Mechanisms Of Vascular Endothelial Cells Counteracting Vasoconstriction Induced By Tentacle Extract From Jellyfish Cyanea Capillate

Objective:To investigate the mechanisms of vascular endothelial cells counteractingvasoconstriction induced by tentacle extract from jellyfish Cyanea capillate and offera new idea and targets for clinical of jellyfish stings.Method:1. Test the toxic effects of TE on HUVEC cell:1.1Measuring cell survival rate with MIT method: collect coastal c. capillataand extract C. capillata TE sample after identification.①Take concentration ascontrol factors, add different concentrations (0μg/ml、0.625μg/ml、1.25μg/ml、2.5μg/ml、5μg/ml、10μg/ml、20μg/ml、40μg/ml、80μg/ml、160μg/ml、320μg/ml) ofTE,incubate HUVEC cells with cell density of4×105, and then detect the survivalrate with MTT method.②Use different concentrations (1μg/ml，2.5μg/ml，5μg/ml，10μg/ml) of TE and different time (6h，12h，24h，48h) incubate HUVEC cells withcell density of4×105, and detect the survival rate of HUVEC cells.1.2Adopt flow cytometryto examine the TE effect on apoptosis rate of HUVECcells: add different concentrations (0μg/ml、1μg/ml，2.5μg/ml，5μg/ml，10μg/ml) ofTE incubate HUVEC cells6hours, and then test the apoptosis rate of HUVEC cellswith flow cytometry.2. Detect the vasomotor factors such as Prostacyclin (PGI2),Thromboxane(TXA2) andAngiotensin Ⅱ(AngⅡ)2.1Add different concentration of TE(0μg/ml、1μg/ml，2.5μg/ml,5μg/ml) treatHUVEC cells for6h, and detect the PGI2and TXA2, AngⅡlevels in differentconcentration of TE with ELISA.2.2Put the TE of5μg/ml into the HUVEC cells, after0min、5min、30min、2h、6h、12h respectively, then detect the PGI2and TXA2, AngⅡlevels in different timewith ELISA.3. Observe the change of SD rat femoral artery Blood pressure (BP) After anesthesia for SD rats (280～320g), adoptaorta intubation and jugular veinintubation method, put in the four solutions respectively:①control group ((0.9%saline);②1.4mg/ml TE;③10μM epoxy synthase inhibitors (indomethacin)+1.4mg/ml TE;④10μMangiotensin converting enzyme inhibitors (captopril)+1.4mg/mlTE. And then observe the change of SD ratfemoral artery Blood pressure in1h, inorder to make clear drug invention effect and protective effect of VECs on TEvasoconstrictor toxicity process., and eventually pick out the effective drug for theclinical treatment of jellyfish stings.4. The impact on cardiac and renal function of SD rat.After the experiment of testing BP change for the animals, extractarterial bloodof them from intubated place of the femoral artery, and detect myocardial enzymes,kidney (LDH, CK, sCr and BUN).Result:1. The effect of TE on HUVEC cells’survival rate①If the concentration of TE is in the range of0～2.5μg/ml, It has nosignificant toxic effect on HUVEC cells, the survival rate is100%(P＞0.05), if theconcentration is above5μg/ml, the death begins, the cell survival rate is97.55%, andwhen the concentration is10μg/ml, the death rate is50%, and if the concentrationreaches320μg/ml, the survival rate is only4.83%, almost all of HUVEC cells aredead, the results these are statistically significant. Therefore, when the concentration isin the range of5～320μg/ml, the toxic effect of TE on HUVEC cells depends on theconcentration, the effect increases with the concentration get greater.②The longer of incubation time, the greater toxic effect TE has on HUVECcells with the same concentration, which means the toxic effect depends on time(P＜0.05). If the concentration is1μg/ml, incubating the HUVEC cells for6h and12hrespectively, we could find that, the cell survival rate is greater than100%, that is, TEhas growth promoting effects on HUVEC cells in the condition of lowconcentration(<1mu g/ml) and a short time (<12h). And the survival rate is100%in the concentration of2.5μg/ml for6h, while when the concentration reaches5μg/mlfor6h, the cells begin to die, and the concentration reaches10μg/ml, a large numberof HUVEC cells is dead, the survival rate＜50%(P<0.05). Accordingly, the lowest TE concentration is5μg/ml which C.capillata TE willcause cell death in6h, so we choose5μg/ml of TE concentration for the follow-upexperiments. What’s more, in the condition of10μg/ml for6h, the cells are obviouslyreduced.2.TE lead to the apoptosis and necrosis of the HUVEC cells.The HUVEC cells apoptosis is greater with the increasing of TE concentration,when the concentration is1～10μg/ml（P＞0.05）, there is no obvious necrosis ofHUVEC cells caused by TE. And when the concentration is1μg/ml and2.5μg/ml, theapoptosis by TE is not significant either（P＞0.05）. But the concentration of5μg/mlcould lead to the apoptosis of some cells, and that of10μg/ml will lead to largenumber of cells（P<0.05）.3.Detection of vasomotor factor PGI2,TXA2andAngⅡ①PGI2increase with the concentration of TE (0μg/ml、1μg/ml，2.5μg/ml,5μg/ml), TXA has little increase with concentration of TE (0μg/ml、1μg/ml，2.5μg/ml,5μg/ml),Therefore PGI2/TXA2increase with the concentration ofTE. Also, Ang II increase with concentration of TE ((0μg/ml、1μg/ml，2.5μg/ml,5μg/ml), the results these are statistically significant. So, PGI2, Ang II and PGI2/TXA2depends on the dose, which means they have increase with the concentration ofTE.②TE（5μg/ml）incubateing HUVEC cells for some time (5min～12h) couldchange the level of vasomotor factor PGI2,TXA2and Ang II. PGI2decrease with thetime of TE stimulation. The production may reach the highest level in5min, and thelowest level in6h, but there is no obvious difference in6h and12h. TXA increasewith the stimulation time, and reach the highest level in6h, and there is no obviousdifference in6h and12h either.Ang II increase first and then decrease in5min～6h,and reaches the highest level in2h, and then decrease to the lowest level in6h, andthere is no difference in6h and12h, the results these are statistically significant.4. The change of SD rats femoral BPDuring the observation time (60min), comparing with the control group (0.9%saline group), SD rat femoral artery BP of the1.4mg/ml TE group fall significantlyin the first10min, reach the lowest level in10min, and then gradually rise, after 60min it back to the beginning BP level, so the average BP is lower than the controlgroup. For the group of10mM epoxy synthase inhibitors (indometacin)+TE, the BPlevel has no significant difference with control group in the observation time. For the10mM angiotensin converting enzyme inhibitors (captopril)+TE, the BP level fallfirst and increase later in10min, and return to the BP level of control group in20～30min, and then continues to rise, higher than the control group, the results these arestatistically significant.5.Tests of SD rats’cardiac and renal functionAfter the intact animal femoral artery BP monitoring experiments, we extract thearterial blood from the femoral artery of experimental animals, and then getsupernatant with ultracentrifugation in low temperature. The blood biochemical testresults show that comparing with the control group (0.9%saline), for the1.4mg/mlTE group, SD rat myocardial enzyme spectrum (LDH and CK) increased significantly,and renal function (sCr, BUN) is serious damaged. For10μM epoxy synthaseinhibitors (indomethacin)+1.4mg/ml TE group and10μMangiotensin convertingenzyme inhibitors (captopril)+1.4mg/ml TE group, the myocardial enzymes (LDHand CK) increase not so much comparing with1.4mg/ml TE group, and renalfunction(sCr, BUN) damage is lighter than1.4mg/ml TE group, the results these arestatistically significant.Conclusions:1. According to the above research, we have proved that TE has direct and toxiceffect on the HUVEC cell, certain concentration of TE incubation for some time could lead to HUVEC cell necrosis and apoptosis, and then reduce the survival rate.2. TE working on HUVEC cell, which could cause the change of product of PGI2and TXA2, Ang II. HUVEC cell regulates product of the vasomotor factor so as to resist vasoconstriction response caused by TE.3. TE can reduce the average arterial BP of animals and cause serious cardiacand renal functionimpairment. Epoxy (indomethacin) synthase inhibitors, angiotensinconverting enzyme inhibitors can regulate the vasomotor factors PGI2and TXA2,AngⅡproduced by VECs, so that resisting specific vasoconstriction reaction causedby TE, therefore TE has the effect of reducing BP,and in order to mitigate the impairment of heart and kidney.