To investigate the effects of different concentrations and different administration time of salvianolic acid B (SalB) on regulating TLR4-NFκB-TNFα inflammatory pathway in hypoxia neonatal rat cardiomyocytes.Methods:Cardiomyocytes of Wistar neonatal rats that were born in 1 to 3 days were isolated and purified,and hypoxia model was established in vitro.High,medium,and low doses of SalB concentrations were 10-5 mol/L,10-6 mol/L and 10-7 mol/L,and were administered at the same time of,and 6 hours before and after hypoxia respectively.TLR4 and NFκB mRNA expressions were detected by qRT-PCR; TLR4 and NFκB protein expressions were detected by immunohistochemical method; the concentration of supernatant TNFα was detected by ELISA.Results:Compared with normal group,the expressions of TLR4 and NFκB mRNA and protein,and the concentration of TNFα in supernatant were significantly increased in hypoxia model group (P<0.05 or P<0.01).Compared with the hypoxia model group,the expressions of TLR4 and NFκB mRNA and protein,and the concentration of TNFα in supernatant were significantly decreased in high and medium doses of salvianolic acid B administered before and meantime of hypoxia group (P<0.05 or P<0.01).The most significant decrease was observed in the high-dose group administered before hypoxia (P<0.01).Conclusion:TLR4-NFκB-TNFα inflammatory pathway was significantly activated 6 h after hypoxia injury.Salvianolic acid B administered before and the same time of hypoxia protected cardiomyocytes tremendously.This effect related to the inhibition of TLR4-NFκB-TNFα inflammatory pathway.Salvianolic acid B showed dose-effects and time-effects,and the best effect was observed in the high-dose group administered before hypoxia.