It exists in the cytoplasm as a dimer predominantly formed by the p65/p50 complex in an inactive state combined with users of the NF-B inhibitor (I-B) family. thrombin group, cells were pre-incubated with baicalin (5, 10, 20 M) for 2 h before exposed to thrombin (40 U/ml) for 6 h. Data are expressed as mean SEM of 3 impartial experiments. ##mRNA expression, which was partly attenuated by baicalin pre-treatment. Similarly, baicalin pre-treatment also attenuated thrombin induced PAR-1 protein expression (Physique 4). Open Darusentan in a separate window Physique 3 Baicalin suppressed PAR-1 mRNA expression following thrombin-induced injury. mRNA expression was determined by the quantitative real-time PCR system. Data are expressed as mean SEM of 3 impartial experiments. *versus thrombin group. Open in a separate window Physique 4 Baicalin suppressed PAR-1 protein expression following thrombin-mediated injury. Anti–actin antibody was utilized for normalization in the Western blotting analysis. The intensity of bands was quantified by densitometric analysis. All values represent mean SEM of three impartial experiments. ## em P /em 0.01 compared with control group; * em P /em 0.05, ** em P /em 0.01 versus thrombin group. Baicalin inhibited thrombin induced NF-B Darusentan and Caspase-3 protein expression The effects of baicalin around the NF-B and Caspase-3 protein expression were determined by western blotting. Compared with the control group, thrombin increased NF-B protein expression, which was significantly attenuated by medium or high dose of baicalin (10, 20 M) (Physique 5). In addition, thrombin also induced Caspase-3 protein expression, which was significantly attenuated by high dose of bacailin (Physique 6). Open in a separate window Physique 5 Effects of baicalin around the protein level of NF-B in thrombin-stimulated SH-SY5Y cells. Histograms symbolize mean SEM of the relative intensity of NF-B protein bands normalized to -actin. ##P 0.01 compared with control group; *P 0.05, **P 0.01 versus Rabbit Polyclonal to OAZ1 thrombin group. Open in a separate window Physique 6 Effects of baicalin around the expression of Caspase-3 protein in thrombin-treated SH-SY5Y cells. The -actin acts as the internal standard. Data are expressed as mean standard deviation. ## em P /em 0.01 compared with control group; * em P /em 0.05, ** em P /em 0.01 versus thrombin group. Darusentan Conversation In the present study, we exhibited that baicalin attenuated thrombin induced cell injury in SH-SY5Y cells. This protective effect of baicalin is usually associated with the inhibition of PAR-1, NF-B and Caspase-3 expression. As a serine protease, thrombin is an essential component of the coagulation cascade, which is usually produced by the cleavage of pro-thrombin. Evidence showed that brain may also be a source of pro-thrombin. Pro-thrombin mRNA is not only expressed in the cells of the nervous system but also up-regulated after cerebral ischemia and spinal cord injury [26-28]. Thrombin is usually generated in the brain either immediately after cerebral hemorrhage or after the blood brain barrier (BBB) breakdown that induced by many kinds of brain damages [26,29]. In the present study, we showed that thrombin (40 U/L) caused obvious cell injury in SH-SY5Y cells, which was significantly attenuated by pre-treatment with baicalin in a dose-dependent manner. Our results were consistent with previous studies showing that baicalin was neuroprotective following cerebral ischemia in animal models [30-32]. It was proposed that this extra-vascular effects of thrombin were mediated by a family of PARs [10,11]. PARs are a family of seven transmembrane G protein-coupled receptors that include PAR-1, PAR-2, PAR-3 and PAR-4. Of these different receptors, PAR-1, PAR-3, and PAR-4 can be activated by thrombin, whereas PAR-2 is usually activated by trypsin . PAR-1 is usually predominantly expressed in the brain and has been suggested to mediate the thrombin toxicity in cerebral ischemia-reperfusion damage . To explore the possible mechanism by which baicalin reduces thrombin-induced cell injury, we determined the effect of baicalin around the PAR-1 expression. Our results showed that this PAR-1 expression was significantly increased after thrombin activation within 6 h at both mRNA and protein levels, which were attenuated by baicalin in a dose-dependent manner. NF-B is usually a critical regulator of inflammation. It exists in the cytoplasm as a dimer predominantly formed by the p65/p50 complex in an inactive state combined with users of the NF-B inhibitor (I-B) family. In an external activation pathway, I-B is usually phosphorylated by I-B kinases (IKKs), which results in its degradation, and thus.