| Neuroprotective agents had been used to save neurons from irreversible injuries in nervous system diseases and had been an aroused general study interest. Evidence-based medical research showed there are still no agents with neuroprotective efficacy. Nimodipine had been proven to possess no efficacy on acute ischemic stroke mortality in patients; a randomized controlled clinical trial on treatment with stroke of tirilazad had been stopped because its effect was not observed; edaravone is not suitable as neuroprotective agent. Natural compounds are major origin of active compounds. Terpenes are a large class of second products of plants,neuroprotection of diterpines from the fruiting body of Antrodia camphorate had been reported. Taxols are diterpines compounds from Taxus cuspidate, we discovered some of them with certain structures and antitumor activities and some of them presenting no antitumor activity. We screened taxane compounds with antioxdation activity and discussed the mechanisms and structure-activity relationship.Experiment 1: Stablishment of oxidative stress cell modelObjective: To establish oxidative Sk-N-Sh cell model with hydrogen peroxide.Methods:SK-N-SH cells were divided into four groups. Each group involved 3 wells of cells in 96 well plate. 100μL experimental cells in logarithmic phase after adjusted concentration of 1*105/L were vaccinated in the 96 well plate and cultured in saturated humidity at 37℃and under 5 % CO2 and 95 % air. 12 hrs later, 100μL different concentrations of hydrogen peroxide were added. The final concentrations of hydrogen peroxide of groups were successively 0, 50, 100 and 150μmol/L. After 24hrs of treatment, the cell viabilities were evaluated using 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT) assay. Results: Cell viabilities in different hydrogen peroxide concentrations(50,100,150μmol/L) are respectively(99 + 3.9)%, (70.6 + 2.3)%(p<0.05), (26.1 + 1.1)%(p<0.05) , cells viabilities of two groups in concentrations of 100,150μmol/L decreased obviously comparing with normal control group(0μmol/L hydrogen peroxide).Conclusion: Hydrogen peroxide can decrease cell viability dose-dependently within 150μmol/L. After treated with 150μmol/L hydrogen peroxide for 24hrs, cell viability was 26.1 + 1.1%, so we chose the concentration of hydrogen peroxide to establish the oxidative cell model. Experiment 2: Screening of taxane compounds using antioxidation activities using MTT assay.Objective: To screen taxane compounds with antioxidation Structure-Activity Relationship.Methods: To screen the type and concentration of taxane compounds with anti-oxidative effects, SK-N-SH cells were divided into blank control group, negative control group, treated groups, positive control group, and normal group, each of them including cells in 3 wells. 160μL cells in logarithmic phase after adjusted concentration of 1×105/L were vaccinated in the 96 well plate, and cultured in saturated humidity at 37℃and under 5 % CO2 and 95 % air. 12 hrs later, agents or PBS were added in. In blank control group, 40μL PBS was added in; in negative control group, 20μLculture medium and 2 hrs later, 20μL hydrogen peroxide (1 mmol/L) was added in. The final concentrations of hydrogen peroxide was 100μmol/L; In treated groups, there are 12 subunits including 3 concentrations of taxine B, 7-deacetyl-taxine B, 5-cinnamoyloxy-taxin B, and 7,10-diacetyl-2'-deoxyl-taxine A groups. Cells were pretreated with 20μL different types and concentrations of taxane compounds and 2 hrs later, 20μL hydrogen peroxide (1 mmol/L) was added in. The final concentrations of every agents were 1, 10, 100μmol/L, and that of hydrogen peroxide was 100μmol/L; in positive control group, 20μL Epigallocatechin Gallatte (EGCG) (100μmol/L) and 2 hrs later, 20μL hydrogen peroxide (1 mmol/L) were added in. The final concentrations of EGCG was 10μmol/L, and that of hydrogen peroxide was 100μmol/L; in normal control groups, 20μL PBS and 20μL 7-Deacetyl-taxine , 5-Cinnamoyloxy-taxin B, Taxine B, 7,10-Diacetyl-2'-deoxyl-taxine were added in. The final concentrations of four taxanes were 100μmol/L. After 24 hrs of treatment, the cell viabilities of groups were evaluated using 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT) assay and the nuclear morphological changes were observed after Hoechst 33258 treated.Results: cell viability of the negative group was 27.3±0.85%. Viabilities of oxidative model cells after treated with 1, 10,100μmol/L 7-deacetyl-taxine B are 25.9±1.609%,31.2±1.253%,57.6±3.74%(p<0.05) respectively, after treated with 1, 10, 100μmol/L 5-cinnamoyloxy-taxin B are 25.9±1.609%,31.2±1.253%,57.6±3.74%(p<0.05) respectively,after treated with 1, 10, 100μmol/L Taxine B are 28.4±0.603%,30.5±1.967%,31.5±1.308% respectively,and after treated with 1, 10, 100μmol/L 7,10-diacetyl-2'-deoxyl-taxine A are 27.4±0.751%,32.4±0.971%,34.4±1.852% respectively. Cell viability of the positive group (EGCG 10μmol/L) was 45.4±1.168% (p<0.05). 100μmol/L 7-deacetyl-taxine B, 5-cinnamoyloxy-taxin B and 10μmol/L EGCG improved viability of oxidative stress cell model obviously.Cell viabilities of the normal group were respectively 102±3.02%,95.4±7.73% , 98.4±5.98% , 97.8±6% , after treated with 100μmol/L 7-Deacetyl-taxine , 5-Cinnamoyloxy-taxin B, Taxine B, 7,10-Diacetyl-2'-deoxyl-taxine. Comparing with viability of blank group cells(100±11%), the four taxanes didn't decrease or increase cell viability. After treated with Hoechst 33258, cell chromatin condensation was observed in the negative group. After treated by 10μmol/L EGCG, 100μmol/L 7-Deacetyl-taxine B and 5-Cinnamoyloxy-taxin B, the number of cell with condensed chromatin decreased, but not Taxine B, 7,10-Diacetyl-2'-deoxyl-taxine A .Conclusion: Taxine B , 7-Deacetyl-taxine B, 5-Cinnamoyloxy-taxin B, 7,10-Diacetyl-2'-deoxyl-taxine A are diterpine compounds from Taxus cuspidate with most the same ring structure. Comparing with taxol, they have the same substitutes: the side chain at the thirteenth carbon atom and the OBZ radical at the second carbon atom were substituted by acetyl radicals, the hydroxide radical at the fifth carbon atom induced the disapearance of oxetane ring, and the acetyl radical at the fourth carbon atom, hydroxide radical at the first carbon atom disappeared. 7-Deacetyl-taxine B has two hydroxide radicals at the fifth and the seventh carbon atom, Taxine B has a hydroxide radical at the fifth carbon atom and an acetyl radical at the seventh carbon atom. 5-Cinnamoyloxy-taxin B has a fat-soluble side chain radical at the fifth carbon atom and an acetyl radical at the seventh carbon atom. 7,10-Diacetyl-2'-deoxyl-taxine A has an alkalescence side chain radical at the fifth carbon atom and an acetyl radical at the seventh carbon atom.The result discovered that taxanes with different substitutes comparing with taxol did not present anticancer activity. It maybe attributed with that side chain at the thirteenth carbon atom and OBZ radical at the second carbon atom were substituted by acetyl radicals, acetyl radical at the fourth carbon atom, hydroxide radical at the first carbon atom disappeared, and the hydroxide radical at the fifth carbon atom induced the disappearance of oxetane ring.7-Deacetyl-taxine B and 5-Cinnamoyloxy-taxin B improve cell viabilities obviously and present powerful activity of anti-oxidative. 7-Deacetyl-taxine B is better than 5-Cinnamoyloxy-taxin B. Taxine B, 7, 10-Diacetyl-2'-deoxyl-taxine A have not the ability. The strongest antioxidative activity of 7-Deacetyl-taxine B may be due to the two adjacent hydroxide radicals having the ability of stabilizing the phenoxy radicals.After treated with hydrogen peroxide, cell chromatin condensation was observed in the negative group. After treated by 10μmol/LEGCG, 100μmol/L 7-Deacetyl-taxine B and 5-Cinnamoyloxy-taxin B, the number of cell with condensed chromatin decreased, but not Taxine B, 7,10-Diacetyl-2'-deoxyl-taxine A . So, the anti-oxidative effect of taxines may be due to that they can alleviate chromatin damage induced by oxidative stress.Experiment 3: Studying of mechanisms of taxanes against oxidative stress induced by hydrogen peroxide.Objective: To study the mechanisms of taxanes against oxidative stress induced by hydrogen peroxide.Methods: SK-N-SH cells were divided into control group and treated groups, each of them including cells in 3 wells. 160μL cells in logarithmic phase after adjusted concentration of 1×105/L were vaccinated in the 96 well plate, and cultured in saturated humidity at 37℃and under 5 % CO2 and 95 % air. 12 hrs later agents or PBS were added in; In control group, 20μL culture medium and 2 hrs later, 20μL hydrogen peroxide (1 mmol/L) were added in. The final concentrations of hydrogen peroxide was 100μmol/L; In treated groups, there are 2 subunits respectively treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B. Cells were pretreated with 20μL two different compounds and 2 hrs later, 20μL hydrogen peroxide (1 mmol/L) was added in. The final concentration of compounds was 100μmol/L, and that of hydrogen peroxide was 150μmol/L . After 24 hrs of treatment, intracellular ROS content, mitochondrial membrane potential and apoptosis rate of groups were detected using flow cytometry assay and the micro-morphology was detected with electron microscopy.Results: Ultramicromorphology of control group cells showed that chromatin margination, endoplasmic reticulum swelling with some dense matter inside, mitochondrial dilation, apoptotic body appeared and 7-Deacetyl-taxine B(100μmol/L), 5-Cinnamoyloxy-taxin B(100μmol/L)obviously alleviated the dilation of mitochondrials and decreased apoptosis cells induced by oxidative stress.SK-N-SH cells after treated with showed features of apoptosis including nucleus shrinkage, dense aggregation of chromatin and chromatin margination chromatin margination and appearance of apoptotic body, endoplasmic reticulum swelling with some dense matter inside and mitochondrial dilation. After treared with 7-Deacetyl-taxine B, 5-Cinnamoyloxy-taxin B, the dilation of mitochondrials was obviously alleviated, but the endoplasmic reticulum swelling didn't change. After annexin-v/pi staining, the livability rate and apoptosis rate of the control group were 73.62±2.14% and 22.06±3.08%. After treated with 5-Cinnamoyloxy-taxin B the two rates were respectively 83.55±2.34% (p<0.05) and 12.68±2.36% (p<0.05); and after treated with 7-Deacetyl-taxine B, they were 84.78±0.75% (p<0.05) and 5.84±2.39% (p<0.05). After treated with taxines the livability rate increased and the apoptosis rate decreased obviously comparing with control group.Fluorescence intensities of DCFH-DA stained cells after treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B were 1.77±0.12(p<0.05), 2.57±0.05(p<0.05) showed obviously decreased comparing with 47.37±2.32 that of control group.Fluorescence intensities of rhodamine123 stained cells after treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B were 15.5±0.44 (p<0.05), 14.93±0.40 (p<0.05) showed obviously decreased comparing with9.77±1.29 that of control group.Conclusion:1. Hydrogen peroxide can induce SK-N-SH cell to present chromatin margination, endoplasmic reticulum swelling with some dense matter inside, mitochondrial dilation and apoptotic body. The oxidative stress damage induced by hydrogen peroxide is associated with apoptosis.2. After treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B, the dilation of mitochondrial was alleviated, the mitochondrial membrane potential was improved, the livability rate increased and the apoptosis rate decreased obviously comparing with control group. The anti-oxidative activity of taxanes may be due to protecting mitochondrial and decreasing apoptosis.3. After treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B, intracellular ROS content decreased obviously comparing with control group cells. The antioxidative activity of taxanes may be due to eliminating intracellular ROS. |
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