| Geniposide is a key bioactive compound isolated from Gardenia Jasminoides Ellis, which is widely used for treatment of stroke in Chinese medicine. Recent studies show that geniposide has protective activities against inflammation and oxidative stress in ischemic stroke. However, the molecular mechanism of geniposide on anti-inflammatory role has not yet been fully studied. In this study, we investigate the effect of geniposide on the expression of P2Y14 receptor and downstream ERK1/2 signaling pathway in brain microvascular endothelial cells (BMECs). P2Y14 receptor, a Gi protein-coupled purinergic receptor for UDP-glucose, is widely expressed in the many brain regions and plays a role in the regulation of immune responses. Our previous studies show that geniposide has protective effects against inflammation and oxidative stress in ischemic stroke. It indicates that P2Y14 receptor may be activated in OGD-induced brain microvascular endothelial cells. Therefore, this study provides the evidence to support that geniposide as a potential drug for protecting the BMECs and inhibiting inflammation response in ischemic diseases.Objective:To investigate the protective effect of geniposide on P2Y14 receptor expression in OGD-induced BMECs and the underlying mechanism of action, the one can provide some new pharmacological targets and experimental basis for prevention and treatment of ischemic damage.Mothods:1. Isolation and culture of rat primary CMECs and establishment of in vitro oxygen-glucose-deprivation (OGD) model.We establish the OGD model that the primary BMECs at third passage are cultured in 93% N2 and 7% CO2 environment for 6h to imitate cerebral ischemia in vitro. The BMECs ischemic model are simulated by OGD according to a method previously described by Li et al.2. The effect of geniposide on P2Y14 receptor expression in OGD-induced BMECs. The third passages of BMECs are used for this experiment;We first establish a OGD model, and the BMECs are harvested four groups:1) control group:the normal cultured BMECs without treatment; 2) OGD group:the ischemic BMECs by OGD; 3) geniposide group:the ischemic BMECs by OGD with 33.2μg/ml geniposide pretreatment for 6h; 4) PTX group:the BMECs by OGD with 100ng/ml PTX pretreatment which is completely abolished UDP-glc agonist responses.3. Effect of Geniposide on Raf-1/MEKl/2/ERK1/2 signaling pathways by OGD-induced injury.4. Effect of Geniposide on IL-8, IL-1β and MCP-1 levels in BMECs culture media.5. Effect of Geniposide on P2Y14 receptor and downstream ERK1/2 signaling pathways by UDP-glc stimulation.To further examine the effect of geniposide on P2Y14 receptor in BMECs. Then we set up a UDP model, and the BMECs are harvested three groups:1) control group:the normal cultured BMECs; 2) UDP group:100μM UDP-glucose treats with the normal BMECs. Results:1. P2Y14 receptor is expressed in rat BMECs and up-regulated by exposure to OGD in mRNA and protein level.Expression of the P2Y14 receptor in normal and OGD-injured BMECs is analyzed using real-time PCR and Western Blot. The rat BMECs express the mRNA and protein for P2Y14 receptor, and the expression of P2Y14 mRNA and protein are increased in OGD group.2. The effect of geniposide on P2Y14 receptor expression in OGD-induced BMECs.Geniposide 33.2μg/ml is the optimal drμg concentrations. We first show expression of P2Y14 mRNA and Protein in the rat cerebral microvascular endothelial cells(CMECs),which is up-regulated particularly in OGD-induced injury. Moreover, geniposide significantly decreases the expression of P2Y14 receptor in OGD-induced BMECs.3. Effect of Geniposide on Raf-1/MEKl/2/ERKl/2 signaling pathways by OGD-induced injury.Following 6h OGD, the model group results in a significantly up-regulate in Raf-1 phosphorylation, which is blocked by pretreatment with geniposide and PTX overnight. Geniposide inhibits MEK1/2 phosphorylation in OGD-induced BMECs, and similar data are observed by immunofluoresence staining. Geniposide and PTX decrease both p44 and p42 phosphorylation.4. Effect of Geniposide on IL-8, IL-1β and MCP-1 levels in BMECs culture media.Geniposide and PTX inhibit the level of IL-8, IL-1β and MCP-1 in OGD-induced BMECs:OGD markedly induced the secretion of IL-8, IL-1β and MCP-1, whereas 33.2μg/ml geniposide and 100ng/ml PTX markedly declined the level of IL-8, IL-1β and MCP-1.5. Effect of Geniposide on P2Y14 receptor and downstream ERK1/2 signaling pathways by UDP-glc stimulation:UDP-glc significantly increase P2Y14 receptor expression in BMECs which is in agreement with a previous report. The UDP-stimulated over expression of P2Y14 receptor is significantly decreased by geniposide treatment. Phosphorylation of Raf-1, MEK1/2 and ERK1/2 is enhanced in UDP-glc induced group. Geniposide treatment blocks activation of p-Raf-1, p-MEK1/2 and p-ERK1/2. Immunofluorescence staining of p-ERK1/2 also shows consistent results with Western Blot.Conclusion:1. In the present study, we detect P2Y14 receptor expression in BMECs via RT-PCR and Western Blot analysis. We first show expression of P2Y14 mRNA and Protein in BMECs.2. Geniposide can inhibit the expression of P2Y14 receptor and the level of IL-8, IL-1β and MCP-lin OGD-induced BMECs, which is related with Raf-1/MEK1/2/ERK1/2 phosphorylation in OGD-induced BMECs.3. Geniposide decreases P2Y14 receptor and downstream ERK1/2 signaling pathways by UDP-glc stimulation. |
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