Protection effect of endomorphins on advanced glycation end products induced injury in endothelial cells

Abstract

Endomorphins (EMs) have a very important bridge-function in cardiovascular, endocrinological, and neurological systems. This study is to investigate the effects of EMs on the synthesis and secretion of vasoactive substances induced by advanced glycation end products in primary cultured human umbilical vein endothelial cells (HUVECs). Firstly, HUVECs were stimulated with AGEs-bovine serum albumin (AGEs-BSA), bovine serum albumin (BSA), or both AGEs-BSA and EMs together, respectively. Then, HUVEC survival rate was calculated by MTT assay, the levels of NO, endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were detected by colorimetric analysis, and the contents of endothelin-1 (ET-1) were detected by ELISA. The mRNA levels of eNOS and ET-1 were measured by RT-PCR. The expression of p38 mitogen-activated protein kinase (p38 MAPK) was detected by immunofluorescence assay. The results showed that the mRNA expression and secretion of eNOS were significantly enhanced after incubation with EMs compared to those with AGEs-BSA, while the secretion of NO and iNOS, mRNA expression, and secretion of ET-1 had opposite changes. The fluorescence intensity of p38MAPK in nuclear was decreased after pretreatment with EMs compared to incubation with AGEs-BSA. Conclusion. The present study suggests that EMs have certain protection effect on AGEs-BSA-induced injury in HUVEC.

Figure 1

Effect of AGEs-BSA on cell viability determined by MTT test. HUVECs were treated with AGEs-BSA (100 mg/L) or BSA (100 mg/L) for 6 h, 24 h, 48 h. Viability was calculated as the percentage of living cells in treated cultures compared to those in control cultures. Each value represents the mean ± SD (n = 3). *P < 0.05,  **P < 0.01 versus BSA group.

Figure 2

Effect of EM1, EM2 on cell viability determined by MTT test. HUVECs were treated with EMs (10 μM, 1 μM, 0.1 μM, 10 nM) for 2 h before treatment with AGEs-BSA (100 mg/L) for 6 h, 24 h, 48 h. Viability was calculated as the percentage of living cells in treated cultures compared to those in control cultures. Each value represents the mean ± SD (n = 3). Statistical analysis compared with AGEs-BSA group by ANOVA. *P < 0.05,  **P < 0.01.

Figure 3

Effect of EM1, EM2 on NO concentration determined by Griess reaction test in HUVEC. Each data is expressed as mean ± SD (n = 3). *P < 0.05,  **P < 0.005 versus AGEs-BSA group, ^# P < 0.05,  ^## P < 0.005 versus control (BSA) group.

Figure 4

Effect of EM1, EM2 on iNOS level in HUVEC. Each data is expressed as mean ± SD (n = 3). *P < 0.05,  **P < 0.005 versus AGEs-BSA group, ^# P < 0.05,  ^## P < 0.005 versus control (BSA) group.

Figure 5

Effect of EM1, EM2 on eNOS secretion determined by ELISA test in HUVEC (a). HUVECs were incubated according to the aforementioned grouping. Each value represents the mean ± SD (n = 3); mRNA expression level of eNOS after treatment with AGEs-BSA and EM1 or EM2, using BSA treated cells as reference control (b). The parameter Ct was derived for each cDNA sample and primer pair; for a given sample, Ct values for β-actin were subtracted from the Ct of each candidate gene reaction to arrive at a ΔCt value. The mean ΔCt from all control reactions was then subtracted from the ΔCt of each treated sample to arrive at ΔΔCt. The relative fold change was calculated by the expression 2^−ΔΔCt. Each data is expressed as mean ± SD (n = 3). *P < 0.05,  **P < 0.005 versus AGEs-BSA group, ^# P < 0.05,  ^## P < 0.005 versus control (BSA) group.

Figure 6

Effect of EM1, EM2 on ET-1 secretion determined by ELISA test in HUVEC (a). HUVECs were incubated according to the aforementioned grouping. Each value represents the mean ± SD (n = 3); mRNA expression level of ET-1 after treatment with AGEs-BSA and EM1 or EM2, using BSA treated cells as reference control (b). The parameter Ct was derived for each cDNA sample and primer pair; for a given sample, Ct values for β-actin were subtracted from the Ct of each candidate gene reaction to arrive at a ΔCt value. The mean ΔCt from all control reactions was then subtracted from the ΔCt of each treated sample to arrive at ΔΔCt. The relative fold change was calculated by the expression 2^−ΔΔCt. Each data is expressed as mean ± SD (n = 3). *P < 0.05,  **P < 0.005 versus AGEs-BSA group, ^# P < 0.05,  ^## P < 0.005 versus control (BSA) group.

Figure 7

Immunofluorescence studies of EM1 on p38 MAPK in HUVECs. Cells were fixed, and incubated with p38 MAPK antibody and a FITC-conjugated second antibody. Pictures were taken at 400x magnification. ((a) BSA, (b) AGEs-BSA, (c) 10 μM, (d) 1 μM, (e) 0.1 μM, (f) 10 nM).

Figure 8

Immunofluorescence studies of EM2 on p38 MAPK in HUVECs. Cells were fixed and incubated with p38 MAPK antibody and a FITC-conjugated second antibody. Pictures were taken at 400x magnification. ((a) AGEs-BSA, (b) BSA, (c) 10 μM, (d) 1 μM, (e) 0.1 μM, (f) 10 nM).