The endoplasmic reticulum (ER) is in charge of the synthesis, folding, and processing of secretory and transmembrane proteins. ER stress is caused by the accumulation of misfolded or unfolded proteins in the ER, mostly caused by environmental factors such as reactive oxygen species (ROS), major inflammatory mediators in the disease pathogenesis [1
]. Then, calcium-dependent protein folding enzymes release calcium from the ER lumen and increase the concentration of calcium influx into the cytoplasm, worsening the level of ER stress, oxidant reactions, and inflammatory reactions [2
]. This process activates three endoplasmic reticulum transmembrane domains: IRE1, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and ATF6. In the resting state, the molecular chaperone, BiP (78 kDa glucose control protein GRP78), a central regulator of ER stress, maintains IRE1, PERK, ATF6 in an inactive state by binding to their luminal domains. In the stressed state, BiP dissociates from these ER stress receptors and binds to an overwhelming misfolded protein, unfolded proteins. Then, BiP-free ER stress receptors become active. Activated PERK causes the translational attenuation of the protein machinery involving in the cell cycle and producing cell cycle arrest in the G1 phase [4
]. Unsolved ER stress induces the unfolded protein response (UPR)-related genes, primarily chaperones, such as BiP, to prevent the further accumulation of unfolded/misfolded proteins [5
]. Activated IRE1α activates an ER-related associated degradation (ERAD) system, consisting of ubiquitin-dependent proteasomes [7
]. IRE1α is self-phosphorylated to activate the RNase activity, initiating the removal of the 26-base intron from the mRNA encoding X-box-binding protein 1 (XBP1) [8
]. This results in translational frameshifting and translation of the XBP1 isoform with potent activity as a transcription factor, resulting in activation of the NF-kB pathway. Under the conditions of prolonged stress, the proapoptotic protein CHOP (CCAAT/enhancer-binding protein homologous protein) is upregulated causing a proapoptotic drive at the mitochondria [9
Approximately 20–40% of inflammatory bowel disease (IBD) patients in Western countries are obese and it has been reported that there is correlation between the incidence of IBD and a high-fat diet rich in cholesterol and animal fat [10
]. ER stress and UPR induction are critical to intestinal stem cells, and it has been reported that damage to UPR signaling leads to chronic inflammatory diseases such as IBD and irritable bowel syndrome (IBS) [12
]. Goblet cells secrete mucin to form a protective luminal mucus layer and Paneth cells secrete defensin and other antimicrobial peptides to keep the crypt of the small intestine in a sterile state [13
]. The depletion of goblet cells results in a failure to protect against and invasion of pathogenic bacteria is a common pathological phenomenon in IBD [10
]. In addition, intestinal epithelial cells, which are functionally impaired in IBD, can trigger an inflammatory response [5
), also known as Goji berry, has attracted attention as a superfood in Western countries. It has been used mainly as an edible and traditional medicinal plant for a long time in Eastern countries, such as China and Korea [14
]. Lycium barbarum
contains many polysaccharides, flavonoids, and polyphenol compounds such as chlorogenic acid, p-coumaric acid, caffeic acid, ferulic acid and Gentisic acid [15
]. While the fruits are widely used worldwide, their leaves are usually wasted. However, if any functional components or effects in the leaves were well studied with the clear mechanisms, it would be most appreciated in the field of agriculture. So far, the main active flavonoid of the L. barbarum
leaf (LL) was identified as rutin [16
]. Hypoglycemic, antimicrobial, antioxidant, anti-aging, anticancer and lipid-lowering effects have been reported [17
]. However, little is known about its effects and the mechanism on the intestine. Previously, we investigated L. barbarum
fruits extract and found that it enhanced the gut barrier function and reduced inflammation and ER stress. Therefore, this study investigated the antioxidant and anti-inflammatory effects of LL through the ER stress and oxidative stress pathway.
2. Materials and Methods
The dried leaves of L. barbarum were purchased from Ningxi Hui in China. The leaves were extracted using 70% ethanol at room temperature for seven days. After filtration through filter paper Advantech No.3 (Toyo Roshi Kaisha, Japan), the extract was concentrated with a rotary evaporator A-1000S (EYELA, Tokyo, Japan) and used in the experiment. The leaf extract of L. barbarum was dissolved in 70% DMSO (Sigma-Aldrich Co., Saint louis, MO, USA) at 25 mg/mL to be used as the stock solution.
2.2. Liquid Chromatography (LC)–Mass Spectrometry
LC-mass were performed SYNAPT G2 Si HDMS QTOF (Waters Corporation, Milrord, Massachusetts, USA). LC conditions were performed using a C18 column (ACQUITY UPLC® HSS T3 C18, 1.8 µm, 2.1 × 100 mm, Waters, Worcester, MA, USA); the temperature at 35 °C with UV detector at 265 nm. The injection volume was set to 5 μL and analyzed. The mobile phase was analyzed using water (A) and methanol (B) diluted with 0.1% formic acid. As for the solvent gradient conditions, the initial flow of A and B at a ratio of 90:10 for 0 to 5 min, followed by 13 to 18 min of B 100%. After that, it was set to run for 18–20 min in the same way as the initial conditions. The ionization condition was set to positive, and the scan range was set to 50~1550 m/z. The source of the mass spectrometer was analyzed using electrospray ionization (ESI).
2.3. Cell Culture
Human intestinal epithelial cells, Caco-2, were purchased from the American Type Culture Collection (Manassas, VA, USA). The MEF cells were a generous gift from David Ron (University of Cambridge, Cambridge, UK). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) with 1% Penicillin-Streptomycin (Sigma-Aldrich Co., Saint louis, MO, USA) and 10% heat-inactivated fetal bovine serum (Gibco, USA) at 37 °C under an atmosphere containing 5% CO2. The Caco-2 cells were polarized on Transwell (SPL Life Sciences, Pocheon, Korea) coated with 396 μg/cm2 Type I collagen (Corning, VA, USA), in which monolayers were grown for 10 days. The electrical resistance was measured using trans epithelial electric resistance measurements (EVOM2, World Precision Instruments Inc., Sarasota, FL, USA). When the transepithelial/ transendothelial electrical resistance (TEER) reached 500–600 Ωcm2, the apical side of the cells was treated with LL and further stimulated with Thapsigargin (TG, Sigma-Aldrich Co., Saint louis, MO, USA) for 1 h or 4 h to induce ER stress or stimulated with cytokine cocktails (CT, 50 ng/mL TNFα + 50 ng/mL IFN-γ + 25 ng/mL IL1β + 10 μg/mL LPS) for 16 h to induce inflammation.
2.4. Experimental Animals
Five-week-old male BALB/c mice were purchased from DaHanBioLink Co., Ltd. (Eumseong, Korea). After one week of quarantine, the mice were fed a 60% high-fat diet (Research Diet, New Brunswick, NJ, USA) for 46 days. After 23 days, the mice were divided randomly into four groups (each group n = 8): HFD diet (H), HFD diet + LPS injection (HL), HFD diet + LPS injection + 150 mg/kg LL (HLL) oral gavage, and HFD diet + LPS injection + 300 mg/kg (HLH) oral gavage. The mice were administered 150 mg/kg and 300 mg/kg LL orally for 20 days daily. The mice were injected intraperitoneally (i.p.) with 5 mg/kg Lipopolysaccharides (LPS, InvivoGen, San Diego, CA, USA) and sacrificed after 1 hr. All animal experiments were approved by the Committee of Animal Care and Experiment of Chungnam National University (201909A-CNU-134) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).
Zebrafish were maintained at 28 °C for 14 h in the light cycle and 10 h in the dark cycle. Brine shrimp were raised in the laboratory and fed to the fish four times a day. To collect the fertilized embryos, individual males and females were separated in a mating cage overnight and spawned in the next morning. To evaluate the toxicity to the zebrafish embryos, the fertilized eggs were washed five to six times with a solution of 4 ppm methylene blue in 0.1% egg water and washed again with a solution of 1 ppm in concentration. The washed fertilized eggs were observed under a microscope, and the normal fertilized eggs were then selected and used in the experiment. The selected fertilized eggs were placed 10 per well in a 24-well plate (Hyundai Micro Co., Seoul, Korea) and treated with 2 mL of different concentrations of LL (0, 12.5, 25, 50, 100, and 250 μg/mL) in 0.1% egg water. The egg water with different concentrations of LL was changed every 24 h. The developmental process and toxicity were evaluated at 2, 4, 8, 14, 22, 30, 48, and 75 h using a DM2000 (Leica Co., Wetzlar, Germany) and a SZ2-ILST microscope (Olympus, Tokyo, Japan). All experiments on the zebrafish were performed according to the protocol approved by the Animal Care and Use Committee of Chungnam National University (CNU-01027).
2.5. Cell Viability Assay
Water-soluble tetrazolium (WST) analysis was performed to determine the cell viability using an EZ-cytox (Daeil lap service Co. Ltd., Korea). The cells were pretreated with various concentrations of LL (0, 12.5, 25, 50, 100, and 250 μg/mL) for 24 h followed by the WST assay according to the manufacturer’s instructions. The absorbance was measured at 450 nm on the xMarkTM Microplate Absorbance Spectrophotometer (Bio-Rad Inc., Hercules, CA, USA) after 3 h.
2.6. Nitric Oxide (NO) Assay
Polarized Caco-2 cells on transwell (SPL Life Sciences, Pocheon, Korea) were pretreated with LL (0, 12.5, 25, 50, and 100 μg/mL) for 24 h and stimulated with/without the cytokine cocktail (CT, 50 ng/mL TNFα + 50 ng/mL IFN-γ + 25 ng/mL IL1β + 10 μg/mL LPS) for a further 16 h to induce inflammation. IL1β and tumor necrosis factor-alpha (TNF-α) from JW Creagene Co. (Seongnam, Korea), IFN-γ from R&D Systems Inc. (Minneapolis, Minnesota, USA), and LPS from InvivoGen (San Diego, CA, USA) were purchased. NO production of the conditional media from the apical side of the chamber was measured using a Griess reagent system (Promega Co., Madison, WI, USA) according to the manufacturer’s instructions. The absorbance was measured at 540 nm using the xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Inc., Hercules, CA, USA).
2.7. Paracellular Permeability
Polarized Caco-2 cells were pretreated with LL on the apical side for 24 h, and stimulated with/without CT for a further 16 h to induce inflammation. The apical and basal sides were washed with Hanks’ balanced salts (HBSS, Sigma-Aldrich Co., Poole, UK) supplemented with 10 mM Hydroxyethyl piperazine Ethane Sulfonic acid (HEPES, Sigma-Aldrich Co, Saint Louis, MO, USA). One mg/mL of 4 kDa Fluorescein isothiocyanate dextran (FITC—dextran, Sigma-Aldrich Co., Uppsala, SWEDEN) in a HBSS/HEPES solution was added to the apical side and incubated for 72 h. The absorbance of the HBSS/HEPES solution on the basal side was measured every 0, 1, 2, 3, 6, 24, 30, 48, and 72 h. The fluorescence signals were measured using a DTX 800 multimode detector (Beckman Coulter, Inc., Brea, CA, USA) at 490 nm for excitation and 520 nm for emission.
2.8. RNA Extraction and cDNA Synthesis
The total RNA was extracted from the cells. Briefly, after removing the cell medium, Tri-reagent (MRC Inc., Cincinnati, OH, USA) was added to each well, and the resulting lysates were harvested. Chloroform was then added, and the mixture was centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was separated, mixed with isopropanol, and centrifuged at 12,000× g at 20 °C for eight minutes. The RNA pellets were harvested, and the RNA concentration was measured using Nano Drop ONE (Thermo Scientific Inc., Madison, WI, USA). The cDNA was synthesized using an RT-Kit (BioFACT Co., Daejeon, Korea).
2.9. Real-Time Polymerase Chain Reaction
Real-time qPCR was performed with cDNA synthesized using 2X Real-time PCR Master Mix Kit (BioFACT Co., Daejeon, Korea). Experimental steps using real-time qPCR machine (Agilent Co., Palo Alto, CA, USA) are as follows: the denaturation step took 15 min at 95 °C; the denaturing step took 20 s at 95 °C; the Anneal and Extension step took 40 s at 60 °C; one amplification cycle was performed for 45 cycles. The data were analyzed using the Agilent AriaMX 1.0 program. Table 1
lists the primer sequences used in the study.
2.10. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed to measure the RNA expression of the target gene using the 2X Taq Basic PCR Master Mix2 (BioFACT Co., Daejeon, Korea). Table 2
lists the primer sequences used in the experiment. The PCR mixture was loaded on 3% agarose gel for 45 min and then exposed to UV Light using the AE-9000 E-graph (ATTO, Japan).
2.11. Enzyme-Linked Immunosorbent Assay (ELISA)
The blood of mice was collected by cardiac puncture after anesthesia and allowed to clot for 30 min at room temperature. The serum was then centrifuged at 1000× g for 10 min. The supernatant was collected to perform the ELISA assay for interleukin 6 (IL6) according to the manufacture’s instruction (BD Biosciences, Franklin Lakes, NJ, USA).
2.12. Tissue Histology
The colon tissues of mice were fixed in a 10% formaldehyde solution, processed into paraffin blocks using standard methods, and then they were sectioned. The tissue sections were stained with hematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS) (T&P bio, Gwangju, Gyeonggi-do, Korea), and all stained tissues were taken with an optical microscope (OLYMPUS, Tokyo, Japan).
2.13. Flow Cytometry for Cell Cycle
After the entire experiment was completed, the mice were sacrificed, and the spleens were isolated for the ex vivo splenocytes culture, as described previously [21
]. For cell cycle analysis, the mouse splenocytes were minced and isolated using a cell strainer with 100 µm diameter pores (SPL Life Sciences, Korea) and washed twice with RPMI1640. The splenocytes were cultured in RPMI1640 media with 10% FBS, stimulated with 10 µg/mL of LPS for 72 h, and fixed with 70% ethanol. The cells were stained with a cell cycle kit (Millipore, Billerica, MA, USA) for 30 min at room temperature in the dark, vortexed gently, and read on the Muse Cell analyzer (Millipore, Darmstadt, Germany).
2.14. Statistical Analysis
Statistical processing of all experiments was analyzed using the SPSS/Windows 24.0 (SPSS Inc., Chicago, IL, USA) program. All experiments were conducted three or more times, and the experimental results are expressed as the mean ± standard deviation. A student’s t-test was used to examine the difference between the mean values between the two groups. One-way ANOVA was performed to analyze the difference between the mean values of three or more groups. After one-way ANOVA, the difference between the independent variables was confirmed using Duncan’s multiple range test, and the statistical significance was defined as being statistically significant when p < 0.05.
Lycium barbarum, also known as Goji berry, contains many phenolic compounds, such as rutin, chlorogenic acid, O-Coumaric acid, caffeic acid, ferulic acid and gentisic acid. This study showed the antioxidant function of Lycium barbarum leaf (LL) related anti-inflammation with the ER stress mechanism in vitro and in vivo. Although rutin is known to be a main antioxidant component of Lycium barbarum, many other compounds related to anti-oxidant functions were found in Lycium barbarum leaf. It contained many polyphenolic compounds such as O-coumaric acid, apocynin B, dendrocandin C, citrusin C, 2-O-rhamnosyl vitexin, benzoyl oxypaeoniflorin, kushenol Q, shikimic acid, wedeloactone, rhein, dihydrocaffeic acid and procyanidin B2 gallate. They seem to be individually, synergically or additively worked more likely by different mechanisms to achieve the antioxidant and anti-inflammatory function examined in our study. Interestingly, LL inhibited inflammation mediated by an IRE1α-XBP1-dependent ER stress pathway, which might be linked to its antioxidant activity. On the other hand, only a high dose of LL might suppress the adaptive immune response represented as allergies in this study, which might involve PERK pathway related to cell apoptosis and arrest of the cell cycle. In conclusion, a proper concentration of LL intake would be beneficial to prevent excessive adaptive immunity and inflammation related to the ER stress, and it would be worth developing as a functional food to enhance gut health.