Effects Of Hypertriglyceridemia And Fenofibrate On CD40L Expression Of Platelets

Backgrounds and Research StrategyAdvances in recent research have provided greater understanding of the underlying pathophysiology of atherosclerosis. Evidence now supports that atherosclerosis is a progressive, dynamic, inflammatory process. Blood lipid is recognized as a major cause of atherosclerosis. Previous viewpoints support that the Triglyceride was a weaker factor for initiating and progressing atherosclerosis. However, some latest clinic research indicated that triglyceride itself have closely relations with atherosclerosis (AS) and coronary heart disease (CHD), it's an important factor for initiating and progressing AS. TG also have close relationship with inflammation, hypertriglyceridemia patients generally have up-regulated inflammatory cytokines such as high C-reactive protein (CRP), inteluekin-6(IL-6) and monocyte chemo-attractant protein-1(MCP-1).It had been regarded that the platelets don't actively participate in immune responses in past decades, But in recent years, it has been found that the activated platelets actively participate in inflammatory reactions and immune responses via secreting TNF-a, IL-1β, IL-3, IL-6 and adhesion molecules(e.g. CD36); Platelet's micro-particles facilitate the interaction of mononuclear cells with endothelial cells and stimulate the mononuclear cells secreting adhesion molecules; Importantly, the discovery that platelets express CD40 and CD40 Ligand on its surface makes sure that the platelets may play important role in inflammatory reaction and immune response, for it's widely conceded that CD40-CD40L is part of whole organic immune network. Now it is conceded that activated platelets involve in atherosclerotic vascular disease, inflammatory reaction and immune response via expressing CD40 and CD40L on its surface. Through CD40-CD40L pathway, platelets participate in initiation and progression of atherosclerosis and it becomes a new hotspot in research field now.Synthetic drug of fenofebrate is an activator of peroxisome proliferator activated receptorα(PPARα) and is widely used as lipid regulator; it could decrease triglyceride concentration and slightly increase high density lipoprotein. Latest researches indicated that fenofibrate have effect of anti-inflammation beyond lowing the triglyceride.Basing on the important effects of TG on atherosclerosis discovered in recent years; basing on the further researched effects of CD40L expressed on immune activated platelets on initiation and progression of atherosclerosis, we suppose what changes of CD40L on platelets and soluble CD40L in plasma would be in hypertriglyceridemia patients and in the patients treated with fenofibrate for 4 weeks, what changes of CD40L expressed on platelets and CD40L protein expressed in platelets would be if platelets are placed in the hypertriglyceridemia plasma and what the changes would be if interfered by Wy14643 of PPARαactivator. These contents haven't been directly researched until now, so we prepared to study these as follows.Detect CD40L positive platelets and plasma sCD40L for clinic simple hypertriglyceridemia patients before and after 4 weeks treatment of fenofibrate. Culture platelets with simple hypertriglyceridemia patient's plasma in vitro and interfere with Wy14643, observe the effects of hypertriglyceridemia plasma and Wy14643 on the expression of CD40L on platelets and the expression of CD40L protein in plateletsSection 1 CD40L expression on platelets and sCD40L concentration in plasma in hypertriglyceridemia patients before and after 4 weeks treatment of fenofibrateObjectiveTry to study CD40L expression and sCD40L concentration in clinic simple hypertriglyceridemia patients by detecting the platelets and plasma before and after 4 weeks treatment of fenofibrate.Methods Select 20 cases of clinic simple hypertriglyceridemia patients, treat these patients with fenofibrate 200 mg per day; detect the expression of CD40L on platelets by Flow Cytometry and measure the concentration of sCD40L in plasma before and after 4 weeks of treatment.Results(1) Compared pre-treatment group with post-treatment group and control group, TG concentration is significantly higher in post-treatment group ( 4.57±0.64 VS 1.23±0.32,4.57±0.64 VS 3.54±0.73,Pall<0.05),but the concentration of TC is no statistical difference among three groups (3.70±0.88 VS 3.80±0.79,3.70±0.88 VS 3.94±0.89,Pall>0.05).(2) Compared pre-treatment group with control group, percentage of CD40L expressed platelets is apparently higher in pre-treatment group (2.65±0.75 VS1.53±0.66,P<0.01); Compared the post-treatment group with pre-treatment group, the percentage of CD40L expressed platelets decreased significantly in post-treatment group(1.89±0.56 VS 2.65±0.75,P<0.05).(3) Compared the pre-treatment group with control group, the concentration of sCD40L is higher in pre-treatment group (3.72±0.94 VS 2.27±0.72,P<0.05);Compared the pre-treatment group with the post-treatment group, the concentration of sCD40L decreased significantly in the post-treatment group(2.85±0.74 VS 3.72±0.94,P<0.05).(4) The concentration of TG has linear correlation with the percentage of CD40L expressed platelets(r=0.403, n=60, P<0.01).(5) The concentration of TG has linear correlation with sCD40L concentration (r=0.451, n=60, P<0.01).(6) The concentration of sCD40L has significant linear correlation with the percentage of CD40L expressed platelets (r=0.643, n=60, P<0.01).Section 2 effects of hypertriglyceridemia plasma and Wy14643 on CD40L expression of platelets ObjectiveTry to observe the direct effect of hypertriglyceridemia plasma and Wy14643 on CD40L expression of platelets in vitroMethods1. Firstly take further leukocytes filtration of aphaeresis platelets before preservation and culture of platelets.2. Collect the patient's plasma and healthy plasma according to design requirements, culture healthy platelets with the hypertriglyceridemia plasma and interfere with Wy14643 in needed group. Detect CD40L expression of platelets by Flow cytometry and Western blot.3. grouping(1) Control groupUncultured healthy platelets brought from blood center are detected in 12 hours.(2) Normal TG groupHealthy platelets are cultured in Normal TG plasma at 22±2℃and gently oscillating state for 4 days. Chose 3 cases of normal TG plasma from healthy person and do 6 times culture per plasma.(3)High TG groupHealthy platelets are cultured in High TG plasma and at the same culture condition. Chose 3 cases of high TG plasma from hypertriglyceridemia patients and do 6 times culture per plasma.(4) Wy14643-added high TG groupHealthy platelets are cultured in Wy14643-added high LDL-C plasma and at the same condition. The high TG plasma comes from group 3.Results(1) In normal TG group, percentage of CD40L expressed platelets increased significantly compared with control group (1.89±0.41 VS 0.75±0.41, P<0.01); Compared high TG group with normal TG group, the percentage increased apparently in high TG group(2.81±0.48 VS 1.89±0.41,P<0.01), in Wy14643-added high TG group, the percentage didn't change obviously compared with high TG group (2.73±0.45 VS 2.81±0.48,P>0.05).(2) Linear regression analysis of concentration of TG with percentage of CD40L expressed platelets indicated that the concentration of TG has linear correlation with the percentage of CD40L expressed platelets (P<0.05).(3) Compared Normal TG group with control group, CD40L protein expression in platelets increased significantly (1.51±0.15 VS 0.97±0.09,P<0.01); in high TG group, the CD40L protein expression increased significantly Compared with Normal TG group (1.79±0.13 VS 1.51±0.15,P<0.01); in Wy14643-added high TG group, the CD40L protein expression didn't change obviously compared with high TG group (1.80±0.12 VS 1.79±0.13,P>0.05).ConclusionsCD40/CD40l widely engaged in immune response and inflammatory reaction, as platelet is stimulated by some cytokines or activators, it will significantly increase the expression of CD40L on its surface, and then it's called immune activation. Our study demonstrated that the percentage of CD40L expressed platelets and the concentration of sCD40L were higher in pre-treatment group Compared with control group, which indicated platelets are partly immune activated in Hypertriglyceridemia patients. Linear correlation analysis of TG concentration with percentage of CD40L expressed platelets or with sCD40L concentration demonstrated the concentration of TG has linear correlation with the percentage of CD40L expressed platelets and with the sCD40L concentration, which indicated the TG may be correlated with the immune activation of platelets. After 4 weeks treatment of fenofibrate, the percentage of CD40L expressed platelets and the sCD40L concentration decreased followed the decreased concentration of TG, which indicated the fenofibrate could decrease the concentration of LDL-C; at the same time, partly inhibit the immune activation of platelets and inflammatory cytokines. The significantly correlation of sCD40L concentration with percentage of CD40L expressed platelets may indicated most sCD40L come from the releasing of CD40L expressed on platelets.By culturing healthy platelets in plasma contained TG in vitro, discovered that percentage of CD40L expressed platelets increased not only in high TG group but also in normal TG group, but the percentage was higher in high TG group than in normal TG group. Linear regression analysis found the concentration of TG associated with the percentage of CD40L expressed platelets. Some latest study demonstrated that the TG could activate leukocyte in vitro. All these results may indicate TG or VLDL which carrying TG in plasma directly or indirectly prompt the immune activation of platelets. Immune activated platelets also have upregulated CD40L protein in platelets. Wy14643 has no effect of inhibiting the CD40L expression of platelets in vitro.