The current experiment explored the rapid effects of the 3 currently prescribed lipid-lowering drugs, pitavastatin, ezetimibe and Xuezhikang, alone and in combination, on PCSK9 and lipid profile concentrations in rats in a very short period. The main finding of this study is that ezetimibe, Xuezhikang and pitavastatin alone and Xuezhikang plus ezetimibe as well as pitavastatin plus ezetimibe, all could significantly increase the PCSK9 levels at day 3, when the lipid profile had no obvious decreases. In addition, combination of ezetimibe 10 mg/kg and Xuezhikang 1200 mg/kg daily did not further enhance PCSK9 levels compared to the monotherapy, while ezetimibe 10 mg/kg and pitavastatin 10 mg/kg daily in combination induced higher PCSK9 levels than monotherapy with pitavastatin or co-therapy with ezetimibe plus Xuezhikang.
It has been well established that PCSK9 promotes the degradation of LDLR and could limit the beneficial effects of lipid-lowering drugs . Several studies indicated that lipid-lowering drugs could elevate plasma PCSK9 concentrations and may have rapid impact on PCSK9 levels . In our present study, we confirmed previous studies and provided novel additional important information regarding the lipid-lowering drugs on plasma PCSK9. This study, for the first time, indicated that the enhancement of PCSK9 levels preceded the changes of lipid profile. Therefore, it is reasonable to deduce that patients with dyslipidemia may get more benefits from PCSK9 inhibitors, such as monoclonal antibody , if it be used before the application of lipid-lowering drugs in clinical practice. Although commonly prescribed lipid-lowering drugs may up-regulate both the LDLR and PCSK9 genes by activating SREBP-2 , the PCSK9 gene might respond much earlier than the LDLR gene, that may partly of the reason why the changes of PCSK9 levels preceded the lipid profile.
Currently, combination of two different lipid-lowering drugs is frequently prescribed in controlling dyslipidemia. Statin plus ezemtibe in combination is the most commonly used style. In recent years, several researchers have described the combination of statin and ezetimibe on the increase of PCSK9 levels in humans. A recent study conducted by Davignon et al.  demonstrated that patients treated with statins alone had a 45% increase in PCSK9 levels and those treated with statin plus ezetimibe showed an approximately 77% increase in PCSK9 concentrations. In our study, the PCSK9 levels increased by 111% in rats administrated with pitavastatin 10 mg/kg daily, while added ezetimibe 10 mg/kg daily to pitavastatin, the PCSK9 levels enhanced by 204%. However, little was known about the reason why ezetimibe causes a further increase in PCSK9 levels. Since ezetimibe exerts its effect by binding to Niemann-Pick C1-Like 1, and thereby inhibiting intestinal cholesterol absorption and subsequently results in reduced hepatic cholesterol , it may be possible that this process is accompanied by a feedback mechanism on transcription factor SREBP-2. Furthermore, in mice models, Brandon Ason et al.  applied qRT-PCR to analyze the expression of 361 genes involved in hepatic lipid metabolism and found that many genes within the SREBP-2 pathway were induced following ezetimibe treatment (2.5-fold average induction relative to control) and were even further induced by ezetimibe plus rosuvastatin combination treatment (11-fold average induction relative to control). This study provides theoretical evidences about the impact of ezetimibe on plasma PCSK9 levels. In contrast, Heiner K. Berthold et al.  indicated that when added to simvastatin, ezetimibe does not cause an incremental increase in PCSK9 concentrations, which conflicted with our observations. However, this study explored the 14 days effect of ezetimibe-statin in combination on plasma PCSK9 levels in humans, and different kinds of statins were used in the two studies. Even so, for the sake of exploring a thorough explanation of the mechanism, further studies are warranted.
In our study, we also observed that combination of ezetimibe 10 mg/kg and Xuezhikang 1200 mg/kg daily did not bring a greater increase in PCSK9 levels than monotherapy, and the PCSK9 levels was significantly lower than co-therapy with ezetimibe plus pitavastatin. Therefore, for patients who need an intensive lipid-lowering therapy, the combination of Xuezhikang and ezetimibe would be a better alternative therapy. This combination could be a better strategy not only for exerting good lipid-lowering effect on LDL-C, but also for mitigating the adverse effects of lipid-lowering drugs on PCSK9 in controlling dyslipidemia and atherosclerosis. At the present time, with regarding to the mechanism by which ezetimibe plus Xuezhikang causes an attenuated increase in PCSK9 levels compared to monotherapy and co-therapy with ezetimibe plus pitavastatin is unclear, but it may likely associated with the chemical constituents of Xuezhikang. Xuezhikang, a Chinese traditional medicine, is widely used as a lipid-lowering drug, and its main components contain lovastatin, as well as other useful substances [16–18]. Recent studies including our data have shown that Xuezhikang has many pleiotropic effects, which could effectively modify not only lipid profile but also inflammatory markers . The interactions among different constituents of Xuezhikang and ezetimibe may influence the effect on PCSK9 levels, but clinical trials and molecular mechanisms are needed in the further studies.
For the past decade, PCSK9 has gained tremendous attention. It is well-established that statin treatment elevates plasma PCSK9 levels and, on the contrary, inhibition of PCSK9 function enhances the lipid-lowering effects of statins [25, 26]. Among the various PCSK9 inhibitors, human data are available for monoclonal antibodies against PCSK9 of which the two most advanced are SAR236553/REGN727 and AMG 145. In a 12-week phase II study of patients with LDL-C levels ≥ 100 mg/dl on a stable dose of atorvastatin (10, 20, or 40 mg/day), add-on of SAR236553/REGN727 (doses 50–150 mg) administered subcutaneously every 2 weeks resulted in LDL-C reductions of 40–72% . The LAPLACE-TIMI trial evaluated patients with documented hypercholesterolemia, while on a statin with or without ezetimibe treatment. The data had shown reductions of LDL-C by 41.8% to 66.1% when administrated AMG 145 at 70, 105 and 140 mg every 2 weeks, with acceptable safety and tolerability . Combining our findings and previous studies together may suggest that, not only statins, but also ezetimibe and Xuezhikang could increase plasma PCSK9 levels, and this effects occurred before the changes of lipid-profile, which strongly suggest that PCSK9 inhibitors should be combined with the currently prescribed lipid-lowering drugs to result in further LDL-C lowering effect, and it would be better to be used prior to lipid-lowering drugs. Moreover, our data also provides additional information with respect to combination of ezetimibe plus Xuezhikang may be a better choice than ezetimibe plus pitavastatin in clinical practice.
There are several limitations in the present study. First, we did not observe the long-term effect of different lipid-lowering drugs, alone and in combination, on plasma PCSK9 and lipid-profile concentrations. Second, we did not explore the molecular mechanisms of the phenomenon. Hence, further studies are needed.