Nutr Res Pract.  2019 Apr;13(2):95-104. 10.4162/nrp.2019.13.2.95.

Anti-inflammatory effect of Lycium barbarum on polarized human intestinal epithelial cells

• Lee SR
• Hwang HJ
• Yoon JG
• Bae EY
• Goo KS
• Cho SJ
• Cho JA

• KMID: 2442366
• DOI: http://doi.org/10.4162/nrp.2019.13.2.95

Abstract

BACKGROUND/OBJECTIVES
Inflammatory Bowel Disease (IBD) has rapidly escalated in Asia (including Korea) due to increasing westernized diet patterns subsequent to industrialization. Factors associated with endoplasmic reticulum (ER) stress are demonstrated to be one of the major causes of IBD. This study was conducted to investigate the effect of Lycium barbarum (L. barbarum) on ER stress.
MATERIALS/METHODS
Mouse embryonic fibroblast (MEF) cell line and polarized Caco-2 human intestinal epithelial cells were treated with crude extract of the L. chinense fruit (LF). Paracellular permeability was measured to examine the effect of tight junction (TJ) integrity. The regulatory pathways of ER stress were evaluated in MEF knockout (KO) cell lines by qPCR for interleukin (IL) 6, IL8 and XBP1 spliced form (XBP1s). Immunoglobulin binding protein (BiP), XBP1s and CCAAT/enhancer-binding homologous protein (CHOP) expressions were measured by RT-PCR. Scanning Ion Conductance Microscopy (SICM) at high resolution was applied to observe morphological changes after treatments.
RESULTS
Exposure to LF extract strengthened the TJ, both in the presence and absence of inflammation. In polarized Caco-2 pretreated with LF, induction in the expression of proinflammatory marker IL8 was not significant, whereas ER stress marker XBP1s expression was significantly increased. In wild type (wt) MEF cells, IL6, CHOP and XBP1 spliced form were dose-dependently induced when exposed to 12.5-50 µg/mL extract. However, absence of XBP1 or IRE1α in MEF cells abolished this effect.
CONCLUSION
Results of this study show that LF treatment enhances the barrier function and reduces inflammation and ER stress in an IRE1α-XBP1-dependent manner. These results suggest the preventive effect of LF on healthy intestine, and the possibility of reducing the degree of inflammatory symptoms in IBD patients.

Keyword

Lycium barbarum; ER stress; inflammation

MeSH Terms

Animals
Asia
Carrier Proteins
Cell Line
Diet
Endoplasmic Reticulum
Epithelial Cells*
Fibroblasts
Fruit
Humans*
Immunoglobulins
Inflammation
Inflammatory Bowel Diseases
Interleukin-6
Interleukin-8
Interleukins
Intestines
Lycium*
Mice
Microscopy
Permeability
Tight Junctions
Carrier Proteins
Immunoglobulins
Interleukin-6
Interleukin-8
Interleukins

Figure

• 

  Fig. 1 Effect of LF on cell viability in vitro and in vivo. Human intestinal epithelial cells (Caco-2) were pretreated with different concentrations of LF (0, 12.5, 25, 50, 100, 250 µg/mL) for 24 h (A) to evaluate for cell viability. Zebra fish eggs were pretreated with same concentrations for 75 h to evaluate toxicity (B) and survival rate (C). The experiments were performed individually at least three times. Data are expressed as mean ± SD. In Fig. 1A, the inter-concentration statistics were analyzed by One-way ANOVA. White bar represents the negative control.

• 

  Fig. 2 Protective effect of LF on cell permeability of Caco-2 cells. Polarized Caco-2 cells were pretreated with indicated concentrations of LF for 24 h, followed by the absence (A) or presence (B) of cytokine cocktail (50 ng/mL TNF-α + 50 ng/mL IFN-γ + 25 ng/mL IL-1β + 10 µg/mL LPS) stimulation apically for additional 24 h. Cells were washed and wells were refilled with fresh HBSS buffer. The apical side contains 1 mg/mL of FITC-dextran fluorescein. Absorbance was measured in the basal solution at the indicated time point. The experiments were performed individually at least three times. Data are expressed as mean ± SD. Significant differences between the different concentrations at each time point were analyzed by One-way ANOVA. #P < 0.00001

• 

  Fig. 3 Effect of LF on expression of IL8 and XBP1s by ER stress in polarized human intestinal epithelial cell. Polarized Caco-2 cells was pretreated with indicated concentrations of LF for 24 h, followed by 3 µM TG stimulation apically for additional 4 h (A and B) or 1 h (C and D). Total RNA was extracted and cDNA prepared for real-time qPCR. Human IL8 (A, B) and XBP1s (C, D) were normalized by human GAPDH. The fold change of IL8 or XBP1s expression induced by TG was calculated at each LF concentration (B, D). The experiments were performed individually at least three times. Data are expressed as mean ± SD. The significant differences between the LF concentrations were analyzed by One-way ANOVA. The TG effect at each concentration was analyzed by student's t-test. *P < 0.05. IL8, interleukin 8; XBP1s, X-box binding protein 1 spliced form; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TG, triglyceride; LF, L. chinense fruit.

• 

  Fig. 4 Effect of LF on proinflammatory response in MEF cells. Wt MEF (A and B), XBP1 KO MEF (C and D) and IRE1α KO MEF (E and F) cell lines were pretreated with LF for 24 h, followed by 3 µM TG for additional 4 h. Total RNA was extracted and cDNA prepared for real-time qPCR. mRNA expression of mouse IL6 (A, C and E) and the fold change of IL6 expression induced by TG (B, D and F) were normalized by mouse β-actin. The experiments were performed individually at least three times. Data are expressed as mean ± SD. Significant differences between the different concentrations were analyzed by One-way ANOVA. The TG effect at each concentration was analyzed by student's t-test. *P < 0.05. IL6, interleukin 6; LF, L. chinense fruit; TG, triglyceride; XBP1s, X-box binding protein 1 spliced form.

• 

  Fig. 5 Effect of LF on the ER stress in MEF cells. MEF cell lines were pretreated with LF for 24 h, followed by 3 µM TG for additional 4 h (A, B) or 1 h (C, D and E). Total RNA was extracted and cDNA prepared for RT-PCR (A and E) and real-time qPCR (C, D). Mouse CHOP, XBP1s mRNA expression were normalized by mouse β-actin (B, C and D); CHOP mRNA expression was quantified using the ImageJ software (B). The experiments were performed individually at least three times. Data are expressed as mean ± SD. Significant differences between the different concentrations were analyzed by One-way ANOVA. The TG effect at each concentration was analyzed by student's t-test. *P < 0.05. BiP, Immunoglobulin binding protein; CHOP, CCAAT/enhancer-binding homologous protein; XBP1s, X-box binding protein 1 spliced form.

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