Pien Tze Huang Attenuates Neuro-inflammatory Injury in BV2 Microglial Cells Induced by Lipopolysaccharide via Inhibition of TLR4/MAPK Signaling Pathway

Abstract

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%CO2 and 37℃in a humidified incubator.BV2 microglial cells were seeded at a density of 5×104 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 5×104 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 1.6×105 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-αwere determined using real time-qPCR.BV2 microglial cells were seeded at a density of 1.6×105 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(P>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(P<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(P<0.05,P<0.05,P<0.01).3)The result of RT-qPCR showed that the transcriptional levels of IL-1β(P<0.001),IL-6(P<0.001)and TNF-α(P<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(P<0.05,P<0.01,P<0.001),IL-6(P>0.05,P<0.01,P<0.001)and TNF-α(P<0.01,P<0.01,P<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(P>0.05,P<0.05,P<0.05),p-ERK1/2(P>0.05,P<0.05,P<0.01),p-p38(P<0.05,P<0.01,P<0.01),COX-2(P<0.01,P<0.01,P<0.001)and iNOS(P<0.01,P<0.01,P<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.