Monascus vinegar alleviates high-fat-diet-induced inflammation in rats by regulating the NF-κB and PI3K/AKT/mTOR pathways

Hunmei Meng, Ji Song,*, Bingqin Fn, Yingqi Li, Jiojio Zhng,Jinping Yu, Yu Zheng, Min Wng,*

a State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology, Tianjin Engineering Research Centre of Microbial Metabolism and Fermentation Process Control, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

b Shandong Engineering Research Centre of Condiment Fermentation Technology, Shandong Yutu Co., Ltd, Zibo 255300, China

Keywords:

Monascus vinegar

Hyperlipidemia

In flammation

NF-κB

PI3K/AKT/mTOR

A B S T R A C T

Monascus vinegar (MV) is a typical fermented food with various health-promoting effects. This study aimed to evaluate the role of MV in alleviating high-fat-diet-induced inflammation in rats with hyperlipidemia and elucidate the possible regulatory mechanisms. In the study, serum lipid profiles, liver pathology and liver inflammatory cytokines were analyzed in hyperlipidemia rats with MV (0.5 mL/kg mb, 2 mL/kg mb). Results showed that the administration of MV alleviated dyslipidemia by decreasing the serum and liver levels of triglyceride and total cholesterol. Increase in hepatic lipase and carnitine palmitoyl transferase 1 (CPT-1)levels and decrease in hepatocyte steatosis, nephritis, and intestinal tissue injury in the HD group showed that high-dose MV can significantly suppress hepatic lipid accumulation and steatosis. In addition, compared with the model (MOD) group, the HD group showed significantly down-regulated the level of serum or hepatic alanine aminotransferase (ALT), aspartate aminotransferase (AST), CPT-1, interleukin (IL)-2, IL-6, IL-12,and tumor necrosis factor α (TNF-α). Moreover, the HD group showed repressed hepatic nuclear factor κB(NF-κB) pathway and inactivated phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway mitigated liver inflammation. Similar results were obtained from cell experiments. Collectively, these findings revealed that MV might attenuate high-fat-diet-induced inflammation by inhibiting the NF-κB and PI3K/Akt/mTOR pathways.

1. Introduction

Hyperlipidemia is a common metabolic disease caused by a highfat diet (HFD). The pathological feature of the disease is increase in serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) and excessive accumulation of TG in the liver [3,4]. The formation of hyperlipidemia is related to lipid disorders and accompanied by high levels of inflammatory cytokines in circulation. It has been shown that lipid accumulation induced by HFD can be alleviated by reducing the levels of pro-inflammatory factors inducible nitric oxide (iNOS) and interleukin 6 (IL-6), as well as the level of lipid metabolism enzymes [3]. Reducing the level of circulating inflammatory factors promotes the regulation of atherosclerosis caused by lipid metabolism disorder.

Studies have shown that hyperlipidemia caused by HFD can be prevented by reducing lipid accumulation and inhibiting inflammatory response [4,5]. In addition, the regulation of peroxisome proliferators-activated receptors (PPAR) and carnitine palmitoyl transterase 1 (CPT-1) could reduce lipid accumulation and prevent obesity. Hyperlipidemia is a metabolic disease, accompanied by the development of chronic inflammation, mainly due to the excessive accumulation of lipids, resulting in fatty liver, leading to inflammatory reaction. It has been found that hyperlipidemia mice can mediate inflammation by regulating phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) and nuclear factor κB(NF-κB) signaling pathways [3,6]. Rice bran phenolic extract treatment can repress the hepatic endotoxin- Toll-like receptor 4 (TLR4)-NF-κB pathway, mitigate liver inflammation [7].Li et al. [8]demonstrated that supplementation withLonicera caeruleaL. polyphenols alleviates the nuclear translocation of NF-κB p65 and decreases the level of intestinal anti-inflammatory factors, such as tumor necrosis factor α (TNF-α), IL-6, and cyclooxygenase 2, and regulate lipid accumulation by affecting the activities of fatty acid synthase and catabolism in the liver.In addition, the PI3K/Akt pathway plays an important role in the inflammatory process, and the activation of the pathway can regulate the expression of inhibitor of NF-κB kinase α/β(IKKα/β), which is regulated by the NF-κB pathway. Moreover,the inhibition of the PI3K/Akt- or NF-κB-induced apoptosis of inflammatory cells, and levels of TC, TG and LDL-C decreased [9,10].It is suggested that the intervention of active substances can prevent the occurrence of hyperlipidemia by reducing lipid metabolism and suppressing inflammation caused by HFD-induced. Four kinds of drugs are commonly used in the treatment of hyperlipidemia, namely,statins, bates, nicotinic acid, and cholic acid chelators. However,some of these drugs have harmful side effects [11]. Therefore, the development of functional food with natural derivatives will play an important role in the treatment of hyperlipidemia.

Monascusvinegar is a traditional Chinese functional food. As a saccharifying starter,Monascusis widely used in the wine- and vinegar-making industries. The distinct characteristics of the vinegar is the utilization of red yeast rice as fermentation starter and liquidstate fermentation. Various beneficial metabolic components are produced, along with numerous pigments, enzymes, lovastatin,polyphenols, and other metabolites. These metabolites offer a healthcare functionality to the vinegar. The use of MV for edible and medicinal purposes has been considered for a long time. Many kinds of primary and secondary metabolites are produced duringMonascusfermentation, and their functions in regulating lipid metabolism have attracted considerable attention. Zhou et al. [12]found that oralMonascuspigment significantly attenuates serum lipids(P＜ 0.05) and ameliorates lipid metabolic disorders and gut microbiota dysbiosis in Wistar rats fed on a HFD.Monascusfermentation extract can induce the mRNA expression of PPARα target genes and enhance lipid metabolism. Additionally, the extract can ameliorate atherosclerosis induced by HFD by inhibiting intestinal inflammation [3,13]. However, the underlying mechanisms by whichMonascusvinegar alleviates HFD-induced inflammatory in rats remain poorly understood.

Therefore, the aim of this research was to analyze the potential of the supplementation of MV for the amelioration of lipid metabolic disorders in rats with hyperlipidemia and explore the mechanism of the effect of MV on inflammation. The levels of inflammatory cytokines and the expression of pro-inflammatory markers were measured. The results were further proved by cell experiments. Our findings suggest that MV not only alleviates dyslipidemia caused by hyperlipidemia but also regulates inflammation, providing theoretical evidence for developing functional foods based on MV.

2. Materials and methods

2.1 Chemicals and reagents

MV was obtained from Shandong Yutu Co., Ltd. (Shandong,China), and its total acid (calculated by acetic acid) is 5.0 g/100 mL.Commercial kits (Nanjing Jiancheng Bioengineering Institute,Nanjing, China) were used in determining TC, TG, LDL-C, HDL-C,alanine aminotransferase (ALT), aspartate aminotransferase (AST),hepaticlipase (HL), CPT-1, IL-1β, nitric oxide (NO) IL-2, IL-6,IL-12, and TNF-α. The antibodies against PI3K, phosphorylated protein-kinase B (p-Akt), mammalian target of rapamycin (mTOR),p-mTOR, p-IKKα/β, inhibitor α of NF-κB (IκBα), p-IκBα, and p-NF-κB p65 were procured from Cell Signaling Technology(Shanghai, China). The antibodies against IKKα/β, NF-κB p65,Akt were purchased from Abcam (Cambridge, UK). The antibodies against p-PI3K was purchased from Signalway Antibody (Maryland,USA). All analytical-grade chemicals were obtained from Sigma(Shanghai, China) unless otherwise noted.

2.2 Animals and treatments

This work complied with the National Guidelines for Experimental Animal Welfare (MOST of PR China, 2006). All animal procedures were performed according to the protocol approved by the Animal Ethics Committee of Tianjin University of Science & Technology. The Sprague-Dawley rats (6 weeks old, male,n= 40, (180 ± 10) g) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were housed in a controlled environment (humidity 55%-60%, temperature(23 ± 2) °C) and a 12 h daylight cycle). After a 1 week of acclimatization, all the rats were randomly divided into two groups:normal diet (containing 59% carbohydrate, 21.1% protein, 4.2% fat,4.9% fiber, 8% ash, 1% phosphorus, and 1.8% calcium) group (CON,n= 8), and the HFD (containing 42.95% carbohydrate, 15.36% protein, 3.05% fat, 3.56% fiber, 5.82% ash, 0.73% phosphorus, 1.31% calcium, 2% cholesterol, 10% lard oil, 0.2% bile salt, 10% yolk powder, and 5% sugar) group (n= 32). After 8 weeks of feeding,the HFD group were randomly divided into four groups (n= 8 in each group): untreated model (MOD), positive control (PC, HFD +2 mg/kgmblovastatin), low-dose (LD, HFD + 0.5 mL/kgmbMonascusvinegar), high dose (HD, HFD + 2 mL/kgmbMonascusvinegar) groups. MV or lovastatin were given daily by oral gavage for 8 weeks, and body weight was recorded once a week.

2.3 Sample collection

After 8 weeks of experimental administration, the rats were anesthetized with pentobarbital sodium (1 mg/kg). whole blood was drawn from the heart and then centrifuged (3 000 ×g, 10 min)to obtain serum. Approximately 0.1 g of liver tissue was used in preparing a homogenate with 0.9 mL of phosphate buffer saline.The liver tissue homogenate was centrifuged (3 500 ×g, 10 min,4 °C), and the supernatant was collected. Kidney, colon, and whole liver tissues were removed, washed with normal saline, and stored at ?80 °C. Some liver, kidney and colon tissues were fixed in 4% paraformaldehyde for histological examination.

2.4 Cell culture and treatments

The murine macrophage cell line RAW264.7 was cultured in Eagle’s minimal essential medium (DMEM) (Hyclone, Logan, Utah,USA) medium at 37 °C and 5% CO2, 10% fetal bovine serum (FBS,Hyclone). Cells were treated with MV at different concentration for 30 min, and then exposed to 1 mg/L lipopolysaccharide (LPS) for 24 h.The levels of NO, IL-1β and TNF-α were detected using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the protocols from the manufacturer. Culture medium was changed every 24 h.

2.5 Cell viability analysis

Cell viability was detected by Cell Counting Kit-8 (CCK-8, Dojin Laboratories, Kumamoto, Japan). Cells were plated on a 96-well microplate for 24 h and treated with MV at different concentration as previously described. The culture medium was totally removed and 10 μL CCK-8 solution was added and incubated for 2 h at 37 °C. The ELISA reader (Tecan Austria GmbH, Salzburg, Austria) was used to detect absorbance of each well at 450 nm.

2.6 Serum or hepatic biochemical assays

The levels of TC, TG, LDL-C, HDL-C, AST, ALT, HL, CPT-1, IL-1β, NO, IL-2, IL-6, IL-12, and TNF-α were determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute, China)according to the manufacturer’s instructions.

2.7 Histological examination

Pieces offixed liver, kidney, and colon tissues were embedded in paraffin and cut into 5 μm sections, then stained with hematoxylineosin (HE). The stained sections were examined under a light microscope (Olympus, Tokyo, Japan) equipped with a digital camera(Olympus, Tokyo, Japan) at a magnification of × 100.

2.8 Western blot analysis

Western blot analysis was measured as previously described.Briefly, the liver tissue or RAW264.7 cells were placed in RIPA lysis buffer at 4 °C and homogenized in homogenates. The extracts were centrifuged at 12 000 ×gfor 10 min at 4 °C for 30 min on ice. The content of total protein in the supernatant was determined using a BCA protein detection kit. Equal amounts of protein per sample were loaded onto 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes, which were blotted with specific primary antibodies. Blots were developed through an enhanced chemiluminescence method and incubated with horseradish peroxidase-conjugated secondary antibody obtained from Signalway Antibody (Maryland, USA).

2.9 Statistical analysis

Experimental data were presented as means ± standard deviation(mean ± S.D.). All statistical analyses were performed using a oneway ANOVA followed by Tukey’s multiple comparison in SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).P＜ 0.05 was considered statistically significant.

3. Results

3.1 Effect of MV on body and tissue weights of hyperlipidemia rats

Changes in body and tissue weights of the groups are shown in Table 1. The initial body weight was equal among groups. The results indicated that body weight in the MOD group was significantly increased compared with those of the CON after intake HFD for 8 weeks (P＜ 0.05). Differences of the body weight in each group continued to increase after intervention for 4 weeks (8-12 weeks).Compared with the MOD group, the PC and HD groups showed significant decreases in body weight and liver weight (P＜ 0.05) at 12 weeks. Compared with the MOD group, the animals in the HD group showed 7.42% decrease in body weight (P＜ 0.05), the liver weight of HD group decreased by 15.82% (P＜ 0.05) at the end of the experiment. These results indicated that supplementation with highdose MV obviously decreased liver and body weights (P＜ 0.05).

Table 1Effect of MV treatment on the body and liver weights of hyperlipidemic rats (n = 8).

3.2 Effect of MV on serum lipid profiles of hyperlipidemia rats

The effects of MV on HFD-induced serum lipid profiles are shown in Fig. 1. Compared with the CON group, the MOD group exhibited hyperlipidemia, as evidenced by increased concentrations of TC and TG (13.73%, 20.75%,P＜ 0.001), which indicated a successful model was established. However, after administration of MV, the serum levels of TC, TG, and LDL-C decreased in a dosedependent manner, whereas HDL-C levels increased compared with the MOD group. Serum TC and TG levels in the HD group decreased by 16.7% and 28.3%, respectively compared with MOD group(P＜ 0.001). In addition, no significant difference was observed between the HD and PC groups.

Fig. 1 Effects of MV treatment on the levels of serum TC (A), TG (B), HDL-C (C), LDL-C and (D) of hyperlipidemia rats. Each value represents the mean ± S.D.(n = 8). ### P ＜ 0.001 for MOD group vs. CON group; *** P ＜ 0.001 for PC, LD and HD group vs. MOD group.

3.3 Effect of MV on hepatic lipid accumulation of hyperlipidemia rats

The liver plays a critical role in lipid metabolic regulation. The hepatic lipid levels of TC and TG are presented in Fig. 2A-B. After 12 weeks of MV treatment, liver TC and TG levels in the MOD group was remarkably increased compared with CON group (P＜ 0.001), but the increases were significantly inhibited by treatment with high-dose MV (P＜ 0.01). As shown in Fig. 2C-D, The levels of hepatic HL and CPT-1 were significantly increased in HD and PC group (P＜ 0.05),but there was no significantly increase in LD group, compared with the MOD group.

Fig. 2 Effect of MV treatment on the levels of hepatic TC (A), TG (B), HL (C) and CPT-1 (D) of hyperlipidemia rats. Each value represents the mean ± S.D. (n = 8).## P ＜ 0.01, ### P ＜ 0.01 for MOD group vs. CON group; * P ＜ 0.05, ** P ＜ 0.01 and *** P ＜ 0.001 for PC, LD and HD group vs. MOD group.

3.4 Effect of MV on liver, kidney and colon histopathology

The representative images of HE-stained liver, kidney, and colon histological sections and pathological changes were shown in Fig.3A-C. The liver cells in the MOD group were arranged unmorally and characterized by excessive lipid accumulation and fatty degeneration(ballooning) in hepatocyte compared with those in the CON group(Fig. 3A). The MOD group exhibited degeneration or necrosis,revealing that HFD intake induced liver steatosis. The kidney sections showed glomerular atrophy and tubular epithelial degeneration in the MOD group (Fig. 3B). The colon of the MOD group exhibited inflammation-induced damage and had shorter intestinal villi than that of the CON group. Additionally, the intestinal villi of the MOD group lost their normal structures and had a large number of necrotic cells (Fig. 3C).

Fig. 3 Effect of MV treatment on histopathology of hyperlipidemic rats. Morphological sections of liver (A), kidney (B) and colon (C) tissues dyed with HE (× 100).

Nevertheless, compared with the MOD group, the PC, LD, and HD groups showed ameliorated liver lipid accumulation and relieved HFD-induced hepatic steatosis. Notably, the PC and HD groups showed better performance in inhibiting hepatic lipid accumulation than the LD group. The HD group exhibited a significant improvement in HFD-induced pathological damage, and the morphology was closer to the CON group (Fig. 3A). Similarly, the improvement in renal pathological examination in the HD group was better than the PC group (Fig. 3B). The colon sections showed the same recovery trend,and the HD group exhibited normal epithelial cells and decreased inflammatory cell inflltration (Fig. 3C).

3.5 Effect of MV on liver injury and inflammatory cytokines of hyperlipidemia rat

ALT and AST are the sensitive markers of liver injury. In this study, compared with the CON group, the MOD group showed dramatic increases in serum ALT (1.95-fold) and AST activities(1.91-fold) (P＜ 0.001, Table 2). Treatment with lovastatin or highdose MV significantly decreased the activities of serum AST and ALT (P＜ 0.01). Moreover, the activity of the enzymes in the PC and HD groups were not significantly different from those of the CON group.

Table 2Effect of MV on liver injury and inflammatory cytokines of hyperlipidemic rat (n = 8).

Liver injury often causes inflammatory reaction. Therefore,hepatic inflammatory factors levels, including TNF-α, IL-2, IL-6,and IL-12 were measured. The results in Table 2 showed that the levels of these inflammatory factors were increased by HFD-induced challenge compared with the levels in the CON group (P＜ 0.01 orP＜ 0.001). By contrast, the HD group showed considerably reduced hepatic TNF-α, IL-2, IL-6, and IL-12 levels, decreasing by 23.27%,27.46%, 28.39%, and 19.15%, respectively (P＜ 0.05). No difference was observed between the PC and HD groups. These results demonstrated that high-dose MV effectively alleviated hepatic pro-inflammatory factors,the efficacy of the HD group was similar to that of the PC group.

3.6 Effect of MV on hepatic NF-κB signalling of hyperlipidemia rat

To assess the effect of decreased of hepatic lipid accumulation on inflammatory response, we analysed the expression of the proinflammatory markers IKKα/β, p-IKKα/β, IκBα, p-IκBα, NF-κB p65,and p-NF-κB p65 in the liver tissues (Fig. 4A-D). NF-κB signaling increases the expression of inflammatory factors by promoting the phosphorylated levels of IKKα/β and IκBα. The results exhibited that the ratios of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and p-IKKα/β/IKKα/β was significantly higher in the MOD group than that in the CON group (P＜ 0.01), but decreased by 35.35% (P＜ 0.01), 39.06%(P＜ 0.05), and 38.64% (P＜ 0.01) respectively in the HD group compared with the MOD group. This experiment showed that MV might prevent HFD-induced inflammatory response by modulating NF-κB signaling cascades.

Fig. 4 Effect of MV on the expression levels of p-IκBα/IκBα, p-IKKα/β/IKKα/β and p-NF-κB p65/NF-κB p65 of hyperlipidemia rats. (A) The expression levels of proteins were detected through Western blot analysis. (B) The ratios of p-NF-κB p65/NF-κB p65 protein expression. (C) The ratios of p-IκBα/IκBα protein expression. (D) The ratios of p-IKKα/β/IKKα/β protein expression. Each value represents the mean ± S.D. (n = 8). ## P ＜ 0.01 for MOD group vs. CON group;* P ＜ 0.05, ** P ＜ 0.01 for PC, LD and HD group vs. MOD group.

3.7 Effect of MV on hepatic PI3K/AkT/ mTOR signalling of hyperlipidemia rat

PI3K/Akt signaling pathway also plays an important role in inflammatory diseases. To explore the regulatory effects of MV on the PI3K/Akt/mTOR signaling pathway, we examined the phosphorylation levels of PI3K, Akt, and mTOR within this signaling pathway (Fig. 5A-D). The MOD group showed significant elevation in the ratios of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR compared with the CON group (P＜ 0.05). Moreover, compared with the MOD group, the HD group showed statistically increases in the ratios of hepatic p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR by 31.62%, 40.31%, and 34.21%, respectively (P＜ 0.05). Additionally,no significant difference between the HD and PC groups. The results indicated that HD could alleviate HFD-induced inflammation by triggering PI3K/AKT/mTOR pathway.

Fig. 5 Effect of MV on the expression levels of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR of hyperlipidemic rats. (A) The expression levels of proteins were detected through Western blot analysis. (B) The ratios of p-PI3K/PI3K protein expression. (C) The ratios of and p-Akt/Akt protein expression. (D) The ratios of p-mTOR/mTOR protein expression. Each value represents the mean ± S.D. (n = 8). ## P ＜ 0.01 for MOD group vs. CON group; * P ＜ 0.05, ** P ＜ 0.01 for PC,LD and HD group vs MOD group.

Fig. 5 (Continued)

3.8 Effect of MV on NO and pro-inflammatory cytokines in RAW264.7 cells treated by LPS

In this part, in order to further con firm our hypothesis of MV in regulating inflammation response, LPS was used to induce RAW264.7 cells to NO and pro-inflammatory cytokines, and the role of MV in inflammation response was elucidated. As shown in Fig. 6A,cell viability was determined with different concentrations of MV for 24 hours. The results showed that there was no significant change in cell viability after 10, 20, 40 and 50 μL/mL MV intervention. And 100, 200 and 400 μL/mL MV treatment on RAW264.7 cells displayed difference. However, the cytotoxicity of 400 μL/mL MV was stronger(P＜ 0.01). Furthermore, 100 and 200 μL/mL MV were used to analyse the role of MV in regulating in inflammation response.RAW264.7 cells were treated with 1 mg/L LPS. Then the contents of NO and pro-inflammatory cytokines in the supernatant of cultured cells were determined. In Table 3, compared with CON group, the contents of NO, IL-1β and TNF-α were significantly increased after LPS-induction. However, pretreatment with MV (100 μL/mL)inhibited the increase of NO, IL-β and TNF-α levels (51.79%, 39.87% and 32.29%, respectively). Thein vitrodata indicated that MV regulated inflammation response by inhibiting inflammatory factors.

Fig. 6 Effect of MV on RAW264.7 cells viability and expression of inflammatory protein. (A) The cell viability was evaluated after different concentrations of MV. (B) Western blot assays of NF-κB and PI3K/Akt/mTOR signaling pathway in RAW264.7 cells. The relative expression levels of (C) p- NF-κB p65/ NF-κB p65, (D) IKKα/β, (E) p-Akt, (F) PI3K, (G) mTOR based on western blot assays. Each value represents the mean ± S.D. (n = 8). # P ＜ 0.05, ## P ＜ 0.01, ### P ＜ 0.001 for LPS group vs. CON group; ** P ＜ 0.01 and *** P ＜ 0.001 for LPS + 100 μL/mL MV group and LPS + 200 μL/mL MV group vs. LPS group.

Fig. 6 (Continued)

3.9 Effect of MV on protein expression of p-NF-κB p65/NF-κB p65, IKKα/β, PI3K and p-Akt in RAW264.7 cells treated by LPS

Table 3The determination of NO and pro-inflammatory cytokines in the supernatant of RAW264.7 cells.

Having further con firmed that MV treatment regulated NF-κB and PI3K/Akt signaling pathways, a series of experiments were conducted to investigate the expression of inflammationassociated proteins in the two pathways. As shown in Fig. 6B-G,LPS significantly increased the p-NF-κB p65/NF-κB p65 and the expression levels of IKKα/β, PI3K, p-Akt and mTOR proteins in RAW264.7 cells compared with the CON group (P＜ 0.001).However, MV administration reversed the increase in the expression of LPS-induced inflammation-associated proteins.Compared with the 1 mg/L LPS group, the expression of p-NF-κB p65/NF-κB p65, IKKα/β, p-Akt, PI3K and mTOR in 1 mg/L LPS + 100 μL/mL MV group were significantly down-regulated by 46.68%, 23.53%, 35.58%, 23.77% and 25.60%, respectively(P＜ 0.05). However, there was no significant change between LPS + 200 μL/mL MV group and 1 mg/L LPS group. The results were consistent with the expression trend of liver inflammationassociated proteins in animal experiments, suggesting that MV has the effect of regulating the inflammation response.

4. Discussion

MV is a typical fermented health food. The use of MV for edible and medicinal purposes has been considered for a long time, and their functions in regulating hyperlipidemia have attracted considerable attention [13-16]. Hyperlipidemia is generally believed to be related to inflammatory diseases closely [17,18]. Long-term HFD directly causes excessive weight gain, and imbalance between energy intake and expenditure leads to obesity. Obesity is often the result of lipid accumulation in the body, eventually leading to hyperlipidemia [19,20].In the present study, the rats in MOD group had significantly increase of body and liver weights compared with the CON group. After feeding with HFD for 8 weeks, serum and liver TC and TG levels were significantly up-regulated, and serum LDL-C showed the same trend in the MOD group (P＜ 0.05). The results suggested that the increase in body and liver weights can cause hyperlipidemia. In addition, severe hepatitis, nephritis, and colon injury were found in the MOD group. These results are consistent with the significant increase in the levels of inflammatory cytokines IL-2, IL-6, IL-12,and TNF-α in the liver. Thus, we can conclude that hyperlipidemia is accompanied by inflammation.

The liver is the main organ for the synthesis and metabolism of endogenous lipids and lipoproteins. Increased triglyceride synthesis and accumulation in hepatocytes are the main causes of hepatic steatosis [21]. The NF-κB signaling pathway is a classical pathway associated with inflammatory metabolism. Activated NF-κB signals enhance inflammation response by increasing the expression of inflammatory factors. TNF-α induces liver inflammation by activating the NF-κB signaling pathway [17], and IKKα/β is the main kinase that induces NF-κB activation. The activation of IKKα/β kinase leads to the phosphorylation and degradation of IκB and promotes the release of the NF-κB dimer. It is further activated by post-translational modification and transferred to the nucleus to bind with a target gene and promote the transcription of pro-inflammatory factors, such as IL-2,IL-6, and IL-1β. Li et al. [8]reported that six types of tea (green,yellow, white, black, raw pu-erh, and oolong) extracts significantly lower iNOS and IL-6 levels, as evidenced by reduced inflammation.Furthermore, rice bran phenolic extract supplementation inactivates the endotoxin-TLR4-NF-κB pathway, remarkably elevates the expression of NF-κB p65 and p-IκBα/IκBα ratio in the liver, and represses inflammatory responses in the liver [7]. However, in our study, we found that the HD group had significantly increased ratios of p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and p-IKKα/β/IKKα/β in the liver of hyperlipidemic rats, which was in accordance with previous studies [7,17]. Moreover, the levels of TG, TC, and LDL-C in sera and the liver were significantly decreased, whereas HDL-C level significantly increased in the HD group (P< 0.05), which reduced the lipid accumulation in liver. The liver histological sections also showed that the accumulation of lipid droplets in the liver was significantly reduced (Fig. 3A). Excessive accumulation of lipids in the liver can cause liver injury and trigger inflammatory reaction [21].The NF-κB signaling pathway is a classical pathway associated with inflammatory metabolism and activated by increasing the expression of inflammatory factors [8]. Therefore, inflammatory factors are the key of NF-κB signaling pathway triggered by lipid accumulation.Studies have demonstrated that sea cucumber saponins can inhibit the progression of atherosclerotic lesions via their anti-inflammatory and lipid-lowering biological properties [22,23]. Our results showed that the inflammatory factors IL-2, IL-6, IL-12, and TNF-α were significantly decreased after high-dose MV intervention (Table 2),the same trend was observed in cell experiment. In LPS+100 μL/mL MV group, the inflammatory factors IL-1β and TNF-α decreased by 39.87% and 32.29%, respectively (P< 0.05) (Table 3). Thus,the inhibitive activity of MV on the NF-κB-mediated inflammation contributed to its protection against the liver injury caused by lipid accumulation.

Hepatitis is the result of liver function damage, which is mainly manifested by the destruction of the integrity of the liver cell membrane. This damage accelerates the release of liver enzymes,increasing AST and ALT levels in systemic circulation [24]. Hepatic function damage leads to increase in pro-inflammatory factors, such as IL-2, TNF-α and IL-6, which cause inflammatory reactions [1,18].Beh et al. [25]confirmed that high-dose Nipa vinegar supplements inhibit the expression of inflammatory cytokines and reduce lipid deposition effectively in HFD-induced rats, playing a role in weight loss and anti-inflammatory processes. Red yeast rice, the starter of MV, potentially ameliorates inflammatory cytokines and systemic serum inflammatory levels induced by HFD [3].The above results are similar to those of this study. In the present study, compared with MOD group, the HD group showed significantly reduced liver ALT (48.0%) and AST (34.0%) levels. The levels of liver IL-2, IL-6,IL-12, and TNF-α in HD group decreased by 27.46%, 28.39%,19.15%, and 23.27%, respectively. The stained sections showed that the HFD-induced pathological damage in the liver in the HD group was significantly reduced. Moreover, inflammation caused by hyperlipidemia leads to the loss of intestinal barrier integrity,directly affecting the function of the intestinal barrier and activates a series of diseases [26,27]. Our studies showed that high-dose MV not only improves liver lipid accumulation and steatosis, but also reduces nephritis and colon injury (Fig. 3). Lipid accumulation causes hyperlipidemia. The occurrence of hyperlipidemia is frequently accompanied by increases in TC, TG, and LDL-C levels and decrease in HDL-C level. Vinegar plays a regulatory role in lipid metabolism.For example, the daily intake of 15 or 30 mL of apple cider vinegar can reduce the blood lipid levels of obese subjects, and after 10 weeks of treatment, 2 mL/kg mb of coconut water vinegar can reduce serum TC and TG levels [25,27]. In our present study, the serum levels of TC and TG in the HD group decreased by 16.7% and 28.3%(P< 0.001, Fig. 1). Meanwhile, the liver TC and TG levels remarkably decreased in the HD group (P< 0.05, Fig. 2A–B). The results of the serum and liver biochemical indexes were consistent with those of HE staining (Fig. 3). We found that the liver levels of HL and CPT-1 in the HD group were significantly higher than those in the MOD group. The results demonstrated that MV regulates lipid metabolism by regulating the activity of lipase.

Different vinegars have varying effects on lipid profile regulation mainly because of the diversity of raw materials and technology.Thus, the bioactive components produced by fermentation vary as well. MV is an acid condiment brewed by a traditional technology in China and is fermented using red yeast rice; it has a unique flavor and health function. Red yeast rice potentially regulates lipid metabolism [12]. A variety of bioactive components produced during fermentation can promote the excretion of cholesterol through the intestine and then reduce cholesterol levels in plasma and tissues [12,15,16,28]. The bioactive components of MV likely promote steatolysis, inhibit lipid accumulation, and improve the balance of lipid metabolismin vivo.

Additionally, inflammatory stress can cause changes in a variety of inflammatory signaling pathways. The NF-κB signaling pathway regulates inflammation response and is affected by other signaling pathways. The PI3K/Akt pathway is involved in many activities in cells and plays an important role in a variety of inflammatory diseases,such as atherosclerosis and inflammatory bowel disease [29,30]. The PI3K family is closely related to intracellular signal transduction and pathogenesis of inflammation, obesity, tumor, and immune diseases.The secretion of pro-inflammatory cytokines can be inhibited, and the secretion of anti-inflammatory factor IL-10 can be increased by inhibiting PI3K signal in the TLR-mediated pathways in inflammatory diseases [31,32]. Akt is a downstream regulatory protein of the PI3K signaling pathway, which regulates cell survival and apoptosis, and the inhibitor can inhibit Akt activation by inhibiting PI3K [21].mTOR, as a member of the PI3K family, is a serine threonine kinase that participates in cell growth and proliferation [33]. In this study,we found that high-dose MV decreased the ratios of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR in the livers of hyperlipidemic rats compared with those in the MOD group. In addition, activated PI3K/Akt signaling enhanced IKKα/β activation and then promoted the phosphorylation and degradation of IκB by enhancing NF-κB, which stimulated the NF-κB activation [34]. Finally, we found that the transcription of inflammatory cytokines such as IL-2, IL-6, and TNF-α also changed (Table 2 and Table 3). These results demonstrated that the inflammatory response induced by hyperlipidemia was affected by the PI3K/Akt/mTOR pathway, which was inhibited by MV, and then regulated inflammatory stress.

Overall, NF-κB and PI3K/Akt signaling can be inhibited by MV. The expression of inflammation-associated proteins (such as p-NF-κB p65/NF-κB p65, IKKα/β, PI3K and p-Akt) was detected by cell experiment, which further proved that NF-κB and PI3K/Akt signaling pathways can be regulated by MV. The mechanism of these two pathways is shown in the Fig. 7. MV can reduce the levels of inflammatory cytokines, such as TNF-α, which inhibit IKKα/β activation by multistage stimulation modification. The inhibition of IKKα/β kinase attenuated the phosphorylation and degradation of IκB protein and prevented the release of NF-κB dimer. Finally, the transcription levels of IL-2, IL-6, and TNF-α decreased. Our research was based on the inflammatory mechanism and analyzed the effect of MV on inflammatory stress. The results showed that MV plays an anti-inflammatory role by regulating NF-κB and PI3K/Akt signaling pathways. Hyperlipidemia and inflammation are closely related to insulin resistance, which is prevalent in obese individuals and plays a key role in the type 2 diabetes. Obesity is not only related to excessive fat accumulation, but also related to chronic inflammation.The increased expression of pro-inflammatory cytokines is one of the molecular mechanisms of insulin resistance. Studies have shown that insulin resistance can be improved by regulating PI3K signaling pathway [35,36]. Thus, in the following studies, we will establish high-fat-high-fructose diet-induced obese mice to explore the possible underlying molecular mechanism of MV regulating insulin resistance and provide theoretical support for the function of MV.

Fig. 7 Mechanism of MV on inflammatory stress regulation. In the diagram, a black arrow stands for direct stimulus modification, and two consecutive arrows stand for multiple stimulus modifications.

5. Conclusion

The present study suggests that MV ameliorates HFD-induced inflammation in a dose-dependent manner. This function further inhibits the deposition of liver fat, attenuates hepatic steatosis and liver injury, and reduces HFD-induced hyperlipidemia finally. In addition,the PI3K/Akt/mTOR and NF-κB pathways were found to be involved in the anti-inflammatory effect of MV. To the best of our knowledge,this study shows that MV potentially treats or prevents hyperlipidemia by improving lipid metabolism disorder and inflammation response.These findings suggest that MV has the potential to improve HFD-induced inflammation, so it can be considered as a potential functional food for the treatment or prevention of hyperlipidemia.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This research was supported by the National Key R&D Program of China (2016YFD0400505), the National Natural Science Foundation of China (81600126), the Tianjin Municipal Education Commission (TD13-5013), the Key Research and Development Program of Shandong Province (2019YYSP011, and the Tianjin Graduate Research Innovation Project (2020YJSB132)).