Effect of catheterin-related antimicrobial peptides on the high glucose induced cardiac microvascular endothelial cell injury / 中华老年医学杂志

ABSTRACT

Objective:To investigate the effect of catheterin-related antimicrobial peptides(CRAMP)on the damage of cardiac microvascular endothelial cells induced by high glucose.Methods:Adult mouse heart microvascular endothelial cells were isolated and cultured.A model of microvascular endothelial cell injury was established by high glucose culture.The endothelial cells were randomly divided into 4 groups as the following.In the control group, 27.5 mmol/L mannitol was given as isoosmotic control as compared with the high glucose group.In the high glucose group(HG group), cells were cultured with 33.3 mmol/L high glucose for 48 h, and then treated without CRAMP.In 0.15 mg/L CRAMP treatment group, cells were cultured with 33.3 mmol/L high glucose for 48 h, followed by 0.15 mg/L CRAMP treatmen for 24 h. In the 0.5 mg/L CRAMP treatment group, cells were cultured with 33.3 mmol/L high glucose treatment for 48 h, and then treated with 0.5 mg/L CRAMP for 24 h. Cell proliferation was examined by staining with CKK-8 cell counting kit.The secretion of inflammatory factors in microvascular endothelial cells was detected by ELISA kit.Reactive oxygen species assay kit detects the level of reactive oxygen species in cells.Cell apoptosis was detected by apoptosis kit.Tubule formation and tubule number were measured by cells cultured on the matrix glue membrane, then detected by microscopic observation.The nitric oxide(NO)test kit measures levels of NO.The expression of nitric oxide synthase(eNOS)was detected by western blotting.Results:The cell proliferation activity was significant lower in the HG group than in control group[(52.2±5.4)% vs.(100.0±7.3)%]. The cell proliferation activity was higher in the 0.15 and 0.5 mg/L CRAMP groups than in the HG group[(72.0±3.4)% vs.(52.2±5.4)%; and(84.2±5.8)% vs.(52.2±5.4)%( F=75.300, P<0.001)]. The expression of tumor necrosis factor-α was significantly higher in the HG group than in the control group and in 0.5 mg/L CRAMP group[HG group of(239.1±32.1)μg/L, the control of(22.1±3.7)μg/L, 0.5 mg/L CRAMP of(84.6±9.4)μg/L]( F=197.300, P<0.001). The level of reactive oxygen species was significantly higher in the HG group than in control group and in 0.5 mg/L CRAMP group[(20.8±2.4)in HG group, (4.8±1.7)in control group, (10.2±1.5)in CRAMP group]( F=105.700, P<0.001). The number of apoptotic cells was significantly higher in the HG group than in control group and in 0.5 mg/L CRAMP group[(21.2±3.1)% in HG group, (2.2±0.6)% in control group(9.5±1.2)% in CRAMP group]( F=141.900, P<0.001). The length and number of tubules were lower in the HG group than in control group and in CRAMP group[for the length (87.8±9.1)μm in HG group, (337.0±37.2)μm in control group(206.5±16.3)μm in CRAMP group( F=160.800, P<0.001); for the number (9.1±1.9)in HG group, (22.0±3.4)in control group, (16.8±2.2)]in CRAMP group( F=36.200, P<0.001)]. The level of NO was lower in the HG group than in control group and in CRAMP group[(0.25±0.05)in HG group, (1.05±0.16)in control group, (0.75±0.06)in CRAMP group( F=83.200, P<0.001)]. The protein expression and mRNA levels of endothelial nitric oxide synthase(eNOS)were lower in the HG group than in the control group and in CRAMP group[for eNOS protein (0.07±0.03)in HG group, (0.81±0.05)in control group, (0.54±0.07)in CRAMP group, F=275.700, P<0.001; and for eNOS mRNA (0.11±0.07)in HG group, (1.00±0.22)in control group, (0.57±0.12)in CRAMP group, F=50.600, P<0.001]. Conclusions:CRAMP protein can inhibit the damage of cardiac microvascular endothelial cells by increasing eNOS-mediated NO signal pathway.