Effects Of Ginkgolide B On Inhibition Of CD40Ligand Expression In Collagen-induced Platelet Activation

Objective:To investigate the effects of ginkgolide B on the CD40Ligand (CD40L) expression in collagen-induced platelet activation and the related mechanism.Materials and Methods:Anti-coagulated blood was collected from health donor. Platelets were isolated centrifugation. Platelets were pre-incubated by the various concentration of ginkgol ide B for5min, then platelets were stimulated by collagen. Protein expression was detected by Western Blot. First, take the normal blood,1100rpm centrifugal15min separation of platelet-rich plasma, to join TYRODE/with HEPES buffer, the ACD(1:9),2mmolL-1EDTA, centrifuged again, supernatant was removed, suspended cells, repeated twice, to remove plasma proteins and other blood cells, to obtain a platelet suspension. Next, use the Chromo-Log platelet aggregat ion analyzer to analyze platelet aggregation rate by the method of turbid metric. The platelet suspension were incubated in Ginkgolide B5min with0.2g·L-0.4g·L-1,0.6g·L-1respectively and then use10g·L-1collagen/1U·ml-1thrombin stimulate platelet10min, analysis the platelet aggregation rate. Add SDS to terminate the reaction of suspension, to detect protein by Western Blot. Sample electrophoresis, transferred to a membrane, closed with5%bovine serum albumin at room temperature for2h, washing the membrane three times. Antibody(PI3K, β-actin, Akt, P-Akt, CD40L, GAPDH, P-p38, p38,4G10, Syk) were added and incubated at4℃overnight. After washing the membrane three times, adding the secondary antibody and incubated at room temperature for1h and washing the membrane five times. Use the ECL chemiluminescence to make membrane laminate then picture it by using gel imaging instrument.Results:1. The effects of ginkgolide B on platelet aggregation10mg·L-1of collagen induced platelet aggregation, and0.2g·L-1,0.4g·L-0.6g·L-1of Ginkgolide B potently inhibited platelet aggregation in a dose-dependent manner, with Ginkgolide B in0.2g·L-1,0.4g·L-1,0.6g·L-pretreatment of platelet, aggregation rates were77.07±13.82%,60.65±18.08%,48.39±14.28%respectively. The differences were statistically significant compared with the pure collagen stimulation group, n=6, P<0.05.1U·ml-1thrombin induced platelet aggregation, and0.2g·L-1,0.4g·L-1,0.6g·L-of Ginkgolide B potently inhibited platelet aggregation in a dose-dependent manner. With Ginkgol ide B in0.2g·L-1,0.4g·L-1,0.6g·L-1pretreatment of platelet, aggregation rates were88.20±7.75%,72.28±6.36%,57.82±6.15%respectively. The differences were statistically significant compared with the pure thrombin stimulation group, n=4, P<0.05.2. The effects of ginkgolide B on CD40L expressionThe expression of CD40L was increased in collagen-induced platelet activation. Ginkgolide B significantly attenuated the increase of CD40L expression in activated platelets. Western Blot results showed that in static platelet CD40L expression was100.47±31.10, after collagen stimulates platelet, CD40L expression was significantly increased to130.7±24.14, with Ginkgolide B in0.2g·L-1,0.4g·L-1,0.6g·L-1pretreatment of platelet, CD40L expression was inhibited in a dose-dependent manner, expressions were117.73±18.83,104.94±26.38,82.16±25.03respectively. The differences were statistically significant compared with the pure collagen stimulation group, n=6, P<0.05.The expression of CD40L was increased in thrombin-induced platelet activation. Ginkgolide B significantly attenuated the increase of CD40L expression in activated platelets. Western Blot results showed that static platelet CD40L expression was103.12±38.12, after thrombin stimulates platelet, CD40L expression increased significantly to142.01±39.23, with Ginkgolide B in0.2g·L-1,0.4g·L-1,0.6g·L-1pretreatment of platelet, CD40L expression was inhibited in a dose-dependent manner, expressions were123.70±35.42,108.71±38.12,87.65±32.00respectively. The differences were statistically significant compared with the pure thrombin stimulation group, n=8, P<0.05.3. The effects of ginkgolide B on PI3K expressionGinkgolide B has no effect on PI3K expression in collagen-induced platelet activation. Western Blot results showed that the expression of PI3K in static platelet were112.37±28.22, after collagen stimulates platelet, PI3K expression increased slightly to115.72±26.04, with Ginkgolide B in0.2g·L-1,0.4g·L-0.6g·L-1pretreatment of platelet, PI3K expression was not significant changing, expressions were114.83±22.34,115.08±24.27,114.16±27.03respectively. Compared with the pure collagen stimulation group, the difference was not statistically significant, n=9, P>0.05. Ginkgolide B has no effect on PI3K expression in thrombin-induced platelet activation. Western Blot results showed that the expression of PI3K in static platelet we re111.78±18.22, after thrombin stimulates platelet, PI3K express ion increased slightly to114.72±20.04, with Ginkgolide B in0.2g±g·L-1,0.4g·L-0.6g·L-1pretreatment of platelet, PI3K expression was not significant changing, expressions were110.64±22.34,115.08±20.74,111.46±19.33respectively. Compared with the pure thrombin stimulation group, the difference was not statistically significant, n=10, P>0.05.4. The effects of ginkgolide B on Akt phosphorylationGinkgolide B obviously abolished Akt phosphorylat ion in activated platelets induced by collagen. Western Blot results showed that in static platelet P-Akt expression was41.30±19.38, after collagen stimulates platelet, P-Akt expression increased significantly to88.14±7.85, with Ginkgol ide B in0.2g·L-0.4g·L-1,0.6g·L-1pretreatment of platelet, P-Akt expression was inhibited in a dose-dependent manner, expressions were79.84±30.53,39.67±15.73,14.11±7.08respectively. The differences were statistically significant compared with the pure collagen stimulation group, n=3, P<0.05.Ginkgolide B obviously abolished Akt phosphorylation in activated platelets induced by thrombin. Western Blot results showed that in static platelet P-Akt expression was37.21±12.93, after thrombin stimulates platelet, P-Akt expression increased significantly to84.77±10.43, with Ginkgolide B in0.2g·L-0.4g·L-1,0.6g·L-1pretreatment of platelet, P-Akt expression was inhibited in a dose-dependent manner, expressions were77.21±11.03,58.14±7.83,38.59±9.69respectively. The differences were statistically significant compared with the pure thrombin stimulation group, n=4, P<0.05.5. The effects of ginkgoiide B on the phosphorylation of p38Ginkgolide B obviously abolished p38phosphorylation in activated platelets induced by collagen. Western Blot results showed that in static platelet P-p38expression was51.43±11.74, after collagen stimulates platelets, the expression of P-p38increased significantly to90.24±8.05, with Ginkgolide B in0.2g·L-0.4g·L-1,0.6g·L-1pretreatment of platelet, P-p38expression was inhibited in a dose-dependent manner, expressions were77.36±19.51,68.92±12.63,51.22±7.85respectively. The differences were statistically significant compared with the pure collagen stimulation group, n=6, P<0.05.Ginkgolide B obvious ly abol ished p38phosphorylat ion in activated platelets induced by thrombin. Western Blot results showed that in static platelet P-p38expression was32.18±11.92, after thrombin stimulates platelets, P-p38expression was significantly increased to89.68±10.23, with Ginkgolide B in0.2g·L-1,0.4g·L-1,0.6g·L-1pre treatment of platelet, P-p38expression was inhibited in a dose-dependent manner, expressions were76.19±18.34,75.82±12.97,39.75±9.81respectively. The differences were statistically significant compared with the pure thrombin stimulation group, n=5, P<0.05.6. The effects of ginkgolide B on phosphotyrosine of SykGinkgol ide B obviously abol ished Syk phosphotyros ine in activated platelets induced by collagen. Western Blot results showed that in static platelet4G10expression was38.55±10.48, after collagen stimulates platelet,4G10expression was significantly increased to88.47±8.24, with Ginkgolide B in0.2g·L-0.4g·L-1,0.6g·L-1pretreatment of platelet,4G10expression was inhibited in a dose-dependent manner, expressions were74.32±13.35,60.91±14.04,48.81±6.76respectively. The differences were statistically significant compared with the pure collagen stimulation group, n=6, P<0.05.Ginkgolide B obviously abolished Syk phosphotyrosine in activated platelets induced by thrombin. Western Blot results showed that in static platelet4G10expression was50.95±11.98, after thrombin stimulates platelet,4G10expression significantly increased to85.45±8.76, with Ginkgolide B in0.2g·L-1,0.4g·L-10.6g·L-1pretreatment of platelet,4G10expression was inhibited in a dose-dependent manner, expressions were77.24±14.68,70.42±15.12,53.49±8.19respectively. The differences were statistically significant compared with the pure thrombin stimulation group, n=6, P<0.05.Conclusion:1. Ginkgolide B can effectively inhibit platelet aggregation in collagen/thrombin-induced platelet activation.2. Ginkgolide B can effectively inhibit CD40L expression in collagen/thrombin-induced platelet activation.3. Ginkgolide B can significantly decrease Akt phosphorylation.4. Ginkgolide B can effectively decrease p38phosphorylation.5. Ginkgolide B can effectively decrease Syk phosphotyrosine. These suggesting that the mechanism of ginkgolide B on inhibition of CD40L expression is associated with PI3K/Akt and Syk/p38pathway.