Of CCN1 knockdown on the apoptosis of PAinduced HUVECs. Apoptosis of HUVECs treated with PA was detected by TUNEL assay; the results revealed that PA promoted apoptosis of PAinduced HUVECs compared with that within the handle group (Fig. 3A). Additionally, the expres sion levels of apoptosisassociated proteins had been detected by western blotting; the Protein tyrosine phosphatases Proteins Source expression levels of Bax and cleaved caspase3 had been enhanced right after PA therapy, whereas the expression degree of Bcl2 have been inhibited. Furthermore, CCN1 knockdown reversed the aforementioned effects triggered by PA compared with inside the PA group (Fig. 3B). General, theseGAN et al: INFLAMMATION AND APOPTOSIS OF HUVECs ARE REGULATED BY DKK1/CCN1 SIGNALINGFigure 2. (A) NO production in every single group. (B) Protein expression levels of peNOS and eNOS in each and every group. (C) Protein expression levels of pIKK , IKK, pNF B and NF B in every group. (D) Concentrations of TNF, IL1 and IL6 inside the cell medium. Cells have been treated with 0.eight mM PA. P0.001 vs. manage group; ##P0.001, ###P0.001 vs. PA + control siRNA group. NO, nitric oxide; p, phosphorylated; eNOS, endothelial nitric oxide synthase; PA, palmitic acid; CCN1, cysteinerich angiogenic inducer 61; siRNA, little interfering RNA.benefits recommended that CCN1 knockdown could inhibit cell apoptosis induced by PA. Expression levels of DKK1 and catenin in PAinduced HUVECs. It has previously been reported that inhibition from the Wnt/catenin signaling pathway can ameliorate endothelial cell injury (19); thus, DKK1, which has been considered to obstruct the Wnt/ catenin signaling pathway (20), was investigated inside the present study. The results revealed that the expression levels of catenin had been progressively elevated, whereas these of DKK1 had been decreased when HUVECs had been exposed to escalating concentrations of PA compared withthose inside the control group (Fig. 4A), as a result suggesting that PA may inhibit the expression levels of DKK1 and activate the Wnt/ catenin signaling pathway. Subsequently, OEDKK1 and DKK1 siRNA were transfected into HUVECs, in order to KIR2DS1 Proteins Formulation regulate the expression of DKK1 (Fig. 4BF); transfection of was effective, and siRNADKK1#1 exhibited an enhanced knockdown impact. Effects of DKK1 on the expression of CCN1 in PAinduced HUVECs. The effects of overexpression or silencing of DKK1 on the expression levels of CCN1 and catenin were subsequently assessed. The outcomes of RTqPCR and westernMOLECULAR MEDICINE REPORTS 23: 122,Figure 3. (A) TUNEL assay was performed to stain apoptotic cells (magnification, x100). (B) Protein expression levels of Bax, Bcl2, cleavedcaspase3 and caspase3 in every single group. Cells were treated with 0.eight mM PA. P0.001 vs. handle group; ###P0.001 vs. PA + control siRNA group. PA, palmitic acid; CCN1, cysteinerich angiogenic inducer 61; siRNA, tiny interfering RNA.blotting revealed that overexpression of DKK1 decreased the expression levels of CCN1 and catenin compared with those inside the PA group; on the other hand, the opposite effects have been detectedwhen DKK1 was silenced (Fig. five). These benefits recommended that activation and inhibition in the Wnt/catenin signaling pathway may possibly regulate the expression of CCN1.GAN et al: INFLAMMATION AND APOPTOSIS OF HUVECs ARE REGULATED BY DKK1/CCN1 SIGNALINGFigure 4. (A) Protein expression levels of DKK1 and catenin in HUVECs exposed to 0.two, 0.4 and 0.eight mM PA for 24 h. P0.01, P0.001 vs. handle group. (B) Transfection efficiency of OEDKK1. P0.001 vs. OENC group. (C) Protein and (D) mRNA expression levels of DKK1 in HUVECs exposed.