Polyphenol-Rich Loquat Fruit Extract Prevents Fructose-Induced

Article Cite This: J. Agric. Food Chem. 2019, 67, 7726−7737

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Polyphenol-Rich Loquat Fruit Extract Prevents Fructose-Induced Nonalcoholic Fatty Liver Disease by Modulating Glycometabolism, Lipometabolism, Oxidative Stress, Inflammation, Intestinal Barrier, and Gut Microbiota in Mice Wenfeng Li,† Hongyan Yang,*,‡ Qiang Zhao,*,§,⊥ Xv Wang,† Jing Zhang,† and Xin Zhao¶ †

School of Life Science and Biotechnology, Yangtze Normal University, Chongqing 408100, China School of Aerospace Medicine, Fourth Military Medical University, Xi’an 710032, China § Department of Cardiology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, China ⊥ Xinjiang Key Laboratory of Cardiovascular Disease Research, Urumqi, China ¶ Chongqing Collaborative Innovation Center for Functional Food, Chongqing University of Education, Chongqing 400067, China

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‡

S Supporting Information *

ABSTRACT: Fructose as a daily sweetener is widely recognized as a risk catalyst for nonalcoholic fatty liver disease (NAFLD). The aim of current study is to evaluate the effects and molecular mechanism by which polyphenol-rich loquat fruit extract (LFP) prevents NAFLD in mice fed 30% fructose water (HF) for 8 weeks. Administration of LFP to HF-fed mice mitigated abnormal body weight, disordered lipid metabolism, oxidative stress, and inflammation through a mechanism regulated by the AKT, ChREBP/SREBP-1c, Nrf2, and TLR4/MyD88/TRIF pathways. LFP caused a significant decrease in the endotoxin content (16.67−12.7 EU/mL) in the liver of HF-fed mice. LFP not only improved HF-induced breakage of the intestinal barrier via interacting with tight junction proteins (ZO-1, occludin), mucin, and immunoreaction in the colon but also maintained normal colonic Firmicutes/Bacteroidetes ratios and the relative abundance of Veillonella in HF-fed mice. Our results suggest that LFP may serve as a nutritional agent for protecting liver in HF-fed mice. KEYWORDS: loquat fruit, polyphenol, fructose, nonalcoholic fatty liver disease, gut microbiota

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INTRODUCTION Dietary fructose, a monosaccharide, is commonly added to foods as a sweetener, both in personal consumption and in the food industry.1 It is noteworthy that global changes in dietary habits have caused the distinct elevation of fructose consumption over the past 200 years, specifically, the wide application of high-fructose corn syrup (HFCS), which comprises 55−90% fructose.1,2 Generally, production of HFCS requires the following four manufacturing steps: (1) extracting starch from corn by wet milling; (2) saccharification and liquefaction to hydrolyze polymer starch to monomer dextrose; (3) isomerization to convert dextrose to fructose; (4) fractionation to enrich the concentration of fructose in the isomerization product stream.3 Epidemiological investigations have indicated a strong link between the intake of a high-fructose diet (HF) and nonalcoholic fatty liver disease (NAFLD), hepatic de novo lipogenesis, obesity, type II diabetes, and cardiovascular disease.4−6 NAFLD is defined as steatosis affecting >5% of hepatocytes in the absence of excessive significant alcohol consumption, other liver disease, or the consumption of steatogenic drugs.7 Previous studies have documented that the genesis of HF-induced NAFLD is associated with the disorder of lipid metabolism, imbalance of the oxidant/antioxidant system, and inflammation in the liver.8,9 These risk factors are caused by the primary metabolites and byproducts of fructose, including glucose, lactate, free fatty acid, very low-density lipoprotein (VLDL), © 2019 American Chemical Society

triglyceride, uric acid, and methylglyoxal, metabolizing in the liver and extrahepatically.10 Recently, increasing evidence has also suggested that HF-induced NAFLD is linked to microecological disturbance in the intestine that damages the gut barrier and increases endotoxin production.11,12 Today, 15% of the total calories in the modern adolescent diet is composed of refined sugar (including fructose), while 10% of the population derives at least 25% of their total calories from refined sugar.6 Therefore, an effective strategy to prevent liver injury due to HF dietary ingestion is urgently required. Interestingly, polyphenolrich extracts from fruits or vegetables have shown extraordinary abilities to protect against chronic liver injury.13 Loquat fruit (Eriobotrya japonica (Thunb.) Lindl.) is a delicious fruit native to the southeast of China.14,15 In China, loquat fruit is not only consumed as food but is also a traditional Chinese medicinal material with anti-inflammation, antioxidant (against DPPH· and ABTS+, etc. in vitro), anticancer, antihyperlipidemia, antidiabetic, and gastro-protection activities.16,17 These health benefits of loquat fruit might be attributed to its polyphenol compounds18,19 including phenolic acids, flavonoids, lignans, stilbenes, and tannins.20,21 Polyphenols with strong antihyperlipidemia, antihyperglycemia and Received: Revised: Accepted: Published: 7726

April 24, 2019 June 14, 2019 June 16, 2019 June 16, 2019 DOI: 10.1021/acs.jafc.9b02523 J. Agric. Food Chem. 2019, 67, 7726−7737

Article

Journal of Agricultural and Food Chemistry

saline once daily; high-fructose group, the mice received 30% fructose water and were administered i.g. with 0.4 mL of normal saline once daily; HF+LLFP group, the low-dose LFP-treated mice received 30% fructose water and were administered LFP at 25 mg/kg·bw (i.g. 0.4 mL) once daily; HF+HLFP group, the mice received 30% fructose water and were administered LFP at 50 mg/kg·bw (i.g. 0.4 mL) once daily. All treatments were performed for 8 consecutive weeks. The mice in this study were allowed free access to the standard chow diet and corresponding water during the treatment period. Distilled water and 30% fructose water were renewed at 9:00 am daily, and the body weight of the mice was weighed once a week. After the last administration, all the mice were fasted but given distilled water for 12 h. Subsequently, the mice were anesthetized in chambers filled with isoflurane (gaseity) and then sacrificed to obtain blood, liver, colon, and epididymal fat plate. Serum and Hepatic Biochemical Assessments. Serum was obtained by the centrifugation of blood at 1500 rpm for 15 min and was stored at −80 °C until further analysis. Serum glutamic-pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) levels were measured using commercial kits, according to the manufacturers’ instructions. Liver tissue (0.3 g) was homogenized in 2.7 mL of cold normal saline (0.9%) with a homogenizer and then was centrifuged at 1500 rpm for 15 min. The supernatant was collected for the determination of the endotoxin malonaldehyde (MDA) and glutathione (GSH) levels as well as total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities. Western Blot Analysis. The liver or colon specimen (100 mg) was homogenized in 1 mL of lysis buffer. After 10 min, the lysate was centrifuged at 15 000 rad/min for 10 min and the supernatant was collected. The protein concentration of the lysates was determined using a commercial kit (BestBio, Shanghai, China). Equal amounts of lysate proteins were separated by SDS-PAGE and were transferred to a PVDF membrane. After blocking nonspecific binding sites with 5% defatted milk in Tris-buffered saline at 25 °C for 1 h, the transferred membrane was incubated with antibodies against AKT, p-AKT, JNK, pJNK, IκBα, MyD 88, TRIF, ZO-1, and occludin at 4 °C for 12 h. The membrane was then washed with PBST and incubated with horseradish peroxidase-conjugated goat antimouse or rabbit secondary antibody for 1 h at 37 °C. The immunoreactive bands were visualized using the ChemiDocXRS System (Bio-Rad Laboratory, Hercules, CA, USA), and the blot densities were analyzed using Quantity One (Bio-Rad Laboratory, Hercules, CA, USA). Quantitative Real-Time PCR. The real-time polymerase chain reaction (RT-PCR) assay was performed as described previously.26 The total RNA of the liver or colon was isolated using TRIzol (Invitrogen, USA), and cDNA synthesized from 1 μg of RNA using the reverse transcription reagent kit (TaKaRa, China). The mRNA expression was performed using a PCR detection kit (TaKaRa) and RT-PCR Detection Systems (Bio-Rad Laboratories, Shanghai, China). Relative mRNA expression was measured by relative quantification with β-actin as the internal reference. 16S rRNA Gene Amplification and MiSeq Sequencing. Intestinal microorganisms were analyzed using pyrosequencing targeting the 16S rRNA V3−V4 regions (515F, 5′-GTGYCAGCMGCCGCGGTAA-3′; 806R, 5′-GGACTACHVGGGTWTCTAAT3′). Total bacterial DNA in the colonic luminal content was extracted using the E.Z.N.A. Mag-Bind Soil DNA Kit according to the manufacturer’s instructions. The DNA was confirmed using 0.8% agarose gel electrophoresis. The PCR reaction included 10 ng of template, 1× PCR buffer, 1.5 mmol/L of MgCl2, 0.4 μmol/L of dNTPs, 1.0 μmol/L of primer and 0.5 U of KOD-Plus-Neo enzyme. PCR was carried out under the following conditions: 94 °C for 1 min, followed by 30 cycles of 94 °C for 20 s, 54 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The PCR products were tested by 2% agarose gel electrophoresis. Amplicons were purified using the QIAquick Gel Extraction Kit (QIAGEN). After quantitative analysis (Qubit@ 2.0 Fluorometer, Thermo Scientific), the purified gene from the same sample was mixed

antioxidant capabilities were found in the loquat fruit or leaf, including chlorogenic acid, neochlorogenic acid, rutin, naringin and quercetin glucosides.15−17 In this regard, the preventive effects of loquat fruit polyphenols on HF-induced NAFLD are clear, although the molecular mechanisms involved require further investigation. Wang and colleagues suggested that phenolic compounds might affect the gut microbial composition to improve obesity, hepatic fat deposition, insulin resistance, oxidative stress and intestinal inflammation in mice.22,23 Whether the polyphenols of loquat fruit is linked to the alleviation of HF-induced NAFLD through the regulating of the gut microbiota and intestinal barrier has not been clarified. Therefore, the aim of this work was not only to investigate the mechanism via the classical pathway but also to study the role of intestinal barriers and gut microbiota, in which polyphenol-rich loquat fruit extract (LFP) protects mice against HF-induced NAFLD. First, the effects of LFP on glycometabolism, lipid metabolism, oxidative stress, and inflammation in HF-fed mice were evaluated by biochemical, enzyme-linked immunosorbent assay (ELISA), real-time qRT-PCR, and Western blot analyses. Subsequently, we assessed whether LFP-prevented NAFLD was related to the modulation of the intestinal barrier and gut microbial composition in HF-fed mice.

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MATERIALS AND METHODS

Chemicals. The polyphenol extract of loquat fruit was prepared in our laboratory according to our previous method, and its polyphenolic composition was analyzed by UHPLC-QqQ-MS/MS.24 The main polyphenol compound in the extract of loquat fruit was chlorogenic acid (63.7%), followed by cryptochlorogenic acid (18.0%), oleanolic acid (5.1%), protocatechuic acid (3.9%), phloretin (3.3%), hesperidin (1.5%), and 27 additional polyphenols detected that accounted for less than 1.5% of total polyphenols detectable based on analysis of the extract (Figure S1). Food-grade crystallographic fructose (purity >98%) was purchased from the local market. C57BL/6J male mice and standard rodent chow25 were obtained from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, China). The serum and hepatic biochemical parameters were analyzed by commercial kits, and their information is shown in the Supporting Information. Monoclonal antibodies against protein kinase B (AKT), phosphorylated AKT (p-AKT), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), inhibitor of nuclear factor κ-B kinaseα (IκBα), myeloid differentiation factor 88 (MyD 88), and TIRdomain-containing adapter-inducing interferon-β (TRIF), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were obtained from Cell Signaling Technology, Inc. (Shanghai, China). Anti-TIRdomain-containing adapter-inducing interferon-β (TRIF), antizonula occludens-1 (ZO-1), and antioccludin were purchased from ABCAM (Shanghai, China). The primers used for real-time qRT-PCR were obtained from Sangon Biotech Co., Ltd. (Shanghai, China), and their sequences are presented in Table S1. All other chemicals used were of analytical grade. Animal Care and Experimental Protocols. All experimental protocols (SCXK20170002) were approved by the Committee on Care and Use of Laboratory Animals of the Fourth Military Medical University, China. These experiments were executed according to the Guidelines of Experimental Animal Administration published by the Publishing House of Science and Technology of Shanghai (Eighth Edition, ISBN-10:0−309−15396−4). C57BL/6J male mice (weighing approximately 20 ± 2 g) were housed in a controlled environment (12/12-h light-dark cycle at 22 °C ± 2 °C) with a standard chow diet and distilled water ad libitum during the adaptive phase. After 1 week of acclimatization on a normal-chow diet, the mice were randomly divided into four groups (n = 10) and received distilled water or 30% high-fructose water (120 kcal/100 mL): control group, this normal group of mice received daily distilled water and were administered intragastrically (i.g.) with 0.4 mL of normal 7727

DOI: 10.1021/acs.jafc.9b02523 J. Agric. Food Chem. 2019, 67, 7726−7737

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Figure 1. Administration of polyphenol-rich loquat fruit extract (LFP) prevents abnormal body weight gain and glycometabolic disorder in mice fed a high-fructose diet (HF). (A) Body weight gain; (B) body weight at the eighth week; (C) weight of the abdominal fat plate; (D) liver weight; (E) blood glucose concentration in the oral glucose tolerance test (OGTT); (F) area under the curve (AUC) of OGTT; (G) ratio of p-AKT (phosphorylated AKT) to AKT (protein kinase B); (H) protein abundance of AKT and p-AKT in the liver, with glyceraldehyde 3 phosphate dehydrogenase (GAPDH) applied as a loading control; (I) periodic acid Schiff (PAS)-stained liver. The data are expressed as the means ± SD (n = 8−10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, significant difference compared with the HF-fed mice. at a ratio of 1:1. The library was constructed using the TruSeq DNA PCR-Free Sample Prep Kit (Illumina Inc., Shanghai, China).

Sequencing was obtained by Hiseq 2500 (Rhonin Biosciences Co., Ltd., Chengdu, China). Using QIIME pipeline v1.9.1, the sequences 7728


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Figure 2. LFP inhibits lipid metabolism disorder in HF-fed mice. (A) Serum total cholesterol (TC); (B) serum total triglyceride (TG); (C) serum lowdensity lipoprotein cholesterol (LDL); (D) serum high-density lipoprotein cholesterol (HDL); (E) images of Oil red O-stained liver; (F) relative mRNA expression of carbohydrate-responsive element binding protein (ChREBP) and sterol regulatory element-binding proteins-1c (SREBP-1c). The data are expressed as the means ± SD (n = 8−10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, significant difference compared with the HF-fed mice. were further demultiplexed and analyzed. Operational taxonomic unit (OTU) clustering was performed at 97% sequence identity. The oral glucose tolerance test (OGTT) and histopathological observation of the liver and colon were described in the Supporting Information. Statistical Analysis. The results of the biochemical, ELISA, and RT-PCR analyses are expressed as the means ± SD. Significant differences between groups were assessed by the unpaired two-tailed t test.27 Differences among the four groups were analyzed using one-way ANOVA with Tukey’s multiple comparison tests.28 Statistical analysis was performed using GraphPad Prism 6.0 (La Jolla, CA, USA), and P < 0.05 was considered to indicate a significant difference. Plots were drawn using Prism and R (×64, 3.4.3).

treatment groups might be approximately the same. We next assessed whether the ingestion of LFP could improve the glucose response to an OGTT. After the fasting mice orally received a glucose solution (0.4 mL, 2 mg/mL), the peak value of the blood glucose concentration was shown at 30 min (Figure 1E). Areas under the glucose concentration curve (AUC) in HFfed mice were increased significantly compared with those in the control mice (Figure 1F). Nonetheless, a decrease in the AUC in mice treated with LFP at 25 and 50 mg/kg·bw was found (Figure 1F). To explore whether LFP improved insulin signaling, we analyzed the expression levels of AKT and its phosphorylation level in the liver of mice. As expected, feeding with HF inhibited AKT phosphorylation, whereas LFP treatment effectively enhanced the hepatic phosphorylation reaction of AKT in a dose-dependent manner (Figure 1G,H). PAS staining showed that the consumption of HF or HF + LFP did not influence the accumulation of glycogen in the liver of mice (Figure 1I). These results suggested that LFP has positive effects on alleviating HFinduced abnormalities of glycometabolism in mice by activating the AKT signaling pathway. LFP Maintains Lipid Homeostasis. We observed that HF ingestion for 8 continuous weeks in mice significantly induced increases in serum TC, TG, and LDL levels by 129%, 42%, and 14% (Figure 2A−C), respectively, whereas the serum HDL levels were reduced by 24% (Figure 2D), indicating that the long-term intake of HF disturbed lipid homeostasis. Interestingly, the treatments of low and high doses of LFP in HF-fed mice dose dependently inhibited the increases in the serum TC and TG levels (Figure 2A,B). Administration of LFP at high

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RESULTS LFP Prevents Abnormal Body Weight Gain and Glycometabolism. Daily oral administration of LFP at 25 and 50 mg/kg·bw continuously for 8 weeks prevented HFinduced abnormal gain of body weight (Figure 1A,B). Meanwhile, the ventral fat weight was slightly, but not significantly, decreased in 25 mg/kg·bw LFP-administered mice (Figure 1C), whereas HF-induced excessive gain of fat weight and liver weight were fully inhibited by LFP treatment at 50 mg/kg·bw (Figure 1C,D). Food intake of the control mice was observably greater than that in other mice, which consumed the 30% fructose water (Figure S2A). However, fructose-fed mice had higher water consumption (35.27 ± 1.20−35.93 ± 1.40 mL/day/group) than the control mice (32.31 ± 1.31 mL/ day/group), although no significant difference was found (Figure S2B). Therefore, energy intake among the four 7729


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Figure 3. LFP prevents oxidative stress in HF-fed mice. (A, B) Hepatic content of malondialdehyde (MDA) and glutathione content (GSH); (C, D) activities of glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) in the liver; (E) protein abundance of c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK) and nuclear factor like 2 (Nrf2) in the liver, with GAPDH applied as a loading control; (F) ratio of p-JNK to JNK; (G) fold change of Nrf2 protein expression. The data are expressed as the means ± SD (n = 8−10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001, significant difference compared with the HF-fed mice.

administration of LFP (Figure 3C, D). Subsequently, we used Western blotting to explore the effects of LFP on key regulatory proteins concerning oxidative stress. As described in Figure 3E, consumption of HF for eight continuous weeks obviously increased the phosphorylation level of JNK in the liver of mice. More importantly, LFP significantly decreased the HF-induced increase in the p-JNK/JNK ratio in the liver of mice (Figure 3F). We also found that HF inhibited the nuclear translocation of Nrf2 expression in the liver of mice, while LFP supplementation effectively induced Nrf2 nuclear translocation (Figure 3E,G). These results indicated that LFP prevents HF-induced hepatic oxidative stress by inhibiting lipid peroxidation and activating the Nrf2 pathway in mice. LFP Ameliorates Inflammation and Hepatocyte Injury. Because the deterioration of NAFLD is closely related to inflammation, we assessed systemic inflammation. The results of ELISA clearly demonstrated that, in mice fed HF, the MCP-1 and TNF-α levels were observably increased compared with those in control mice (Figure 4A,B). Interestingly, treatment with LFP at 50 mg/kg·bw significantly decreased the MCP-1 and TNF-α levels by 15% and 20%, respectively (Figure 4A,B). LFP also slightly reduced the serum IL-1 and IL-6 levels in HFfed mice (P > 0.05, Figure S2C,D). Notably, LFP administration effectively inhibited the HF-induced up-regulation of TLR4, MyD 88 and TRIF protein in the liver of mice (Figure 4C−F). Compared with HF-fed mice, LFP treatment elevated the expression level of PPARα mRNA and reduced the expression levels of PPARα and NF-κB mRNA in the liver of HF-fed mice

doses also effectively prevented the HF-induced elevation of serum LDL levels and reduction of serum HDL levels in HF-fed mice (Figure 2C,D). The images of Oil red O staining for liver sections presented that HF caused widespread fat accumulation inside hepatic cells (Figure 2E). Compared with the accumulation of fat droplets of mouse liver from HF-fed mice, the liver of LFP-treated mice distinctly alleviated the abnormal situation of fat deposition (Figure 2E). To further reveal the mechanism by which LFP inhibited HF-caused lipid homeostasis, we analyzed the key genes for lipogenesis.29 As shown in Figure 2F, the mRNA expression levels of hepatic sterol regulatory element-binding proteins-1c (SREBP-1c) and carbohydrate responsive element binding protein (ChREBP) were lower in the LFP-treated mice than in the HF-fed mice. These findings suggested that LFP mitigated lipid homeostasis by inhibiting hepatic lipogenesis in HF-fed mice. LFP Antagonizes Oxidative Stress. MDA, a landmark product of cell membrane lipid peroxidation,30 was overproduced in the liver of mice by feeding HF for 8 continuous weeks (Figure 3A). However, the elevation of the MDA level was markedly decreased by 31% and 53% in mice treated with LFP at 25 and 50 mg/kg·bw, respectively. Meanwhile, chronic supplementation of LFP caused a dose-dependent increase in the GSH content in the liver of HF-fed mice (Figure 3B). Next, we assessed hepatic GSH-Px and T-SOD activities, which were identified as markers of an inherent antioxidant defense system in vivo.31 The results presented that HF-reduced activities of GSH-Px and T-SOD were significantly elevated in mice by 7730


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Figure 4. LFP mitigates systemic inflammation and hepatocyte injury in mice fed HF. (A) Monocyte chemoattractant protein-1 (MCP-1); (B) tumor necrosis factor-α (TNF-α); (C−E) fold change of myeloid differentiation factor 88 (MyD 88), Toll-like receptor 4 (TLR4) and TIR-domaincontaining adapter-inducing interferon-β (TRIF) proteins in the liver; (F) protein abundance of TLR4, MyD 88 and TRIF in the liver, with GAPDH applied as a loading control; (G) relative mRNA expression of peroxisome proliferator-activated receptor α (PPARα), nuclear factor-κB (NF-κB), TLR4 and MyD88 in the liver; (H, I) activities of serum glutamic-pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT); (J) HEstained liver. The data are expressed as the means ± SD (n = 8−10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, significant difference compared with the HF-fed mice.

(Figure 4G). We also found that the IκBα level in the liver of HF-fed mice was enhanced by LFP supplementation; however, this influence showed no significant difference (Figure 4F, Figure S3). To reflect the protective effects of LFP on hepatocytes in HFfed mice, we assessed serum transaminase activities. As presented in Figure 4H, the serum GPT activity in HF-fed mice markedly increased to 2.52 ± 0.09 U/mL from 2.27 ± 0.07 U/mL in control mice. Although a slight elevation was found in the serum GOT activity following feeding with HF for 8 continuous weeks, there was no statistically significant difference relative to the control group (Figure 4I). Interestingly, the HFfed mice receiving the oral administration of LFP at 50 mg/kg· bw showed a significant decrease in serum GPT and GOT activities by 37% and 46%, respectively, compared with HF-fed mice (Figure 4H, I). Additionally, the serum GPT activity was significantly reduced in mice treated with LFP at 25 mg/kg·bw. To verify these results of biochemical analyses for hepatocyte damage and protection, histopathological observation of

hematoxylin and eosin (HE) staining of the liver was performed. As shown in Figure 4J, the liver tissue of HF-fed mice presented much vacuolation, fuzzy cell boundaries and severe cellular degeneration. Co-consumption of HF together with LFP prevented hepatic injury in mice, showing a near normal appearance with little cytoplasmic vacuolation, legible cell boundaries, a clear nucleolus and a visible nucleus. These results indicate that LFP, as an effective nutritional ingredient, prevented HF-induced NAFLD by inhibiting inflammation. LFP Strengthens the Intestinal Barrier. To reveal whether LFP-prevented HF-induced NAFLD is associated with breakage of the intestinal barrier, we measured the hepatic endotoxin level, which could translocate into the blood recycle with a leaky gut. The hepatic endotoxin content was significantly increased by 28% following HF feeding for 8 continuous weeks (Figure 5A). Conversely, the endotoxin level in the liver of LFPtreated mice decreased to 12.70 ± 0.64 EU/mL from 16.67 ± 0.86 EU/mL of HF-fed mice. Next, we found that HF feeding down-regulated the expression levels of ZO-1 and occludin 7731


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Figure 5. LFP consolidates the intestinal barrier in HF-fed mice. (A) Hepatic endotoxin; (B, C) fold change of zonula occludens-1 (ZO-1) and occludin proteins in the colon; (D) protein abundance of ZO-1 and occludin in the colon, with GAPDH applied as a loading control; (E) relative mRNA expression of ZO-1 and occludin; (F) relative mRNA expression of TLR4, TLR5 and NF-κB in the colon; (G) relative mRNA expression of mucin-2 (MUC-2) and MUC-4 in the colon; (H) crypt area in the colon; (I) area of Alcian blue (AB)-stained and high-iron diamine (HID)-stained (AB+/HID+) cells in the colon; (J) area of AB-stained cells that was negative for HID staining in the colon; (K) images from the AB- and HID-stained colon. The data are expressed as the means ± SD (n = 8−10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001, significant difference compared with the HF-fed mice.

LFP Regulates Composition of Gut Microbiota. In the present study, we investigated whether LFP protection of the liver against HF-caused injury was associated with the regulating effect on the composition of the gut microbiota. In 16S rRNA sequencing analysis, we discovered that 473 OTUs were picked in the colon content of all the mice in this study. The Venn diagrams showed 44.8% of OTU similarity among the four treatment groups, but there was 55.7% of the OTU difference between the control diet-fed and HF-fed mice (Figure 6A). We also found that only 13.2% and 9.4% of an OTU difference in the mice fed with LFP at 25 mg/kg·bw and 50 mg/kg·bw relative to that in the HF-fed mice, respectively, suggesting that LFP mainly changed the abundance of microorganisms than the microorganism species in the gut of mice (Figure 6A). The PCA score plot showed that mice clustered together according to the diets with a clear separation among the control, HF, and LFP administration, suggesting that HF and LFP have a firm effect on the gut microbial in the colon of mice (Figure 6B). The first component distinguished the HF group versus the control group and LFP-treated groups, while the second component divided

proteins, and their mRNA levels in the colon of mice (Figure 5B−E). However, this down-regulation was reversed by LFP consumption in HF-fed mice. We also found that consumption of LFP could inhibit HF-induced up-regulation of the mRNA expression of colonic TLR4, TLR5 and NF-κB in mice (Figure 5F). Notably, HF feeding for 8 weeks significantly downregulated the expression of mucin synthesis genes (MCU-2 and MUC-4) in the mouse colon (Figure 5G). Interestingly, the expression levels of MUC-2 and MUC-4 mRNA in the colon were increased by LFP treatment for HF-fed mice. Alcian blue staining and the histological score of the colon revealed that the long-term ingestion of HF caused noteworthy reduction in the crypt area, AB+/HIF+ cells and AB+ cells in the colon of mice (Figure 5H−K). As shown in Figure 5H, coconsumption of LFP combined with HF effectively prevented the crypt area in the colon. Furthermore, LFP treatment slightly increased the area of AB+/HIF+ cells and AB+ cells in the colon of HF-fed mice, with no significant difference (Figure 5I, J). These results indicated that LFP could maintain the intestinal barrier. 7732


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Figure 6. LFP regulates gut microbiota in HF-fed mice. (A) Venn diagram for OTUs; (B) score plot for principal component analysis of OTUs; (C) bacterial taxonomic analysis of intestinal microorganisms at the phylum level; (D) top 50 genera in abundance in gut microflora; (E) correlation analysis between intestinal barrier parameters and intestinal microorganisms concerning the top 50 genera; (F) ratio of Firmicutes to Bacteroidetes; (G, H) relative abundance of the potential key microbes in the colon. vs: versus.

the low-dose LFP-treated group versus high-dose LFP-treated group (Figure 6B), indicating that LFP supplement prevented HF-induced changes in the composition of intestinal flora. To investigate the effects of HF and LFP on the richness, evenness, and diversity of intestinal microflora in mice, the α-diversity index was calculated in this work. As displayed in Figure S4A,

feeding of HF caused an increase in the Chao-1 value, whereas this elevation was slightly decreased by LFP administration. A similar change was found in the value of Faith’s phylogenetic diversity. Particularly, the value of Shannon and Simpson indices indicated that LFP could effectively inhibit the excessive proliferation of gut microflora induced by the chronic 7733


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carboxylase 1, fatty acid synthase, citrate lyase) and glycolysis (glucokinase and L-pyruvate kinase).34 We found that LFP administration resulted in significant decreases in serum TC, TG, and LDL levels and regulated the hepatic SREBP-1c and ChREBP mRNA expression in HF-fed mice. Therefore, LFP inhibited the HF-induced “first hit” of NAFLD via regulating glycometabolism and the de novo lipogenesis pathway. Similarly, a previous study has shown that polyphenols from loquat leaves could prevent hyperlipidemia.16 The cause might be that extracts from loquat fruit or leaf involve abundant chlorogenic acid and its isomers, which possess antihyperglycemia and antihyperlipidemia activities.38,39 The second “hit” leading to NAFLD proposes that the formation of proinflammatory cytokines caused by oxidative stress related to reactive oxygen species (ROS) and bacterial endotoxin.33,34 Compared with glucose, fructose produces more than 100 times ROS,37 which can attack the unsaturated bonds of fatty acids and initiate lipid peroxidation to generate MDA.40 We found that LFP decreased the generation of hepatic MDA in mice fed HF, which suggested that LFP inhibited the formation of ROS. ROS can activate the stress-sensitive signaling pathways mediated by mitogen activated protein kinases (MAPK).41 Here, our work demonstrated that LFP inhibited HF-activated MAPK pathways, as evidenced by the reduced liver ratio of pJNK/JNK, which are important members of MAPK.42 Fang and Kwon reported that MAPK participated in cell survival against oxidative stress through the Nrf2 signaling pathway.43,44 ROStriggered activation of Nrf2 resulted in its translocation into the cell nucleus, thereby mediating the transcriptional induction of SOD and GSH-Px.43 Strikingly, administration of LFP elevated the nuclear Nrf2 level, GSH content, GSH-Px activity, and TSOD activity in the liver of HF-fed mice. These data demonstrated that LFP prevented HF-induced oxidative stress through regulating the MAPK/Nrf2 pathway. Furthermore, antioxidant enzymes were affected by PPARα,45 which could antagonize the NF-κB-controlled transcription of proinflammatory mediators and equilibrated inflammatory cytokine homeostasis.46 We observed that LFP increased PPARα mRNA expression and reduced NF-κB mRNA expression and proinflammatory cytokines levels (MCP-1 and TNF-α), indicating that LFP prevented HF-induced inflammation via inhibiting oxidative stress and the NF-κB pathway. It has been demonstrated that altered copper levels can promote the occurrence of oxidative stress as well as related dysfunctions, such as obesity, NAFLD and intima-media tickess.47 Interestingly, chlorogenic acid, which is the main polyphenol in LFP, could react with exogenous CuII ions resulting from weak or strong oxidative stress, thus promoting either normal cell survival or carcinoma cell death.48 Additionally, increasing evidence has extensively proposed that bacterial endotoxin is another trigger for oxidative stress and inflammation.49 Although low doses of fructose are cleared by the small intestine, superabundant fructose alters the gut microbiota composition and enhances the risk of exposure to endotoxin.28 Endotoxin could activate the innate immune system through pattern recognition receptors such as Toll-like receptors, particularly TLR4.27,50 Current research has shown that HF leads to a significant increase in the hepatic endotoxin content, as well as up-regulation of TLR4 protein expression in line with the mRNA levels in mice. TLR signaling could break the balance that MyD88 and TRIF interact with Beclin-1 and Bcl-2, a process that is crucial to induce autophagy and cell apoptosis.50 Interestingly, we not only found that LFP reduced the hepatic

consumption of HF. Therefore, a series of analyses was executed to reveal the critical intestinal microorganisms, which were suppressed by LFP in HF-fed mice (Figure 6C−H). We found that the long-term consumption of HF induced a significant decrease in the relative abundance (RA) of Bacteroidetes, but elevated the RA of Firmicutes, Proteobacteria, and Actinobacteria, in the colon of mice (Figure 6C). Notably, the RA of Bacteroidetes was higher in the colon of mice treated with LFP, and the proportions of Firmicutes, Proteobacteria, and Actinobacteria in the colon were both higher in these mice (Figure 6C). Moreover, while the Firmicutes/Bacteroidetes (F/B) ratio was elevated in the colon of HF-fed mice, feeding with LFP effectively inhibited this increase in the F/B ratio (Figure 6F). For the top 50 genera in abundance, HF consumption for 8 weeks significantly altered 41 genera in the colon of mice (Figure 6D). Among them, 32 genera were also influenced by LFP treatment in HF-fed mice. Detailed analysis of the 32 genera revealed by correlation analysis indicated that Ruminococcaceae UCG-014 (R. UCG-014), Lactobacillus, Ruminiclostridium 9 (R. 9), Lachnospiraceae UCG-008 (L. UCG-008), Aeromonas, [Eubacterium] f issicatena group ([E.] f. group), Anaerotruncus, Veillonella, Selenomonas 4, Ruminococcaceae NK4A214 group (R. NK4A214 group), Ruminococcaceae UCG-003, RB41, and Clostridium sensu stricto 1 were related to intestinal barrier function (Figure 6E). Subsequently, we performed a combining analysis of the Bonferroni posthoc test (P < 0.001, Figure 6D) and coefficient of variation (>50%, Figure S4B) to find that LFP significantly decreased the relative abundance of R. UCG-014, R. 9, L. UCG-008, [E.] f. group, Veillonella, and R. NK4A214 group against HF-induced excessive proliferation (Figure 6G,H).

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DISCUSSION Fructose, an important sweetener in daily dietary intake,32 is identified as one of critical risk factors in the development of NAFLD, which is closely related to glucose intolerance, liver steatosis, oxidative stress, inflammation, hepatocyte injury, and others.33 The “two-hit” progress is widely recognized as the pathogenic mechanism of NAFLD.33 The first “hit” includes abnormal blood glucose metabolism and triglyceride accumulation in hepatocytes.9,34 Our study showed that a crude extract from loquat fruit containing a rich mixture of chlorogenic acid, cryptochlorogenic acid, and other polyphenols (LFP) prevents abnormal weight gain and glucose tolerance induced by the long-term consumption of a high-fructose diet (HF) in mice. Although the food intake of the control mice was significantly higher (doubled the amount) than that of the HF-treated mice, the latter were able to obtain additional energy from fructose water. This difference in food intake did not, however, cause a significant difference in energy ingestion (495−521 kcal). This suggests that the decrease in the body weight of the HF-fed mice was due, not to reduced energy ingestion but rather to the contributing effect of the administration of LFP. Phosphorylation of AKT translocates intracellular vesicles containing glucose transporter-4 proteins to the plasma membrane that promotes saccharide utilization in the tissues.35 Current work has shown that LFP inhibited the HF-induced decrease in the pAKT/AKT ratio, suggesting that LFP could enhance the utilization of blood glucose by activating the PI3K/Akt/ GLUT-4 signaling pathway. However, excessive fructose cannot be utilized completely in vivo36 and therefore promotes the de novo lipogenesis by activating key transcription factors (SREBP1c, ChREBP).32,37 SREBP-1c and ChREBP can increase the expression of key enzymes involved in lipogenesis (acetyl-CoA 7734


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produce endotoxin and caused immunoreaction.55 It is widely recognized that bacterial endotoxin is mainly generated by Gram-negative bacteria.28 Interestingly, Ruminiclostridium commonly showed Gram-negative staining.56 Accordingly, the LFP-inhibited excessive proliferation of Veillonella and R. 9 might reduce the production of endotoxin in HF-fed mice. An increased abundance of Ruminococcaceae characterizes dysbiosis in patients with inflammatory bowel syndrome.57 Similarly, Konturek and colleagues observed that rats with colitis and exposed to stress showed an increased abundance of Lachnospiraceae,58 including L. UCG-008 and [E.] f. group. However, some studies have suggested that R. UCG-014 is beneficial to health by the production of butyrate,59 and its RA was decreased in rats with hypertriglyceridemia-related acute necrotizing pancreatitis.60 Additionally, Lachnospiraceae was suggested as a protective gut commensal strain to prevent intestinal barrier dysfunction.61 This might suggest that the role of R. UCG-014, L. UCG-008, [E.] f. group, and R. NK4A214 group was vague, even contradictory, regarding LFP’s action against an HF-induced leaky gut. Although probiotics are usually considered secure and beneficial to health, some clinical trials and experimental models have shown that superabundant probiotics might cause systemic infections, deleterious metabolic activities, excessive immune stimulation, and other side effects.62 In this regard, we speculated that LFP-inhibited excessive proliferation of intestinal bacteria contributes to protect the damaging effects of dietary exposure to high fructose sweeteners added to foods. That an extract of the loquat fruit might prevent the onset and progression of fructose-induced NAFLD warrants further research. In conclusion, the current study provides firm evidence that extracts enriched in polyphenols from loquat fruit exerted a beneficial effect against fructose-induced NAFLD in mice. Concerning the potential mechanism, we suggested that polyphenols of loquat fruit regulate blood glucose and lipid metabolism, inhibit oxidative stress, and modulate inflammation to play pivotal roles in preventing hepatic injury. Furthermore, this is the first study to indicate that this protective effect of loquat fruit polyphenols was associated with decreased bacterial endotoxin, enhancing the intestinal barrier and inhibiting the excessive proliferation of Veillonella. These findings suggest that the consumption of loquat fruit polyphenols might be an effective strategy to protect the liver and provides useful information for polyphenol prevention of fructose-caused NAFLD.

protein and mRNA levels of TLR4, MyD88 and TRIF but also showed that LFP distinctly decreased the serum activities of liver injury markers (GPT and GOT) in HF-fed mice, as confirmed by histological observations. Thus, we demonstrated that the TLR4/MyD88/TRIF pathway contributes to the well-established link between bacterial endotoxin and the consumption of LFP against HF-induced hepatocyte injury. It is worth noting that chlorogenic acid, the main polyphenol in LFP, has the capacity to regulate glucose and lipid metabolisms, to exert antioxidant and anti-inflammatory effects, as well as to regulate gut microbiota.38,39,51 Thus, the abovementioned preventive actions of LFP on “the first and second hit” of HF-induced NAFLD might have been induced by chlorogenic acid. In this regard, therefore, the current study might lack sufficient novelty. Interestingly, however, it has been shown that the gut microbiota and intestinal barriers also played a key role in HF-induced NAFLD,11,12 thus providing a novel pathway for this study to reveal the mechanism with which LFP protects the liver in HF-fed mice. A damaged intestinal integrity is a critical risk factor to increase endotoxin translocation to the liver.37 The intestinal barrier comprises four components, microbiological, chemical, physical, and immunological barriers.52 The chemical barrier is the first defense against microorganisms invading the mucosal membrane.52 Mucins, the main structural components of the mucus layer, comprise the chemical barrier to prevent contact between bacteria and the epithelium.53 Acidic mucin-producing goblet cells were visualized by Alcian blue and high-iron diamine staining in the present study. The results suggested that fructose decreases the number of cells that can produce sulfomucin (AB +/HID+), while this reduction is inhibited by LFP. MUC2 and MUC4 are the best characterized secreted and membranebound mucins of gastrointestinal cells, respectively.53 We observed that LFP up-regulated colonic MUC2 and MUC4 gene expression in HF-fed mice. These data indicated that LFP maintained the strength of the intestinal chemical barrier, which was damaged by HF. Recently, the physical intestinal barrier was widely studied in fructose-caused increased gut permeability, which enhanced the translocation of endotoxin from the gut to the blood circulation.28,37 The tight junctions of intestinal epithelial cells, as a dynamic permeability barrier, have double functions to prevent potential harmful substances and allow nutrients to enter the body.52 We found that the mRNA and protein expression levels of the tight junction proteins (ZO-1 and Occludin) in the colon were increased following LFP administration in HF-fed mice, indicating that LFP could avoid endotoxin accumulation in blood by protecting the gut physical barrier. When the intestinal chemical and physical barriers break, the sensitized intestinal immune system will be the last barrier to resist bacteria.52 This work showed that LFP down-regulated HF-induced increases in mRNA expression of colonic endotoxin receptor Toll-like receptors and nuclear transcription factor, indicating that LFP enhanced the intestinal immunological barrier. On the basis of the above discussion, we speculated that the protection effects of LFP on the chemical, physical and immunological barriers of the gut were due to LFP regulating the components of the gut microbiota changed by HF. The overall gut microbiota balance could be effectively assessed by the F/B ratio, which is associated with obesity and a worse glucose tolerance.54 Notably, HF-induced increase in the F/B ratio was significantly decreased by LFP, suggesting that LFP maintains the intestinal flora balance. Veillonella has been to

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02523.

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Commercial kits; OGTT method; method of histopathological observation; primers used in real-time PCR; composition of polyphenol from loquat fruit; effects of LFP on food intake, water intake, serum IL-1 and serum IL-6 levels in HF-fed mice; expression of IκBα protein in liver; α-diversity and CV of gut microbiota (PDF)

AUTHOR INFORMATION

Corresponding Authors

* E-mail: [email protected]; yanghongyan2013@163. com. Fax: + 86 29 83246270. *E-mail: [email protected]; Fax: + 86 991 4363579. 7735


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Journal of Agricultural and Food Chemistry ORCID

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Wenfeng Li: 0000-0002-3250-3062 Hongyan Yang: 0000-0001-6966-4700 Funding

This work was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission (KJQN201801436) and the Construction project of the Chongqing Collaborative Innovation Center for Functional Food at the Chongqing University of Education. Notes

The authors declare no competing financial interest.

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Polyphenol-Rich Loquat Fruit Extract Prevents Fructose-Induced

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