The Estrogen-like Protective Effects Of Ginsenoside Rb3on Vascular Endothelial Cells

Objective: Human umbilical vein endothelial cells (HUVECs) were separated, incubatedand identified to provide solid foundation for the futher research in vitro. To explorethe protective mechanism of ginsenoside Rb3on impaired endothelial cells and compare theeffects with estrogen to provide experimental basis for the clinical application of Panaxquinquefolius saponin of stem and leaf (PQS) of replenishing Qi and nourishing Yin.Method: Newborn umbilical cord were taken from healthy parturient, then0.1%collagenaseⅠwere used to digest the endothelia cells from umbilical vein. HUVECs werecultured in DMEM culture containing20%fetal bovine serum（FBS）and10ng/ml EGFor in M199culture containing8%FBS and2%LSGS, observing the growth of HUVECsin the two medium. HUVECs was passaged by0.25%trypsin solution. The method ofimmunocytochemistry of Ⅷ factor were used to identify the HUVECs. HUVECs werecultured in vitro for the study, ox-LDL stimulated endothelial cell injury model is established.There are eight groups: normal control group (NOR), model group (MOD),17-β estrodiolgroup (E2,0.01μM),17-β estrodiol+ICI182780group (E2I, E2:0.01μM, ICI:1μM),ginsenoside Rb3low-dose group (RL,1μM), ginsenoside Rb3middle-dose group (RM,10μM), ginsenoside Rb3high-dose group (RH,100μM), ginsenoside Rb3+ICI182780group(RMI, RM:10μM, ICI:1μM). According to the different groups, HUVECs werepretreated for1hour with ICI182780, and followed by treatment with E2or ginsenoside Rb3for1hour before the24hour exposure to ox-LDL. The NO, ET-1, sE-selectin and sICAM-1concentrations in the cell culture supernatant were measured by ELISA method. The activitiesof SOD, NOS and the contents of MDA in the cell lysate were examined by enzyme methodor spectrophotometry. HUVECs apoptosis rate was measured by flow cytometry usingAnnexinV-FITC/PI staining. HUVECs iNOS and eNOS expression were measured byRT-PCR, while p-Akt, p-ERK1/2, p-p38MAPK expression were measured by Western-blot.Results:①The culture of HUVEC: The primary cultured endothelia cells(ECs) began toadhere to the flask in4h after inoculation, the ECs arrayed like pitching stone under light microscopy. Cells incubated in M199culture containing8%fetalbovine serum and2%LSGS can passage to6~7generation. The cells exhibited brownishyellow granules in the cytoplasm after factor VIII-related antigen indirectimmunocytochemistry staining, thus the cells were identified as HUVECs.②Cellular oxidative damage: Compared with NOR group, MOD group’s SODactivity was obviously lower (P<0.01), and MDA content increased significantly (P<0.01);compared with MOD group, E2, RL, RM, RH group’s HUVECs SOD activity was obviouslyincreased (P<0.01or P<0.05), and MDA content decreased obviously (P<0.01). Pretreatedwith ICI182780，compared with the corresponding medication group, HUVECs SOD activitydecreased obviously (P<0.05), MDA content significantly increased (P<0.01).③Proinflammatory cytokines: In comparison with NOR group, the level ofsE-selectin and sICAM in MOD groups were all significantly increased(P0.01). Incomparison with MOD group, E2, RM and RH group were all significantly decreased（P0.01）, the level of sICAM in RL group were also decreased(P0.01). Pretreated withICI182780，compared with the corresponding medication group, the level of sE-selectin andsICAM were all significantly increased(P0.01). In addition, compared with RL group, thelevel of sICAM-1in RH group is further decreased(P<0.05).④Endothelial function: Compared with NOR group, the level of NOS, NO, ET-1, andiNOSmRNA expression in MOD group were significantly increased (P<0.01), eNOSmRNAexpression in MOD group were decreased significantly (P<0.01). Compared with MODgroup, these were all significantly increased or decreased respectively in E2, RL, RM and RHgroup(P<0.01). Pretreated with ICI18278，compared with the corresponding medication group,the level of NOS, NO, ET-1, and iNOS mRNA expression were significantly increased(P<0.01or P<0.05), and eNOS mRNA expression were decreased significantly (P<0.01orP<0.05).⑤Apoptosis: In comparison with NOR group, the rate of early apoptosis (AnnexinVpositive/PI negative), later apoptotic and necrosis cells (AnnexinV positive/PI positive)increased significantly in MOD group(P<0.01). Compared with MOD group, the rate of earlyapoptotic cells was significantly decreased in E2, RL, RM and RH group (P<0.01), and the rate of later apoptotic and necrosis cells was significantly decreased in E2, RM and RHgroup(P<0.01), but the RL group had no statistical difference. Pretreated with ICI182780,compared with the corresponding medication group, the rate of early apoptosis, laterapoptotic and necrosis cells increased significantly (P<0.01).⑥Related proteins expression: In comparison with NOR group，the phosphorylationlevels of Akt, ERK1/2in MOD groups were all increased significantly(P0.01), meanwhilethe phosphorylation levels of p38MAPK were decreased significantly (P<0.01). Comparedwith MOD group, the phosphorylation levels of Akt in E2, RH and RM groups were decreasedsignificantly (P0.05), the phosphorylation levels of ERK1/2in E2and RH groups weredecreased significantly (P0.01), and the phosphorylation levels of p38MAPK in E2, RH, RMand RL groups were increased significantly (P0.01). Pretreated with ICI182780，comparedwith the corresponding medication group, the phosphorylation levels of Akt, ERK1/2were allincreased significantly(P0.01or P<0.05), and the phosphorylation levels of p38MAPK weredecreased in E2I group significantly (P<0.05); but compared with RM group, thephosphorylation levels of p38MAPK in RMI groups had no statistical difference.Conclusions:①HUVECs are isolated and cultured successfully which provides solid foundation forthe research on vascular endothelial cell injury and protection mechanism of the medicines.②Ginsenoside Rb3has estrogen-like vascular protective effects of alleviatingoxidative stress and inflammatory reaction, reducing ET-1,NO excessive release, decreasingthe apoptosis and inhibiting Akt, ERK1/2protein phosphorylation by binding to estrogenreceptors and these effects are relatively weaker than estradiol.③The effect that Ginsenoside Rb3increases the phosphorylation level of p38MAPKdemonstrates ginsenosides Rb3also play a protective role through other pathways.④Both deficiency of Qi and Yin syndrome is the pathogenesis of the falling levelof estrogen, and there is estrogen-like effects of replenishing Qi and Nourishing Yin.