Anti-neuroinflammatory activity of <i>Humulus japonicus</i> extract and its active compound luteolin on lipopolysaccharide-induced inflammatory response in SIM-A9 microglia via NF-κB and MAPK signaling pathways

Humulus japonicus (HJ) is administered to patients with pulmonary disease and skin disease owing to its anti-inflammatory and antioxidant effects. Lately, HJ has been shown to exert impacts on protection against neurodegenerative sickness. Nevertheless, its impacts on microglia have not been discovered in depth. As a result, anti-inflammatory impacts of HJ ethanol extract (HJE) and its active compound luteolin on lipopolysaccharide-stimulated SIM-A9 cells were investigated. The results showed that HJE luteolin inhibited generation of interleukin-1β, tumor necrosis factor-α, nitric oxide, interleukin-6, together with prostaglandin E₂, iNOS, COX-2 along with HO-1 induction. Furthermore, HJE and luteolin reduced degrees of NF-κB pathway components within cytoplasm and nucleus, while reducing cytoplasmic MAPK levels and increasing Nrf2 levels in the cytoplasm and nucleus. In conclusion, our results demonstrate that HJE might exhibit neuroprotective effects and could be used as a novel drug for various neurological diseases.

Keywords:
Humulus japonicus; anti-neuroinflammation; NF-κB; MAPK; microglia

Authorship

Feng WANG

Department of Health Management, Jeonju University, Jeonju-si, Jeollabuk-do, Republic of KoreaJeonju UniversityRepublic of KoreaJeonju-si, Jeollabuk-do, Republic of KoreaDepartment of Health Management, Jeonju University, Jeonju-si, Jeollabuk-do, Republic of Korea

Department of Life Sciences, Yuncheng University, Yuncheng, Shanxi Province, PR, ChinaYuncheng UniversityPR, ChinaYuncheng, Shanxi Province, PR, ChinaDepartment of Life Sciences, Yuncheng University, Yuncheng, Shanxi Province, PR, China

Byoung Ok CHO

Institute of Health Science, Jeonju University, Jeonju-si, Jeollabuk-do, Republic of KoreaJeonju UniversityRepublic of KoreaJeonju-si, Jeollabuk-do, Republic of KoreaInstitute of Health Science, Jeonju University, Jeonju-si, Jeollabuk-do, Republic of Korea

Research Institute, Ato Q&A Co., LTD, Jeonju-si, Jeollabuk-do, Republic of Korea Ato Q&A Co., LTDRepublic of KoreaJeonju-si, Jeollabuk-do, Republic of KoreaResearch Institute, Ato Q&A Co., LTD, Jeonju-si, Jeollabuk-do, Republic of Korea

Jae Young SHIN

Suping HAO

Seon Il JANG ^**Corresponding author: sonjjang@jj.ac.kr

https://orcid.org/0000-0001-6259-5124

*Corresponding author: sonjjang@jj.ac.kr

Conflict of interest

The authors declare no conflict of interests.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (6)

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Figure 1
The cell viability of Humulus japonicus extract (HJE) and Luteolin toward microglial cells. Microglial cells (5 ×10⁵ cells/mL) were cultivated and disposed by HJE solely at mentioned concentrations (A), HJE at the indicated concentrations and disposed 2 µg/mL LPS (B), Luteolin alone at indicated concentrations (C), and Luteolin at 0, 15, 30, or 60 μM and treated 2 µg/mL LPS (D) lasting twenty-four hours before cell viability research conduction. Outcomes are displayed with the mean ± standard deviation (n = 3). ^###P < .001 versus the control group.

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Figure 2
The effects of HJE and luteolin NO production (A), iNOS expression levels (B), PGE₂ generation (C), and COX2 expression degrees (D) in LPS-stimulated microglia cells. Microglial cells were disposed by HJE (25 or 50 µg/mL), Luteolin (30 µM) lasting one hour,after that stimulated with LPS (2 µg/mL) lasting twenty-four hours. The measurement of NO and PGE₂ production degrees in cell culture supernatants was conducted through Griess assay kit and ELISA kit. The iNOS and COX2 expression degrees were discovered through western blot analysis of whole-cell protein extract. Error bars stand for means ±SD; ^###P < .001 versus the control group; **P < .01, ***P < .001 versus only LPS team. Results from three researches of independence are symbolic.

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Figure 3
Impacts of HJE and luteolin on IL-1β(A), TNF-α(B) along with IL-6 (C) production in the microglial cells stimulated by LPS. Cells were pretreated with HJE (25 or 50 µg/mL), Luteolin (30 µM) and stimulated with LPS (2 µg/mL). Microglial cells were disposed by HJE (25 or 50 µg/mL), Luteolin (30 µM) lasting 1 h,and subsequently stimulated through 2 µg/mL LPS for one day. We made the collection of culture media supernatants and utilized for ELISA. Error bars stand for means ±SD; ^###P < .001 versus the control group; **P < .01, and ***P < .001 versus only LPS team. Outcomes from three researches of independence are symbolic.

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Figure 4
HJE and luteolin regulate the activation of NF-κB signal pathways (A), inhibited the nuclear translocation of NF-κB (B), on AKT expression levels (C)in the microglial cells induced by LPS. Microglial cells (5×10⁵ cells/mL) were cultivated and disposed by HJE or luteolin in advance and then stimulated with LPS lasting thirty minutes. The protein expression degrees of cell signaling kinases along with activated NF-κB from nuclear extract and AKT were discovered through western blot assays and the analysis of band densities was conducted with the assistance of ImageJ analysis software and normalized to β-actin and Lamin b degrees. Error bars represent the means ±SD; ^###P < .001 versus control team; **P < .01, and ***P < .001 versus only LPS team. Outcomes from three researches of independence are symbolic.

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Figure 5
HJE and luteolin control the MAPK signal pathways activation in microglial activation induced by LPS. 5×10⁵ cells/mL of microglial cells were cultivated and disposed by HJE or luteolin (A) in advance and then stimulated with LPS lasting thirty minutes. The protein expression degrees of cell signaling kinases were discovered through western blot assays and the analysis of band densities was conducted with the assistance of ImageJ analysis software and normalized to the β-actin levels (B-D). Error bars represent the means ±SD; ^###P < .001 versus control team; **P < .01, and ***P < .001 vs. only LPS group. Outcomes are representative of three independent experiments.

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Figure 6
HJE and luteolin regulate the activation of Nrf2 signal pathways (A), increased the nuclear translocation of Nrf2 (B), and HO-1 expression levels (C) in the microglial activation induced via LPS. Microglial cells (5 × 10⁵ cells/mL) were cultivated and disposed by HJE or luteolin in advance for 1 hour and subsequently stimulated through LPS for half an hour. The levels of protein expression of Nrf2 in the cytosol, nuclear extract and HO-1 expression levels were discovered through western blot assay and the analysis of band densities was conducted with the assistance of ImageJ analysis software, with respect to β-actin and Lamin b. Outcomes stand for mean ±SD. ^##P < .01, and ^###P < .001 versus the control team. **P < .01, and ***P < .001 versus only LPS group. Outcomes from three researches of independence are symbolic.

Figure 1 The cell viability of Humulus japonicus extract (HJE) and Luteolin toward microglial cells. Microglial cells (5 ×10⁵ cells/mL) were cultivated and disposed by HJE solely at mentioned concentrations (A), HJE at the indicated concentrations and disposed 2 µg/mL LPS (B), Luteolin alone at indicated concentrations (C), and Luteolin at 0, 15, 30, or 60 μM and treated 2 µg/mL LPS (D) lasting twenty-four hours before cell viability research conduction. Outcomes are displayed with the mean ± standard deviation (n = 3). ^###P < .001 versus the control group.

Figure 2 The effects of HJE and luteolin NO production (A), iNOS expression levels (B), PGE₂ generation (C), and COX2 expression degrees (D) in LPS-stimulated microglia cells. Microglial cells were disposed by HJE (25 or 50 µg/mL), Luteolin (30 µM) lasting one hour,after that stimulated with LPS (2 µg/mL) lasting twenty-four hours. The measurement of NO and PGE₂ production degrees in cell culture supernatants was conducted through Griess assay kit and ELISA kit. The iNOS and COX2 expression degrees were discovered through western blot analysis of whole-cell protein extract. Error bars stand for means ±SD; ^###P < .001 versus the control group; P < .01, *P < .001 versus only LPS team. Results from three researches of independence are symbolic.

Figure 3 Impacts of HJE and luteolin on IL-1β(A), TNF-α(B) along with IL-6 (C) production in the microglial cells stimulated by LPS. Cells were pretreated with HJE (25 or 50 µg/mL), Luteolin (30 µM) and stimulated with LPS (2 µg/mL). Microglial cells were disposed by HJE (25 or 50 µg/mL), Luteolin (30 µM) lasting 1 h,and subsequently stimulated through 2 µg/mL LPS for one day. We made the collection of culture media supernatants and utilized for ELISA. Error bars stand for means ±SD; ^###P < .001 versus the control group; P < .01, and *P < .001 versus only LPS team. Outcomes from three researches of independence are symbolic.

Figure 4 HJE and luteolin regulate the activation of NF-κB signal pathways (A), inhibited the nuclear translocation of NF-κB (B), on AKT expression levels (C)in the microglial cells induced by LPS. Microglial cells (5×10⁵ cells/mL) were cultivated and disposed by HJE or luteolin in advance and then stimulated with LPS lasting thirty minutes. The protein expression degrees of cell signaling kinases along with activated NF-κB from nuclear extract and AKT were discovered through western blot assays and the analysis of band densities was conducted with the assistance of ImageJ analysis software and normalized to β-actin and Lamin b degrees. Error bars represent the means ±SD; ^###P < .001 versus control team; P < .01, and *P < .001 versus only LPS team. Outcomes from three researches of independence are symbolic.

Figure 5 HJE and luteolin control the MAPK signal pathways activation in microglial activation induced by LPS. 5×10⁵ cells/mL of microglial cells were cultivated and disposed by HJE or luteolin (A) in advance and then stimulated with LPS lasting thirty minutes. The protein expression degrees of cell signaling kinases were discovered through western blot assays and the analysis of band densities was conducted with the assistance of ImageJ analysis software and normalized to the β-actin levels (B-D). Error bars represent the means ±SD; ^###P < .001 versus control team; P < .01, and *P < .001 vs. only LPS group. Outcomes are representative of three independent experiments.

Figure 6 HJE and luteolin regulate the activation of Nrf2 signal pathways (A), increased the nuclear translocation of Nrf2 (B), and HO-1 expression levels (C) in the microglial activation induced via LPS. Microglial cells (5 × 10⁵ cells/mL) were cultivated and disposed by HJE or luteolin in advance for 1 hour and subsequently stimulated through LPS for half an hour. The levels of protein expression of Nrf2 in the cytosol, nuclear extract and HO-1 expression levels were discovered through western blot assay and the analysis of band densities was conducted with the assistance of ImageJ analysis software, with respect to β-actin and Lamin b. Outcomes stand for mean ±SD. ^##P < .01, and ^###P < .001 versus the control team. P < .01, and *P < .001 versus only LPS group. Outcomes from three researches of independence are symbolic.

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Anti-neuroinflammatory activity of <i>Humulus japonicus</i> extract and its active compound luteolin on lipopolysaccharide-induced inflammatory response in SIM-A9 microglia via NF-κB and MAPK signaling pathways

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[1] *Corresponding author: sonjjang@jj.ac.kr