Effects Of Celastrol On Platelet Function And The MIF And MMP-9 Expressions In The Aorta Of ApoE Gene Knockout Mice

Objective Celastrol is one of the active components extracted from T. wilfordii Hook F.,"Thunder of God Vine", which has been used as a traditional Chinese medicine for hundreds of years. Its anti-oxidant, anti-inflammatory, anti-cancer, immunosuppressive effects has been confirmed. But the effect of celastrol on atherosclerosis has not been researched. Here we investigate the effects of celastrol on platelet function, then the MIF and MMP-9 expressions in the aorta of apoE gene knockout mice with earlier atherosclerosis, thus provide a strong evidence for prevention and treatment of atherosclerosis by celastrol.Methods Blood was collected by venipuncture with a 21-gauge butterfly needle from healthy volunteers taking no medications, and blood was anticoagulated. Platelet-rich plasma (PRP) was obtained by centrifugation. Gel filtered platelets (GFP) were prepared from citrated PRP using a sepharose CL-2B column. Aggregation of platelets:After incubation of PRP with different concentrations of celastrol (DMSO as vehicle control) at 37℃for 10minutes, aggregation was induced by 10μmol/L ADP (final concentration), Aggregometry was performed in a Biodata PAP-4 aggregometer. Platelet activation assay:GFP were incubated with increasing concentrations of celastrol (0.1-10μmol/L) in the dark at room temperature for 20minutes. DMSO was used as vehicle control. After that, ADP was added to 20μmol/L final concentration, mouse anti-human CD62P-PE or PAC-1-FITC was used for detecting the activation of human platelets. samples were analysed on a FACSCalibre flow cytometer using Cell Quest Pro software. Adhesion of platelets to fibrinogen under static condition: Glass cover slips (12 mm circular) were incubated with fibrinogen (30μg/ml) at 4℃overnight. GFP were incubated with 1, 2, 5μmol/L celastrol, using DMSO as control, for 10minutes at room temperature, then added ADP to 20μmol/L final concentration, incubated 30minutes at 37℃. Slides were checked with DIC (×100) model under LEICA DM IRB microscope, the image was dealt with MCID software.Animal experiment: Twelve ApoE gene knockout male mice, 8-week old, were divided randomly into control group and celastrol treantment group( n=6 in each group) . All the mice were fed with western food (21%fat and 0.21% cholesterol) for further 8 weeks. In the last 4 weeks, the mice in Celastrol group were given celastrol (2mg.kg-1.d-1) by intraperitoneal injection; the mice in control group were only given equivalent amount of dimethyl sulfoxide (DMSO). The platelet phosphatidylserine (PS)-exposure was assayed by flow cytometry using annexin V. The fibrinogen binding on platelets were also detected by flow cytometry using polyclonal antibody against fibrinogen (rabbit anti-human). HE staining of root aorta were used to observe the histomorphological change and measure the plaque area in ApoE-/- mice. The expression of MIF and MMP-9 were detected by immunological histochemical staining.Results Celastrol at high concentration range (0.05-0.5mmol/L) can inhibit 10μmol/L ADP-induced platelet aggregation significantly. Lower celastrol concentration (<0.01mmol/L) did not have obvious effects on the platelets aggregation comparing with appropriate volume of DMSO. Celastrol inhibits ADP-induced platelet activation in vitro. The 50% inhibiting concentration(IC50) for inhibiting 20μmol/L ADP-induced P-selectin expression on platelets is 1.62μmol/L. The IC50 for PAC-1 is 1.86μmol/L. Celastrol at 5μmol/L significantly inhibited 20μmol/L ADP-induced platelets adhesion on fibrinogen-coated coverslips.The PS exposure on mouse platelets in Celastrol group is lower than in DMSO group, though without statistical difference between the two groups. The fibrinogen binding to the mouse platelets stimulated by 2μmol/L or 20μmol/L ADP in celastrol group was much lower than that in control group(P<0.01 and <0.05 respectively).The area of lipid plaque in Celastrol group was 4947.63±1277.42μm~2 , significantly smaller than that in the control group 8403.87±3535.73μm2 (P < 0.01). The ratio of plaque area/aorta cross section area were 0.05047±0.01653 in Celastrol group and 0.08224±0.02056 in the control group(P<0.01). The expression of MIF in aorta was significantly decreased in Celastrol group compared with the control group,the mean optical density was 0.02182±0.00813 and 0.05363±0.01474 respectively ( P<0. 01). The expression of MMP-9 was also significantly decreased in Celastrol group compared with the control group,the mean optical density was 0.02182±0.00813 and 0.05363±0.01474 respectively ( P < 0. 01). The expression of MIF in plaque was significantly decreased in Celastrol group compared with the control group , the mean optical density was 0.0114±0.00163 and 0.02270±0.00390 respectively ( P < 0. 01). The expression of MMP-9 was also significantly decreased in Celastrol group compared with the control tgroup,the mean optical density was 0.00536±0.00202 and 0.02630±0.00800 respectively ( P < 0. 01).Conclusions Celastrol can inhibit ADP-induced platelet activation in vitro or in vivo, which may play a key role in celastrol's anti-inflammation effect. It also indicates that celastrol may inhibit thrombi formation. Celastrol can inhibit the progress of atherosclerotic plaque in ApoE-/- mice, the reason may be relative to its inhibition effects on the expression of MIF and MMP-9 in the aorta.