基于TLR4-NF-κB信号通路的槐果碱改善人支气管上皮细胞炎症和黏液高分泌的机制

doi:10.3969/j.issn.1006-5725.2023.19.009

Abstract

Abstract:

Objective To investigate the anti-inflammatory effect of sophocarpine （SC） in vitro and the potential mechanism of its anti-asthma effect based on the TLR4-NF-κB signaling pathway. Methods NCI-H292 cells were stimulated by Lipopolysaccharide （LPS） to induce inflammation model in vitro， and SC was given in different concentrations. The safe concentrations of LPS and SC on NCI-H292 cells were screened by MTT method， and the control group， LPS group （10 μg/mL LPS） and SC group （10 μg/mL LPS+ different concentrations of SC） were set up. The expression of mucin 5AC （MUC5AC） in cells and the secretion of Interleukin-6（IL-6） and Interleukin-8（IL-8） in the supernatant were detected by ELISA. The mRNA expression of MUC5AC， IL-6， IL-8， myeloid differentiation factor 88（MyD88） and nuclear factor-kappa B p65（NF-κB p65） in cells was detected by RT-PCR. The protein expression of TLR4， tumor necrosis factor receptor-associated factor 6 （TRAF6）， p-p38 and p-p65 in cells and NF-κB p65 in nucleus was detected by Western blotting. Results MTT results showed that LPS and SC had no effect on cell viability in the concentration range of 0 ~ 10 μg/mL and 0 ~ 40 μg/mL. The results of ELISA showed that the content of MUC5AC in LPS group was significantly higher than that in control group （P < 0.01）， and the levels of IL-6 and IL-8 in the supernatant of cells were also extremely significant increase （P < 0.01）. Compared with the model group， the content of MUC5AC in SC group extremely significant reduction （P < 0.01）， and the content of IL-6 and IL-8 in cell supernatant also significantly reduced （P < 0.01）. RT-PCR results showed that the expressions of MUC5AC， IL-6， IL-8， MyD88 and NF-κB p65 mRNA in LPS group were extremely significant higher than those in control group （P < 0.01）， however， the expression of MUC5AC， IL-6 and IL-8 mRNA in SC group decreased significantly （P < 0.05）， and the expression of MyD88 and NF-κB p65 mRNA extremely significant reduction （P < 0.01）. Western blotting results showed that SC could extremely significant reduction the expression of TRAF6， TLR4， p-p38 and p-p65 in NCI-H292 cells induced by LPS and the expression of NF-κB p65 protein in nucleus （P < 0.01）. Conclusion SC may play an anti-asthmatic role by inhibiting TLR4-NF-κB signaling pathway， improving airway inflammation and mucous hypersecretion.

Key words: sophocarpine, NCI-H292 cells, TLR4-NF-κB signal pathway, inflammation, mucus hypersecretion

CLC Number:

R285.5

Shengnan JIANG,Wenbing ZHI,Hong ZHANG,Xiaoting WANG,Tingting SUN,Zongren XU,Jing CHEN,Ye LI,Yang. LIU. Mechanism of Sophocarpine on improving inflammation and mucous hypersecretion of human bronchial epithelial cells based on TLR4⁃NF⁃κB signaling pathway[J]. The Journal of Practical Medicine, 2023, 39(19): 2461-2468.

Figures/Tables 6

Tab.1

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References 28

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基因	引物序列
GAPDH	F 5′-TGTTCGTCATGGGTGTGAACC-3′
GAPDH	R 5′-CATGAGTCCTTCCACGATACC-3′
MUC5AC	F 5′-TCCACCATATACCGCCACAGA-3′
MUC5AC	R 5′-TGGACGGACAGTCACTGTCAAC-3′
IL-6	F 5′-GACAGCCACTCACCTCTTCA-3′
IL-6	R 5′-AGTGCCTCTTTGCTGCTTTC-3′
IL-8	F 5′-AAACCACCGGAAGGAACCAT-3′
IL-8	R 5′-GCCAGCTTGGAAGTCATGT-3′
MyD88	F 5′-GATGGTAGCGGTTGTCTCTGAT-3′
MyD88	R 5′-GATGCTGGGGAACTCTTTCTTC-3′
NF-κB p65	F 5′-AGTACCCTGAGGCTATAACTCGC-3′
NF-κB p65	R 5′-TCCGCAATGGAGGAGAAGTC-3′

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Mechanism of Sophocarpine on improving inflammation and mucous hypersecretion of human bronchial epithelial cells based on TLR4⁃NF⁃κB signaling pathway

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