Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway
Objective:To investigate the protective effect and the molecular mechanism of Pien Tze Huang (PZH) on Lipopolysaccharide (LPS) -induced neuro-inflammatory injury in BV2 microglial cells.Methods:BV2 microglial cells were cultured with RPMI-1640 medium supplemented with 10%fetal bovine serum and 1%pen...
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Editorial Office of Rehabilitation Medicine
2021-02-01
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| Series: | 康复学报 |
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| Online Access: | http://kfxb.publish.founderss.cn/thesisDetails#10.3724/SP.J.1329.2021.01007 |
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| author | Zhenwei HUANG Qing ZHANG Lili HUANG Xiaoqin ZHANG Mingqing HUANG |
| author_facet | Zhenwei HUANG Qing ZHANG Lili HUANG Xiaoqin ZHANG Mingqing HUANG |
| author_sort | Zhenwei HUANG |
| collection | DOAJ |
| description | Objective:To investigate the protective effect and the molecular mechanism of Pien Tze Huang (PZH) on Lipopolysaccharide (LPS) -induced neuro-inflammatory injury in BV2 microglial cells.Methods:BV2 microglial cells were cultured with RPMI-1640 medium supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin at 5%CO<sub>2</sub>and 37℃in a humidified incubator. BV2 microglial cells were seeded at a density of 5×10<sup>4</sup>cells/mL in 96-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. And then its absorbance at 490 nm was detected after the treatment of MTT for 4 h. BV2 microglial cells were seeded at a density of 5×10<sup>4</sup>cells/mL in 24-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Then supernatant was collected and the concentration of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA) kit. BV2 microglial cells were seeded at a density of 1.6×10<sup>5</sup>cells/mL in 6-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Total RNA was extracted using TRIzol reagent and it was reverse-transcribed to synthesize the complementary DNA. And then the mRNA expression of IL-1β, IL-6 and TNF-α were determined using real time-qPCR. BV2 microglial cells were seeded at a density of 1.6×10<sup>5</sup>cells/mL in 6-well plate. The protein extracts were isolated from each group of cells using RIPA protein lysis buffer after the administration of LPS(100 ng/mL) and different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Then the protein expression of TLR4, ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, COX-2, iNOS in BV2 microglial cells were detected by Western blot.Results:1) The result of MTT assay showed that PZH at 0.05-0.15 mg/mL concentration and LPS at 100 ng/mL concentration had no significant cytotoxicity on BV2 microglial cells (<italic>P</italic>> 0.05).2) The result of ELISA showed that the secretion of IL-6 was significantly increased in BV2 microglial cells induced by LPS compared with the normal control group (<italic>P</italic>< 0.001); compared with the LPS group, different concentrations of PZH significantly decreased the expression level of IL-6 in LPS-induced BV2 microglial cells (<italic>P</italic>< 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.01).3) The result of RT-qPCR showed that the transcriptional levels of IL-1β(<italic>P</italic>< 0.001), IL-6(<italic>P</italic>< 0.001) and TNF-α(<italic>P</italic>< 0.01) were significantly increased after the induction of LPS compared with normal controal group; different concentrations of PZH significantly inhibited the LPS-induced IL-1β(<italic>P</italic>< 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001), IL-6(<italic>P</italic>> 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) and TNF-α(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) mRNA upregulation.4) The result of Western blot showed that PZH significantly inhibited the LPS-induced TLR4/MAPK signaling pathway activation, and decreased the protein expressions of TLR4(<italic>P</italic>> 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.05), p-ERK1/2(<italic>P</italic>> 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.01), p-p38(<italic>P</italic>< 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.01), COX-2(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) and iNOS(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.01), but not p-JNK.Conclusion:PZH can significantly improve the LPS-induced neuro-inflammatory injury, which may be related to the inhibition of the activation of TLR4/MAPK signaling pathway. |
| format | Article |
| id | doaj-art-2a0d0f502e5f4ffe92a0e65e4edace4e |
| institution | Kabale University |
| issn | 2096-0328 |
| language | English |
| publishDate | 2021-02-01 |
| publisher | Editorial Office of Rehabilitation Medicine |
| record_format | Article |
| series | 康复学报 |
| spelling | doaj-art-2a0d0f502e5f4ffe92a0e65e4edace4e2025-01-14T10:06:58ZengEditorial Office of Rehabilitation Medicine康复学报2096-03282021-02-0131445123133326Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling PathwayZhenwei HUANGQing ZHANGLili HUANGXiaoqin ZHANGMingqing HUANGObjective:To investigate the protective effect and the molecular mechanism of Pien Tze Huang (PZH) on Lipopolysaccharide (LPS) -induced neuro-inflammatory injury in BV2 microglial cells.Methods:BV2 microglial cells were cultured with RPMI-1640 medium supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin at 5%CO<sub>2</sub>and 37℃in a humidified incubator. BV2 microglial cells were seeded at a density of 5×10<sup>4</sup>cells/mL in 96-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. And then its absorbance at 490 nm was detected after the treatment of MTT for 4 h. BV2 microglial cells were seeded at a density of 5×10<sup>4</sup>cells/mL in 24-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Then supernatant was collected and the concentration of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA) kit. BV2 microglial cells were seeded at a density of 1.6×10<sup>5</sup>cells/mL in 6-well plate. The inflammatory response in BV2 microglial cells was induced by LPS(100 ng/mL), while the cells were treated with different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Total RNA was extracted using TRIzol reagent and it was reverse-transcribed to synthesize the complementary DNA. And then the mRNA expression of IL-1β, IL-6 and TNF-α were determined using real time-qPCR. BV2 microglial cells were seeded at a density of 1.6×10<sup>5</sup>cells/mL in 6-well plate. The protein extracts were isolated from each group of cells using RIPA protein lysis buffer after the administration of LPS(100 ng/mL) and different concentrations of PZH(0.05, 0.10 and 0.15 mg/mL) for 12 h. Then the protein expression of TLR4, ERK1/2, p-ERK1/2, p38, p-p38, JNK, p-JNK, COX-2, iNOS in BV2 microglial cells were detected by Western blot.Results:1) The result of MTT assay showed that PZH at 0.05-0.15 mg/mL concentration and LPS at 100 ng/mL concentration had no significant cytotoxicity on BV2 microglial cells (<italic>P</italic>> 0.05).2) The result of ELISA showed that the secretion of IL-6 was significantly increased in BV2 microglial cells induced by LPS compared with the normal control group (<italic>P</italic>< 0.001); compared with the LPS group, different concentrations of PZH significantly decreased the expression level of IL-6 in LPS-induced BV2 microglial cells (<italic>P</italic>< 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.01).3) The result of RT-qPCR showed that the transcriptional levels of IL-1β(<italic>P</italic>< 0.001), IL-6(<italic>P</italic>< 0.001) and TNF-α(<italic>P</italic>< 0.01) were significantly increased after the induction of LPS compared with normal controal group; different concentrations of PZH significantly inhibited the LPS-induced IL-1β(<italic>P</italic>< 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001), IL-6(<italic>P</italic>> 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) and TNF-α(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) mRNA upregulation.4) The result of Western blot showed that PZH significantly inhibited the LPS-induced TLR4/MAPK signaling pathway activation, and decreased the protein expressions of TLR4(<italic>P</italic>> 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.05), p-ERK1/2(<italic>P</italic>> 0.05, <italic>P</italic>< 0.05, <italic>P</italic>< 0.01), p-p38(<italic>P</italic>< 0.05, <italic>P</italic>< 0.01, <italic>P</italic>< 0.01), COX-2(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.001) and iNOS(<italic>P</italic>< 0.01, <italic>P</italic>< 0.01, <italic>P</italic>< 0.01), but not p-JNK.Conclusion:PZH can significantly improve the LPS-induced neuro-inflammatory injury, which may be related to the inhibition of the activation of TLR4/MAPK signaling pathway.http://kfxb.publish.founderss.cn/thesisDetails#10.3724/SP.J.1329.2021.01007neuro-inflammatory injuryPien Tze HuangBV2 microglial cellsTLR4/MAPK signaling pathway |
| spellingShingle | Zhenwei HUANG Qing ZHANG Lili HUANG Xiaoqin ZHANG Mingqing HUANG Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway 康复学报 neuro-inflammatory injury Pien Tze Huang BV2 microglial cells TLR4/MAPK signaling pathway |
| title | Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway |
| title_full | Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway |
| title_fullStr | Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway |
| title_full_unstemmed | Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway |
| title_short | Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway |
| title_sort | pien tze huang attenuates neuro inflammatory injury in bv2 microglial cells induced by lipopolysaccharide via inhibition of tlr4 mapk signaling pathway |
| topic | neuro-inflammatory injury Pien Tze Huang BV2 microglial cells TLR4/MAPK signaling pathway |
| url | http://kfxb.publish.founderss.cn/thesisDetails#10.3724/SP.J.1329.2021.01007 |
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