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Scutellarin Exerts Anti-Inflammatory Effects in Activated Microglia/Brain Macrophage in Cerebral Ischemia and in Activated BV-2 Microglia Through Regulation of MAPKs Signaling Pathway

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Abstract

Background

Scutellarin, an herbal compound, can effectively suppress the inflammatory response in activated microglia/brain macrophage(AM/BM) in experimentally induced cerebral ischemia; however, the underlying mechanism for this has not been fully clarified. We sought to elucidate if scutellarin would exert its anti-inflammatory effects on AM/BM through the MAPKs pathway.

Materials and Methods

Western blot and immunofluorescence labeling were used to determine the expression of the MAPKs pathway in AM/BM in rats subjected to middle cerebral artery occlusion (MCAO) also in lipopolysaccharide (LPS)-activated BV-2 microglia in vitro. Furthermore, expression of p-p38 along with that of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta(IL-1β), and inducible nitric oxide synthase (iNOS) in LPS-activated microglia subjected to pretreatment with p38 inhibitor SB203580, p38 activator sc-201214, scutellarin, or a combination of them was evaluated.

Findings

Scutellarin markedly attenuated the expression of p-p38, p-JNK in AM/BM in MCAO rats and in vitro. Conversely, p-ERK1/2 expression level was significantly increased by scutellarin. Meanwhile, scutellarin suppressed the expression of proinflammatory mediators including iNOS, TNF-α, and IL-1β in AM/BM. More importantly, SB203580 suppressed p-p38 protein expression level in LPS-activated BV-2 microglia that was coupled with decreased expression of proinflammatory mediators (TNF-α, iNOS) in LPS-activated BV-2 microglia. However, p38 activator sc-201214 increased expression of proinflammatory mediators TNF-α, iNOS, and IL-1β. Interestingly, the decreased expression of both proinflammatory markers by p38 MAPK inhibitor and increased expression of proinflammatory markers by p38 MAPK activator were compatible with that in BV-2-activated microglia pretreated with scutellarin.

Conclusions

The results suggest that scutellarin down-regulates the expression of proinflammatory mediators in AM/BM through suppressing the p-JNK and p-p38 MAPKs. Of note, the anti-inflammatory effect of p38 MAPK inhibitor and scutellarin is comparable. Besides, p38 MAPKs activator reverses the effect of scutellarin. Additionally, scutellarin increases p-ERK1/2 expression that may be neuroprotective.

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Abbreviations

BCA:

Bicinchoninic acid

CNS:

Central nervous system

COX-2:

Cyclooxygenase-2

DAPI:

4′, 6-diamidino-2-phenylindole

DMEM:

Dulbecco’s modified Eagle’s medium

ERK1/2:

Extracellular signal-regulated kinase1/2

FCS:

Fetal calf serum

GEB:

Gastrodia elata Blume

GSK-3β:

Glycogen synthase kinase-3 beta

HRP:

Horseradish peroxidase

IL-1β:

Interleukin-1 beta

iNOS:

Inducible nitric oxide synthase

JNK:

c-Jun N-terminal kinase

LPS:

Lipopolysaccharide

MAPKs:

Mitogen-activated protein kinases

MA-5:

Mitochonic acid 5

MCAO:

Middle cerebral artery occlusion

NMDA:

N-methyl-d-aspartate

PBS:

Phosphate buffered saline

p-JNK:

Phosphorylated c-Jun N-terminal kinase

p38 MAPK:

p38 mitogen-activated protein kinase

p-p38:

Phosphorylated p38

p-ERK1/2:

Phosphorylated extracellular signal-regulated kinase1/2

PVDF:

Polyvinylidene difluoride

SD rats:

Sprague-Dawley rats

TNF-α:

Tumor necrosis factor-alpha

AM/BM:

Activated microglia/brain macrophage

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Acknowledgements

This study was supported by National Natural Science Foundation of China (Project Number 31760297, Y Yuan), Applied Basic Research Projects of Yunnan Province (Project Number 2018FE001(-189)). It was also supported by Applied Basic Research Program Key Projects of Yunnan Province (Project Number 2015FA020, C-Y Wu), National Natural Science Foundation of China (Project Number 31260254, C-Y Wu).

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Author notes

  1. Hao-Lun Chen and Wen-Ji Jia have contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Histology/Embryology, School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Kunming, 650500, People’s Republic of China

    Hao-Lun Chen, Hong-E Li, Hong Han, Juan-Juan Li, Yun Yuan & Chun-Yun Wu

  2. Department of Neurology, No. 2 Affiliated Hospital, Kunming Medical University, 374 Dianmian Road, Kunming, 650101, People’s Republic of China

    Wen-Ji Jia

  3. Department of Experiment Center for Medical Science Research, Kunming Medical University, 1168 West Chunrong Road, Kunming, 650500, People’s Republic of China

    Fan Li

  4. Department of Pain Management, No1 Affiliated Hospital, Kunming Medical University, 295 Xichang Roda, Kunming, 650032, People’s Republic of China

    Xiao-Li-Na Zhang

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  1. Hao-Lun Chen
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12017_2019_8582_MOESM1_ESM.tif

Supplementary Fig. 1 ERK2 immunofluorescence in activated microglia in MCAO rats given scutellarin treatment was not noticeably changed. Confocal images ERK2 immunofluorescence (red) was hardly detected in lectin positive AM/BM (green) in MCAO rats. Note that it is comparable to that of the same cells following scutellarin pretreatment. DAPI–blue. Scale bars = 20μm. Supplementary Material 1 (TIFF 1386 kb)

12017_2019_8582_MOESM2_ESM.tif

Supplementary Fig. 2 p38 and JNK immunofluorescence remained relatively unchanged in activated AM/BM in MCAO rats given scutellarin treatment. Confocal images showing p38 and JNK expression (red) was hardly detected in lectin positive AM/BM (green) in MCAO rat brain. Note it is comparable to that in AM/BM in MCAO rats with scutellarin treatment. DAPI–blue. Scale bars = 20μm. Supplementary Material 2 (TIFF 21040 kb)

12017_2019_8582_MOESM3_ESM.tif

Supplementary Fig. 3 Scutellarin pretreatment did not affect p38, JNK and ERK2 expression in LPS-activated BV-2 microglia. Immunofluorescence labeling and bar graphs depicting p38, JNK and ERK2 expression in LPS-activated BV-2 microglia and those with LPS + scutellarin pretreatment was comparable to that of the control BV-2 cells. DAPI – blue. Scale bars = 50μ. Supplementary Material 3 (TIFF 1126 kb)

12017_2019_8582_MOESM4_ESM.tif

Supplementary Fig. 4 Protein expression of p-p38 and p-JNK was decreased but that of p-ERK1/2 was up-regulated in MCAO 1d rat given scutellarin treatment. Western blot shows the expression level of p-p38 and p-JNK in MCAO 1d was depressed significantly at 1 day; however, the expression of p-ERK1/2 was obviously increased following treatment with scutellarin when compared with the MCAO 1d rats not treated with scutellarin. * and # represent significant differences in protein levels. P < 0.05; * when sham group is compared with MCAO 1d; # when MCAO 1d group is compared with MCAO+S 1d group. The values represent the mean ± SD in triplicate. Supplementary material 4 (TIFF 13685 kb)

12017_2019_8582_MOESM5_ESM.tif

Supplementary Fig. 5 MCAO 1d rats treated with scutellarin showed reduced p-p38 and p-JNK expression in AM/BM. Confocal images showing p-p38 and p-JNK immunofluorescence (red) in lectin positive AM/BM (green) of MCAO 1d rats and MCAO 1d rats given scutellarin treatment. A marked increase in p-p38 and p-JNK expression was evident in the AM/BM (b2-b3) in MCAO 1d rat brain; however, it was noticeably attenuated in AM/BM(c2-c3) by scutellarin treatment. Bar graph shows increased immunofluorescence in MCAO 1d rats was suppressed by scutellarin. DAPI–blue. Scale bar=75μm. * and # represent significant differences. (P < 0.05); * when sham group is compared with MCAO 1d; # when MCAO 1d group is compared with MCAO+S 1d group. The values represent the mean ± SD in triplicate. Supplementary Material 5 (TIFF 6898 kb)

12017_2019_8582_MOESM6_ESM.tif

Supplementary Fig. 6 MCAO 1d rats given scutellarin treatment showed up-regulated p-ERK1/2 expression in activated AM/BM. Confocal images showing p-ERK1/2 expression (red) in lectin positive AM/BM (green) in MCAO 1d rats (b2-b3) and following treatment with scutellarin (c2-c3). Increase in p-ERK1/2 expression was evident in the AM/BM (b3) in MCAO 1d rat. Note p-ERK1/2 expression was further augmented in AM/BM (c3) at 1 days following treatment with scutellarin. Bar graphs show expression changes of p-ERK1/2. Note its suppression in scutellarin treatment group. DAPI - blue. Scale bar = 75μm. * and # represent significant differences. (P < 0.05); * when sham group is compared with MCAO 1d; # when MCAO 1d group is compared with MCAO+S 1d group. The values represent the mean ± SD in triplicate. Supplementary Material 6 (TIFF 2392 kb)

12017_2019_8582_MOESM7_ESM.tif

Supplementary Fig. 7 Protein expression of p-p38 and p-JNK was decreased but that of p-ERK1/2 was up-regulated in MCAO 7d rat given scutellarin treatment. Western blot shows the expression level of p-p38 and p-JNK in MCAO 7d was depressed significantly at 7 day; however, the expression of p-ERK1/2 was obviously increased following treatment with scutellarin when compared with the MCAO 7d rats not treated with scutellarin. * and # represent significant differences in protein levels. P < 0.05; * when sham group is compared with MCAO 7d; # when MCAO 7d group is compared with MCAO+S 7d group. The values represent the mean ± SD in triplicate. Supplementary Material 7 (TIFF 13530 kb)

12017_2019_8582_MOESM8_ESM.tif

Supplementary Fig. 8 MCAO 7d rats treated with scutellarin showed reduced p-p38 and p-JNK expression in activated AM/BM. Confocal images showing p-p38 and p-JNK immunofluorescence (red) in lectin positive AM/BM (green) of MCAO 7d rats and MCAO 7d rats given scutellarin treatment. A marked increase in p-p38 and p-JNK expression was evident in the AM/BM (b2-b3) in MCAO 7d rat brain; however, it was noticeably attenuated in AM/BM(c2-c3) by scutellarin treatment. Bar graph shows increased immunofluorescence in MCAO 7d rats was suppressed by scutellarin. DAPI–blue. Scale bar = 75μm. * and # represent significant differences. (P < 0.05); * when sham group is compared with MCAO 7d; # when MCAO 7d group is compared with MCAO+S 7d group. The values represent the mean ± SD in triplicate. Supplementary Material 8 (TIFF 6421 kb)

12017_2019_8582_MOESM9_ESM.tif

Supplementary Fig. 9 MCAO 7d rats given scutellarin treatment showed up-regulated p-ERK1/2 expression in activated AM/BM. Confocal images showing p-ERK1/2 expression (red) in lectin positive AM/BM (green) in MCAO 7d rats (b2-b3) and following treatment with scutellarin (c2-c3). Increase in p-ERK1/2 expression was evident in the AM/BM (b3) in MCAO 7d rat. Note p-ERK1/2 expression was further augmented in AM/BM (c3) at 7 days following treatment with scutellarin. Bar graphs show expression changes of p-ERK1/2. Note its suppression in scutellarin treatment group. DAPI - blue. Scale bar = 75μm. * and # represent significant differences. (P < 0.05); * when sham group is compared with MCAO 7d; # when MCAO 7d group is compared with MCAO+S 7d group. The values represent the mean ± SD in triplicate. Supplementary Material 9 (TIFF 3657 kb)

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Chen, HL., Jia, WJ., Li, HE. et al. Scutellarin Exerts Anti-Inflammatory Effects in Activated Microglia/Brain Macrophage in Cerebral Ischemia and in Activated BV-2 Microglia Through Regulation of MAPKs Signaling Pathway. Neuromol Med 22, 264–277 (2020). https://doi.org/10.1007/s12017-019-08582-2

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