Lycorine inhibited LPS-induced primary microglial inflammatory response via TLR4/NF-κB signaling pathway

doi:10.11958/20220323

Abstract

Abstract:

Objective To investigate the effect and potential mechanism of lycorine (LYC) on inflammatory response and phenotype of primary microglia induced by lipopolysaccharide (LPS). Methods The cultured primary microglia cells were divided into the control group, the LYC group (0.1, 0.5, 1 and 5 μmol/L LYC), the LPS group (0.1 mg/L LPS) and the LPS+LYC group (0.1 mg/L LPS+5 μmol/L LYC). The morphological changes of cells were observed under inverted microscope. Flow cytometry was used to detect the purity of primary microglia cells and the effect of LPS-induced cell proportion on the two phenotypes of primary microglia cells. Cell activity was detected by CCK-8 method. The mRNA expressions of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and microglia surface markers of different phenotypes were detected by real-time fluorescence quantitative PCR (qPCR). The content of nitric oxide (NO) was determined by Griess method. The expression levels of TLR4 and p-NF-κB p65 protein were detected by Western blot assay. Results The purity of primary microglia was over 95%. There was no significant difference in the cell activity between the control group and the LYC group. The concentration of LYC 5 μmol/L was used in subsequent experiments. Compared with LPS group, the number of amoeba-like microglia was significantly reduced in the LPS+LYC group, expression levels of IL-1β, IL-6, TNF-α mRNA and NO content decreased, the expression level of CD86 mRNA and phenotype ratio decreased, the expression level of CD206 mRNA and phenotype ratio increased, and expression levels of TLR4 and p-NF -κB P65 protein were also decreased (P<0.01). Conclusion Treatment with LYC inhibits LPS-induced primary microglial inflammatory response and promotes the anti-inflammatory polarization through TLR4/NF-κB signaling pathway.

Key words: Lycorine, microglia, lipopolysaccharides, Toll-like receptor 4, NF-kappa-B, inflammatory response

CLC Number:

R392.12

Figures/Tables 8

References 19

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基因名称	引物序列（5′→3′）	产物大小（bp）
IL-1β	上游：AAGCCTCGTGCTGTCGGACC	140
IL-1β	下游：TGAGGCCCAAGGCCACAGG	140
IL-6	上游：GCTGGTGACAACCACGGCCT	107
IL-6	下游：AGCCTCCGACTTGTGAAGTGGT	107
TNF-α	上游：CAAGGGACAAGGCTGCCCCG	109
TNF-α	下游：GCAGGGGCTCTTGACGGCAG	109
CD86	上游：GACCGTTGTGTGTGTTCTGG	148
CD86	下游：GATGAGCAGCATCACAAGGA	148
CD206	上游：TTCGGTGGACTGTGGACGAGCA	108
CD206	下游：ATAAGCCACCTGCCACTCCGGT	108
GAPDH	上游：GCCAAGGCTGTGGGCAAGGT	112
GAPDH	下游：TCTCCAGGCGGCACGTCAGA	112

基因名称	引物序列（5′→3′）	产物大小（bp）
IL-1β	上游：AAGCCTCGTGCTGTCGGACC	140
IL-1β	下游：TGAGGCCCAAGGCCACAGG	140
IL-6	上游：GCTGGTGACAACCACGGCCT	107
IL-6	下游：AGCCTCCGACTTGTGAAGTGGT	107
TNF-α	上游：CAAGGGACAAGGCTGCCCCG	109
TNF-α	下游：GCAGGGGCTCTTGACGGCAG	109
CD86	上游：GACCGTTGTGTGTGTTCTGG	148
CD86	下游：GATGAGCAGCATCACAAGGA	148
CD206	上游：TTCGGTGGACTGTGGACGAGCA	108
CD206	下游：ATAAGCCACCTGCCACTCCGGT	108
GAPDH	上游：GCCAAGGCTGTGGGCAAGGT	112
GAPDH	下游：TCTCCAGGCGGCACGTCAGA	112

组别	IL-1β mRNA		IL-6 mRNA
对照组	1.026±0.137		1.047±0.118
LYC组	1.135±0.507		1.032±0.156
LPS组	1 357.000±186.300^a		3 644.000±391.400^a
LPS+LYC组	631.900±83.950^ab		1 549.000±267.200^ab
F	159.500^＊＊		212.000^＊＊
组别		TNF-α mRNA		NO含量
对照组		1.036±0.128		1.185±0.366
LYC组		1.136±0.508		2.238±0.949
LPS组		397.700±62.410^a		28.770±2.472^a
LPS+LYC组		248.800±45.230^ab		18.830±4.847^ab
F		103.100^＊＊		93.640^＊＊

组别	IL-1β mRNA		IL-6 mRNA
对照组	1.026±0.137		1.047±0.118
LYC组	1.135±0.507		1.032±0.156
LPS组	1 357.000±186.300^a		3 644.000±391.400^a
LPS+LYC组	631.900±83.950^ab		1 549.000±267.200^ab
F	159.500^＊＊		212.000^＊＊
组别		TNF-α mRNA		NO含量
对照组		1.036±0.128		1.185±0.366
LYC组		1.136±0.508		2.238±0.949
LPS组		397.700±62.410^a		28.770±2.472^a
LPS+LYC组		248.800±45.230^ab		18.830±4.847^ab
F		103.100^＊＊		93.640^＊＊

组别	CD86 mRNA	CD206 mRNA	CD86（%）	CD206（%）
对照组	0.921±0.062	1.190±0.419	9.150±0.387	8.463±0.697
LYC组	0.741±0.207	0.972±0.278	9.020±0.486	8.850±0.265
LPS组	6.003±1.094^a	3.682±0.584^a	36.150±2.525^a	16.680±1.396^a
LPS+LYC组	3.427±0.542^ab	6.200±0.695^ab	22.400±2.754^ab	25.150±1.234^ab
F	63.910^＊＊	89.730^＊＊	186.600^＊＊	246.500^＊＊