Regulatory Effect Of Extract Of Eupolyphaga On LPS-induced NOS In Vascular Endothelial Cells

Objective: To investigate the effect of extract of Eupolyphaga onlipopolysaccharide (LPS) induced endothelial nitric oxide synthase (eNOS)and inducible nitric oxide synthase (iNOS) expression of human umbilicalvein endothelial cells (HUVEC)Methods:1The effects of extract of Eupolyphaga on the proliferation of Humanumbilical Vein Endothelial Cells1.1The cultivation of Human umbilical Vein Endothelial CellsHuman umbilical vein endothelial cells (HUVECs) were purchased fromATCC, HUVECs were cultured in DMEM medium with100mL/L fetal bovineserum (FBS),100kU/L penicillin and100kU/L streptomycin, and werepassaged with0.25%trypsin.1.2The effects of extract of Eupolyphaga on the proliferation of HUVECHUVECs were treated with different concentration of extract ofEupolyphaga (0.0625,0.125,0.25,0.5,1.0,2.0,4.0mg/ml) for24hours; Cellviability was determined by MTT assay, absorbance intensity measured at490nm..1.3The effects of extract of Eupolyphaga on the proliferation of HUVECinduced by LPSExperiment is divided into blank control group (control), LPSintervention group (LPS100μg/ml for12hours), LPS and extract ofEupolyphaga treated group (0.0625,0.125,0.25,0.5,1.0,2.0,4.0mg/mlextract of Eupolyphaga for24hours plus LPS100μg/ml for12hours), Cellviability was determined by MTT method.2The effects of extract of Eupolyphaga on the NO and MDA content andSOD activity of HUVEC treated with LPS Experiment is divided into blank control group (control), LPSintervention group (LPS100μg/ml for12hours), LPS and extract ofEupolyphaga treated group (0.0625,0.125,0.25,0.5,1.0,2.0,4.0mg/mlextract of Eupolyphaga for24hours plus LPS100μg/ml for12hours). Thesupernatant of culture medium were collected to measure NO and MDAcontent and SOD activity with spectrophotometrically using NO,SOD,MDAkit.3Comparison of the expression of eNOS and iNOS protein in HUVEC treatedwith LPS for different hoursExperiment is divided into blank control group (control), LPS(100μg/ml)induced for different hours group, the incubation times with LPS were4h,8h,12h,16h,24h. Whole protein was extracted by RIPA lysate. eNOS and iNOSprotein expressions were detected by Western blot technology.4Comparison of the expression of eNOS and iNOS protein in HUVEC treatedwith LPS and different concentration extract of EupolyphagaExperiment is divided into blank control group (control), LPSintervention group(LPS100μg/ml for12hours), LPS and extract ofEupolyphaga treated group (0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus LPS100μg/ml for12hours). Whole protein was extracted by RIPAlysate. eNOS and iNOS protein expressions were detected by Western blottechnology.Results:1The effects of extract of Eupolyphaga on the proliferation and differentiationof HUVEC1.1Cell morphology observationUnder the inverted phase contrast microscope，the HUVEC was adherentgrowth after about half an hour by cultivation in vitro. The adherent cells wereround, a single or gather in groups, gradually transformed into a polygon orfusiform. non-adherent cells were globular and float in culture medium, andthen, more and more cells were adherent growth. After three days, the cellswere growth gradually intensive, and mutual connections between each other. The cells became polygon, fusiform, border clear, they were loosearrangement abundant cytoplasm and round or oval nuclei, often have one ortwo nucleolus. After8days, the cells are closely related with each other,merging into a continuous monolayer cells, boundary is fuzzy, appearance wascharacteristic of pebbles shape.1.2The effects of extract of Eupolyphaga on the proliferation of HUVECExtract of Eupolyphaga could significantly promote HUVECproliferation at the range of0.0625-4.0mg/ml concentration, compared withblank control group (P<0.01), The concentration of4.0mg/ml contrasted2.0mg/ml concentration OD value is decreased (P<0.01), The resultsdemonstrated that its effect on promoting proliferation of HUVEC wasdecreased when the concentration of extract of Eupolyphaga was greater than4.0mg/ml.1.3The effects of extract of Eupolyphaga on the proliferation of HUVECincubated with LPSOD value of LPS intervention group (LPS100μg/ml for12hours) wasdecreased significantly compared with blank control group (P <0.01), HUVECproliferation was decreased in LPS intervention group. OD value of The LPSplus extract of Eupolyphaga with different concentration content group(0.0625,0.125,0.25,0.5,1.0,2.0,4.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPS for12hours) was promoted significantly comparedwith LPS intervention group (P<0.01). The concentration of between0.0625-4.0mg/ml concentration extract of Eupolyphaga could promote theproliferation of HUVEC treated with LPS at the the range of0.0625-4.0mg/mlconcentration.1.4Determination of the optimal concentration of extract of EupolyphagaThe HUVEC was treated with different concentrations（0.0625,0.125,0.25,0.5,1.0,2.0,4.0mg/ml）LPS， the optimal concentration were measuredby MTT method, it was0.25,0.5,1.0mg/ml.2The effects of extract of Eupolyphaga on the NO and MDA content andSOD activity of HUVEC incubated with LPS 2.1The effects of extract of Eupolyphaga on NO contentThe NO content in LPS intervention group (LPS100μg/ml for12hours)was increased significantly compared with blank control group (P<0.01)，TheLPS plus extract of Eupolyphaga with different concentration content group(0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPSfor12hours) compared with LPS intervention group (LPS100μg/ml for12hours), the NO content decreased significantly (P<0.01)，and the NO contentwas significantly decreased as the concentration increase of extract ofEupolyphaga (P<0.05).2.2The effects of extract of Eupolyphaga on SOD activityLPS intervention group (LPS100μg/ml for12hours) compared withblank control group，the SOD activity decreased significantly (P <0.01)，theLPS plus extract of Eupolyphaga with different concentration content group(0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPSfor12hours) compared with LPS intervention group (LPS100μg/ml for12hours), the SOD activity increased significantly (P<0.01)，and the SODactivity was significantly increased as the concentration increase of extract ofEupolyphaga (P <0.05).2.3The effects of extract of Eupolyphaga on MDA contentLPS intervention group (LPS100μg/ml for12hours) compared withblank control group， the MDA content increased significantly (P <0.01)，theLPS plus extract of Eupolyphaga with different concentration content group(0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPSfor12hours) compared with LPS intervention group (LPS100μg/ml for12hours)，the MDA content decreased significantly (P<0.01), the MDA contentwas no significantly difference as extract of Eupolyphaga concentrationincrease (P﹥0.05).3Comparison of the expression of eNOS and iNOS protein in HUVEC treatedwith LPS for different hoursCompared with blank control group，eNOS and iNOS protein expressionincreases over time (P <0.01), and reaches the maximum when12hours，after declining gradually.4Determination of the optimal time of LPS interventionInterference time was set as follows:4,8,12,16,24hours，the optimaltime was measured12hours by Western blot method.5Comparison of the expression of eNOS and iNOS protein in HUVEC treatedwith LPS and different concentration gradient extract of Eupolyphaga5.1Comparison of the expression of eNOSThe expression of eNOS in LPS intervention group (LPS100μg/ml for12hours) was increased significantly compared with blank control group (P <0.01). The LPS plus extract of Eupolyphaga with different concentrationcontent group (0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPS for12hours) compared with LPS intervention group (LPS100μg/ml for12hours)，the expression of eNOS decreased significantly(P<0.01)，and the expression of eNOS significantly decreased as the extract ofEupolyphaga concentration increase (P <0.05).5.2Comparison of the expression of iNOSLPS intervention group (LPS100μg/ml for12hours) compared withblank control group, the expression of iNOS increased significantly (P <0.01)，The LPS plus extract of Eupolyphaga with different concentrationcontent group (0.25,0.5,1.0mg/ml extract of Eupolyphaga for24hours plus100μg/ml LPS for12hours) compared with LPS intervention group (LPS100μg/ml for12hours) expression of iNOS decreased significantly (P<0.01)，and the expression of iNOS significantly decreased as the extract ofEupolyphaga concentration increase (P <0.01).Conclusion:1Extract of Eupolyphaga (0.0625-4.0mg/ml) can significantly promotethe proliferation of HUVEC and HUVEC treated with LPS. The effect ofextract of Eupolyphaga was the most significant (the two effect no difference)at dose of1.0and2.0mg/ml.2HUVEC was treated with LPS, NO and MDA content was increased,the activity of SOD was reduced, in the supernatant of culture medium. extract of Eupolyphaga could decreased due to the LPS effect of NO, MDA contentincrease, and increased SOD activity.3Extract of Eupolyphaga could down regulate the expression of eNOS,iNOS protein content, induced with the LPS and the regulation of NO.Summary: Extract of Eupolyphaga could improve the LPS inducedHUVEC lesion by increasing the proliferation of HUVEC and play aprotective role by reducing LPS elevated eNOS, iNOS protein expressionchanges of cell, descreasing the content of free radicals in the vascularendothelial cells, so as to achieve anti-inflammatory, antioxidant, preventionof vascular wall damage and antiatherosclerosis.