Nat Prod Sci.  2017 Sep;23(3):183-191. 10.20307/nps.2017.23.3.183.

Luteolin 5-O-glucoside from Korean Milk Thistle, Cirsium maackii, Exhibits Anti-Inflammatory Activity via Activation of the Nrf2/HO-1 Pathway

• Jung HA
• Roy A
• Abdul QA
• Kim HR
• Park HJ
• Choi JS

• KMID: 2393799
• DOI: http://doi.org/10.20307/nps.2017.23.3.183

Abstract

Luteolin 5-O-glucoside is the major flavonoid from Korean thistle, Cirsium maackii. We previously reported the anti-inflammatory activities of luteolin 5-O-glucoside in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. In this study, we determined the anti-inflammatory mechanisms of luteolin 5-O-glucoside through the inhibition of nitric oxide (NO) production in vitro and in vivo. Results revealed that luteolin 5-O-glucoside dose-dependently inhibited NO production and expression of iNOS and COX-2 in LPS-induced RAW 264.7 cells. Luteolin 5-O-glucoside also significantly inhibited the translocation of NF-κB, the activation of MAPKs, and ROS generation in LPS-induced RAW 264.7 cells. In addition, protein expressions of Nrf-2 and HO-1 were also upregulated by luteolin 5-O-glucoside treatment. Moreover, luteolin 5-O-glucoside inhibited λ-carrageenan-induced mouse paw edema by 65.34% and 48.31% at doses of 50 and 100 mg/kg body weight, respectively. These findings indicate potential anti-inflammatory effect of luteolin 5-O-glucoside particularly by downregulating NF-κB and upregulating HO-1/Nrf-2 pathway.

Keyword

Luteolin 5-O-glucoside; Cirsium maackii; Anti-inflammation; HO-1/Nrf-2; NF-κB; λ-carrageenan

MeSH Terms

Animals
Body Weight
Cirsium*
Edema
In Vitro Techniques
Luteolin*
Mice
Milk Thistle*
Milk*
Nitric Oxide
RAW 264.7 Cells
Luteolin
Nitric Oxide

Figure

• 

  Fig. 1 Cell viability of luteolin 5-O-glucoside measured by the MTT assay. Values represent the mean ± SD of three independent Experiments.

• 

  Fig. 2 Inhibitory effects of luteolin 5-O-glucoside on the production of NO in LPS-stimulated RAW 264.7 cells. Cells were pretreated with different concentrations of luteolin 5-O-glucoside for 2 h and then stimulated with LPS (1 µg/ml) for 24 h. The culture media were used to measure the amount of nitrite to determine NO production. Data are presented as mean ± SD of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

• 

  Fig. 3 Inhibitory effects of luteolin 5-O-glucoside on the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentration of luteolin 5-O-glucoside for 2 h and stimulated with LPS (1 µg/ml) for 18 h. The expression of iNOS, COX-2, and β-actin was detected by Western blotting using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

• 

  Fig. 4 Inhibitory effects of luteolin 5-O-glucoside on the expression of NF-κB in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentration of luteolin 5-O-glucoside for 2 h and stimulated with LPS (1 µg/ml) for 18 h. The expression of NF-κB and β-actin was detected by Western blot using the corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

• 

  Fig. 5 Inhibitory effects of luteolin 5-O-glucoside on the expression of MAPKs in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentration of luteolin 5-O-glucoside for 2 h and stimulated with LPS (1 µg/ml) for 18 h. The expression of MAPKs was detected by Western blotting using corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates significant differences from the control group. *p < 0.05 indicates significant differences from the LPS-treated group.

• 

  Fig. 6 Inhibitory effects of luteolin 5-O-glucoside on the production of ROS in LPS-stimulated RAW 264.7 cells. Cells were pretreated with different concentration of luteolin 5-O-glucoside for 2 h and then stimulated with LPS (1 µg/ml) for 24 h. ROS levels were measured by fluorescence analysis of DCFH-DA. Data are presented as means ± SDs of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

• 

  Fig. 7 Inhibitory effects of luteolin 5-O-glucoside on the expression of HO-1 and Nrf-2 in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentration of luteolin 5-O-glucoside for 2 h and stimulated with LPS (1 µg/ml) for 18 h. Expression of HO-1 and Nrf-2 was detected by Western blotting using the corresponding antibodies. The results presented are representative of three independent experiments. #p < 0.05 indicates a significant difference from the control group. *p < 0.05 indicates a significant difference from the LPS-treated group.

• 

  Fig. 8 Effects of luteolin 5-O-glucoside on carrageenan-induced hind paw edema in mice. Luteolin 5-O-glucoside was administered at doses of 25 mg/kg and 50 mg/kg 1 h before injection of carrageenan into the mice to alleviate acute inflammation. Paw volume was measured using a plethysmometer at 2 h, 5 h, and 7 h after carrageenan injection. The increase in paw volume was calculated based on the difference in volume between before carrageenan injection and at different time intervals after carrageenan injection. Values are expressed as the mean ± SD; n = 6 mice per group. *p < 0.05 indicates a significant difference from the vehicle control group. Indomethacin was used as a positive control.

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