In recent years, the prevalence of obesity has increased dramatically and has become a severe health problem worldwide. In 2016, 39% (more than 1.9 billion) of adults were overweight, and 13% were obese (1). The worldwide prevalence of obesity nearly tripled between 1975 and 2016 (2). It has been estimated that by 2030, the obesity rate will reach up to 20% of the adult population. Approximately 500 million people are obese, and 1.4 billion are overweight globally (3). The main characteristics of obesity are excessive weight gain and fat accumulation due to various factors, such as genetic predisposition, high-calorie energy intake, and sedentary lifestyle (4). Obesity can cause chronic low-grade inflammation, hyperglycemia, hyperlipidemia, and insulin resistance (5, 6). Accumulating evidence suggests that obesity is associated with various metabolic disorders, such as cardiovascular disease, type II diabetes, non-alcoholic fatty liver disease, and various kinds of cancers (3). With the recognition that obesity is responsible for a growing prevalence of chronic diseases, obesity has been recognized as “the greatest threat to global public health in this century” (7). Reducing and curbing obesity is one of the most important health challenges in the modern world. Depending on the severity of the disease, there are several ways to intervene or treat obesity, including lifestyle changes, pharmacotherapy, and bariatric surgery (8). However, the use of drugs or bariatric surgery has been shown to be associated with significant side effects, some of which could increase the risk of developing chronic diseases (6, 9). Therefore, the search for natural compounds to treat obesity and its related diseases has attracted much research interest.

Despite significant research efforts to combat obesity-induced metabolic syndrome in the last decade, there has been slow progress in understanding the causes and mechanisms that regulate its development. In recent years, accumulating evidence suggests that gut microbiota dysbiosis is strongly associated with host metabolism and obesity development (10–12). Studies have shown that the high-fat diet can change the structure of gut microbiota, disrupt the intestinal microenvironment, and lead to dysbiosis of gut microbiota (13, 14). Conversely, it has been shown that gut microbiota play an important role in the dynamic balance of metabolism by regulating intestinal endothelial barrier function, glucose metabolism, and chronic inflammation associated with obesity. Therefore, it has been proposed that altering the composition of gut microbiota by dietary or other means can confer beneficial effects, restoring the integrity of intestinal function and reversing the characteristics of obesity (15).

In recent years, polysaccharides have attracted attention for their role in weight loss. Polysaccharides are macromolecular polymers composed of at least ten monosaccharides linked by glycosidic bonds (16), and they are natural macromolecular active substances widely found in animals, plants, algae, and microbial cells (17). Polysaccharides are well-known for their health benefits, such as immunomodulatory (18), anti-tumor (19), anti-inflammatory (20), anti-oxidant (21), and hypolipidemic effects (22). Previous studies have suggested that polysaccharides from plants, such as Angelica sinensis (23), Schisandra (24), and Ophiopogon (25), show great lipid-lowering effects. Polysaccharides can reduce body weight, leaky gut, and low-grade inflammation in various tissues by physicochemical properties, such as water retention, and/or by probiotic activity, such as altering gut microbiota and production of microbiota-derived metabolites (26). A study showed that Polygonatum odoratum polysaccharides modulated gut microbiota and mitigated obesity induced by a high-fat diet (HFD) in rats (27). It has also been shown that the phylum Firmicutes was 40% higher than the phylum Bacteroidetes in HFD-fed mice, and supplementation with Ophiopogon polysaccharides increased Bacteroidetes by 28% and decreased Firmicutes by 15% (25). Thus, dietary polysaccharides are considered one of the effective regulators of gut microbiota, which may help the host regulate metabolism.

Moringa oleifera Lam. is a perennial plant of the Moringaceae family, also known as the “drumstick tree,” “miracle tree,” or “tree of life” (28). M. oleifera is rich in nutrients, including protein, vitamins, essential amino acids, minerals, dietary fiber, and bioactive compounds (29). In 2011, the Ministry of Health of the People's Republic of China issued a notice to consider M. oleifera leaves as a new food resource. It has been demonstrated that M. oleifera has many biological functions such as anti-microbial, anti-oxidant, anti-inflammatory, anti-cancer, hepatoprotective, hypoglycemic, hypolipidemic, and other effects (30–32). However, the biological activities of M. oleifera polysaccharides (MOP) in regulating gut microbiota and alleviating obesity have not yet been reported. We hypothesized that MOP could positively modulate the gut microbiota and attenuate HFD-induced obesity. Therefore, to verify this hypothesis, we investigated the effects of MOP on body weight, fat accumulation, blood lipid levels, insulin resistance, chronic inflammation, and gut microbiota in a HFD-induced obese mice, and further investigated the correlation of gut bacteria with obesity-related parameters including host obesity phenotype, blood lipids, glucose homeostasis, and pro-inflammatory cytokines.

The prevalence of overweight and obesity is an increasing chronic disease worldwide, and has become a growing global health problem. In recent years, some studies have shown that extracts from M. oliefera leaves could regulate lipid metabolism. For example, Syamsunarno et al. (43) explored the effects of ethanol extract from M. oleifera leaves on modulating brown adipose tissue differentiation in mice fed an HFD. Mabrouki et al. (44) evaluated the effect of the methanolic extract of M. oleifera leaves on HFD-induced obesity and cardiac damage in rats. Sha et al. (45) investigated the efficacy and mechanism of new resource medicinal material M. oleifera leaves against hyperlipidemia. Our team has also investigated M. oleifera leaf petroleum ether extract inhibits lipogenesis (46). In these studies, the main components of M. oleifera leaf extracts were phenolic compounds (44) and flavonoid glycosides (46), without even elucidating their main components (43, 45). Therefore, the activity of MOP in regulating lipid metabolism is currently unclear. Furthermore, there has been growing evidence that the occurrence of obesity is closely related to alteration in the gut microbiota. Thus, growing interests have been aimed to modulate gut microbiota as a therapeutic strategy against obesity and its related diseases (11, 12). Many studies have shown that bioactive components of plants, including kale (47), Ganoderma lucidum (42), Lobelia chinensis (48), and Dictyophora indusiata (3), can inhibit the development of obesity in experimental animals by regulating gut microbiota. However, the biological activities of MOP in modulating gut microbiota is also unclear. The present study demonstrated that crude polysaccharides extracted from M. oleifera leaves could effectively prevent weight gain, fat accumulation, lipid increase, and chronic inflammation in HFD-induced obese mice, which were associated with modulation of the gut microbiota. This is the first report to assess the activity of MOP for obesity prevention, and to investigate the regulation of the gut microbial community by MOP. These findings suggested that easily available MOP could be served as an alternative strategy for preventing obesity and obesity-related metabolic diseases.

Our study found that MOP supplementation significantly reduced fat weight and adipocyte size in HFD-induced obese mice (Figures 2A–D). Meanwhile, the size of fat vacuoles in the liver gradually decreased with the increase of MOP dose, suggesting that MOP helped reduce fat accumulation to protect the liver (Figure 2F). Previous studies have shown that the feeling of satiety is increased due to the swelling effect of natural polysaccharides, and thus reduced feeding occurs during the ingestion of undigested polysaccharides (49). Our results showed that MOP supplementation did not reduce daily food intake or caloric intake in mice (Figures 1C,D), suggesting that the protective effect of MOP on HFD-induced weight gain was not achieved by suppressing food or energy intake. Abnormal lipid metabolism is one of the most common characteristics of obesity and related chronic diseases. Our study found that MOP significantly reduced serum TG, T-CHO, and LDL-C concentrations, and increased HDL-C concentrations in a dose-dependent manner, compared to HFD-fed mice (Figures 3A–D). This is consistent with the results of previous studies (50, 51). These results suggest that MOP probably plays an influential role in reducing obesity-induced abnormal lipid metabolism and dyslipidemia.

Obesity is a major cause of impaired insulin signaling and, therefore, insulin resistance development (52). Insulin resistance leads to impaired glycogen synthesis and proteolytic metabolism in skeletal muscle, and inhibits lipoprotein lipase activity in adipocytes, resulting in the release of free fatty acids and inflammatory cytokines. In addition, insulin resistance could lead to impaired glucose output and fatty acid metabolism, resulting in increased triglyceride levels and hepatic VLDL secretion (53). Hyperglycemia is a hallmark of insulin resistance (54). In this study, MOP supplementation reduced fasting blood glucose and fasting serum insulin in HFD-fed mice (Figures 3E–G), indicating a positive effect of MOP on improving insulin resistance.

Previous studies have shown that obesity is associated with inflammation. Obesity is regarded as a chronic low-grade inflammatory state (55). To assess the anti-inflammatory effects of MOP, the levels of representative pro-inflammatory cytokines including TNF-α and IL-1β in serum, and the mRNA expression of TNF-α, IL-1β, IL-6, and MCP-1 in the liver were examined. The results showed that these pro-inflammatory cytokines were significantly reduced by MOP intervention in a dose-dependent manner (Figure 4), indicating that MOP supplementation could reduce HFD-induced systemic inflammation and liver inflammation. This is similar to the findings of Sang et al. (42), who found that Ganoderma lucidum polysaccharides significantly inhibited HFD-induced inflammatory response, manifested in the reduction of inflammatory factors (TNF-α, IL-1β, and MCP-1) in serum and adipose tissue, and reduced macrophage infiltration in adipose tissue.

As previously reported, HFD can promote obesity by altering the expression of lipid-related genes in the liver (56). Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily (57). PPARs have been considered potential therapeutic targets for treating several metabolic syndromes (58). PPARs, including PPARα, PPARγ, and other subtypes, could regulate the expression of many genes, involved in lipid metabolism, adipogenesis, inflammation, apoptosis, immune response, oxidative stress, and adipocyte differentiation (59). PPARα is highly expressed in tissues associated with increased fatty acid oxidation (e.g., liver, skeletal muscle, and heart), and its activation leads to lower lipid levels and the elimination of TG in plasma, resulting in high levels of HDL-C (60). In contrast, PPARγ is a transcription factor involved in adipocyte differentiation and adipogenesis, which includes the processes of preadipocyte recruitment, differentiation, and TG synthesis (50). In the present study, we found by RT-qPCR analysis that MOP treatment resulted in increased expression of PPARα and decreased expression of PPARγ in the liver, compared to the HFD group (Figure 5A). SREBP-1c could regulate the expression of fatty acid synthase, participate in hepatic adipose differentiation and adipogenesis (61, 62). In this study, MOP supplementation decreased the expression of the SREBP-1c gene (Figure 5A). Fasting-induced adipose factor (Fiaf) is a multifunctional protein involved in plasma triglyceride (TG) metabolism, energy metabolism, cancer metastasis, angiogenesis, wound healing, inflammation, and nephrotic syndrome (63). Increased levels of Fiaf have been shown to be protective against diet-induced obesity (64). In the present study, MOP treatment significantly decreased the gene expression of Fiaf compared to HFD (Figure 5A). The Cide (cell death-inducing DFF45-like effector) family of proteins has been shown to play a critical role in lipid metabolism and energy metabolism, including lipolysis, lipid oxidation, and lipid droplet formation (65). The expression levels of Cidea and Cidec genes were examined in this study, and it was found that MOP treatment decreased their expression levels (Figure 5A). Bile acids are critical to lipid digestion, cholesterol metabolism, and other lipid-related pathways (66). Increased primary bile acid biosynthesis has been shown to be associated with obesity (67). Cyp7a1 is a rate-limiting enzyme for cholesterol catabolism and bile acid synthesis (68). Cyp7b1 is a microsomal enzyme involved in various physiological functions, including bile acid biosynthesis (69). Several studies have confirmed that an HFD significantly reduces the gene expression of Cyp7a1 and Cpy7b1 in the liver (70). In this study, MOP supplementation increased Cyp7a1 and Cyp7b1 expression in the liver compared to the HFD group, suggesting that MOP may prevent obesity by regulating bile acid metabolism (Figure 5B). This is consistent with the results of a previous study (71), in which a significant increase was found in the mRNA expression levels of Cpy7a1 and Cyp7b1 in the liver after theabrownin treatment. In conclusion, our findings suggested that MOP regulated the expression levels of genes related to hepatic fatty acid synthesis and lipid metabolism, thereby preventing obesity.

The gut microbiota depends heavily on plant-derived dietary fiber and polysaccharides as energy sources. Besides, dietary polysaccharides significantly impact the host's gut microbial ecology and health (42). Some studies have shown that the gut microbiota is an environmental factor associated with obesity (72, 73). There is growing evidence that dysbiosis of the gut microbiota is associated with the development of obesity. However, emerging evidence supports the benefits of gut microbiota in weight management (74). Many natural products, including plant foods and phytochemicals, have been found to be effective in weight management by modulating gut microbiota (75). Several studies have also found that the prevention of obesity by plant polysaccharides is related to the regulation of gut microbiota (3, 42, 76). The decrease in microbial diversity and abundance is one of the main features of human obesity-related gut malnutrition (77). The present study showed that HFD reduced the diversity indices of gut microbiota, including Sobs, Chao, and Ace, while high concentrations of MOP (400 mg/kg/d) reversed the HFD-induced changes in these indices to some extent. Moreover, MOP supplementation increased the species diversity of the gut microbiota (Figures 6A–D). It is probably explained that dietary polysaccharides, which are usually indigestible in the stomach and small intestine, reach the large intestine, where they are available for fermentation, thereby enhancing the growth of beneficial bacteria and improving gut microbial diversity.

The human gut microbiota is dominated by two phyla, Firmicutes and Bacteroidetes, which account for more than 90% of all bacterial species in the gut (78). These two bacteria contribute to the host's energy absorption and metabolism associated with gut microbiota. The F/B ratio was confirmed to be higher in obese individuals compared with the healthy population (56). Although many studies point toward a relative increase in the F/B ratio as a characteristic of the “obese microbiome,” the findings are inconsistent. For example, some studies reported that polyphenols or herbal extracts with anti-obesity bioactivity could downregulate the F/B ratio (9), whereas the protective effect of berberine against HFD-induced obesity was not associated with any significant change in the F/B ratio (79). In the current study, MOP supplementation did not significantly reduce the abundance of Firmicutes, but significantly increased the abundance of Bacteroidetes and the F/B ratio (Figures 7C–E). The results of this study showed that MOP treatment helped maintain the F/B ratio at a level similar to that of the NCD group, thereby helping to control energy absorption and body weight in the HFD mice.

Our results showed that MOP treatment altered the abundance of some bacterial genera (Figure 7F). For instance, the abundance of Bacteroides and Oscillibacter was increased. Bacteroides is a major genus in the human microbiota with a broad ability to use various types of dietary polysaccharides (42). Kaoutari et al. reported that members of the Bacteroidetes encode a proportionally higher number of carbohydrate-active enzymes (CAZymes) that are key enzymes for digesting polysaccharides than bacteria of other phyla (80). Oscillibacter is considered a potentially beneficial bacterium (81). Studies have shown that Oscillibacter is a valeric acid producer that enhances the differentiation of interleukin 10 (IL-10)-producing Tregs in vivo (82). Kim et al. (83) investigated the effect of Ephedra sinica on gut microbiota composition, and found that Oscillibacter in Firmicutes was negatively correlated with body weight and body mass index (BMI). These studies support our findings that MOP treatment increased the abundance of Bacteriodes and Oscillibacter. Furthermore, our results showed that MOP treatment reduced the relative abundance of Blautia compared to HFD (Figure 7F). Blautia is a commonly present genus in the gut microbiota. However, the results of studies on the relationship between Blautia and obesity are inconsistent. In a Japanese weight loss study, the genus Blautia was observed as the only gut microbe that was inversely associated with visceral fat, independent of sex (84). In contrast, some studies have suggested that Blautia is a harmful bacterium (85). In the current study, the results of LFSE analysis showed that Blautia was an important factor in distinguishing HFD and NCD groups (Figure 7G). Moreover, our findings showed that Blautia was positively associated with most obesity markers, including body weight, perirenal fat, mesenteric fat, T-CHO, HDL-C, blood glucose, and HOMA-IR (Figures 8A–C). Our findings are consistent with those of Goffredo et al., who reported a positive correlation between the abundance of Blautia and obesity in American youth and verified that the level of acetate, which is the product by Blautia, was associated with body fat partitioning and hepatic lipogenesis (86). Alistipes is considered to be an obesity-associated bacterium. Previous studies showed that HFD significantly increased the abundance of Alistipes in feces (87). In this study, MOP treatment reduced the abundance of Alistipes (Figure 7F). This is consistent with the previous study, in which the probiotic-fermented blueberry juice treated mice showed relatively low abundances of obese-related gut bacteria (Oscillibacter and Alistipes) (88). In a randomized controlled trial, overweight and obese adults consuming avocado daily for 12 weeks were enriched in the relative abundance of Alistipes compared to the control group (89). Tyzzerella is considered a pathogenic bacteria in the intestine (90). Huang et al. investigated the protective effects of sodium alginate on the gut microbiota, immunity, and intestinal mucosal barrier function in cyclophosphamide-induced immunosuppressed BALB/c mice, and found that sodium alginate significantly decreased the pathogenic bacteria Tyzzerella in the intestine (90). However, few studies have reported the correlation between Tyzzerella and obesity. The current study showed that MOP treatment reduced the relative abundance of Tyzzerella (Figure 7F). Furthermore, Tyzzerella was positively correlated with the majority of the obesity indicators, including body weight, liver weight, perirenal fat, mesenteric fat, TG, T-CHO, blood glucose, HOMA-IR, TNF-α, and IL-1β (Figure 8). These results suggested that Tyzzerella is a “harmful indicator” closely related to obesity.

In conclusion, this study demonstrated that MOP could effectively prevent HFD-induced weight gain and lipid accumulation, ameliorate blood lipid levels and insulin resistance, and reduce the secretion of inflammatory cytokines in mice. In addition, MOP supplementation also modulated gut microbiota composition, increasing the abundance of some beneficial bacteria and decreasing the abundance of some harmful bacteria. These results might be attributed to the change in gut microbial composition and regulation of lipid metabolism induced by the prebiotic ability of polysaccharides. This study suggests that MOP possesses great potential to be utilized as a dietary supplement for obesity management. In future, it would be important to confirm the composition of polysaccharides in M. oleifera leaves and to identify specific ones with anti-obesity properties.

The original contributions presented in the study are publicly available. This data can be found here: NCBI, PRJNA759102.

The animal study was reviewed and approved by the Animal Care and Use Committee of Yunnan Agricultural University.

JS, YT, and LM conceived and designed the experiments. LL, LM, YW, JX, LY, AJ, and YZ performed the experiments. YT, LL, and LM analyzed the data. LL and LM wrote the paper. All authors contributed to the article and approved the submitted version.

This work was supported by Major Project of Science and Technology Department of Yunnan Province (2018ZI001 and 202002AA100005), YEFICRC Project of Yunnan Provincial Key Programs (2019ZG009), and Yunnan Province Young and Middle-Aged Academic and Technical Leaders Reserve Talents Project (2018HB040).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.