rhamnaceae). Even if antioxidant and anti-inflammatory properties have been attributed to some medicinal plants from the same species or genus [
24-
26], there is still a lack of data regarding their effect on adipose cell biology.
Our objective was to evaluate for the first time the antioxidant and anti-inflammatory properties of polyphenol-rich extracts from A. borbonica, D. apetalum and G. mauritiana medicinal plants on preadipocytes exposed to H2O2, TNFα or LPS. We determined their effects on cell viability, the production of ROS, IL-6 and MCP-1 pro-inflammatory markers, as well as on the expression of genes coding for SOD and catalase antioxidant enzymes, and for NF-κB transcription factor.
Discussion
Few studies have focused on the redox status and inflammatory response of preadipocytes during obesity despite the fact that preadipose cells play an important role by governing the development of adipose tissue and fat mass. Some dietary polyphenols such as phenolic acids have been shown to affect preadipocyte viability by inducing apoptosis through a Fas- and mitochondrial-mediated pathway [
36]. Moreover, our recent data demonstrated that the ability of polyphenols to modulate preadipocyte growth depended on their chemical nature and concentration [
23]. Thus, it was important to evaluate polyphenol levels in medicinal plant extracts in order to assess a potential cytotoxic effect depending on the dose used. It appeared that all plant extracts exhibited a high polyphenol content ranging from 1-7% GAE (w/w) with
D. apetalum extract identified as the most abundant source. Major flavonoids like quercetin and catechin as well as phenolic acids derived from caffeic acid such as chlorogenic acids, known to be abundant in foods and medicinal plants [
18,
37], were identified. Similar polyphenols have been reported for cocoa seed (6-8%, w/w) as well as for green coffee (3%, w/w) [
18,
38]. Results from Hsu et al. [
39] demonstrated that such polyphenols caused a cell cycle arrest of 3T3-L1 preadipocytes in the G
1 phase. Here, none of the polyphenol-rich plant extract affected preadipocyte viability measured by MTT and LDH assays, suggesting the absence of cytotoxic effect.
In order to explore the impact of polyphenol-rich plant extracts against oxidative stress, preadipocytes were co-exposed to H
2O
2. All polyphenol-rich extracts were able to reverse H
2O
2-mediated anti-proliferative action and ROS production. This protective effect could be attributed to the presence of high levels of polyphenols exhibiting a strong free radical-scavenging activity as demonstrated by using both DPPH and ORAC methods. Such antioxidant properties of polyphenols have been largely reported and could be explained not only by the scavenging of free radicals, but also by the modulation of the function of mitochondria which constitutes the major cellular source of ROS [
4]. Indeed, several polyphenols including the family of phenolic acids detected here in medicinal plant extracts have been reported to affect the activity of mitochondrial components mainly involved in the regulation of free radical generation, by increasing levels of SOD, glutathione, glutathione peroxidase and glutathione S-transferase [
40]. In the present study, the elevation of ROS production induced by H
2O
2 was accompanied by a significant reduction of SOD gene expression which was counteracted by both
A. borbonica and
D. apetalum extracts. H
2O
2 also induced an increase in the production of IL-6 pro-inflammatory cytokine which was down-regulated only by the polyphenol-rich extract from
D. apetalum plant. Such a pro-inflammatory action of H
2O
2 was associated with an elevation of the expression of NF-κB, established as a key transcription factor involved in the regulation of IL-6 secretion in several cell types [
41-
44]. Interestingly, all polyphenol-rich plant extracts significantly down-regulated NF-κB gene expression induced by H
2O
2. Concerning the inhibitory effect of
D. apetalum extract on IL-6 secretion, it may be mediated by the presence of specific compounds. Our work led to identify polyphenols such as chlorogenic acid at a very high level in
D. apetalum extract. The ability of such plant polyphenols to down-regulate NF-κB gene expression may partly explain their effect on the decrease in pro-inflammatory cytokine production. However, other mechanisms including the regulation of NF-κB protein translocation or Inhibitor-κB (IκB) phosphorylation cannot be excluded. Interestingly, several literature data have reported that chlorogenic acid is very effective against inflammation due to its ability to decrease IκB phosphorylation as well as the nuclear translocation of NF-κB [
45-
47]. It is also noteworthy that
D. apetalum extract exhibited a 6-fold higher level of flavonoids as compared to other plant extracts. Such compounds could also contribute to explain
D. apetalum extract anti-inflammatory action. For illustration, quercetin which is one of the most antioxidant flavonoids commonly found in plants, has been reported to strongly down-regulate H
2O
2-induced IL-6 secretion [
23,
48].
Importantly, the present work highlights that H
2O
2 as well as TNFα and LPS provoked oxidative and pro-inflammatory damages on preadipose cells. Noticeably, the inflammatory response of preadipocytes exposed to these major obesity-related inflammatory mediators depended on the nature of the mediator considered. Indeed, whereas H
2O
2-induced inflammation was characterized by an increase in IL-6 secretion without any modification of MCP-1 level, TNFα and LPS up-regulated the production of both IL-6 and MCP-1 pro-inflammatory markers. This result is in agreement with literature data reporting the ability of TNFα and LPS to change the expression and secretion of several adipokines. Indeed, it was nicely described that 34 genes including those encoding for IL-6 and MCP-1 were induced in a dose dependent manner in TNFα-treated human and mouse adipocytes. Such pro-inflammatory effects were also reported to be time-dependent [
40,
49]. Regarding LPS action, Chirumbolo et al. [
50] recently demonstrated that IL-6 secretion was constantly up-regulated by prolonging incubation with LPS for 24 h, whereas the production of other inflammatory markers such as the chemokine Macrophage Inflammatory Protein-1α (MIP-1α) was induced within the first 2–8 h. Mechanistically, it was established that the expression of these cytokines was regulated predominantly by NF-κB protein [
40,
49,
50]. Accordingly, our present work provided evidence that TNFα and LPS induced a significant increase in NF-κB gene expression, similarly to H
2O
2. Such TNFα or LPS pro-inflammatory response pattern associated with their oxidative damages through ROS generation may play a role in the regulation of the immune phenotype of preadipose cells, and affect their normal differentiation. Indeed, ROS, IL-6 and TNFα were reported to impair the normal differentiation of preadipocytes and lipid accumulation contributing to inflammation-induced adipose tissue dysregulation and insulin resistance [
1,
5]. Our study also showed that TNFα and LPS significantly increased the secretion of MCP-1, which is known as a major chemokine involved in macrophage infiltration into the adipose tissue. Here, taking into account the preadipocyte inflammatory state in response to major mediators such as H
2O
2, TNFα or LPS, our data contributed to support the demonstration of the crucial impact of inflammation on preadipocyte capacity to proliferate/differentiate as well as on their ability to recruit other cells such as macrophages. Several literature data have established a link between adipose tissue and immuno-competent cells. This link is illustrated by the great cellular plasticity exhibited by preadipocytes to be very efficiently and rapidly converted into macrophages in an inflammatory environment. This preadipocyte phenotype conversion disappears when preadipose cells stop proliferating and differentiate into adipocytes. Thus, such an ability of preadipocytes to function as macrophage-like cells may play a crucial role in the involvement of adipose tissue in inflammatory processes [
8,
9,
51]. Moreover, through the preadipocyte secretion of MCP-1 which participates to the recruitment of macrophages, immune processes in adipose tissue may be reinforced in response to TNFα and LPS mediators [
10].
Our data provided the first evidence that polyphenol-rich extracts from
A. borbonica,
D. apetalum and
G. mauritiana plants inhibited ROS production and the down-regulation of SOD gene expression mediated by H
2O
2, TNFα and LPS mediators. In agreement with literature data, the absence of effect of such mediators on catalase gene expression may reflect a specific action on ROS level regulation through SOD enzyme modulation [
40]. This antioxidant effect was also associated with a decrease in preadipocyte pro-inflammatory state through the reduction of IL-6 and MCP-1 secretion as well as NF-κB gene expression. Thus, such an anti-inflammatory action of antioxidant polyphenols from medicinal plants emphasizes their potential capacity to modulate the link between adipose tissue and immune processes during obesity, by regulating key signalling pathways involving SOD and NF-κB proteins. There is a growing literature data regarding the evaluation of anti-inflammatory therapeutic strategies based on phenolic acids like caffeic acid derivatives or flavonoids such as those present in green tea, blueberry, grape seed and several other plants derived from traditional medicine [
52-
56]. Interestingly, our results showed that polyphenol-rich plant extracts exerted different antioxidant and anti-inflammatory effects depending on the plant considered. As cells were treated with the same dose of plant polyphenols (25 μM GAE), the different pattern of the regulation of ROS and cytokine production, SOD and NF-κB gene expression may result from the activity of some specific compounds present in the plants. For illustration,
D. apetalum plant extract identified as the most bioactive extract contained procyanidins which were not detected in other plants. Moreover, a synergistic action of polyphenols depending on the plant extract considered cannot be excluded. This is in accordance with our previous work showing that antioxidant and anti-inflammatory properties of polyphenols may depend on their chemical nature, dose and ability to target cells according to their bio-accessibility extent [
23].
In this study, we were interested in the effect of plant polyphenols on the production of three major pro-inflammatory cytokines, namely TNFα, IL-6 and MCP-1. However, TNFα was not detectable in 3T3-L1 preadipocytes. This agrees with previous data from authors who detected TNFα mRNA in 3T3-L1 preadipocytes, but were unable to measure any secreted TNFα [
57]. A similar observation was reported by Fain et al., who found significant amounts of TNFα secreted by stroma vascular cells, with little or no detectable TNFα secreted by adipocytes obtained from human adipose explants [
58]. The hypothesis suggested by the authors is that TNFα secretion by adipocytes would depend on signalling events from their
in vivo environment, where they are exposed to macrophage-derived TNFα [
57]. Collectively, our results led to suggest that the molecular mechanisms involved in the inflammatory state of adipose tissue during obesity could constitute relevant candidates for therapeutic agents such as antioxidant polyphenols. By demonstrating that polyphenol rich-extracts from medicinal plants increased cellular antioxidant defence system and down-regulated the release of pro-inflammatory molecules from preadipose cells, our data highlight their ability to interfere in the cross-talk between adipose cells and major immuno-competent cells like macrophages. This may result in an attenuation of the deleterious inflammatory process that occurs during obesity and related disorders such as type 2 diabetes. In order to assess the beneficial properties of polyphenols derived from medicinal plants tested here, it will be important to evaluate their effects in animal models, then in obese subjects. Such studies will also help to precise all systemic and tissular benefits, taking into account the bioavailability extent of polyphenols which constitutes a key factor governing their ability to efficiently target cells in
in vivo condition.
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Competing interests
The authors have no competing financial interests to declare in relation to the work described. The authors alone are responsible for the content and writing of the manuscript.
This work was supported by the European Union, the French Ministry of Education and Research, the Région Réunion and the Federative Structure for Environment, Biodiversity and Health from the University of Réunion Island. MM and FLS are recipients of Région Réunion and French Ministry of Education and Research fellowships, respectively.
Authors’ contributions
MM, FLS, MPG and CRDS contributed to the study design, data collection and analysis, and the writing of the manuscript. JS and CLDH contributed to data analysis and the writing of the manuscript. All authors read and approved the final manuscript.