Anti-inflammatory effects of two lupane-type triterpenes from leaves of <i>Acanthopanax gracilistylus</i> on LPS-induced RAW264.7 macrophages

Acanthopanax gracilistylus W.W.Smith (AGS) is an traditional Chinese medicine and its leaves that have rich biological activities has been long used as a fresh vegetable in Chinese folk. There are two lupane-type triterpenoids, 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid (1) and 3α,11α,23-trihydroxy-lup-20(29)-en-28-oic acid (2), were isolated as the active constituents from leaves of AGS. However, the anti-inflammatory effects and potential mechanisms of 1 and 2 on LPS-induced RAW264.7 Macrophages have not been evaluated. In this study, the results shown that compounds 1 and 2 reduced the levels of early pro-inflammatory cytokines TNF-α, IL-1β, the secretion of late pro-inflammatory cytokine HMGB1, as well as the activation of transcription factor NF-κB in RAW 264.7 macrophages induced by LPS. In conclusion, two lupane-type triterpenes (1 and 2) have anti-inflammatory activities in LPS-induced RAW264.7 cells through inhibiting expression of proinflammatory cytokines and NF‐κB activation and could be potentially used in treatment of inflammatory-related diseases in the future.

Keywords:
Acanthopanax gracilistylus W.W. Smith; lupane-type triterpenes; RAW 264.7 macrophages; proinflammatory cytokines; HMGB1; NF-κB

Authorship

Jiao LUO

School of Pharmacy, Hunan University of Chinese Medicine, Changsha, ChinaHunan University of Chinese MedicineChinaChangsha, ChinaSchool of Pharmacy, Hunan University of Chinese Medicine, Changsha, China

Xiao-jun LI

College of Pharmaceutical Sciences, Gannan Medical University, Ganzhou, Jiangxi, ChinaGannan Medical UniversityChinaGanzhou, Jiangxi, ChinaCollege of Pharmaceutical Sciences, Gannan Medical University, Ganzhou, Jiangxi, China

Geon-ho LEE

Kyunghee Korean Pharmacy, Kwanak goo, Seoul city, KoreaKyunghee Korean PharmacyKoreaKwanak goo, Seoul city, KoreaKyunghee Korean Pharmacy, Kwanak goo, Seoul city, Korea

Jian-jun HUANG

Department of Clinical Biochemistry, Xiangya Medical College, Central South University, Changsha, ChinaCentral South UniversityChinaChangsha, ChinaDepartment of Clinical Biochemistry, Xiangya Medical College, Central South University, Changsha, China

Wan-kyunn WHANG

College of Pharmacy, Chung-Ang University, Seoul, KoreaChung-Ang UniversityKoreaSeoul, KoreaCollege of Pharmacy, Chung-Ang University, Seoul, Korea

Xiao-dan ZHANG

College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, ChinaZhejiang Sci-Tech UniversityChinaHangzhou, ChinaCollege of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China

Chang-soo YOOK

College of Pharmacy, Kyung Hee University, Seoul, KoreaKyung Hee UniversityKoreaSeoul, KoreaCollege of Pharmacy, Kyung Hee University, Seoul, Korea

Xiang-qian LIU ^**Corresponding author: lxq0001cn@163.com

https://orcid.org/0000-0002-9713-0498

*Corresponding author: lxq0001cn@163.com

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication of this article.

Author contributions

Jiao LUO and Xiao-Jun LI contributed equally to this work.

SCIMAGO INSTITUTIONS RANKINGS

Kyunghee Korean Pharmacy, Kwanak goo, Seoul city, KoreaKyunghee Korean PharmacyKoreaKwanak goo, Seoul city, KoreaKyunghee Korean Pharmacy, Kwanak goo, Seoul city, Korea

College of Pharmacy, Chung-Ang University, Seoul, KoreaChung-Ang UniversityKoreaSeoul, KoreaCollege of Pharmacy, Chung-Ang University, Seoul, Korea

College of Pharmacy, Kyung Hee University, Seoul, KoreaKyung Hee UniversityKoreaSeoul, KoreaCollege of Pharmacy, Kyung Hee University, Seoul, Korea

Figures

Figures (5)

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Figure 1
Chemical structures of lupane-type triterpenes (1 and 2). 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid (1), 3α,11α,23-trihydroxy-lup-20(29)-en-28-oic acid (2).

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Figure 2
Cell viability in RAW264.7 macrophages treated with lupane-type triterpenes (1 and 2). RAW264.7 cells were exposed to 1 and 2 at the indicated concentrations for 48 h and evaluated by MTT-based EZ4U test. Cell viability of treated samples was expressed as a percentage of surviving cells compared with that in the vehicle control. Values are expressed as mean±S.D. of three individual experiments performed in triplicate. CON stands for vehicle control. *P<0.05 vs. the control group.

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Figure 3
Effects of lupane-type triterpenes (1 and 2) on LPS-induced TNF-α and IL-1β production and mRNA expression in RAW264.7 cells. (A, B) Effects of 1 and 2 on LPS-induced TNF-α (A) and IL-1β (B) production. The concentration of TNF-α and IL-1β secreted by RAW264.7 was measured by commercially available sandwich ELISA kits. Control (CON) values were obtained in the absence of LPS and compounds 1 and 2. LPS values were obtained in the presence of LPS (100 ng/mL) and absence of compounds 1 and 2. The positive control (DEX) was obtained in the presence of LPS (100 ng/mL) and Dexamethasone (10 μM). Three independent experiments were performed with similar results. Data is expressed as mean±S.D. Student’s t-test was used to determine significant differences between two groups. #P<0.05 vs. negative controls (CON); *P<0.05 vs. 100 ng/mL LPS-treated cells; (C, D) Effects of lupane-type triterpenes (1 and 2) on mRNA expression of TNF-α and IL-1β in LPS-stimulated macrophages. RAW 264.7 cells were pre-incubated with various concentrations of lupane-type triterpenes 1 and 2 for 1 h and were then treated with 100 ng/mL LPS for an additional 2 h. mRNA expression of TNF-α (C) and IL-1β (D) was analyzed by real-time RT-PCR and β-actin was used as the internal control.

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Figure 4
Effects of lupane-type triterpenes (1 and 2) on the expression of HMGB1 protein in LPS-induced RAW264.7 cells. (A) Effect of different concentrations of compound 1 on the expression of HMGB1 protein in the supernatant and quantitative densitometric analysis of HMGB1 protein in LPS-treated RAW264.7 cells. (B) Effect of different concentrations of compound 2 on the expression of HMGB1 protein in the supernatant and quantitative densitometric analysis of HMGB1 protein in LPS-treated RAW264.7 cells. Mean values ± SD (n = 3). *P < 0.05 compared with the LPS-treatment group; (C) Effects of 1 and 2 on the expression of HMGB1 protein in intracellular of LPS-induced RAW264.7 cells. The expression level of HMGB1 protein in cellular extracts was normalized by β-actin levels.

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Figure 5
Effects of lupane-type triterpenes (1 and 2) on NF-κB transcriptional activity in LPS-induced RAW264.7 cells. Lupane-type triterpenes 1(A) and 2 (B) reduces NF-κB activity in a dose-dependent manner. RAW264.7 cells were stimulated with or without LPS (100 ng/mL) in the absence or presence of lupane-type triterpenes (1 and 2) (10, 20, 30, 40 or 50 μM) for 2 h. Nuclear proteins were extracted, and NF-κB activity was determined by NF-κB p65 ELISA kit (Cell Signaling Technology, Inc). Control (CON) values were obtained in the absence of LPS and lupane-type triterpenes (1 and 2). LPS values were obtained in the presence of LPS (100 ng/mL) and absence of lupane-type triterpenes (1 and 2). Mean values ±SD (n= 3). #P<0.05 compared with the control group. *P<0.05 compared with the LPS-treatment group.

Figure 1 Chemical structures of lupane-type triterpenes (1 and 2). 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid (1), 3α,11α,23-trihydroxy-lup-20(29)-en-28-oic acid (2).

Figure 2 Cell viability in RAW264.7 macrophages treated with lupane-type triterpenes (1 and 2). RAW264.7 cells were exposed to 1 and 2 at the indicated concentrations for 48 h and evaluated by MTT-based EZ4U test. Cell viability of treated samples was expressed as a percentage of surviving cells compared with that in the vehicle control. Values are expressed as mean±S.D. of three individual experiments performed in triplicate. CON stands for vehicle control. *P<0.05 vs. the control group.

Figure 3 Effects of lupane-type triterpenes (1 and 2) on LPS-induced TNF-α and IL-1β production and mRNA expression in RAW264.7 cells. (A, B) Effects of 1 and 2 on LPS-induced TNF-α (A) and IL-1β (B) production. The concentration of TNF-α and IL-1β secreted by RAW264.7 was measured by commercially available sandwich ELISA kits. Control (CON) values were obtained in the absence of LPS and compounds 1 and 2. LPS values were obtained in the presence of LPS (100 ng/mL) and absence of compounds 1 and 2. The positive control (DEX) was obtained in the presence of LPS (100 ng/mL) and Dexamethasone (10 μM). Three independent experiments were performed with similar results. Data is expressed as mean±S.D. Student’s t-test was used to determine significant differences between two groups. #P<0.05 vs. negative controls (CON); *P<0.05 vs. 100 ng/mL LPS-treated cells; (C, D) Effects of lupane-type triterpenes (1 and 2) on mRNA expression of TNF-α and IL-1β in LPS-stimulated macrophages. RAW 264.7 cells were pre-incubated with various concentrations of lupane-type triterpenes 1 and 2 for 1 h and were then treated with 100 ng/mL LPS for an additional 2 h. mRNA expression of TNF-α (C) and IL-1β (D) was analyzed by real-time RT-PCR and β-actin was used as the internal control.

Figure 4 Effects of lupane-type triterpenes (1 and 2) on the expression of HMGB1 protein in LPS-induced RAW264.7 cells. (A) Effect of different concentrations of compound 1 on the expression of HMGB1 protein in the supernatant and quantitative densitometric analysis of HMGB1 protein in LPS-treated RAW264.7 cells. (B) Effect of different concentrations of compound 2 on the expression of HMGB1 protein in the supernatant and quantitative densitometric analysis of HMGB1 protein in LPS-treated RAW264.7 cells. Mean values ± SD (n = 3). *P < 0.05 compared with the LPS-treatment group; (C) Effects of 1 and 2 on the expression of HMGB1 protein in intracellular of LPS-induced RAW264.7 cells. The expression level of HMGB1 protein in cellular extracts was normalized by β-actin levels.

Figure 5 Effects of lupane-type triterpenes (1 and 2) on NF-κB transcriptional activity in LPS-induced RAW264.7 cells. Lupane-type triterpenes 1(A) and 2 (B) reduces NF-κB activity in a dose-dependent manner. RAW264.7 cells were stimulated with or without LPS (100 ng/mL) in the absence or presence of lupane-type triterpenes (1 and 2) (10, 20, 30, 40 or 50 μM) for 2 h. Nuclear proteins were extracted, and NF-κB activity was determined by NF-κB p65 ELISA kit (Cell Signaling Technology, Inc). Control (CON) values were obtained in the absence of LPS and lupane-type triterpenes (1 and 2). LPS values were obtained in the presence of LPS (100 ng/mL) and absence of lupane-type triterpenes (1 and 2). Mean values ±SD (n= 3). #P<0.05 compared with the control group. *P<0.05 compared with the LPS-treatment group.

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Anti-inflammatory effects of two lupane-type triterpenes from leaves of <i>Acanthopanax gracilistylus</i> on LPS-induced RAW264.7 macrophages

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[1] *Corresponding author: lxq0001cn@163.com