Limonin Ameliorates HFD-Induced Dyslipidemia and Intestinal Barrier Damage by Modulating Gut Microbiota

Limonin treatment significantly improved dyslipidemia induced by HFD by reducing serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels, and increasing high-density lipoprotein cholesterol (HDL-C) levels in HFD-fed mice. Importantly, starting from week 2 until the end of the 16-week experiment, limonin administration significantly reduced weight gain in the HFD+Lim group compared to the HFD group, demonstrating its preventive effect against HFD-induced weight gain. Supplementation of limonin significantly reduced accumulation of epididymal fat and the increase in liver and spleen weight induced by HFD. Moreover, the levels of ALT and AST were significantly lower in the HFD+Lim group compared to the HFD group.

Consistent with previous experiments (Yang et al. 2022), we observed that HFD caused damage to the intestinal barrier. H&E staining showed a significant decrease in villus length and crypt depth in the ileum of mice fed with HFD. Through immunofluorescence and qRT-PCR, we confirmed that HFD significantly reduced the expression of tight junction proteins, namely Occludin and ZO-1. This alteration in tight junctions resulted in weakened barrier function and epithelial integrity (Ramos and Papadakis 2019; Capaldo et al. 2017). Supplementation of limonin significantly increased crypt depth and villus length, as well as the expression of tight junction proteins in the HFD+lim group, indicating a protective effect of limonin against HFD-induced damage to the intestinal barrier.

Previous studies have demonstrated that eliminating HFD-induced microbiota with antibiotics can reverse damage to the intestinal barrier caused by HFD. In our study, analysis of alpha and beta diversity indicated that HFD changed the composition of the gut microbiota. Because of the low solubility and poor oral absorption of limonin, a substantial amount of orally administered limonin remains unabsorbed and lingers in the intestine. Therefore, we speculate that the target of limonin’s effect on HFD-induced dyslipidemia may be associated with changes in the gut microbiota. At the phylum level, HFD caused an increase in Firmicutes, Proteobacteria, and Desulfobacterota, while decreasing Bacteroidota, Verrucomicrobiota, and Actinobacteriota in the gut microbiota. The ratio of Firmicutes to Bacteroidota (F/B ratio) is linked to obesity-related diseases, and limonin significantly decreased the elevated F/B ratio caused by HFD. At the genus level, the increased abundance of Lachnospiraceae, Alistipes, Desulfovibrio, Clostridium, and Ruminococcaceae induced by HFD was restored upon limonin administration.

Alistipes, Desulfovibrio, and Ruminococcaceae were found to be significantly more abundant in HFD-fed mice and were associated with impaired intestinal barrier function, as confirmed by correlation analyses and other research findings. Furthermore, limonin supplementation increased the abundance of beneficial probiotic bacteria, including Lactobacillus, Muribaculaceae, Bifidobacterium, Ligilactobacillus, and Akkermansia. Pearson correlation analysis showed a positive correlation between these probiotic bacteria and the integrity of the intestinal barrier. Faecalibaculum, Lactobacillus, Bifidobacterium, Dubosiella, and Akkermansia have been demonstrated to enhance intestinal barrier function by producing short-chain fatty acids (SCFAs). Additional studies have shown that Bifidobacterium enhances the integrity of tight junction proteins and maintains epithelial integrity. Akkermansia promotes the proliferation of intestinal stem cells (ISCs) and protects against radiation- and methotrexate-induced intestinal damage (Kim et al., 2021). Lactobacillus supports the regeneration of intestinal epithelial cells and repairs damaged intestinal mucosa. Importantly, Lactobacillus is known to stimulate the production of aryl hydrocarbon receptor (AHR) ligands, which enhance metabolic function and strengthen the integrity of the intestinal barrier.

In our experiments, we did not observe a significant increase in the expression of Lactobacillus due to limonin. However, another strain of Lactobacillus called Limosilactobacillus produces a substantial quantity of AHR ligands, which activate ILC3s to stimulate the production of the critical barrier factor IL-22, thereby preventing HFD-induced obesity and metabolic disorders. Supplementation of limonin significantly increased the abundance of Limosilactobacillus.

In conclusion, the changes in gut microbiota caused by limonin alleviate HFD-induced dyslipidemia and intestinal barrier damage, and potentially involve the key barrier pathway ILC3-IL-22-IL-22R.