Protection Effect Of Plantain Seed Polysaccharid On Human Umbilical Vein Endothelial Cells Injury Induced By Oxidized Low Density Lipoprotein

Atherosclerosis (Atherosclerosis, AS) is a major disease which is harmful to human. Research for preventing and treating AS is a main subject in the theory and clinical research of medicine. Hyperlipemia is a main factor in AS, of which oxidized low-density lipoprotein (oxidized low-density lipoprotein, ox-LDL) is an initiating factor and leads to vascular endothelial cell damage, smooth muscle cell proliferation, monocyte adhesion, platelet aggregation and foam cell formation, which then contributes to endothelial cell dysfunction, excretion of a variety of cell activitive factors and growth factors, which eventually leads to plaque formation. Therefore, inhibiting the production of ox-LDL or reducing the damage induced by ox-LDL is a key to prevent the occurrence and development of AS.Nowadays, angiotensin-converting enzyme inhibitor (ACE-I),β-receptor blockers, and lipid-lowering drugs are common in improving endothelial dysfunction. However, two main issues exist: 1, Block the necessary substances which is key to physiological function; 2, most of the drugs have side effects on liver and kidney. In views of the western modern medicine, AS is a disease of unknown etiology, the best treatment is to alleviate symptoms and inhibit medical strategies. Chinese medicine, as an important component of most natural substances for human beings to maintain the health care system, are in line with the "Return to Innocence", "Return to Nature" trend, and is adapted to biological-psychological-social medicine model. Chinese Pharmacopoeia records, Plantago record for the Plantaginceae's dry plant mature seeds. Modern pharmacological research show that Plantago not only plays a key role in clean-up of intestinal, lowering blood lipids, regulating blood sugar and disturbing the immune system, but also inhibited lipid peroxidation and free radical production, and has important role in arterial wall remodeling. However, the mechanism is unclear whether to improve endothelial function.In this experiment, we extracted the Plantago active ingredients polysaccharide by using modern technical means, to explore the effect and mechanism of plantain seed polysaccharide (plantain seed polysaccharide, PSP) on the injury of vascular endothelial cells through the ox-LDL in cultured endothelial cells in vitro, providing theoretical basis for prevention and treatment of AS.Part.1 Improved cultivation and identification of human umbilical vein endothelial cells in vitroObjective: To establish a simple, effective and highly reproducible method for primary and passaged human umbilical vein endothelial cells (HUVEC) in vitro and identify the cultural cells for further study.Methods: (1) Culture medium preparation: VEGF, penicillin and streptomycin were added to 10% and 20% FBS DMEM culture medium respectively and the final concentration of VEGF, penicillin and streptomycin were 15 ng /ml, 100 U/ml and 100 U/ml. (pH 7.0~7.2) (2) Isolation and culture of the primary HUVECs: Fetal umbilical cord (more than 20 cm) were extracted from fetal umbilical cord HUVECs with typeⅠcollagenase perfusion and digestion, then added DMEM culture medium containing 20% FBS, placed in incubator to be cultivated. (3) The passage of HUVECs: The primary HUVECs were 80% confluence, digested with trypsin solution. The DMEM culture medium of 10% FBS was cultured in incubator. (4) Identification of HUVECs:①Morphology, adhesion and growth of cell were observed under the inverted microscope every day.②Cells were detected by immunohistochemistry with factorⅧantigen-antibody.Results: (1) Growth and morphological changes of HUVECs:①Growth time: the primary endothelial cells adherent growth after about 2 h, flat polygonal cells, completely adherent to become fusiform after 24 h, and fusion into a single layer after 7~10 d. The passaged cells distributed more evenly, adhered after 0.5 h around and growed monolayer cells typical cobblestone-like after 3~5 d.②Cell morphology: Endothelial cells can be seen small triangular, circular, the majority of cells agglomerans under the inverted microscope in primary culture. The growth of active cells can be seen oval or round with the clear border, rich cytoplasm and small particles. The cells emerged spindle. Passaged cells which were densely, distributed evenly, growed faster than the primary cells. The passaged cells can be seen short spindle or polygonal, round nuclei under the inverted microscope. These cells showed cobblestone-like arrangement and demonstrated obvious halation near the nucleus, which were the typical characteristics of endothelial cell growth and can be used for identification of endothelial cells.(2)FactorⅧ-related antigen-antibody immunohisto chemical detection: Cells can be seen round, oval or spindle. They were rich in cytoplasm and had brown granular in cytoplasm with no staining in the nuclei, which was positive. But the control group (add PBS solution) had not cytoplasmic brown granular, which was negative. The cultured cells could be further confirmed as the endothelial cells.Conclusion: The human umbilical vein endothelial cells could be isolated with 0.1% typeⅠcollagenase and digested for 10min in the level of shaking bed at 37℃. These cells were primarily cultured and passaged. Clearly halo department near the surrounding cells can be seen under the inverted microscope, which was the typical characteristic of endothelial cell to be identified as endothelial cells. Cultured cells were further confirmed as vascular endothelial cells by immunohistochemistry with factorⅧantigen-antibody. Part.2 Protection effect of plantain seed polysaccharid on human umbilical vein endothelial cell injury induced by oxidized low density lipoproteinObjective: To investigate the protective effect of PSP on the lipid peroxidation injury of cultured HUVECs induced by oxidized low density lipoprotein and study possible mechanism.Methods: Proliferation rate, NO content, NOS activity MDA content, SOD vitality of HUVECs were assessed by MTT assay and chemical methods in the cellular level; ICAM-1 expression and apoptosis-related gene mRNA were assessed by ELISA and RT-PCR in the molecular level. We observed the effect of different concentrations of PSP (25 mg/L, 50 mg/L, 100 mg/L) on the above parameter.Results: (1) The effect on cell proliferation rate: the proliferation rate in ox-LDL injuryed group decreased significantly compared with the control group. The cell proliferation rate in low concentration of PSP group was (9.65±0.89)% and had significant difference compared with ox-LDL injured group; whereas the cell proliferation rate in medium and high concentrations of PSP group were (39.03±3.35)% and (54.78±4.03)% respectively, and had significant difference compared with ox-LDL injured group.(2) The effect on MDA content: The content of MDA in control group was 1.31±0.39 nmol/ml, whereas the ox-LDL injured group was 5.07±0.78 nmol/ml, and had significance difference compared with the control group (P<0.01), after treatment with different concentrations of PSP the content of MDA were 4.61±0.70, 2.83±0.48, 1.29±0.31 nmol/ml, which had significant difference compared with ox-LDL injured group (P<0.05), and was in dose-dependent manner, indicating that PSP has a certain antioxidant.(3) The effect on SOD activity: The activity of SOD in control group was 21.65±1.89 U/ml, whereas the ox-LDL injured group was 13.97±0.97 U/ml and had significance difference compared with the control group (P<0.01). The activity of SOD was 15.76±1.13, 18.58±1.64, 20.92±1.78 U/ml in low, medium and high concentrations of PSP respectively. Different concentrations of PSP groups were higher than the ox-LDL injured group in SOD activity and there was no dose-dependent (P<0.05).(4) The effect on NO content: The PSP increased the content of NO and had a dose-dependent. The content of NO was 69.73±3.35μmol/L in control group, whereas the ox-LDL injured group was 31.71±1.91μmol/L, the NO content in low concentration of PSP group was 46.41±2.25μmol/L, which had significant difference with ox-LDL injured group (P<0.05). The NO content in medium and high concentrations of PSP group were 57.68±3.03, 65.51±3.12μmol/L respectively, and had significant difference compared with ox-LDL injured group (P<0.01).(5) The effect on NOS activity: The activity of NOS was 0.99±0.09 U/ml in control group, whereas the ox-LDL injured group was 0.31±0.02 U/ml and had significance difference compared with the control group (P<0.01). The activity of NOS in low concentration of PSP group was 0.55±0.06 U/ml and had significant difference compared with ox-LDL injured group (P<0.05). The activity of NOS in medium and high concentrations of PSP group were 0.71±0.07 and 0.98±0.10 U/ml, enhanced respectively in a dose-dependent manner, which had significant difference with ox-LDL injured group (P<0.01).(6) The effect on ICAM-1 expression: The expression of ICAM-1 was 4.35±0.64 pg/ml in control group, whereas the ox-LDL injured group was 5.23±0.78 pg/ml, the expression of ICAM-1 after different concentrations of PSP were 5.13±0.58, 4.28±0.31, 4.36±0.52 pg/ml, and they were not in dose-dependent manner, which had significant difference with ox-LDL injured group (P<0.05).(7) The results of RT-PCR: PSP can reduce transcriptional level of apoptosis-associated genes (c-myc, P53) in a concentration-dependent manner after HUEVCs injured by ox-LDL. The relative expressions of c-myc were 0.2029±0.0225, 0.3094±0.0572, 0.2837±0.0482, 0.2792±0.0457 and 0.2705±0.0323 in control group, ox-LDL injured group, low, medium and high concentrations of PSP groups. The relative expressions of P53 were 0.1839±0.0122, 0.2841±0.0203, 0.2594±0.0371, 0.2278±0.0197 and 0.2189±0.0210 in above five groups. Different concentrations of PSP groups had significant difference with ox-LDL injured group (P<0.05). Conclusion: (1)PSP can stabilize endothelial cells membrane structure and protect the cells from injury of free radical, which reduce the production of MDA, and increase the activity of SOD. PSP not only increased proliferate activity of HUVEC but also resisted injury on HUVEC by ox-LDL.(2)PSP can get a balance between the release of NO and the expression of NOS, and resist the expression of ICAM-1 injury in HUVECs induced by ox-LDL.(3)PSP can down-regulate the mRNA level of apopsis-related gene such as c-myc and P53 induced by endothelial cells injury mediated by ox-LDL.