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Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells

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Abstract

Objective

To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.

Methods

RAW264.7 cells were pretreated with 0–200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2−) radical and nitrite scavenging activity were also measured.

Results

The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2− radical and nitrite scavenging activity.

Conclusion

EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

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Authors and Affiliations

  1. Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu Province, 221116, China

    Cheng-liu Yang, Shi-bo Wang, Wen-ping He & Jin-juan Liu

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  1. Cheng-liu Yang
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  2. Shi-bo Wang
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  3. Wen-ping He
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  4. Jin-juan Liu
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Contributions

Liu JJ and Yang CL performed the main experiments. Wang SB and He WP performed the anti-oxidant experiments. Liu JJ designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jin-juan Liu.

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The authors declare no conflicts of interest.

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Yang, Cl., Wang, Sb., He, Wp. et al. Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells. Chin. J. Integr. Med. 29, 905–913 (2023). https://doi.org/10.1007/s11655-023-3602-7

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