Limonin Ameliorates High-Fat Diet Induced Dyslipidemia by Modulating Gut Microbiota and Intestinal Barrier: A Mechanistic Study

| 原始文本 | 修改后文本 | 修改原因 | | -------- | ---------- | -------- | | Dyslipidemia is a general term that encompasses all disorders of lipid metabolism, mainly referring to the increased plasma concentrations of total cholesterol (TC), triglycerides (TG) or low-density lipoprotein cholesterol (LDL-C), or the decreased plasma concentrations of high-density lipoprotein cholesterol (HDL-C) or a combination of these characteristics (Arvanitis and Lowenstein 2023). Crucially, dyslipidemia stands as a significant risk factor for ischemic heart disease (IHD), one of the leading culprits of global mortality (Pirillo et al. 2021). In 2019, ∼4.40 million deaths and ∼98.62 million disability-adjusted life years (DALYs) were attributable to high plasma LDL-C levels (Cieza et al. 2021). The 2017 Global Burden of Disease Study (GBD) suggested that socioeconomic development and the ubiquitous 'Western' high-sugar and high-fat diet intake are considered to be the main reasons for the increase in plasma lipid levels, while high plasma LCL-C levels is one of the main risk factors for all-cause risk attributable burden in 2017 (Jeffrey D Stanaway et al. 2018). While the extensive use of statins in high-income countries has significantly decreased plasma levels and cardiovascular disease-related deaths (Vancheri et al. 2016), their accessibility remains limited in low-income countries(Chow et al. 2020).Consequently, dyslipidemia continues to be an underdiagnosed and undertreated condition globally (Pirillo et al. 2021).Numerous studies have reported that HFD could cause gut microbiota dysbiosis and intestinal barrier function impairment. Dietary lipids are digested in the intestinal lumen and then enter enterocytes, and the proper functioning of intestinal lipid metabolism is the key for the body to obtain energy in the form of lipids. The intestinal barrier is composed of intestinal epithelial cells and immune cells, and its integrity and functional homeostasis provide an important guarantee for intestinal lipid metabolism. The gut microbiota plays an important role in host lipid metabolism by regulating nutrient acquisition, energy regulation, and fat storage (Bäckhed F et al. 2007). Intestinal strains Ruminococcaceae, Enterorhabdus, Lachnospiraceae_NK4A136_group, and Anaerotrucus, which were involved in host lipid metabolism. Gut microbiota can convert complex carbohydrates and sugars into short-chain fatty acids (SCFAs). These SCFAs interact with G protein-coupled receptors, orchestrating the host energy balance (Jandhyala et al. 2015). Furthermore, gut microbiota is also involved in regulating gut barrier integrity and functional homeostasis. Some intestinal microbiota such as Lactobacillus and Bifidobacterium have been shown to promote intestinal barrier function. Short-chain fatty acids have been reported to directly involve in hormone secretion, including GLP-1 and GLP-2, thus promoting the intestinal barrier integrity. Notably, microbiota-related metabolites can stimulate lamina propria lymphocytes to secrete interleukin-22 via the AhR pathway. IL-22 plays a protective role in intestinal barrier by promoting epithelial cell survival, proliferation, wound healing, and inducing the production of two intestinal antimicrobial peptides (RegIIIβ and RegIIIγ) (Kinnebrew et al. 2012; Kinnebrew et al. 2010; Lindemans et al. 2015). Moreover, IL-22 has been found to promote the expression of lipid transporter in intestinal epithelial cells (IECs) (Wang et al. 2017). Shinichiro Sawa et al. proposed most of the IL-22 in the gut is produced by RORγt+ ILCs (Shinichiro Sawa et al.), with earlier studies indicating that commensal bacteria and their metabolites modulate RORγt+ ILCs (Buela et al. 2015). Taken together, both gut microbiota and gut barrier are considered potential therapeutic targets for intestinal lipid metabolism . Limonin, also known as evodin, is a natural tetracyclic triterpenoid compound widely exists in the traditional Chinese herb Euodiae fructus and citrus fruits (Fan et al. 2019). Euodiae fructus, originates from the dried fruit of Euodia rutaecarpa (Juss.) Benth. Its most bioactive components evodiamine (EVO) and rutaecarpine (RUT) have demonstrated abilities to improve serum lipid profiles (Zhou et al. 2017) and effectively combat obesity and visceral fat accumulation in HFD-fed rats (Nie et al. 2016). Limonin, a primary volatile component of Euodiae fructus, possesses extensive pharmacological effects, including anti-cancer, anti-inflammatory, anti-bacterial, anti-viral, antioxidant, anti-obesity, and hepatoprotective effects, etc. (Fan et al. 2019). Studies have shown that limonin can reduce serum TC and TG levels in obese mice (Halder et al. 2014) and diminish the accumulation of lipid droplets in the liver, in addition to downregulating the levels of lipogenic transcription factors FASN and SREBP1 in NAFLD (Li et al. 2021). It has been reported that limonin treatment significantly enriched Bacteroidetes and inhibited Firmicutes. Limonin treatment increased the abundance of the genus Oscillospira, which is associated with a reduced incidence of inflammatory bowel disease and leanness in humans. Yet, the underlying mechanism of how limonin improves lipid profiles remains unclear. Research has demonstrated that a large portion of orally administered limonin is not absorbed and persists in the gut, which may lead to alterations in gut microbiota (Gu et al. 2019; Fan et al. 2019). Therefore, we hypothesized that the mechanism of limonin's improvement of lipid metabolism may be associated with regulating gut microbiota and intestinal barrier. In this study, we utilized C57BL/6 mice subjected to a high-fat diet (HFD), mimicking the Western high-fat diet pattern, to investigate the underlying mechanism of limonin in improving HFD-induced dyslipidemia. Our findings revealed that limonin ameliorated HFD-induced lipid metabolism disorder and investigated the composition of gut microbes by 16S rRNA gene sequencing. Then, the proportion of RORγt+ ILC3s was analyzed by flow cytometry. We further detected the levels of molecules associated with the ILC3-IL-22-IL-22R pathway by immunofluorescence and qRT-PCR. Additionally, we analyzed specific intestinal microbiota and intestinal barrier function biomarkers, aiming to explore the potential mechanism of limonin in improving dyslipidemia by modulating intestinal microbiota and intestinal barrier. This study provides molecular mechanism support for limonin in the prevention and treatment of dyslipidemia.