| Objective:To explore the protective effects of ganoderma lucidum polysaccharides peptide on human umbilical vein endothelial cells induced by oxidative stress and to investigate its possible mechanisms.Methods: Primary HUVEC cells were cultured in vitro.It was definited as HUVECs using CD31 immunofluorescence. HUVEC cells were injured by ROS,derived from the different concentration ( 2.4×10-5,4.8×10-5,9.5×10-5,1.9×10-4, 3.8×10-4, 7.6×10-4 mol·L-1)of t-BOOH.The activity of cells was measured by Cell Counting Kit-8 assay. We selected the injured group by 1.9×10-4 mol·L-1 t-BOOH as study object. And then,we use the different concentration (6.125,12.5,25,50,100 mg·L-1 ) of GLPP to act on the injured group.At the indicated time,we collected cells from different groups. In addition, Hoechst 33258 nucleus staining was used to observe the early apoptosis injury by ROS. And the activity of Caspase-3 was detected by spectrophotometry. Electron microscopy was used to show the morphological change in HUVEC cells. To investigate the change of mitochondrial membrane potential (ΔΨm),the rhodamine123(Rh123) fluorescence intensity of HUVEC cells with Rh123 stain was observed by laser scanning confocal microscope. Western Blot was used to detect protein expression of Bcl-2 in the HUVEC cells.The ROS content was detected by DCFH-DA .Results:1.The cultivation and identification of HUVEC cellsPrimary HUVEC cells were cultured in vitro.It was definited as HUVECs using morphology and immunofluorescence. 2. The oxidative damage of HUVEC cells by different concentrations of t-BOOHCCK-8 results showed that the different dose of t-BOOH (2.4×10-5, 4.8×10-5,9.5×10-5,1.9×10-4,3.8×10-4,7.6×10-4mol·L-1) cultured for 24 hours,the cells injury induced by oxidative damage. The difference of the rate of injured cells between oxidative damage group and normal control group was significant statistically(P <0.05),except for 2.4×10-5 mol·L-1 group (P >0.05)3. GLPP reduced oxidative damage of HUVEC cells induced by 1.9×10-4 mol·L-1t-BOOHCCK-8 assays showed that OD value of control group was 1.30±0.051, 1.9×10-4 mol·L-1t-BOOH group without GLPP was 0.57±0.060. While the different concentration(6.125,12.5,25,50,100 mg ? L-1) of GLPP reduces oxidative damage of HUVEC cells by t-BOOH. OD values were 0.66±0.063,0.74±0.080,0.85±0.060,0.91±0.057,1.07±0.087 respectively . The difference had statistical significance between each GLPP group and 1.9×10-4 mol·L-1t-BOOH group without GLPP (P <0.05).4. GLPP reduced the apoptosis of the injured HUVEC cells by t-BOOHAs shown in Hoechst 33258 staining assay ,the apoptotic rate of control group was (3.2±1.2)%,cells using 1.9×10-4 mol·L-1t-BOOH without GLPP was (57.8±3.0)%. While the different concentration(6.125,25,100 mg·L-1)of GLPP can protect HUVEC cells from apoptosis by t-BOOH .Their apoptotic rate of cells were(47.4±1.1)%,(33.8±3.9)%,(25.8±1.6)% respectively(P <0.05).5. Effect of GLPP on morphology change of the injured HUVEC cells by t-BOOHAs shown in electron microscopy assay, the nucleus and the organelle such as mitochondria injury by ROS could be relieved by GLPP(6.125,100 mg·L-1).6. Effect of GLPP on the caspase-3 activity change of the injured HUVEC cells oxidative damage induced by t-BOOHColorimetry showed Caspase-3 activity of the control group was 0.03±0.005,t-BOOH injury group was 0.09±0.075.GLPP(6.125,25,100 mg·L-1) Caspase-3 activity were 0.07±0.069,0.05±0.087, 0.04±0.041 respectively(P <0.01).7. Effect of GLPP on the△Ψm change of the injured HUVEC cells by t-BOOH The Rh123 fluorescence and MFI in HUVEC cells control group was 122.64±10.03,treated with t-BOOH for 24h was 57.96±8.35. GLPP(6.125,25,100 mg·L-1)MFI were 75.54±9.78,91.3±8.16,106.44±9.00(P <0.05).8. Effect of GLPP on the Bcl-2 protein expression change of the injured HUVEC cellsExpression of Bcl-2 was substantially decreased after t-BOOH 1.9×10-4 mol·L-1 for 24h but prevented by GLPP (6.125,25,100 mg·L-1).9. Effect of GLPP on the ROS change of injured HUVEC cells by t-BOOH DCFH-DA results show that control group MFI was 52.92±11.09,1.9×10-4 mol·L-1t-BOOH group MFI was 112.89±10.37, GLPP (6.125,25,100 mg? L-1) MFI were 91.55±9.10, 79.66±9.86, 64.4±8.12 ( P <0.01).Conclusions:1. GLPP has protective effects against oxidative damage in primary HUVEC cells;2. GLPP reduces cells damage by oxidative damage and the corresponding enzyme and gene expression changes in apoptosis;3.GLPP can prevent the ultra-structure change of cells from oxidative damage;4. GLPP can prevent the mitochondrial membrane damage of cells from oxidative damage;5. The protective effects of GLPP against oxidative damage might be related to the elimination of intracellular free radicals.
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