| Acute coronary artery disease has been one of the leading causes of death in the worldwide. There are many studies about its mechanism and it was accepted that inflammation was the major stimulator in pathlogical progress. Moreover, studies has revealed the interaction of CD40 andCD40L presented on cells involved in plaque formation played an important mediator in the inflammatory reponses,subsquently resulted in plaque reputation and platelet aggeragation, progressing the phthophysiologic condition. Formor studies as well as our recent study also showed an increase of CD40-CD40L in ACS compared with stable angina and healthy volunteers. CD40-CD40L signaling pathway has been considered as a potential theraptic target in ACS.Ginkgo biloba is a traditional phytomedcine in the past centuries, is mainly proposed with two active compositions, flavanoids (ginkgo-flavone glycosides) and terpenoids(ginkgolides and bilobalides). Nowdays, ginkgo biloba was widly used as theraptic phytomedicine in cardiovascular dieases. Studies have indicated ginkgo bilolba effectively removed the ROS to protect mycardio condition and and restrain the progress of atherosclerosis. Ginkgo biloba was also suggested that it improved coronary blood flow and inhibited platelet aggregation. In addition, studies showed it supressed some inflammatory cytokines.Objective:To study ginkgo biloba potential effects on CD40-CD40L on PMBCs in ACS.Methords:Select ACS patient's whole blood as the test sample as well as volunteers as control sample. PBMCs was obtained from whole blood by centrifugation and treated with a serial concentration ginkgo biloba extract, and also added a blank group without treatment of ginkgo biloba. After incubation for 24 hours, the expression of CD40/CD40L on PBMCs was tested by flow cytometer. The secrection of IL-12 was determined by ELISA.Results:1, without ginkgo biloba extract, the exression of CD40/CD40L in ACS was 17.17±9.58%,3.58±2.16% respectivly, and 11.8±9.17%,1.28±0.7% in control group. There is a statistical significance between ACS group and control group (p<0.05)2, compared with the blank group, the expressions of CD40 in ACS group were (13.18±5.56)%(12.5mg/L) (p>0.05), (11.37±4.44)%(25mg/L) (p<0.05) (11.16±5.08)%(50mg/L) (p<0.05) (7.57±4.47)%(1OOmg/L) (p<0.05), and the expressions of CD40L were (2.64±1.48)%(12.5mg/L) (p>0.05), (1.94±1.63)% (25mg/L) (p<0.05), (1.85±1.61)%(50mg/L) (p<0.05) (1.34±1.06)%(1OOmg/L) (p<0.05)3, compared with the blank group, the expressions of CD40 in control group were (11.66±8.71)%(12.5mg/L) (p>0.05), (10.52±5.11)%(25mg/L) (p>0.05) (8.40±5.11)%(50mg/L) (p>0.05), (7.53±4.33)%(1OOmg/L) (p>0.05), and CD40L:(1.17±0.69)%(12.5mg/L) (p>0.05), (0.86±0.78)%(25mg/L) (p>0.05) (0.83±0.67)%(50mg/L) (p>0.05) (0.72±0.61)%(1OOmg/L) (p>0.05)4. compared with the blank group(292.27±77.83)pg/ml, the contents of IL-12 were (168.83±100.51)pg/ml (12.5mg/L) (p<0.05), (116.87±110.48) pg/ml (25mg/L) (p<0.05), (102.41±97.01) pg/ml (50mg/L) (p<0.05) (76.17±47.68) pg/ml (1OOmg/L) (p<0.05)Conclusion:The result of our present study revealed that EGB inhibited expressions pf CD40/CD40L in ACS in vitro, which manifested EGB's potential anti-inflammatory and anti-immune effects in ACS. Proteasome is part of the ubiquitin-proteasome system, and widely exsits in all eukaryotes. It played a major role in the non-lysosomed breakdown of key proteins, subsquently regulated the normal cellular homeostasis including cell cycle control, apoptosis, DNA repair, endocytosis, immune and inflammatory responses etcGinkgo biloba is a traditional phytomedcine in the past centuries. Studies have indicated ginkgo biloba extract (EGB) effectively removed the ROS, improved coronary blood flow and inhibited platelet aggregation. In addition, studies showed it supressed some inflammatory cytokines.A recent study has indicated the inhibitors of proteasome involving bortezomib and lactacystin down-degraded the expression of CD40 and the secrection of IL-12 in the process of DC maturation. Our research also showed ginkgo biloba extract depressed the CD40/CD40L on PBMCs and the content of IL-12 in ACS. Thus, we presented the question whether ginkgo biloba extract influnce the activity of proteasome, which is partially the mechanism about anti-inflammatory effect of ginkgo biloba.Objective:to study ginkgo biloba potential effects on proteasomal activity of PMBCsMethords:PBMCs harvested was degraded by lysis buffer and The supernatant layer was finally obtained. Detected the concentration of protein and prepared a sample solution of 2ug/ul. Then add 1Oul per plate of them into 96-well cell culture plate fixed with a serial concentration of EGB(6.25 mg/L-200mg/L group) dissolved in action buffer.Simultaneously, added only action buffer (without EGB) as the control group. Then, fluorogenicsubstrates were added to a final concentration of 100uM and further incubated for 5 min at 37℃before measuring.PBMCs were incubated in 96-well plates at 1×1O5 cells/well and treated with a serial concentration of EGB, and.added only culture meidium (no EGB)as control. Set up a time-dependent proliferation detection including 2hr,24hr,48hr and 72hr. CCK-8 was added to each well for 2hr and was measured at 450nm in an ELISA plate reader.Results:1,Compared with control, all activity of three catalytic sites on pureified 20S proteasome were inhibited by ginkgo biloba concentration-dependently.2. Compared with control, the activity of ChT-L were 97.15±11.64%(6.25mg/L),84.42±10.49%(25mg/L),66.38±12.33%(50mg/L),55.05±10.86%(100mg/L), 29.39±15.73%(200mg/L) respectively, and IC50 was 127.13 mg/L. there were statistical significance(p<0.01) between control group and test groups except 6.25mg/L and 25mg/L group.3. Compared with control, the activity of T-L were 80.41±17.54%(6.25mg/L). 57.44±9.24%(25mg/L),39.17±8.04%(50mg/L),31.87±5.85%(100mg/L), 15.93±1.25%(200mg/L), IC50%75.06 mg/L. There were statistical significance (p<0.01) between control group and test groups except 6.25mg/L group.4. No change was observed on the catalytic sit of PGPGH-L of PBMCs crude proteasome with the treatment of ginkgo biloba extract.Conclusion:Rare study has indicated the direct effect of ginkgo biloba on proteasome. Our present study that the significant change of protasome activity manifested the hyporthesis that ginkgo biloba extract was an inhibitor of proteasome complex which is partially a mechanism in cardiovascular diseases. we showed new evidences about the GBE effectiveness on biological function and addressed a new point for further GBE research.
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