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Effects of 『Geum-Gwe-Yo-Ryak(金匱要略)』 Prescription for Chest Pain Including Kwaruhaebaekbanha-tang and Kwaruhaebaekpaekju-tang on Macrophage Polarization

Article information

J Korean Med. 2019;40(2):51-62
Publication date (electronic) : 2019 June 30
doi : https://doi.org/10.13048/jkm.19016
Chang-Hyeon Son1orcid_icon, Sang-Min Lee1orcid_icon, Ga-Ram Yu2,3orcid_icon, Seung-Jun Lee3orcid_icon, Dong-Woo Lim4orcid_icon, Hyuck Kim2,3orcid_icon, Won-Hwan Park3orcid_icon, Jai-Eun Kim4,orcid_icon
1College of Korean Medicine, Dongguk University, Goyang
2Institute of Korean Medicine, Dongguk University, Goyang
3Department of Diagnostics, College of Korean Medicine, Dongguk University, Goyang
4Department of Pathology, College of Korean Medicine, Dongguk University, Goyang
Correspondence to: 김재은 (Jai-Eun Kim), 경기도 고양시 일산동구 동국로 32 동국대학교 한의과대학 병리학교실, Tel: +82-31-961-5829, Fax: +82-31-961-5835, E-mail: herbqueen@dongguk.ac.kr
Received 2019 May 13; Revised 2019 May 23; Accepted 2019 May 27.

Abstract

Objectives

This study was designed to evaluate the macrophages polarization of traditional Korean medicine on cardiac pain about Geum-Gwe-Yo-Ryak’s two prescriptions including Kwaruhaebaekbanha-tang (KHB) and Kwaruhaebaekpaekju-tang (KHP).

Materials and methods

Flow cytometry analysis was used to measure the changes in the ratio of M1 type and M2 type macrophages. Protein expression of nuclear factor-like 2 (Nrf2), heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured by Western Blot, and ABCA1 and SR-B1 were detected by real time PCR (RT-PCR). Intracellular lipid accumulation was measured by Oil Red O staining (ORO staining).

Results

KHB and KHP increase anti-oxidative activity related protein levels including Nrf2 and HO-1. Furthermore, KHB and KHP inhibit lipid accumulation on intracellular levels through induction of ATP binding receptor cassette subfamily A member 1 (ABCA1) and scavenging receptor class B member 1 (SR-B1), respectively. Finally, KHB and KHP also blocked pro-inflammatory mediators including tumor necrosis factor-alpha (TNFα) and interleukin-6 (IL-6), iNOS and COX-2 expression.

Conclusion

This study suggests that KHB and KHP potently regulate the M1/M2 macrophage polarization.

Keywords: Geum-Gwe-Yo-Ryak; Kwaruhaebaekbanha-tang; Kwaruhaebaekpaekju-tang; Macrophage polarization
Fig. 1

The effect of KHB (A) and KHP (B) on cell viability in human monocytic THP-1 cell line. Cell viability was determined using the cell viability assay kits (Ez-CyTox). All data represent the means±SD of three different experiments.

Fig. 2

The effect of KHB and KHP on expression of CD86 levels in M1 polarized THP-1 cells. Flow cytometry analysis (A) and CD86 population levels (B) on IFNγ (20ng/ml) and LPS (100ng/ml)-stimulated THP-1 cells. All data represent the means±SD of three different experiments. #P < 0.05 compared to untreated cells, and asterisk indicates statistically significant difference between polarized group (*< 0.05).

Fig. 3

The effect of KHB and KHP on antioxidant-related protein expressions including Nrf2 (A) and HO-1 (B). All data represent the means±SD of three different experiments. Asterisk indicates statistically significant difference between untreated group (* < 0.05).

Fig. 4

The effect of KHB and KHP on oxi-LDL-stimulated lipid accumulation. Microscopic images (A) and resultant solution were measured at 520 nm under microplate reader (B). Rever cholesterol-related genes expression were detected by quantitative real-time PCR including ABCA (C) and SR-B1 (D). All data represent the means ±SD of three different experiments. #P < 0.05, ##P < 0.01 compared to oxi-LDL untreated cells. Asterisk indicates statistically significant difference between ox-LDL-stimulated group (* < 0.05, ** < 0.01).

Fig. 5

The effect of KHB and KHP on anti-inflammation-related mediator expressions including TNF-α (A), IL-6 (B), iNOS (C) and COX-2 (D). #P < 0.05, ##P < 0.01 compared to oxi-LDL untreated cells. Asterisk indicates statistically significant difference between ox-LDL-stimulated group (* < 0.05, ** < 0.01).

The primer sequences of reverse cholesterol transport-related genes for quantitative polymerase chain reaction.

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Article information Continued

Copyright © 2019 The Society of Korean Medicine all rights reserved

Fig. 1

Fig. 1

The effect of KHB (A) and KHP (B) on cell viability in human monocytic THP-1 cell line. Cell viability was determined using the cell viability assay kits (Ez-CyTox). All data represent the means±SD of three different experiments.

Fig. 2

Fig. 2

The effect of KHB and KHP on expression of CD86 levels in M1 polarized THP-1 cells. Flow cytometry analysis (A) and CD86 population levels (B) on IFNγ (20ng/ml) and LPS (100ng/ml)-stimulated THP-1 cells. All data represent the means±SD of three different experiments. #P < 0.05 compared to untreated cells, and asterisk indicates statistically significant difference between polarized group (*< 0.05).

Fig. 3

Fig. 3

The effect of KHB and KHP on antioxidant-related protein expressions including Nrf2 (A) and HO-1 (B). All data represent the means±SD of three different experiments. Asterisk indicates statistically significant difference between untreated group (* < 0.05).

Fig. 4

Fig. 4

The effect of KHB and KHP on oxi-LDL-stimulated lipid accumulation. Microscopic images (A) and resultant solution were measured at 520 nm under microplate reader (B). Rever cholesterol-related genes expression were detected by quantitative real-time PCR including ABCA (C) and SR-B1 (D). All data represent the means ±SD of three different experiments. #P < 0.05, ##P < 0.01 compared to oxi-LDL untreated cells. Asterisk indicates statistically significant difference between ox-LDL-stimulated group (* < 0.05, ** < 0.01).

Fig. 5

Fig. 5

The effect of KHB and KHP on anti-inflammation-related mediator expressions including TNF-α (A), IL-6 (B), iNOS (C) and COX-2 (D). #P < 0.05, ##P < 0.01 compared to oxi-LDL untreated cells. Asterisk indicates statistically significant difference between ox-LDL-stimulated group (* < 0.05, ** < 0.01).

Table 1

The primer sequences of reverse cholesterol transport-related genes for quantitative polymerase chain reaction.

Primer Sequence
ABCA1 Forward 5′-ACCAGTTTGTTTGTGGCCCTTTTG-3′
Reverse 5′-AGTTCCAGGCTGGGGTACTT-3′
SR-B1 Forward 5′-CTGTGGGTGAGATCATGTGG-3′
Reverse 5′-GCCAGAAGTCAACCTTGCTC-3′
GAPDH Forward 5′-GGCCTCCAAGGAGTAAGACC-3′
Reverse 5′-AGGGGTCTACATGGCAACTG-3′