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HPLC analysis followed by individual identification of compounds by comparison with authentic standard compounds was performed. Five compounds were identified: coumaric acid (CA), ferulic acid (FA), gallic acid (GA), caffeic acid (CFA), and vanillic acid (VA) (Table 1). LFBE exhibited significantly higher VA and FA contents compared to those of RBE, which correlated well with the stronger biological actions of LFBE.
Table 1. Phenolic Acid Contents of RBE and LFBE (dry weight)
Component RBE LFBE GA (µg/g) 1.98 ± 0.08b 8.30 ± 0.08a CA (µg/g) 5.39 ± 0.05b 49.99 ± 0.51a VA (µg/g) 22.35 ± 0.21b 117.99 ± 1.15a CFA (µg/g) 2.27 ± 0.07a 1.31 α 0.02b FA (µg/g) 23.52 ± 0.07b 112.79 ± 1.21a Note. Data are expressed as the mean ± SEM, n = 3. Different letters indicate a significant difference (P < 0.05). RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; CA: coumaric acid; GA: gallic acid; VA: vanillic acid; CFA: caffeic acid; FA: ferulic acid. -
In the present study, LFBE and RBE were tested for their effects on glucose consumption in HepG2 cells. LFBE significantly enhanced glucose consumption in HepG2 cells at concentrations of 50-100 µg/mL (no cytotoxicity) (Table 2). Glucose consumption decreased at high concentrations of LFBE, compared with low and intermediate concentrations, due to its ability to inhibit HepG2 cell growth. However, RBE scarcely increased glucose consumption at all concentrations tested.
Table 2. Effect of LFBE and RBE on Glucose Consumption after 24 h Treatment of HepG2 Cells
Item Control (µg/mL) Model (µg/mL) LFBE (µg/mL) RBE (µg/mL) 0 0 200 100 50 200 100 50 Glucose consumption (mmol/L)a 2.64 ± 0.24a 1.26 ± 0.25d 2.02 ± 0.81b 2.51 ± 0.36a 2.18 ± 0.17b 1.42 ± 0.26c 1.34 ± 0.22cd 1.23 ± 0.13d Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1. The effects of FA and VA on glucose consumption in HepG2 cells were also investigated (Table 3). At all concentrations tested from 2 µg/mL to 8 µg/mL, VA showed a significant enhancement of glucose consumption in HepG2 cells (compared with model). FA also enhanced glucose consumption in HepG2 cells, but the effect was not significant. These results indicated that VA showed the greatest enhancement of glucose consumption in HepG2 cells. Therefore, VA may contribute to the glucose-consumption-enhancing activity of LFBE. Since 50-100 µg/mL LFBE did not induce cell toxicity, and 100 µg/mL LFBE enhanced glucose consumption the most, 100 µg/mL LFBE was selected for further studies. At the same time, 2-8 µg/mL VA did not induce cell toxicity, and 4 µg/mL and 8 µg/mL VA had a similar effect on enhancement of glucose consumption activity. Therefore, 4 µg/mL VA was selected for further studies.
Table 3. Effect of FA and VA on Glucose Consumption after 24 h Treatment of HepG2 Cells
Item Control (µg/mL) Model (µg/mL) FA (µg/mL) VA (µg/mL) 0 0 8 4 2 8 4 2 Glucose consumption (mmol/L)a 2.69 ± 0.16a 1.37 ± 0.16e 1.84 ± 0.23cd 1.76 ± 0.23d 1.60 ± 0.21d 2.43 ± 0.41b 2.25 ± 0.16b 2.05 ± 0.21c Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; FA: ferulic acid; VA: vanillic acid. -
Chronic, low-grade inflammation is a major component of obesity[23]. The mRNA expression levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β, etc.) were measured in palmitate-induced HepG2 cells by RT-PCR. As shown in Table 4, compared with the model, the relative mRNA contents of TNF-α were 0.08, 0.07, and 0.52 in HepG2 cells of the control, VA, and LFBE, respectively. Furthermore, the relative mRNA contents of IL-1β were 0.01, 0.01, and 0.09, respectively, and the relative mRNA contents of IL-6 were 0.35, 0.44, and 0.14, respectively. The results showed that palmitate induced changes in inflammatory cytokines, increasing the risk of inflammation. VA and LFBE can significantly reduce the levels of TNF-α, IL-1β, and IL-6.
Table 4. TNF-α, IL-1β, and IL-6 mRNA Expression Analysis in HepG2 Cells
Relative mRNA Expression TNF-α IL-1β IL-6 Control (0 µg/mL) 0.08 ± 0.00c 0.01 ± 0.00c 0.35 ± 0.02c Model (0 µg/mL) 1.00 ± 0.01a 1.00 ± 0.00a 1.00 ± 0.02a VA (4 µg/mL) 0.07 ± 0.00c 0.01 ± 0.00c 0.44 ± 0.02b LFBE (100 µg/mL) 0.52 ± 0.03b 0.09 ± 0.00b 0.14 ± 0.01d Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate. -
The phosphorylation of mitogen-activated protein kinase (MAPK) plays an important role in activating transcription factors that induce inflammatory gene expression. As shown in Figure 1, LFBE and VA treatment led to a significant reduction in phosphorylation of p38 and JNK1 compared with the model in palmitate-induced HepG2 cells.
Figure 1. Inhibitory effects of LFBE and VA on phosphorylation of JNK (A) and p38 (B) in HepG2 cells. Results are expressed as relative ratios of band density of phosphorylated forms of p38 and JNK to the respective total proteins. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; VA: vanillic acid.
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To address how LFBE and VA improve lower levels of glucose improve high levels of glucose and the expression of proinflammatory factors in palmitate-induced HepG2 cells. MiR-212 was upregulated in palmitate-induced HepG2 cells, while the addition of LFBE and VA diminished miR-212 expression relative to the model (Figure 2A). These results prompted us to further clarify the functional and molecular mechanisms of miR-212 dysregulation in glucose consumption and expression of proinflammatory factors. We also measured DUSP9 mRNA levels and confirmed that DUSP9 downregulation was blocked when LFBE and VA treatment increased glucose consumption and reduced proinflammatory cytokine expression (Figure 2B).
Figure 2. The expression of miR-212 (A) and mRNA expression of DUSP9 (B) in HepG2 cells treated with LFBE and VA. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; VA: vanillic acid.
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Previously, we found that high-fat-diet-induced downregulation of miR-212 and LFBE effectively prevented the decrease in miR-212 expression induced by a high-fat-diet in obese rats by microarray and RT-PCR in liver (unpublished data). We performed a luciferase reporter assay to identify genes that were targeted by miR-212. As shown in the miRWalk database, DUSP9 is a validated target gene of miR-212, given that DUSP9 is a central regulator contributing to the inflammatory response. To verify that DUSP9 is a true target of miR-212, we generated Renilla luciferase reporter plasmids containing miR-212 binding sites in the 3a'-UTR of DUSP9. Transfection of the miR-212 precursor oligonucleotide significantly reduced luciferase expression from a reporter plasmid that contained the 3a'-UTR of DUSP9 compared with the transfection of control miRNA (Figure 3).
Figure 3. Target validation for miR-212. A: Top, miR-212 and the miR-212-binding site in the 3a'-UTR of DUSP9. Bottom, miR-212 and the mutated miR-212-binding site in the 3a'-UTR of DUSP9. B: Results are shown as the relative luciferase activity for the combination of miR-212 and reporter construct. NC: negative control. Data are means ± SEM of six replicate experiments. *, P < 0.05, compared with corresponding control.
To further determine whether DUSP9 is a validated target gene of miR-212, HepG2 cells were transfected with miR-212 mimics, inhibitor or their NC. As measured by qRT-PCR, miR-212 mimics upregulated, while miR-212 inhibitor downregulated miR-212 levels in HepG2 cells (Figure 4A), confirming that the inhibitors and mimics used in this study are effective. The miR-212 mimics downregulated DUSP9 mRNA and protein levels (Figure 4B and C). By contrast, treatment with the miR-212 inhibitor enhanced the expression of DUSP9 mRNA and protein in HepG2 cells. These data suggest that miR-212 downregulates the expression of DUSP9 by targeting the DUSP9 3a'-UTR to facilitate translational repression or mRNA degradation in HepG2 cells. In addition, to determine whether LFBE and VA directly modulate miR-212, the results presented in Figure 3 were analyzed again. We found that the expression of miR-212 was decreased, and mRNA expression of DUSP9 was increased, by LFBE and VA treatment (Figure 2). Above all, these data confirmed that LFBE and VA modulate miR-212 and clearly indicate that DUSP9 is a target gene of miR-212 involved in palmitate-induced abnormal glucose consumption.
Figure 4. A: The expression of miR-212 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. B: The mRNA expression of DUSP9 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. C: The protein expression of DUSP9 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. NC: negative control. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05).
doi: 10.3967/bes2018.091
Fermented Barley Extracts with Lactobacillus plantarum dy-1 Rich in Vanillic Acid Modulate GlucoseConsumption in Human HepG2 Cells
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Abstract:
Objective To investigate the effect of fermented barley extracts with Lactobacillus plantarum dy-1 (LFBE) for modulating glucose consumption in HepG2 cells via miR-212 regulation. Methods Hepatocellular carcinoma (HepG2) cells were treated with palmitate. After 12 h, palmitate-induced HepG2 cells were treated with LFBE and its main components. Changes in glucose consumption, proinflammatory cytokine secretion, and miRNA-212 expression in HepG2 cells was observed. Results Treatment with LFBE rich in vanillic acid (VA) increased glucose consumption and reduced proinflammatory cytokine secretion in HepG2 cells. LFBE and VA normalized the upregulation of miR-212, which led to the upregulation of dual-specificity phosphatase-9 (DUSP9), a direct target of miR-212, at both protein and mRNA levels. Downregulation of miR-212 markedly increased glucose consumption and reduced proinflammatory cytokine secretion by enhancing DUSP9 expression. Conclusion The results showed the benefit of LFBE and miR-212 downregulation in modulating glucose consumption and reducing proinflammatory cytokine secretion by targeting DUSP9. VA in LFBE was a strong regulator of palmitate-induced abnormal glucose consumption in HepG2 cells and can be a primary mediator. -
Key words:
- Fermented barley extract /
- Vanillic acid /
- Glucose consumption /
- HepG2 /
- Dual-specificity phosphatase-9
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Figure 1. Inhibitory effects of LFBE and VA on phosphorylation of JNK (A) and p38 (B) in HepG2 cells. Results are expressed as relative ratios of band density of phosphorylated forms of p38 and JNK to the respective total proteins. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; VA: vanillic acid.
Figure 2. The expression of miR-212 (A) and mRNA expression of DUSP9 (B) in HepG2 cells treated with LFBE and VA. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; VA: vanillic acid.
Figure 3. Target validation for miR-212. A: Top, miR-212 and the miR-212-binding site in the 3a'-UTR of DUSP9. Bottom, miR-212 and the mutated miR-212-binding site in the 3a'-UTR of DUSP9. B: Results are shown as the relative luciferase activity for the combination of miR-212 and reporter construct. NC: negative control. Data are means ± SEM of six replicate experiments. *, P < 0.05, compared with corresponding control.
Figure 4. A: The expression of miR-212 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. B: The mRNA expression of DUSP9 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. C: The protein expression of DUSP9 in HepG2 cells treated with miR-212 mimic and miR-212 inhibitor. NC: negative control. Data are means ± SEM of six replicate experiments. Different letters indicate a significant difference in each figure (P < 0.05).
Table 1. Phenolic Acid Contents of RBE and LFBE (dry weight)
Component RBE LFBE GA (µg/g) 1.98 ± 0.08b 8.30 ± 0.08a CA (µg/g) 5.39 ± 0.05b 49.99 ± 0.51a VA (µg/g) 22.35 ± 0.21b 117.99 ± 1.15a CFA (µg/g) 2.27 ± 0.07a 1.31 α 0.02b FA (µg/g) 23.52 ± 0.07b 112.79 ± 1.21a Note. Data are expressed as the mean ± SEM, n = 3. Different letters indicate a significant difference (P < 0.05). RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1; CA: coumaric acid; GA: gallic acid; VA: vanillic acid; CFA: caffeic acid; FA: ferulic acid. Table 2. Effect of LFBE and RBE on Glucose Consumption after 24 h Treatment of HepG2 Cells
Item Control (µg/mL) Model (µg/mL) LFBE (µg/mL) RBE (µg/mL) 0 0 200 100 50 200 100 50 Glucose consumption (mmol/L)a 2.64 ± 0.24a 1.26 ± 0.25d 2.02 ± 0.81b 2.51 ± 0.36a 2.18 ± 0.17b 1.42 ± 0.26c 1.34 ± 0.22cd 1.23 ± 0.13d Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; RBE: aqueous extract of unfermented raw barley; LFBE: aqueous extract of fermented barley with Lactobacillus plantarum dy-1. Table 3. Effect of FA and VA on Glucose Consumption after 24 h Treatment of HepG2 Cells
Item Control (µg/mL) Model (µg/mL) FA (µg/mL) VA (µg/mL) 0 0 8 4 2 8 4 2 Glucose consumption (mmol/L)a 2.69 ± 0.16a 1.37 ± 0.16e 1.84 ± 0.23cd 1.76 ± 0.23d 1.60 ± 0.21d 2.43 ± 0.41b 2.25 ± 0.16b 2.05 ± 0.21c Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate; FA: ferulic acid; VA: vanillic acid. Table 4. TNF-α, IL-1β, and IL-6 mRNA Expression Analysis in HepG2 Cells
Relative mRNA Expression TNF-α IL-1β IL-6 Control (0 µg/mL) 0.08 ± 0.00c 0.01 ± 0.00c 0.35 ± 0.02c Model (0 µg/mL) 1.00 ± 0.01a 1.00 ± 0.00a 1.00 ± 0.02a VA (4 µg/mL) 0.07 ± 0.00c 0.01 ± 0.00c 0.44 ± 0.02b LFBE (100 µg/mL) 0.52 ± 0.03b 0.09 ± 0.00b 0.14 ± 0.01d Note. Data are expressed as the mean ± SEM, n = 8. Different letters indicate a significant difference (P < 0.05). Control: normal cells without palmitate treatment; Model: HepG2 cells treated with palmitate. -
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