2022 Volume 45 Issue 3 Pages 260-267
Non-alcoholic fatty liver disease (NAFLD) has become prevalent worldwide, but sufficient pharmaceutical treatments for this condition are lacking. Previous literature suggests that vitexin offers beneficial effects in the treatment of NAFLD, but the underlying mechanisms are not well understood. In this study, the in vivo effects of vitexin were investigated in high-fat-diet (HFD)-induced NAFLD mice. Liver pathology, biochemical parameters, lipid levels, hepatocyte ultrastructure, and related regulatory proteins were measured at the end of treatment. Treatment consisted of four weeks of daily administration of vitexin at a dose of 6 mg/kg of body weight. This treatment markedly improved hepatic architecture, attenuated lipid accumulation, and regulated lipid abnormalities. In addition, the treatment reduced endoplasmic reticulum (ER) stress, restored mitochondrial biological proteins, and increased autophagy. Furthermore, the treatment increased peroxisome proliferator-activated receptor-γ (PPAR-γ) protein, which was inhibited by HFD. Thus, it was speculated that vitexin degraded lipids in HFD-induced NAFLD mice liver by inducing autophagy and restoring both ER and mitochondrial biological proteins.
Due to the increased rate of economic development along with the improvement of living standards, the percentage of people with high fat intake is increasing every year. A long-term high-fat diet (HFD) affects health in various ways, such as by increasing blood pressure and the percentage of fat contained in blood. This can induce obesity, diabetes, cerebrovascular diseases, and cancer. Excessive accumulation of fats in the liver induces non-alcoholic fatty liver disease (NAFLD), which has become a health problem worldwide.1) NAFLD can progress to serious liver problems, such as fibrosis, cirrhosis, and hepatocarcinoma.2) Hence, it is important to control fat intake in order to prevent chronic diseases that result from long-term high fat intake.
Autophagy, an intracellular regulating mechanism, controls intracellular metabolism by removing damaged organelles.3) Autophagy is inhibited in the liver in HFD intervention models.3) Endoplasmic reticulum (ER) stress is caused by an imbalance of the ER that leads to upregulation of the unfolded protein response (UPR).4) Excessive lipid accumulation can induce proteostasis and trigger ER stress in hepatocytes.5) In order to adapt to lipid packing, the pro-survival UPR enhances ER protein folding and promotes autophagy to clear the aggregate-prone proteins. Next, lipid droplets are degraded by autophagy (i.e., lipid autophagy, or lipophagy). Chronic ER stress stimulates the pro-death UPR, resulting in liver damage.6,7) Both abnormal autophagy and ER stress have been regarded as the underlying mechanism of NAFLD.6)
Vitexin, a flavone C-glycoside, is present in many Chinese herbal medicines, such as Ficus deltoidei and hawthorn. It has been previously reported that vitexin has anti-tumor, anti-oxidative, anti-inflammatory, and antibiosis properties.8) There is evidence that vitexin inhibits lipogenesis, and also activates lipolysis and fatty acid oxidation by activating AMP-activated protein kinase (AMPK). This alleviates NAFLD.9,10) However, it remains unclear whether vitexin is effective in the treatment of NAFLD in mice by activating autophagy and reducing ER stress.
This study was conducted in order to determine the effects of vitexin on HFD-induced NAFLD mice and to elucidate the underlying mechanisms of the disease. The hypothesis of this study was that vitexin alleviates NAFLD by regulating autophagy and ER stress.
Eighteen eight-week-old male C57BL/6J mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China). After one week of adaptive feeding, the mice were randomly divided into three groups of six: a normal control group (CON), a high-fat diet group (HFD), and a vitexin group (VIX).
The CON mice were fed a normal diet. The mice in the other two groups were fed for eight weeks with HFD, which contained 71.6% basal feed, 10% fat, 10% protein, 5% sucrose, and 3% cholesterol. Then, vitexin was injected at a dose of 6 mg/kg per day in the VIX mice for the following four weeks. CON and HFD mice received an equivalent dose of saline per day. At the end of this period, after 12–14 h of fasting, blood samples were collected by removing an eyeball from anesthetized mice with tribromoethanol. All mice were sacrificed by cervical dislocation. Serum was separated from the blood by centrifugation at 5000 × g for 10 min and stored at −80 °C. The liver tissues were quickly picked, weighted, and washed. Part of the liver was snap-frozen in liquid nitrogen and stored at −80 °C. Other liver samples were cut into small pieces and fixed in formaldehyde at 4 °C for later use. Animal experiments were performed in strict accordance with the recommendations in the Guidelines for the Care and Use of Animal Experiments of the Science Council of Youjiang Medical University for Nationalities. All manipulations were carried out under tribromoethanol anesthesia to avoid pain and suffering.
Histological AnalysisThe right lobe of the hepatic tissue was fixed in formaldehyde and was then embedded in paraffin, sectioned, and stained with Hematoxylin and Eosin (H&E). The frozen liver tissue was sectioned into 5 µm portions and stained with Oil Red-O stain in order to analyze the accumulation of fat droplets in the liver. Subsequently, the liver tissue section slides were observed under an optical microscope (Olympus, Tokyo, Japan).
Analysis of Serum BiochemistryThe total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the serum were measured according to the manuals using kits procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Liver Lipidomic AnalysisSample Pretreatment and Lipid ExtractionThe methyl tert-butyl ether (MTBE) method was used to extract liver tissue lipids. Briefly, tissues were homogenized with 200 µL of water and 240 µL of methanol. The mixture was added to 800 µL of MTBE and subjected to ultrasound for 20 min at 4 °C. Samples were then left to sit at room temperature for 30 min. Then, the mixture was centrifuged at 14000 × g for 15 min at 10 °C. The upper layer was separated and then dried under nitrogen.
Instruments for Lipid AnalysisThe lipid extracts were then dissolved in 200 µL of a solution of isopropanol: acetonitrile at a ratio of 9 : 1, v/v, and then centrifuged at 14000 × g for 15 min. The CSH C18 column (1.7 µm, 2.1 × 100 mm, Waters, MA, U.S.A.) was used to facilitate LC separation. Solvent A was a mixture of liquid acetonitrile and water (6 : 4, v/v) with formic acid 0.1%, and ammonium formate 0.1 Mm. Solvent B was a mixture of acetonitrile and isopropanol (1 : 9, v/v) with formic acid 0.1%, and ammonium formate 0.1 Mm. Linear gradient elution was performed as follows: 0–2 min, 30% solvent B, 300 µL/min; 2–23 min, 30–100% solvent B; 23–30 min, 5% solvent B.
After UHPLC separation, a Q-Exactive Plus in positive and negative mode was used to perform mass spectrometry analysis with the following parameters: source temperature, 300 °C; capillary temp, 350 °C; ion spray voltage, 3000 V; S-Lens level, 50%; and scan range, 200–1800 m/z.
Based on MS/MS math, lipid species were searched in the “Lipid Search.” Bohr mass tolerance for precursor and fragment was set to 5 ppm.
Transmission Electron MicroscopyAfter samples were fixed in glutaraldehyde, the liver tissue was then re-fixed, dehydrated, infiltrated, embedded, sectioned, and stained. Images were then collected using a transmission electron microscope (HT7700, HITACHI, Tokyo, Japan).
Protein Extraction and Western Blot AnalysisProteins were extracted from liver tissues by ultrasonic lysis buffer containing 1 mM Phenylmethanesulfonyl fluoride (PMSF) and 1% phosphatase inhibitor cocktail, on ice. Lysate was layered by centrifugation at 12000 × g for 15 min at 4 °C. The supernatant, which contained proteins, was separated. The proteins were assessed with a bicinchoninic acid (BCA) protein quantitative kit. Next, 50 µg aliquots of protein lysates were subjected to electrophoresis on 10 or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in order to detect mitochondrial fission factor (MFF), mitochondrial fusion protein-1 (MFN1), Parkin, PINK1, SQSTM1/p62 (p62), Beclin-1, light chain 3B (LC-3B), GRP78, peroxisome proliferator-activated receptor-γ (PPAR-γ), DDIT3, ATF4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The gels were transferred onto 0.45 or 0.25 µm nitrocellulose (NC) membranes. After blocking, the membranes were incubated with monoclonal antibody (MFF, MFN1, Parkin, PINK1, GRP78, DDIT3, ATF4, PPAR-γ, GAPDH, affinity, product #DF12006; #DF7543; #AF 0235; #DF7742; product #AF5366; #DF6025; #DF6008; #AF6284; #T0004, and p62, LC-3B, Beclin-1, abcam, product #ab109012; #ab192890; #ab62557) at 4 °C overnight. Following washing, secondary antibodies IRDye 800RD Goat anti-rabbit immunoglobulin G (IgG) (LI-COR, product #925-32211) and IRDye 680RD Goat anti-mouse IgG (LI-COR, product #925-68070) were used to incubate the membranes. The bands were visualized by a two-color IR laser imaging system (LI-COR, Lincoln, NE, U.S.A.). The expression of related proteins was determined using ImageJ software (v18.0, NIH, Bethesda, MD, U.S.A.) in order to semi-quantitatively analyze the data.
Statistical AnalysisAll data are represented as mean and standard deviation (S.D.). Statistical differences between groups were determined by one-way ANOVA. p-Values less than 0.05 (p < 0.05) were considered significant.
In order to gain insights into the effects on liver tissue histological changes, the external and internal states of the liver were observed by HE and Oil Red-O staining. The results showed that HFD intervention caused the liver to enlarge and turn yellow (Fig. 1A), and numerous vacuoles and oil droplets appeared in the internal cells (Figs. 1B, C). These changes were barely observed in the liver of CON mice, whereas the liver of VIX mice fluctuated between both states.
(A) representation of the external features of the liver. (B) H&E stained liver of mice. ×100 (C) Oil Red O-stained liver. ×100. (D) Liver weight. (E) Liver index (liver/ body weight ratio). (F) Serum TC concentration. (G) Serum TG concentration. (H) Serum ALT concentration. (I) Serum AST concentration. Data are presented as mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01 vs. normal diet (CON), # p < 0.05 vs. high fat diet (HFD).
In addition, the liver weight and liver index (liver weight/body weight) were obviously increased in the HFD mice compared with the CON mice (p < 0.01, Fig. 1D). The VIX treatment significantly ameliorated the adverse changes compared with the HFD treatment. Hepatic TC, TG, ALT, and AST contents were significantly stimulated in the HFD mice compared to the CON mice (p < 0.01). The VIX treatment reduced the TC, TG, and ALT contents (Figs. 1F–I). In addition, these results indicated that the NAFLD mice model was successfully established in this study.
VIX Treatment Modulates Lipid Species Profiles in LiverLipid analyses were conducted in order to reveal lipid species changes in the liver tissue. The ion peaks of all of the samples were analyzed by PCA, which showed that all of the samples and quality control (QC) samples were closely clustered together, indicating that the experiment had good repeatability (Fig. 2A). Lipid search was used to analyze the data obtained in the positive and negative ion modes. The analysis results identified 34 lipid classes and 1298 lipid species (Fig. 2B).
Seven main lipid classes were analyzed. (A) PCA score plot of all samples and QC samples. (B) Number of the identified lipid number. (C) Hierarchical clustering analysis of based on significant different in lipids. Alterations in lipidomic profiles in the VIX of mice in each group. (D) Glycerolipids, (E) Fatty acyls, (F) Pernol lipids, (G) Serol lipids, (H) Glycerophospholipids, (I) Saccharolipids, (J) Sphingolipids. TG, triglyceride; DG, diglyceride; WE, wax esters; AcCa, acylcarnitine; Co, coenzyme; ChE, cholesterol ester; PI, phosphatidylinositol; PG, phosphatidylglycerol; PA, phosphatidic acid; LPI, lysophosphatidylinositol; LPS, lysophatidylserine; SQDG, sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; SM, sphingomyelin; CerG1, simple glc series 1; CerG2, simple glc series 2; CerP: ceramides phosphate; GM2: gangliosides 2; phSM, phyto sphingosine; GM3, gangliosides 3; GD2, gangliosides 2. * p < 0.05 vs. CON; ** p < 0.01 vs. CON; # p < 0.05 vs. HFD; ## p < 0.01 vs. HFD.
In order to comprehensively and intuitively demonstrate the relationship between samples and the differences in lipid expression patterns in different samples, the expression level of qualitatively significant difference lipids (VIP >1, p < 0.05) was used to perform hierarchical clustering of samples in each group (Fig. 2C).
The results of lipidomic analyses showed that there were significant differences in the seven lipid ion classifications: glycerolipids, fatty acyls, pernol lipids, serol lipids, glycerophospholipids, saccharolipids, and sphingolipids. These lipids were observed in the liver tissues of all three groups. The HFD treatment increased the content of TG, diglyceride (DG), wax exters (WE), acyl carnitine (AcCa), coenzyme (Co), cholesterol ester (ChE), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), sphingomyelin (SM), simple glc series 2 (CerG2), ceramides phosphate (CerP), and gangliosides 3 (GM3). The VIX treatment effectively ameliorated these effects. Furthermore, compared with the CON treatment, the VIX treatment significantly ameliorated the reduction of the levels of lysophosphatidylinositol (LPI), lysophosphatidylserine (LPS), digalactosyldiacylglycerol (DGDG), simple glc series 1 (CerG1), gangliosides 2 (GM2), and phyto sphingosine (phSM) induced by HFD stimulation, but it was unable to address the reductions in phosphatidic acid (PA), sulfoquinovosyldiacylglycerol (SQDG), and gangliosides 2 (GD2).
VIX Treatment Reverses HFD-Induced Subcellular Organelle Damage in the LiverLipid metabolism is mainly regulated by the liver, and subcellular organelles are the main sites of metabolism. Therefore, transmission electron microscopy was performed to evaluate the changes in cell structure and subcellular organelles.
Imaging showed that most of the electron density inside the CON cell cytoplasm was rich, whereas the local low electron density observed was edema. Mitochondria were abundant. The rough ER (RER) had no obvious expansion and was distributed around the mitochondria. In addition, autophagolysosomes were identified in the liver of CON mice.
In the HFD mice, swollen mitochondria were found in the liver. Mitochondrial membrane breakage led to matrix extravasation. The inner membrane matrix evolved to facilitate the fracture and solution of cristae. There was evidence of RER expansion, degranulation, and vacuoles. Meanwhile, lipid droplets were present in large quantities. Small numbers of lipid droplets fused to form large lipid droplets.
Compared with the HFD mice, the state of liver cells improved in the VIX mice. The structure of the mitochondrial membrane was complete, and few mitochondrial cristae were fragmented. Mitochondrial autophagy occurred following mitochondrial matrix extravasation. The RER was intact with no vacuolation, and partially wrapped the mitochondria. Three autophagolysosomes were observed. A few lipid droplets were found to be dispersed throughout the cytoplasm, but none aggregated into larger lipid droplets (Fig. 3).
The hepatocyte subcellular organelles in each group of mice were examined by electron microscopic analysis.
To further investigate the effect of VIX on mitochondrial dynamics and autophagy, the related protein level was detected using Western blotting (Fig. 4A). The HFD treatment was found to significantly reduce MFF expression while increasing MFN1 expression. The mitochondrial autophagy proteins, Parkin and PINK1, were significantly decreased in HFD mice compared to CON mice (Fig. 4A). The administration of VIX treatment alleviated the effects of HFD treatment on the MFF, MFN1, Parkin, and PINK1 proteins. Compared with the HFD treatment, the VIX treatment also inhibited p62 expression and stimulated Beclin-1 and LC-3B expression (Fig. 4A).
(A) Expression of MFF, MFN1, Parkin, PINK1, p62, Beclin-1 and LC-3B. (B) Expression of GRP78, DDIT3, ATF4 and PPRR-γ. Values are expressed as the mean ± S.D. * p < 0.05 vs. CON; # p < 0.05 vs. HFD.
The expression levels of ER chaperone and UPR regulators GRP78, DDIT3, and ATF4 were evaluated in the VIX mice. VIX treatment down-regulated GRP78, DDIT3, and ATF4 expression, which were stimulated by HFD. PPAR-γ was inhibited by the HFD treatment, which was ameliorated by the VIX treatment (Fig. 4B).
Previous studies have reported that vitexin has a protective effect on HFD-induced liver injury.1,10) However, the potential effects of vitexin on ER stress and autophagy associated with hepatic steatosis remain unknown. In the present study, it was found that the regulatory mechanisms of vitexin attenuate liver steatosis in NAFLD mice induced by HFD. The results also suggest that vitexin exerts beneficial effects by promoting autophagy and regulating ER stress.
In this study, the effect of vitexin was obtained by liver tissue structure detection, biochemical indices, and lipidomic analysis. In HFD mice, the liver tissue structure and function and lipid metabolism were improved significantly after vitexin treatment.
Lipid class composition glycerophospholipids and sphingolipids are key components of cellular membranes and are capable of bidirectional homeostatic crosstalk with each other. The dysregulation of both of these often leads to lipotoxicity and induction of metabolic stress. Glycerophospholipids play roles as signaling molecules and as fixators for proteins in cell membranes.11) Moreover, it has been reported that sphingolipids are responsible for mediating recognized toxic effects such as mitochondrial stress, ER stress, and apoptosis.12) In this study, after analyzing the lipidics data, it was speculated that vitexin protected cellular membranes and regulated mitochondrial and ER structure, associating these two lipids’ metabolism.
As shown in Fig. 3, the mitochondria and ER structure of HFD mice liver experienced significant adverse changes, which were effectively alleviated by vitexin. Moreover, concomitant intracellular lipid accumulation, abnormal expression of mitochondrial regulatory proteins, and ER stress characteristic proteins have occurred with corresponding changes.
In this study, the Western blotting results showed that MFN1 increased in the hepatocytes of HFD mice, which may contribute to mitochondria binding to lipid droplets and forming peri droplet mitochondria (PDM). This result is consistent with the literature.13) The PDM and increase of MFN1 were not observed in the CON and VIX mice. Although PDM expands lipid droplets to protect against overnutrition induced lipotoxic liver injury,13) overaccumulation of lipid droplets in the liver leads to a chronic inflammatory state and cirrhosis. However, it was shown in the present study that the protein expression of MFF decreased in HFD mice. According to a comprehensive analysis, the mitochondrial system biased towards fusion over fission, leading to the presence of a small number of large mitochondria. In addition, this phenomenon was observed in the mitochondrial ultrastructure.
Autophagy, which refers to apoptosis and cell aging, is a very important biological phenomenon that removes cytoplasmic proteins or organelles to maintain intracellular homeostasis. It is well noted that autophagy is a double-edged sword. That is, autophagy has dual functions in that it protects cells from a starvation state but causes cell death when excessive autophagy occurs.14) The important mechanism of autophagy is autophagosome-lysosome fusion.15) Although the complete autophagy structure was not observed in the liver ultrastructure, autophagy marker proteins (p62, Beclin-1, and LC3B) were detected in the CON and VIX mice. The lysosome structures were not observed in the HFD mice. This phenomenon is consistent with the literature reporting impaired lysosomal pathways participated in the pathology of NAFLD.16)
Mitochondrial autophagy (mitophagy) is deemed as a cellular adaptive stress response that occurs by purging dysfunctional and damaged mitochondria to maintain mitochondrial homeostasis.10) The PINK1-Parkin axis is the core mechanism of mitophagy.17) The over-expression of PINK1-Parkin activates mitophagy, thereby controlling mitochondrial quality.18) The VIX treatment rescued the PINK1 expression, which was inhibited by HFD intervention.
Autophagy is also an important mechanism of lipid catabolism, by which lipid droplets are degraded via lipophagy. In overnutrition induced NAFLD, lipophagy are significantly decreased, along with lipid droplet accumulation.19) Because the lysosome is an essential structure of autophagy, lysosome deficiency accounts for the decreased levels of lipid components of autolysosomes. This inhibits the progression of lipid degradation and accumulation, resulting in lipotoxicity and apoptosis.20) In this study, vitexin activated lipophagy, which degraded lipid droplets and thus protected hepatocytes by avoiding excessive lipid accumulation.
The PPAR-γ protein, which is a ligand activated nuclear hormone receptor of the RRAR family,21) was also considered in this study. The findings were consistent with previous reports that the PPAR-γ protein level was reduced in the liver of HFD fed-mice22,23) and the advanced glycationed product-treated HepG2 cells.22) In addition, Zhong et al. reported that the stimulation of PPAR-γ ameliorated hepatic steatosis though inhibiting inflammation and activating autophagy.22,24) Elsewhere, up-regulating PPAR-γ has been reported to have a beneficial effect on hepatocytes steatosis by cutting down on free fatty acids (FFA) deposition in the liver, along with increasing the insulin sensitivity of adipose tissue.21,25) In the present study, the VIX treatment ameliorated the decrease of the protein level of PPAR-γ in the HFD disturbed liver, inducing the activation of autophagy and reduction of lipid accumulation. This may be one of the mechanisms underlying the liver protection mechanism of vitexin.
ER stress is an important pathogenic factor of metabolic diseases, such as fatty liver disease and diabetes. Targeting ER stress is an effective means to alleviate some metabolic diseases. Sphingolipids are regarded as the center of ER stress. The levels, localization, and species of sphingolipids all affect ER stress.26) The accumulation of sphingolipid intermediates affects mitochondria by altering mitochondrial membrane potentials, increasing mitochondrial reactive oxygen species (ROS), reducing mitochondrial mass, and initiating mitochondrial apoptosis.19,20,27,28)
Prolonged ER stress results in the increase of markers, including ATF-4, which increases the transcription of pro-apoptotic proteins, as well as CHOP-10 (C/EPBα-homologous protein-10, DDIT-3) and GRP78. Soliman et al.21) demonstrated that PPAR-γ activation reduced ER stress, which in turn reduced oxidative stress. Park et al.29) found that PPAR-γ agonists reduced ER stress to act directly on islet function and insulin resistance in diabetes. The intervention of vitexin reduces the ER stress marker proteins GRP78, DDIT, and ATF4 to normal levels. Hence, the ER stress inhibition effect of vitexin may be associated with the activation of PPAR-γ.
Vitexin has been reported to protect the liver from NAFLD and ethanol-induced liver damage.8–10) The protective mechanisms of vitexin against NAFLD are the inhibition of inflammation and the activation of AMPK. However, the important mechanisms of lipid metabolism, ER reaction, autophagy, and mitochondrial biology have not been studied in relation to vitexin. This study confirmed that the roles of vitexin in the treatment of NAFLD are reducing ER stress, increasing autophagy, and regulating mitochondrial biology (Fig. 5). In this study, HFD mice were treated a single dose of vitexin based on the published literature.10) In the future, multiple dose groups and in vivo experiments are necessary to provide stronger evidence for vitexin’s effects and mechanisms.
Mitochondria (Mit), Swelling mitochondria (SM), Mitochondrial fission (M-fi), Mitochondrial fusion (M-fu), Auto phagolysosome (ASS), Lipid droplets (LD), Swelling endoplasmic reticulum (Ser), mitochondrial autophagy (MA), Lipid autophagy (LA), rough endoplasmic reticulum (RER).
This study provides important evidence that vitexin protects the liver from hepatic steatosis induced by HFD intervention. The molecular mechanism underlying the regulation of liver lipid steatosis by vitexin may be the activation of PPAR-γ. This finding provides new insights about the lipid-lowering mechanisms of vitexin, which can be beneficial for the prevention and treatment of NAFLD.
This study was supported by Special Funding for Guangxi Special Experts (No. GRCT[2019]13), Guangxi Medical High-level Leading Talents Training “139” Project (No. GWKJ[2018]22) Guangxi Clinical Medical Research Center of hepatobiliary Diseases (No. AD17129025) and Guangxi Natural Science Foundation Project (No. 2020JJB140078).
The authors declare no conflict of interest.
