Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

Abstract

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation.

Keywords: Baicalin; neuroprotection; protease-activated receptor-1; thrombin.

Figure 1

Effects of baicalin against cytotoxicity of thrombin stimulation. In thrombin group, cells were pre-incubated with baicalin (5, 10, 20 μM) for 2 h before exposed to thrombin (40 U/ml) for 6 h. Data are expressed as mean ± SEM of 3 independent experiments. ##P<0.01 compared with control group; *P<0.05, **P<0.01 versus thrombin group.

Figure 2

Baicalin inhibited thrombin-induced apoptosis. Four populations was possible to identify: viable cells in the lower-left quadrant (low PI and FITC signals); early apoptotic cells in the lower-right quadrant (low PI and high FITC signals); necrotic cells in the upper-left quadrant (high PI and low FITC signals); and late apoptotic cells in the upper-right quadrant (high PI and high FITC signals). The flow cytometric data in thrombin treated SH-SY5Y cells.

Figure 3

Baicalin suppressed PAR-1 mRNA expression following thrombin-induced injury. PAR-1 mRNA expression was determined by the quantitative real-time PCR system. Data are expressed as mean ± SEM of 3 independent experiments. *P<0.05, **P<0.01 versus thrombin group.

Figure 4

Baicalin suppressed PAR-1 protein expression following thrombin-mediated injury. Anti-β-actin antibody was used for normalization in the Western blotting analysis. The intensity of bands was quantified by densitometric analysis. All values represent mean ± SEM of three independent experiments. ##P<0.01 compared with control group; *P<0.05, **P<0.01 versus thrombin group.

Figure 5

Effects of baicalin on the protein level of NF-κB in thrombin-stimulated SH-SY5Y cells. Histograms represent mean ± SEM of the relative intensity of NF-κB protein bands normalized to β-actin. ##P<0.01 compared with control group; *P<0.05, **P<0.01 versus thrombin group.

Figure 6

Effects of baicalin on the expression of Caspase-3 protein in thrombin-treated SH-SY5Y cells. The β-actin acts as the internal standard. Data are expressed as mean ± standard deviation. ##P<0.01 compared with control group; *P<0.05, **P<0.01 versus thrombin group.