Inonotus hispidus Protects against Hyperlipidemia by Inhibiting Oxidative Stress and Inflammation through Nrf2/NF-κB Signaling in High Fat Diet Fed Mice

Obesity is frequently associated with dysregulated lipid metabolism and lipotoxicity. Inonotus hispidus (Bull.: Fr.) P. Karst (IH) is an edible and medicinal parasitic mushroom. In this study, after a systematic analysis of its nutritional ingredients, the regulatory effects of IH on lipid metabolism were investigated in mice fed a high-fat diet (HFD). In HFD-fed mice, IH reversed the pathological state of the liver and the three types of fat and significantly decreased the levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglycerides (TG), and leptin (LEP) and increased the level of high-density liptein cholesterol (HDL-C) in serum. Meanwhile, IH ameliorated liver damage by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and plasminogen activator inhibitor-1 (PAI-1) levels in the liver and serum. Compared with HFD-fed mice, IH significantly modulated the gut microbiota, changed the relative abundances of microflora at different taxonomic levels, and regulated lipid levels. The results showed that 30 differential lipids were found. Results from Western blotting confirmed that IH regulated the nuclear factor erythroid-2 related factor 2 (Nrf2)/nuclear factor-kappa B (NF-κB) signaling pathway and oxidative stress. This study aimed to provide experimental evidence for the applicability of IH in obesity treatment.


Introduction
Obesity is a pathological condition requiring clinical intervention [1]; it may induce complications involving metabolic disorders, including type II diabetes and hyperlipidemia [2,3]. In China, from 2015 to 2019, the prevalence of obesity was 3.6%, 7.9%, and 16.4% among children under the age of 6 years, adolescents aged 6-17 years, and adults (≥18 years), respectively [4]. The increasing burden of obesity has grave implications for individuals, families, and societies.
Obesity is frequently associated with dysregulated lipid metabolism and lipotoxicity [5], which may induce increased hepatic fat uptake and new fat synthesis in the liver. However, the compensatory enhancement of fatty acid oxidation fails to normalize lipid levels, ultimately triggering oxidative stress and leading to cellular damage and the occurrence of non-alcoholic fatty liver disease [6]. Furthermore, certain gut microbes influence the occurrence and development of obesity by improving intestinal oxidative stress, inhibiting intestinal inflammation, and maintaining intestinal barrier integrity [3,7]. Obesity is associated with elevated levels of markers of oxidative stress and low-grade systemic inflammation [8]. An excessive accumulation of white adipose tissue leads to adipocyte China) or a high-fat diet (HFD; D12492; 60% kcal fat, 20% kcal protein, and 20% kcal carbohydrate; Xiao Shu You Tai Biotechnology Co., Ltd., Beijing, China) under specificpathogen-free (SPF) conditions on a 12 h light/dark cycle at a constant temperature (23 ± 1 • C) and humidity (40-60%) for 8 weeks. To establish the DIO model for the high-fat diet study, 24 randomly selected mice were fed an HFD ad libitum during the entire experimental period. From week 9, the DIO mice were randomly divided into four groups (n = 6/group), including an intragastrically HFD-fed group with 5 mL/kg of normal saline, an intragastrically simvastatin (SV) (SFDA Approval No.: H20093943, Chengdu Hengrui Pharmaceutical Co., Ltd., Chengdu, China)-treated group with 3 mg/kg of SV, and low-and high-dose intragastrically IH-treated groups with 500 and 1000 mg/kg of IH, respectively, daily for 8 weeks. The NCD mice were randomly divided into two groups (n = 6/group), including the vehicle-treated intragastrically NCD-fed group with 5 mL/kg of normal saline and the intragastrically IH-treated NCD-fed group with 500 mg/kg of IH daily for 8 weeks. Weekly measurements of body weight and plasma glucose levels were performed for all mice. After 8 weeks of drug treatment, the mice were euthanized using CO 2 (the CO 2 replacement rate was 30-70% of the container volume per minute). Peripheral blood was obtained by sampling the retro-orbital venous plexus. Organs (heart, liver, spleen, and kidney), epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT), and perirenal white adipose tissue (pWAT) were dissected and weighed. The above tissue parts were stored at −80 • C for further biochemical analysis, and the remaining parts were fixed in a 4% tissue fixative (BL539A, Biosharp, Guangzhou, China) for subsequent pathological analysis.

Histopathological Analysis
Hematoxylin and eosin (H&E) staining and Oil Red O staining were performed as described in our previous study [14]. Fixed adipose tissue (eWAT, iWAT, and pWAT) and organ tissue (heart, liver, spleen, and kidney) were embedded in paraffin, sectioned at 5 µm, and stained with H&E. Frozen liver tissue was sectioned at 10 µm, fixed, and stained with Oil Red O and H&E. All specimens were observed and photographed using an Eclipse Ci-L upright microscope (Nikon Corporation, Tokyo, Japan).

Intestinal Microflora Analysis
A gut microbiota analysis of mouse cecum content in samples from NCD-, HFD-, and IH-treated groups (500 mg/kg) (n = 3/group) was performed by 16S rRNA sequencing using an Illumina NovaSeq platform at Shanghai Personalbio Technology Co., Ltd. (Shanghai, China), as described in our previous study [14]. Sequence sets with 97% identity in 16S rDNA gene sequences were clustered into operational taxonomic units (OTUs). The alpha diversity index included Chao1, observed species, Shannon, Faith's phylogenetic diversity (Faith's PD), Pielou's evenness, and Good's coverage, and the significance of differences was verified by the Kruskal-Wallis rank-sum test and Dunn's test as post hoc tests. The beta diversity was calculated by the Bray-Curtis. When the linear discriminant analysis (LDA) score was >2, the results of the LDA effect size (LEfSe) analysis, performed to identify the biomarkers for each group, were more reliable.

Plasma Lipidome Analysis
A plasma lipidome analysis of mouse serum from the NCD-, HFD-, and IH-treated groups (500 mg/kg) (n = 3/group) was performed by liquid chromatography-mass spectrometry (LC-MS) at Shanghai Personalbio Technology Co., Ltd. (Shanghai, China), as described in our previous study [14]. According to the detected compounds, an orthogonal partial least squares discriminant analysis (OPLS-DA) was used for a metabolite variation analysis. Statistically significant differences (p ≤ 0.05) in metabolite levels and variable importance in projection (VIP) values ≥ 1.0 were regarded as the standard for differential lipids to filter out biomarkers.

Statistical Analysis
All values are presented as means ± SD. Biochemical indices were compared between different groups by one-way analysis of variance (ANOVA) followed by Tukey's test using BONC DSS Statistics 25 (IBM, Armonk, NY, USA). Differences were considered statistically significant at p < 0.05.

IH Regulated Intestinal Microflora in HFD-Fed Mice
Imbalances involving intestinal microflora are related to abnormal lipid metabolism and are involved in the pathogenesis of obesity [24]. In intestinal microflora analysis, alpha diversity was used to reflect the richness, diversity, and uniformity of species within any sample [25]. Between the vehicle-and IH-treated HFD-fed mice, IH showed no effects on alpha diversity ( Figure 3A). Beta diversity was used to represent differences in species composition. IH administration resulted in a distinct separation of the intestinal microflora composition in mice (PCo2, 13.1%, Figure 3B). A Venn diagram was used to represent the intestinal microbial community, and the number of OTUs in each collection was determined based on OTU abundance. Among the 4763 OTUs detected in the experimental groups, 95 were common among all. The number of specific OTUs was 1786 (37.50%) in vehicletreated NCD-fed mice, 995 (20.89%) in vehicle-treated HFD-fed mice, and 1383 (29.04%) in IH-treated HFD-fed mice, indicating a strong influence of IH on the composition of intestinal flora ( Figure 3C). A heatmap analysis of the flora with the top 20 average abundances at the genus level showed that IH treatment increased the average abundance of Allobaculum, Adlercreutzia, Shigella, Dorea, Oscillospira, and Streptococcus and decreased Ruminococcus and Coprobacillus compared with the vehicle-treated HFD-fed mice ( Figure 3D). An LEfSe analysis was performed to identify species with significantly different abundance across experimental groups and identify stable and differential landmark species at all taxonomic levels [26]. IH significantly increased the relative abundances of Burkholderiales, Streptococcaceae, Lactococcus, Dorea, and Oscillospira in HFD-fed mice ( Figure 3E). Oscillospira had the highest LDA value ( Figure 3F). Between the vehicle-and IH-treated HFD-fed mice, 18 significantly altered taxa were noted (Table S2). Differential metabolic pathways were detected by metagenomeSeq (Table S3), and the superpathways of methylglyoxal (MGO) degradation and enterobactin biosynthesis were significantly upregulated by IH in HFD-fed mice ( Figure 3G) and were related to the abundance of Shigella.

Discussion
IH is rich in dietary fiber, which helps to improve hyperlipidemia by affecting lipid metabolism [28] and has a positive effect on weight loss [29], suggesting a material basis for its hypolipidemic effect. IH significantly improved the pathological state and function of adipocytes. Changes in the levels of TC, TG, LDL-C, and HDL-C (related to hyperlipidemia [30]) revealed the anti-hyperlipidemic effect of IH in HFD-fed mice. LEP is an adipokine secreted by adipocytes that reflects the degree of obesity [31]; a decline in the level of LEP revealed the anti-obesity effect of IH in HFD-fed mice. IH significantly suppressed the levels of AST, ALT, and PAI-1 and inhibited hepatic steatosis in HFD-fed mice, confirming its hepatoprotective effect, which is the center of lipid metabolism [32]. The reduction in the levels of ROS and MDA demonstrated the antioxidant activity of IH in HFD-fed mice.
In obese mice, IH increased the abundance of the genera Allobaculum, Dorea, and Oscillospira, facilitating the production of short-chain fatty acids (SCFAs) related to metabolic processes [33][34][35] while decreasing the abundance of the genera Ruminococcus and Coprobacillus. SCFAs such as acetate, propionic acid, and butyric acid can reduce the generation of pro-inflammatory cytokines [36], displaying resistance to intestinal inflammation [37]. SCFAs can suppress appetite, promote lipid oxidation rather than lipid production, and reduce the storage of white adipose tissue [38,39]. Dietary fiber can mitigate the reduction in SCFAs caused by a high-fat diet [40], which has been consistent with our present data. Accordingly, as a beneficial bacteria genus [41], Allobaculum can regulate hepatic lipid metabolic processes [42], shows a negative correlation with obesity [43], and its supplementation can reduce the rapid weight gain caused by a high-fat diet [44].
Allobaculum plays a role in suppressing inflammatory responses by reducing the expression of p-IKK and TNF-α [45], which has been confirmed in our results. As a healthy bacteria genus [41], Dorea is negatively associated with inflammatory diseases [46]. Both Oscillospira and Ruminococcus are inflammatory bacteria associated with inflammatory bowel disease [47,48], and Oscillospira helps to maintain lipid homeostasis [49]. Rosa Roxburghii Tratt, possessing hypolipidemic effects, can reduce the abundance of Coprobacillus [50]. Moreover, Streptococcus may be the main force for decomposing a large amount of cellulose [51], which increased in IH-treated HFD-fed mice. Based on the changes induced by IH on the abundance of intestinal microbes, IH might upregulate the superpathway of MGO degradation and enterobactin biosynthesis. The impairment of enterobactin biosynthesis elevates ROS levels [52]. Meanwhile, MGO can activate the oxidative pathway and induce inflammation [53,54], and the formation and accumulation of MGO are strongly associated with obesity [55].
Metabolites of intestinal flora affect the process of lipid metabolism and host lipid composition [56]. IH could significantly decrease the level of LPC in HFD-fed mice. As a biologically active pro-inflammatory lipid molecule, LPC promotes the expression of genes involved in cholesterol biosynthesis, thereby participating in lipid metabolic processes [57]. LPC is formed by the hydrolysis of PC in LDL and cell membranes via phospholipase A (2) or oxidation [58], and impairment in PC biosynthesis is associated with the occurrence of fatty liver disease [59]. PC has excellent antioxidant properties, can scavenge ROS, prevents lipid peroxidation, and is known as an antioxidant/anti-inflammatory phospholipid [60]. Additionally, changes in the MGO level are, in turn, related to changes in the metabolic processes involving PC [61]. MGO increases oxidative stress levels, which can lead to lipid peroxidation through the production of ROS and ultimately lead to cell damage [54,61]. IH regulated lipid metabolism and showed anti-hyperlipidemic activity related to its modulation of intestinal flora, which further influenced the levels of metabolites.
Oxidative stress leads to increased lipid peroxidation and is closely associated with hyperlipidemia-related tissue damage [62,63]. Nrf2, a key regulator of antioxidant responses and maintaining cellular redox hemostasis, can inhibit lipogenesis [64] and nullify ROS [65] by promoting the expression of a series of downstream antioxidant genes, such as SOD and HO-1 [66]. SOD-1 is one of the three distinct isoforms identified of SOD in mammals. SOD isoforms scavenge superoxide radicals and reduce their toxicity [67]. HO-1, a key target gene of Nrf2, exerts antioxidant effects by resisting endogenous and exogenous stimuli [68]. Nrf2/HO-1 signaling contributes to the scavenging of lipid peroxides [69].
Oxidative stress and inflammation are inextricably associated [70]. Nrf2 can negatively regulate NF-κB signaling during cellular inflammatory responses [10]. Under typical physiological conditions, NF-κB is in a normal binding state with IκBα [71], whereas cellular injury caused by various factors such as pro-inflammatory factors or ROS triggers the activation of the IKK complex, which leads to IκBα phosphorylation, ubiquitination by the ubiquitin ligase system (ULS), and the release of NF-κB dimers. These NF-κB dimers enter the nuclear membrane, thereby initiating the transcription of target genes [72,73]. Activated NF-κB in hepatocytes promotes liver inflammation [74] and induces the transcription of inflammatory cytokine genes such as TNF-α and IL-6 [75]. TNF-α can promote lipolysis and increase the release of free fatty acids, thereby promoting adipogenesis [76]. According to the present data, IH could regulate lipid metabolic processes through the Nrf2/NF-κB pathway and had a relevant effect on oxidative stress as well as the inflammatory response in this process.
The present study had certain limitations. In this study, we first reported the hypolipidemic effects of IH and analyzed the components involved. However, the specific active components possessing hypolipidemic activity were not confirmed; thus, further investigation is needed.

Conclusions
In conclusion, IH regulated lipid metabolism through the Nrf2/NF-κB signaling pathway, which is closely related to oxidative stress and inflammatory responses, in HFDfed mice. Our study provides insights into the application of IH as a hypolipidemic agent and will facilitate its commercial application.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14173477/s1, Figure S1: HPLC chromatograms of IH components; Figure S2: H&E staining of heart, spleen, and kidney of mice; Table S1: Details of antibodies used in Western blotting; Table S2: The taxa with significant differences between vehicle-treated HFD-fed mice and IH-treated HFD-fed mice; Table S3: The differential metabolic pathways between vehicle-treated HFD-fed mice and IH-treated HFD-fed mice; Table S4: Differential lipids between vehicle-treated HFD-fed mice and IH-treated HFD-fed mice.