Rebalancing of the gut flora and microbial metabolism is responsible for the anti-arthritis effect of kaempferol

Abstract

Kaempferol is a natural flavonol that possesses various pharmacological activities, including anti-arthritis effects, yet the underlying mechanisms remain controversial. To evaluate the anti-arthritis efficacy and the underlying mechanisms of kaempferol, collagen-induced arthritis (CIA) mice were treated with kaempferol intragastrically (200 mg · kg⁻¹ · d⁻¹) and intraperitoneally (20 mg · kg⁻¹ · d⁻¹). Pharmacodynamic and pharmacokinetic studies showed that the oral administration of kaempferol produced distinct anti-arthritis effects in model mice with arthritis in terms of the spleen index, arthritis index, paw thickness, and inflammatory factors; the bioavailability (1.5%, relative to that of the intraperitoneal injection) and circulatory exposure of kaempferol (C_max = 0.23 ± 0.06 ng/mL) and its primary metabolite kaempferol-3-O-glucuronide (C_max = 233.29 ± 89.64 ng/mL) were rather low. In contrast, the intraperitoneal injection of kaempferol caused marginal anti-arthritis effects, although it achieved a much higher in vivo exposure. The much higher kaempferol content in the gut implicated a potential mechanism involved in the gut. Analysis of 16S ribosomal RNA revealed that CIA caused imbalance of 14 types of bacteria at the family level, whereas kaempferol largely rebalanced the intestinal microbiota in CIA mice. A metabolomics study showed that kaempferol treatment significantly reversed the perturbation of metabolites involved in energy production and the tryptophan, fatty acid and secondary bile acid metabolisms in the gut contents of the CIA mice. In conclusion, we demonstrate for the first time that the high level of kaempferol in the gut regulates the intestinal flora and microbiotic metabolism, which are potentially responsible for the anti-arthritis activities of kaempferol.

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease accompanied by synovitis, cartilage erosion, and bone destruction [1]. Conventional remedies, such as disease-modifying anti-rheumatic drugs (DMARDs) and non-steroidal anti-inflammatory drugs (NSAIDs), produce therapeutic effects, but serious and widespread adverse reactions to these drugs have significantly restricted their continuous use [2,3,4]. Herbal medicines provide an alternative choice to treat RA due to their definite efficacy with fewer side effects [5,6,7].

Kaempferol is a natural flavonol found in many edible plants and herbal medicines. Kaempferol shows a wide range of pharmacological activities, including anti-inflammatory and antioxidant effects [25]. It has been reported that Bacteroidales_S24-7_group is significantly decreased in CIA mice, [22] and this bacteria are associated with the production of anti-inflammation molecules, such as acetate and propionate [26]. To some extent, kaempferol reshaped the gut flora and primarily modulated the abundance of Lachnospiraceae, Bacteroidales_S24-7_group, Prevotellaceae, Erysipelotrichaceae, Staphylococcaceae, and Alcaligenaceae, suggesting that the anti-arthritis effect of kaempferol involves the intestinal microbiota.

The gut microbiota not only influence the maturation and development of the host immune system but also influence host metabolic homeostasis by cross-communication via microbial metabolites or cometabolites. In this study, a significantly perturbed metabolism was observed in the fecal extracts of CIA mice. For example, tryptophan metabolism plays an important role in microbiota-host crosstalk in health and disease [27]. In the present study, reduced tryptophan and elevated indole-3-acetic acid in the CIA group demonstrated that tryptophan metabolism was enhanced in mice with arthritis. Indole-3-acetic acid, the metabolite of tryptophan in the gut, is a ligand for AhR (aryl hydrocarbon receptor), which regulates the immune response and intestinal homeostasis [27, 28]. Kaempferol treatment efficiently reversed the tryptophan metabolism, decreased the indole-3-acetic acid level and increased the tryptophan level, which further suggests that kaempferol modulates the gut flora.

Glucose, fructose, and the key intermediate of the citric acid cycle α-ketoglutaric acid are important factors that regulate T cell activation [29] and differentiation [30]. Treatment with kaempferol efficiently reduced the levels of fructose, glucose, and α-ketoglutaric acid in the gut, suggesting that kaempferol may modulate energy metabolism and affect T cell properties. In contrast to the upregulated serum fatty acid levels in CIA rats in a previous study [31], the levels of many fatty acids, such as palmitoleic acid, palmitic acid, linoleic acid, and oleic acid, were significantly decreased in the feces of CIA mice. However, kaempferol reversed the decreased levels of these fatty acids, indicating the modulation of the gut flora and the turnover of intestinal lipids.

Bile acids are closely associated with immune inflammation [32, 33]. In addition, their metabolism and turnover exclusively involves the gut microbiota. In addition, primary bile acids are transformed into secondary bile acids by the means of the gut flora. Arthritis significantly increases the levels of cholic acid (DC) and deoxycholic acid (DOCA) and decreases the level of glycocholic acid (GCA). DOCA is a secondary bile acid predominantly metabolized by the intestinal microbiota, and a previous study reported that DOCA induces severe inflammation [34]. Treatment with kaempferol decreased the DOCA level and had little effect on DC and GCA levels, again indicating that kaempferol exerts anti-arthritis effects by modulating the gut microbiota.

Conclusion

Intraperitoneal injection achieves high plasma levels of kaempferol and its primary metabolite, yet marginal effects are observed. Orally administered kaempferol has relatively low bioavailability and in vivo exposure, yet it shows distinct anti-arthritis activity. The high level of kaempferol in the gut after oral administration reshapes the intestinal microbial community and modulates the microbiota-mediated metabolism of tryptophan, fatty acids and secondary bile acids and energy production, which may contribute to the effectiveness of kaempferol in RA.