Agastache rugosa alleviates the multi-hit effect on hepatic lipid metabolism, inflammation and oxidative stress during nonalcoholic fatty liver disease

Background Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and has high rates of morbidity and mortality worldwide. Agastache rugosa (AR) possesses unique antioxidant, anti‑inflammatory and anti-atherosclerosis characteristics. Methods To investigate the effects and the underlying mechanism of AR on NAFLD, we fed mice a high-fat diet (HFD) to establish NAFLD model of mice in vivo experiment and induced lipidosis in AML12 hepatocytes through a challenge with free fatty acids (FFA) in vitro. The contents of total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver homogenates were measured. Pathological changes in liver tissue were evaluated by HE staining. Oil red O staining was used to determine degree of lipid accumulation in liver tissue, and Western blot was used to detect abundance of inflammation-, lipid metabolism- and endoplasmic reticulum stress-related proteins. Supply of AR alleviated accumulation of lipid in hepatocytes induced by HFD in vivo and challenged with free fatty acids (FFA) in vitro. Compared with the HFD group, supplementing AR decreased p-NF-κB/NF-κB and p-IκB/IκB protein and inhibited abundance of PERK, IRE1 and ATF6 ( P < 0.05). Furthermore, AR reduced lipid accumulation within hepatocytes by downregulating abundance of SREBP, ACC1 and FAS ( P < 0.05). Supply of AR significantly attenuated ROS accumulation and MDA production by improving antioxidant enzymatic activity including SOD and GSH ( P < 0.01). Supply of AR attenuates disordered lipid metabolism and enhances the antioxidative defense associated with NAFLD induced by HFD in mice. Results underscore the potential of plants used in traditional Chinese medicine to achieve pharmacological benefits through a multi-tier cellular response. Our studies suggested that AR could alleviate hepatic inflammation, steatosis and oxidative stress in in vivo an in vitro models of NAFLD. The data provide the basis for developing novel bioactive food additives based on AR that can help alleviate complications associated with development of NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is an important component of the metabolic syndrome in states such as obesity and insulin resistance [1]. Inflammatory reactions induced by reactive oxygen species in the liver parenchymal cells characterize the so-called "first-hit" during NAFLD [2].
Dysregulation of adipocyte metabolism in the metabolic syndrome is an independent risk factor for development of NAFLD [3]. Natural substances are not only effective treatment for obesity, diabetes, insulin resistance (IR) and other metabolic diseases, but also relatively safe to consume [4].
Traditional Chinese medicine (TCM) formulas based on plant extracts contain substances capable of eliciting the so-called "multiple organ-multiple hit" effect [5]. Various TCM and supplements offer suitable therapeutic options in the treatment and prevention of NAFLD [6]. For instance, Agastache rugosa (AR), a herbal drug, has been used in humans for the treatment of anorexia, vomiting and other intestinal disorders [7,8]. Studies indicate that AR has anticarcinogenic [9] and protective effects against lung [10] and brain injury [11]. Extracts of AR are also believed to be valuable in the treatment of inflammatory [12] and oxidative stress-induced disorders [13]. Therefore, our general hypothesis was that supply of AR would attenuate the negative effects of NAFLD on hepatic lipid metabolism and oxidative stress. At present, the effect of Agastache rugosa on NAFLD is unknown.
Thus, specific objectives were to induce NAFLD in vivo and in vitro to study the underlying mechanisms whereby supply of AR can have a positive effect.

Materials And Methods
Herbal plant extract AR was purchased from Daqing Fu Rui Bang pharmacy, China. The raw herbs were soaked in distilled water overnight followed by decocting twice in boiling water (60 min each time). The combined aqueous extract was filtered through gauze and then heated until evaporation [14]. Insoluble particles were removed by low-speed centrifugation, the supernatant sterilized by filtration through a 0.22 µm Millipore filter (MILLEX, GP) and stored at 4 °C for use. Main components of AR were analyzed by highperformance liquid chromatography-electrospray ionization/mass spectrometry (LC/MS).

Animals and treatment
Male Kun Ming mice (20-22 g; 8 weeks) were obtained from Harbin Medical University (Daqing, China). Mice were housed in cages with a 12 h light/dark cycle in a temperature-controlled environment. The mice were acclimatized to laboratory conditions for 1 week before the study and then randomly divided into five groups of six: control group fed a standard diet, NAFLD group fed a high fat diet (HFD) (60% kcal fat), low dose group fed HFD + 1.8 g/kg AR given orally (0.1 mL per 10 g body weight), medium dose group (HFD + 4.5 g/kg AR) and high dose group (HFD + 9.0 g/kg AR). HFD feeding was initiated at 8 weeks of age and continued for an additional 8 weeks at which point mice were fasted for 12 h prior to sacrifice with ether. Blood was collected just before sacrifice for serum biochemical analysis. The liver was quickly excised, cleaned completely with ice-cold phosphatebuffered saline (PBS), weighed and preserved in liquid nitrogen until use. All animal studies were approved by the Ethics Committee of Heilongjiang Bayi Agricultural University in accordance with the Chinese guidelines for the care and use of laboratory animals.

Histological Examination
A portion of liver tissue was fixed with 4% paraformaldehyde and embedded in paraffin. For hematoxylin and eosin (H&E) staining [15], rehydration was done in a decreasing ethanol series, and then stained with H&E. Frozen sections were prepared and stained with Oil red O to determine hepatic lipid accumulation. The most severe areas with hepatic inflammation in the representative histology sections were photographed using a microscope. Cells were fixed with 4% paraformaldehyde and stained with freshly diluted Oil Red O solution. Representative photomicrographs were captured using a system incorporated in the microscope.
Cell viability analysis 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Solarbio, M8180) was used to analyze cell viability. Cells were treated with different concentrations of AR for 20 h, and then 10 µL MTT was added for another 4 h. Culture medium was then totally removed and DMSO added, followed by measurement of absorbance using a microplate reader. All MTT assays were performed at least 3 times for each group. Subsequently, results of the MTT assay were used to select 5 different concentrations of AR to add to cell culture fluid. Real-time cell growth curves were measured through the Real-time label-free cell analysis (RTCA) system (ACEA Biosciences).
Cell treatment AML12 cells were seeded in 6-well plates. Hepatic steatosis in vitro was induced according to previously established methods [16] in which AML12 cells were treated for 24 h with a mixture of FFA containing a 2:1 ratio of oleate (SIGMA, O1383-5G) and palmitate (SIGMA, P5585-10G), the final concentration of FFA being 1 mM. For the AR supplementation experiment, the herbal extracts were added to the above medium containing 1 mM FFA for 24 h at a high (25 mg/mL), medium (12.5 mg/mL), or low (6.25 mg/mL) concentration.

Statistical analysis
All results are expressed as the mean ± SD. Statistical analyses were performed using the Student ttest. For multiple comparisons, one-way analysis of variance (ANOVA) was used. P < 0.05 was considered statistically significant.

AR alleviated liver lesions induced by HFD
Compared with the control group, the content of ALT and AST in the liver homogenate of the HFD group increased significantly (P < 0.01); compared with the HFD group, the content of ALT and AST in the AR group decreased with the increase of AR concentration (Fig. 1A).
The structure of hepatocytes based on HE staining indicated that the control group had complete structural features, i.e. hepatocytes were polygonal, boundary was clear, cytoplasm stained red, there were no vacuoles, the nucleus was in the center; the outline of liver lobules was clear, the structure was regular, and the liver cords were arranged radially with the central vein as the axis. However, in the HFD group, there were diffuse vacuoles around the central vein and portal area, the boundary between the cells in the field of vision was unclear, hepatocytes were obviously swollen or inflated like balloons, a large number of nearly round vacuoles could be seen in the cells, even the vacuoles squeezed the nucleus of the liver to one side; the hepatic sinuses were compressed and narrow, and the structures of the hepatic sinuses and hepatic cords were unclear. Compared with the HFD group, liver tissue of the AR group was improved in terms of hepatic sinuses, structural arrangement of hepatic cords, morphology of liver cells, and fat globules and balloon like changes (Fig. 1B).
Changes of oil red O staining in liver tissue showed that the nucleus in the control group was blue, there were no obvious orange lipid droplets, space between liver cells was clear, and the structure of liver sinuses was normal. In the HFD group, however, hepatocytes were enlarged with diffused lipid droplets in the field of vision. In contrast, in the AR group the content of orange lipid droplets was significantly lower than the HFD group, and there was a reduction in lipid accumulation (Fig. 1B).
In the FFA group, oil red O staining showed a large number of lipid droplets in AML12 cells, and some of the fusion showed chain and mass changes. In the AR group, the number of orange lipid droplets in hepatocytes was significantly lower than the model group, and the degree of lipid accumulation was reduced which was consistent with the change observed in liver tissue (Fig. 1C).

AR increased liver antioxidant capacity during NAFLD induced by HFD
Compared with the control group, the activity of SOD and GSH in the liver of HFD group decreased significantly (P < 0.01), while MDA increased significantly (P < 0.01). Furthermore, AR significantly increased GSH level (P < 0.01) and decreased MDA level (P < 0.01) in a dose-dependent manner ( Fig. 2A). In AML12 cells, the content of ROS in the FFA group was significantly greater (P < 0.01) than the control group, while content of ROS in the AR groups was significantly lower (P < 0.01) than the control group (Fig. 2B).

AR suppresses inflammation in liver and AML12 cells
Compared with the control group, the levels of TNF-α,IL-6 and abundance of IKK, p-NF-κB/NF-κB and p-IκB/IκB protein in the liver of HFD group increased significantly (P < 0.01), while they decreased significantly (P < 0.01) in the 9.0 g/kg AR group (Fig. 3). Furthermore, AR significantly decreased p-NF-κB/NF-κB and p-IκB/IκB abundance in AML12 cells (P < 0.01) ( Fig. 4A and B). In AML12 cells, compared with the control group, abundance of NF-κB protein was mainly distributed in the nucleus of the FFA group, while abundance of NF-κB protein increased in the cytoplasm of the AR group (Fig. 4C).
AR reduced fat deposition during NAFLD induced by HFD Compared with the control group, the content of TC and TG in liver homogenate of the HFD group was significantly increased (P < 0.01); compared with the HFD group, the content of TC and TG in liver homogenate of the AR group was significantly lower (P < 0.01) (Fig. 5A). In addition, compared with the control group, abundance of SREBP, ACC1 and FAS were significantly upregulated in the HFD group. Compared with the HFD group, the AR group significantly decreased abundance of SREBP, ACC1 and FAS (Fig. 5B). In addition, protein abundance of SREBP and FAS decreased in a dosedependent manner in the AR groups. Furthermore, the abundance trend of SREBP, ACC1 and FAS in AML12 cells was consistent with that in liver tissue (Fig. 5C).

AR alleviated ER stress during NAFLD induced by HFD
Protein abundance of PERK, IRE1 and ATF6 in liver of the HFD group was increased significantly compared to the normal group (P < 0.05). No difference was observed in the different dose groups of AR (P > 0.05), whereas marked decrease in abundance of PERK, IRE1 and ATF6 was observed in the AR group compared with the HFD group (P < 0.05) (Fig. 6A). Compared with the control cells, cells treated with FFA showed significantly increased levels of PERK and IRE1 (P < 0.05). Compared with FFA-treated cells, cells pretreated with AR showed significantly lowered protein abundance of PERK and IRE1 (P < 0.05) in a dose-dependent manner (Fig. 6B).

Compositions of compounds in the AR
The chemical composition of AR of peak MS spectrum of was showed in Fig. 7. The concentrations of substances were showed in Table 1 and Table 2. Ninety-nine compounds were identified where the concentration of flavonoids was 33%. The full spectrums of constituents were identified based on the database Metlin (https://metlin.scripps.edu).

Discussion
The hallmark of NAFLD is the hepatic accumulation of lipids, which subsequently leads to cellular stress, inflammation and hepatic injury, eventually resulting in chronic liver disease [19,20]. . Our results showed that compared with the HFD group, the levels of SOD and GSH in the liver of AR group were significantly increased, while the levels of MDA and ROS were significantly decreased in hepatocytes (Fig. 2). Thus, we speculate that AR decreased lipid peroxidation directly through increasing antioxidant enzymes activites.
An HFD increase in mitochondrial β oxidation can cause oxidative stress, and the increase of ROS production can activate the inflammatory pathway regulated by IKK/NF-κB Our study showed that AR not only decreased NF-κBinduced transcription of inflammatory cytokines in HFD-induced NAFLD (Fig. 3A), but also inhibited protein abundance of IKK-NF-κB signaling pathway components ( Fig. 3 and Fig. 4). These results suggest that AR might inhibit activation of IKK/IKB/NF-κB signaling to interrupt the inflammatory cascade, and reduce the "second hit" of inflammatory factors on liver.
Serum enzymology and blood lipid are typical indices used for clinical diagnosis of NAFLD, but liver histology is still the "gold standard" [35]. Our results showed that compared with the HFD group, the AR group reduced TC and TG (Fig. 5A), and the AR group reduced lipid accumulation in hepatocytes

Consent for publication
Not applicable.

Availability of data and materials
All the data obtained and materials analyzed in this research are available with the corresponding author.

Competing interests
The authors declare that they have no competing interests

Funding
The work was supported in part by the National Key R&D Program of China (2017YFD0502200); Group control technology and product development and demonstration of important mass production disease groups in dairy cattle (GA16B20); Heilongjiang Bayi Agricultural University Support Program for San Heng San Zong (ZRCLG201904).

Authors' Contribution
YC and RC designed, performed sample preparation and data analysis. YC wrote the manuscript. QW participated in the method development and validation. JJ, YL and CX participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.                  There are significant differences among the groups with different lowercase letters (P < 0.05). Groups: CON, control; HFD, high-fat diet; FFA, free fatty acid; AR, Agastache rugosa. There are significant differences among the groups with different lowercase letters (P < 0.05). Groups: CON, control; HFD, high-fat diet; FFA, free fatty acid; AR, Agastache rugosa. There are significant differences among the groups with different lowercase letters (P < 0.05). Groups: CON, control; HFD, high-fat diet; FFA, free fatty acid; AR, Agastache rugosa.