Study participants
Patients with hyperlipidemia (diagnosed as hypercholesterolemia or hypertriglyceridemia, or mixed hyperlipidemia, and high low-density lipoproteinemia according to 2019 the European Society of Cardiology guidelines) with no other diseases were selected for this study. No statin or other lipid-lowering drugs were prescripted in those patients. All patients provided and signed informed consents. The study was approved by the Ethical Committee of Shanghai Pudong Hospital (Fudan University, Pudong Medical Center, Shanghai, China) and experiments were performed in accordance with the Ethical Committee’s guidelines and regulations. ClinicalTrials.gov number was NCT 03491956.
Exclusion criteria: Patients were excluded if (1) coexistence with diabetes or hypertension. (2) There were tumors, liver and kidney dysfunction, severe edema, cardiac insufficiency, respiratory insufficiency caused by severe lung disease, and pregnant. (3) Age over 80 years old.
Treatment procedure
Plasmapheresis using Plasauto iQ automatic blood purification system, model KM-9000 (Kawasuml, Japan) was used in this study, with primary membrane PE-08 plasma separator and secondary membrane plasma component separator EC-4A20. Cubital elbow median veins on both arms were selected as artery and vein access. Blood flow was 60-100ml/min, plasma separation rate was 25-30ml/min and plasma rejection rate was 15ml/min. Ordinary heparin was used as an anticoagulant, initial dose of 3000 U, followed by additional maintenance dose 20-40U/(kg•h). Calculation of therapeutic target as estimated by weigh (kg) ´40ml (1-1.4 times blood volume). Each session lasted for 3-4 hours.
Laboratory examination
Isolation of PBMCs
PBMCs from hyperlipidemia patients were separated from 5 ml venous blood and isolated by lymphocyte separation medium (Haoyang, China). PBMC were collected and washed with PBS for 3 times for later experimental assays.
Detection of lipids metabolic, ER stress-related and apoptosis-related proteins
Lipids metabolic (CD36, PCSK9, LDLR), ER stress-related (GRP78, CHOP, ATF4, EIF2a) and apoptosis-related (Bcl-2, BAX, Caspase-3) proteins in PBMCs were examined by western blotting analyses. The lysis buffer (Invent, China) used to extract the proteins of PBMC. Proteins were separated and transferred onto polyvinylidene difluoride (PVDF) membrane. In brief, the protein was detected as described previously (19). The primary antibodies against LDLR, CD36, PCSK9, GRP78, CHOP, ATF4, p-EIF2a, Bcl-2, BAX and Caspase-3, and secondary antibodies were obtained from Proteintech (Proteintech Group, Inc, IL, USA) and antibody against EIF2a was purchased from CST (Danvers, MA 01923 USA).
Detection of ROS and serum inflammatory factors
ROS level in PBMCs was examined by reactive oxygen species assay kit (Beyotime, China) and followed by flow cytometry (FCM). Serum inflammatory factors (IL-1b,IL-6, TNF-awere detected by ELISA using commercial kits ( Neobioscience, China ).
Statistical Analysis
Data are expressed as means ± SEM, with the exception of skewed data, which are expressed as median (range). After correcting non-normally distributed data, the differences were calculated by paired t test, p value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 22.0 statistical software (SPSS, Chicago, IL, USA).