| Objective: To investigate the protection effect of Essential Oil from Fructus Alpinia zerumbet(EOFAZ) on ox-LDL-induced human aortic endothelial cells(HAECs) injury, explore crosstalk of mitogen-activated protein kinases(MAPK) and nitric oxide synthase(NOS) in ox-LDL-induced HAECs injury, clarify the mechanism of EOFAZ on ox-LDL-induced HAECs injury, finnaly provide the theortical guide and expriments for EOFAZ and vascular-diseases in clinic. Methods:(1) Protection effects of EOFAZ on ox-LDL-induced HAECs injury: the experiment was designed 7 groups as following: control group(serum free ECM), model group(200 mg/L ox-LDL), high dose group(200 mg/L ox LDL+100 μg/L EOFAZ), low dose group(200 mg/L ox LDL+10 μg/L EOFAZ), aspirin group(200 mg/L ox-LDL+2.5×10-4 mol/L aspirin), carvedilol group(200 mg/L ox-LDL+1×10-6 mol/L carvedilol) and atorvastatin calcium group(200 mg/L ox-LDL+1×10-6 mol/L atorvastatin calcium).(2) Cell migration and repairmentexperiment was divided into 4 groups as following: control group(0.5% of ECM), model group(200 mg/L ox-LDL+0.5% of ECM), EOFAZ high dose group(200 mg/L ox-LDL+100 μg/L EOFAZ+0.5% of ECM)andlow dose group(200 mg/L ox-LDL+10 μg/L EOFAZ+0.5% of ECM).(3) MAPK mechanism research experiment is divided into 5 groups as following: control group(serum free ECM), model group(200 mg/L ox-LDL), EOFAZ group(200 mg/L ox-LDL+100 μg/L EOFAZ), MAPK inhibitor group(200 mg/L ox-LDL+40 μmol/L SB203580 or 5 μmol/L sp600125 or 10 μmol/L PD98059) and EOFAZ with MAPK inhibitor group(200 mg/L ox-LDL+100 μg/L EOFAZ+40 μmol/L SB203580 or 5 μmol/L SP600125 or 10 μmol/L PD98059).(4) NOS mechanism researchexperiment was allotted to 5 groups as following: control group(serum free ECM), model group(200 mg/L ox-LDL), EOFAZ group(200 mg/L ox-LDL+100 μg/L EOFAZ), NOS inhibitor group(200 mg/L ox-LDL+100 μmol/L L-NAME or 300 μmol/L L-NMMA) and EOFAZ with NOS inhibitor group(200 mg/L ox-LDL+100 μg/L EOFAZ+100 μmol/L L-NAME or 300 μmol/L L-NMMA).(5) MAPK inhibitor regulating NOS mechanismexperiment was divided into 7 groups as following: control group(serum free ECM), model group(200 mg/L ox-LDL), EOFAZ group(200 mg/L ox-LDL+100 μg/L EOFAZ), NOS inhibitor group(200 mg/L ox-LDL+100 mol/L L-NAME or 300 μmol/ L-NMMA),p38 MAPK inhibitor group(200 mg/L ox-LDL+40 μmol/L SB203580), JNK1/2/3 inhibitor group(200 mg/L ox-LDL+5 μmol mol/L SP600125), ERK1/2 inhibitor group(200 mg/L ox-LDL+10 μmol/L PD98059).(6) NOS inhibitor regulating MAPK mechanism was divided into 5 groups as following: control group(serum free ECM), model group(200 mg/L ox-LDL), EOFAZ group(200 mg/L ox-LDL+100 μg/L EOFAZ), MAPK inhibitor group(200 mg/L ox-LDL+40 μmol/L SB203580 or 5 μmol/L SP600125 or 10 μmol/L PD98059), NOS inhibitor group(200 mg/L ox-LDL+100 μmol/L L-NAME+300 μmol/L L-NMMA). HAECs were cultured in vitro, pretreated with different drug 1 h and then added ox-LDL for 24 hours in accordance with the designing groups, respectively. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) method was used toanalyze the cell survival rate for determining the optimal concentration and time of ox-LDL induced HAECs injury. The morphology of cell injury was observed by inverted microscope, and then giemsa staining(Giemsa) and hematoxylin-eosin staining(HE) were used to observe cell pathologicalfeature. Wound scratch assay was used for cell migration and repairment analysis and transwell system was used for cell migration rate analysis. Microplate method and Enzyme-linked Immunosorbent Assay(ELISA) were used to detect the lactate dehydrogenase(LDH) or nitric oxide(NO) content, respectively. Endothelial nitric oxide synthase(e NOS) and inducible nitric oxide synthase(i NOS) were determined by chemical method. Quantitative Real-time Polymerase Chain Reaction(QRT-PCR)was used to analyze the m RNA expression of p38 MAPK, JNK1/2/3, ERK1/2, i NOS and e NOS.And western blot analysis was used to detect the protein expression of p38MAPK, p-p38 MAPK, JNK1/2/3, p-JNK1/2/3, ERK1/2, p-ERK1/2, i NOS and e NOS. Results: MTT results showed that EOFAZ could improve the survival rate of HAECs, attenuatecell injury, enhance cell migration rate and repairment. Besides, EOFAZ could alleviate the leakage of LDH and i NOS, increase the release of NO and e NOS.QRT-PCR analysisresultssuggested thatox-LDL significantly up-regulated i NOS m RNA expression, down-regulation e NOS m RNA expression, but EOFAZ reverses themsignificantly(P<0.05). Besides,EOFAZ and inhibitors had no significant effect on total m RNA expression of p38 MAPK, JNK1/2/3, ERK1/2. The results of western blot were consistent with QRT-PCR results, further confirmedthatalthough there was no significant effect on total protein expression of p38 MAPK, JNK1/2/3 and ERK1/2, EOFAZand inhibitorsdown-regulated protein expression of p-p38 MAPK, p-JNK1/2/3, p-ERK1/2 and i NOS, up-regulated protein expression of e NOS,significantly(P<0.05).Conclusion:(1) EOFAZ could protect against HAECs injury-induced by ox-LDL, and ameliorate the HAECs migration and wound health. The mechanism of EOFAZ may be involved in the inhibition of p38 MAPK, ERK1/2 and JNK1/2/3 phosphorylation, and the regulation balance of e NOS and i NOS protein expression.(2) There is acrosstalk regulation between MAPK and NOS signal in HAECs injury. The present conclusion provided the novel theory and experimential basic for vascular dysfunction deasease in clinic. |
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