Hages, neutralizing antibody or tiny interfering RNA (siRNA) could inhibit the activity of CCN1, thereby attenuating oxidized lowdensity lipoprotein (oxLDL)induced lipid accumulation (7). Moreover, CCN1 has been shown to promote apoptosis of endothelial cells within the presence of TNF (2).Correspondence to: Dr YanHong Ding, Department ofAnesthesiology, The first People’s Hospital of Lanzhou City, 1 Wujiayuan West Street, Qilihe, Lanzhou, Gansu 730050, P.R. China E mail: [email protected] Dr DingXiong Xie, Gansu Cardiovascular Institute, 1 Wujiayuan West Street, Qilihe, Lanzhou, Gansu 730050, P.R. China E-mail: [email protected] equallyKey words: Dickkopf1, cardiovascular diseases, cysteinerichangiogenic inducer 61, human umbilical vein endothelial cellsGAN et al: INFLAMMATION AND APOPTOSIS OF HUVECs ARE REGULATED BY DKK1/CCN1 SIGNALINGFatty acids (FAs) could be classified into three main kinds: Quick, medium and longchain FAs (SCFAs, MCFAs and LCFAs, respectively). Several research have demonstrated that, in contrast with SCFAs and MCFAs, LCFAs bear higher dangers for the occurrence of coronary heart disease, which can be one of several main forms of CVD (eight,9). Palmitic acid (PA), which falls below the category of LCFAs, would be the most common Complement Factor H Related 2 Proteins Storage & Stability saturated FA in food, plants and animal items. PA has been reported to become involved in the apoptotic method of various cells, such as cardiomyocytes and endothelial cells (1013). Furthermore, a prior plasma metabolomic study has identified PA as a novel biomarker of atherosclerosis (14). On the other hand, small is presently recognized regarding the function of CCN1 in PAinduced endothelial cell injury. Human umbilical vein endothelial cells (HUVECs) are widely utilised to study the functions of endothelial cells (1517). The present study aimed to discover the mechanism by which CCN1 exerts its effects on the inflammation and apoptosis of PAinduced HUVECs. Materials and techniques Cell culture. The HUVEC line Carboxypeptidase D Proteins Recombinant Proteins utilized within the present study was obtained from Shanghai Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were cultured in Dulbecco’s modi fied Eagle’s medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10 fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37 in an atmosphere containing 5 CO2. PA (SigmaAldrich; Merck KGaA) was dissolved in 0.1 mM sodium hydroxide at 70 and combined with 10 fatty acidfree BSA (Beijing Solarbio Science Technology Co., Ltd.) at 55 for ten min to achieve the final concentrations. The obtained PA (0.two, 0.four and 0.8 mM) was utilized to stimulate HUVECs for 24 h at 37 . Cell transfection. siRNAs targeting CCN1 and Dickkopf1 (DKK1) (CCN1 siRNA#1 and CCN1 siRNA#2; DKK1 siRNA#1 and DKK1 siRNA#2, respectively) in addition to a damaging handle siRNA (handle siRNA) have been synthesized by Guangzhou RiboBio Co., Ltd. DKK1 overexpression plasmids (OEDKK1) and adverse manage plasmids (empty pCEP4 vector; OENC) had been supplied by Shanghai GenePharma Co., Ltd. HUVECs (1×106 cells/well) were incubated at 37 until they reached 7080 confluence, and had been transfected with 30 nM siRNA or 20 plasmids working with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in line with the manufacturer’s guidelines. A total of 48 h posttransfection, cells had been collected to verify transfection efficiency. Transfected cells had been then treated with 0.8 mM PA for 24 h at 37 in subsequent experiments. The sequences are shown in Table SI.