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To establish the cell models of toxin-heat engendering endogenous collateral wind syndrome induced by LPS to activate the TLR4-MyD88 inflammatory pathway of HCASMC, seeking a new breakthrough for the scientific research platform of endogenous collateral wind.
HCASMC were cultured in vitro. And cell viability was determined via methylthiazoletetrazolium (MTT) assay to determine the optimal concentration range of LPS for promoting the growth of HCASMC. Then co-incubated with the safe range of LPS at different points (0,6,24,and48h), the expression of inflammatory cytokines were determined via enzyme-linked immunosorbent assay(ELISA). Besides, semi-quantitative reverse transcription-PCR was performed to detect mRNA expression level of IL-6, TNF-a, IL-1β, TLR4, MyD88 and NF-κB p65. The expressions of TLR4,MyD88 and NF-κB p65 were measured by western blotting to determine the final mold conditions.
Beyond certain density of LPS(5μg/ml,100μg/ml), the smaller action-time of LPS did not have cytotoxicity. The dose of (0.5,1)μg/ml LPS with action-time of (24,48h) could induce the expression of IL-6, IL-1βand TNF-α significantly(P<0.05). The RT-PCR and Western blotting indicated that 1μg/ml LPS with action-time of 48h could maximum activation of TLR4-MyD88 inflammatory pathway.
1μg/ml LPS with action-time of 48h can ensure that the HCASMC is in a sustained high inflammatory activation state, which can be used as the best cell models of toxin-heat engendering endogenous collateral wind syndrome.