紫杉烷類化合物的抗氧化應激作用

段瑞生,董 玫,王維平＊,劉 娜,賈麗景,甄軍麗,黃 曉

1河北醫科大學第二附屬醫院神經內科,石家莊 050000;2河北醫科大學法醫教研室,石家莊 050017; 3石家莊醫學高等?？茖W校外科教研室,石家莊 050081;4保定市第一中心醫院,保定 071000

Oxidative stress is caused by an imbalance be tween the production of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage and can induce diseases especially neurology.

Neuroprotective agents had been used to save neurons from irreversible injuries by various pathologic processes 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[1];a randomized controlled clinical trial on trea tment with stroke of tirilazad had been stopped because its effect was not observed[2]; edaravone is not suitable as neuroprotective agent[3]. Natural compounds are major origin of active compounds.Many natural plant extracts presented neuroprotective effects such asginkgo biloba extracts egb761[4],EGCG[5],Lig-8[6],Sesamin and sasemolin[7],and Huperzine A[8].Hydrogen peroxide is a kind of reactive oxygen species and has been the most commonly used to establish cellmodelwhich can well simulate cell oxidative damages and had been widely used in the study of oxidative stress for its s imple operation, high controllabitity and repeatability.

As anti-tumor compounds,taxanes are studied in many aspects including synthesis and modifications,as well as structure-activity relationship.Our previous studies on anti-tumor agents showed that some new taxane compoundswithout antineuoplastic activity can improve cell viability.So,we study the neuroprotective effects of taxanes fromTaxus cuspidateon hydrogen peroxide-induced oxidative stress.

Materials and methods

Cell culture

SK-N-SH cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences.Cellswere routinely cultured in RPM I 1640 cell culture medium(100 mg/mL),which contained 2 mM L-glutamine,10%fetal bovine serum (FBS),1mM sodium pyruvate,100000 U/L penicillin,100 g/L streptomycin and non-essential amino acids.Cells were cultured in monolayer in 100 mm×20 mm plastic dishes at 37℃and under 5%CO2and 95%air.Medium was changed every 2-3 days,and the cells were passed once they reached approximately 70-80%confluence.

Taxane compounds

4 type taxane compounds,namely Taxine B,7-deacetyltaxine B,5-cinnamoyloxy-taxin B and 7,10-diacetyl-2’-deoxyl-taxineA,their structural formulawere depicted below,were obtained principally according to the method described by reference[9].Air-dried leaves of Taxus cuspidata were extracted with methanol at room temperature.The combined organic extracts were evaporated under reduced pressure.Lipidswere removed by stirring the mixture with hexane,and then the aqueous phase was salted and extracted with CH2Cl2.The methylene chloride extractwas subjected to a flash column chromatography.Successive elution with CH2Cl2-MeOH gradient yielded 32 fractions(Fr D-1 to Fr D-32)on the basis of TLC pattern,sub-fractions was applied to preparative HPLC,finally providing compounds.

Reagents

EGCG,trypxin and Hoechst33258 were purchased from Sigma corporation(USA),hydrogen peroxide,fetal calf serum,RPM I1640 cell culture medium,penicillin,glutamine,L-Glutamine,MTT glutaricdialdehydeand DCFH-DA were obtained from beyot ime institute of biotechnology(china)

I nstruments

Enzyme-labeled instrument(MK3 Multiskan MK3-Thermo labsystems),inverted microscope(Olympus), flow cytometry(FACS 420),inverted fluorescence microscope(Olympus).

Establishment of oxidative stress cellmodel[10]

To deter mine the final concentration of hydrogen peroxide to establish oxidative stressmodel,cellswere divided into four groups.Each group involved 3 wells of cells in 96 wellplate.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%CO2and 95%air.After 12 hrs,100μL different concentration of hydrogen peroxide was added.The final concentrations of hydrogen peroxide of groups were successively 0,50,100 and 150μmol/L.After treatment for 24 hrs,cells were rinsed with phosphate-buffered saline (PBS)three t imes.In each well 20μL MTT(5 g/L) was added and incubated for1 h and then carefully discard theMTT solution.After addition of 150μL DMSO and being shaked for 15 mins,survival rate was calculated from the relative absorbance at 570 nm using enzyme-labeled instrument and expressed aspercentage of 0μmol/L group absorbance value.Select concentration of hydrogen peroxide in which group cell viability decreased obviously to establish oxidative stress cellmodel.

Exper imental grouping

To study the type and concentration of compoundswith anti-oxidative effects,cellswere divided into blank control group,negative control group,treated groups,positive control group,and normal 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%CO2and 95% air.12 hrs later agents or PBS were added in.

In blank control group,40μL PBS was added in.

In negative 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 12 subunits including 3 concentrationsof taxineB,7-deacetyltaxine B,5-cinnamoyloxy-taxin B,and 7,10-diacetyl-2′-deoxyl-taxine A groups.Cells were pretreated with 20μL different types and concentrations of 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 thatof hydrogen peroxide was 100μmol/L.

In positive control group,20μL 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 100 μmol/L.

In normal control groups,20μL PBS and 20μL 7-deacetyl-taxine B,5-cinnamoyloxy-taxin B (1 mmol/ L).The final concentrations of 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B were 100μmol/L.

Screening of compoundswith antioxidation activities usingMTT[11]

Cells of groups,after 24 hrs treatment,were rinsed with PBS three times.MTT(5 g/L)was added to each well and incubated for 1 h and then carefully discarded.After addition of 150μL DMSO and shaking for 15 mins,Survival rate was calculated from the relative absorbance at 570 nm using enzyme-labeled instrument,and expressed as percentage of 0μmol/L group absorbance value.

Observation on nuclearmorphological changes[12]

Cells of groups after adjusted concentration of 1＊105/ L were suspended in 1μg/mL hoechst 33258,incubated 30 mins and rinsed 3 times.Observe DNA condensation and take pictures after cells were contrifugated and smeared on glass slides.

Detecting intracellular ROS content[13]

Cells of groups after adjusted concentration of 1＊105/ L were suspended in 10μmol/L DCFH-DA,After 20 mins incubating in darkness,cellswere rinsed with pbs twice,the mean fluorescence intensities were detected by flow cytometry(count 10000cells)with excitation wavelength of 488 nm and emission wavelength of 530 nm.

Data analysis

Data analysis was perfor med using the software SAS V8.Viabilitywas present asmean with deviation.Comparison of dates from every two groups was done using student-newman-keuls test,Significancewas assumed at P<0.05.

Result

Oxidative stress cellmodel

24 hrs after 50,100 and 150μmol/L hydrogen peroxide trea tment,the viability of sk-n-sh cellswas detected with the ratio of(99±3.9)%,(70.6±2.3)%,(26.1± 1.1)%respectively.Selecting agentswith antioxidation activities usingMTTmethod.

I mprovment of cell viability

7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B dosedependently improve viability of oxidative stress cell modelwith ratio of 57.6%,54.5%respectivelywhen the concentrations were 100μmol/L,obviously higher than negative group 27.35%and positive group 45.4 %.Taxine B,7,10-diacetyl-2′-deoxyl-taxine A have no significant improvement of viability comparingwith that of negative group.Normal control group treated with 100μmol/L 7-Deacetyl-taxine B and 5-Cinnamoyloxytaxin B showed no obviously decreasing of viability comparing with blank control group.

Nuclear morphological changes

Cells in negative group,showed a great number of cells with condensed chromatin.The number of cells with changed neucleis decreased in 7-deacetyl-taxine B,5-cinnamoyloxy-taxin B and positive groups but not in taxine B,7,10-diacetyl-2′-deoxyl-taxine A groups comparing with negative control group.

I ntracellular ROS content

Fluorescence intensities of DCFH-DA stained cells after treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B were 1.77±0.12,2.57±0.05 showed obviously decreasing comparingwith 47.37±2.32 that of negative group.

D iscussion

Taxanes are diterpenes.Some of them present broad spectrum anticancer activity.Structure-activity relationship studies showed that 2′(R),3′(S),hydroxyl groups at C 2′are essential for anticancer activity,but hydroxyl groups on C 1′C7′C9′not[14].Diterpenes isolated from the fruiting bodies ofAntrodia camphorata protected neurons from Aβdamage[15].Our results suggested that 7-deacetyl-taxine B and 5-cinnamoyloxytaxine B could improve cell viability obviously as antioxidative agents.The activity of 7-deacetyl-taxine B and 5-cinnamoyloxy-taxine B are stronger than that of taxine B and 7,10-diacetyl-2′-deoxyl-taxine A.

Fig.5 After sta ined with DCFH-DA,fluorescence intensities of 7-deacetyl-taxine B(B)and 5-c innamoyloxy-taxine B (C)groups(＊P<0.01)

Intracellular ROS play a important role in the process of cell damage[16].Physiological dose ROS generated from mitochondria can protect cell from oxidative stress[17,18].Excessive intracellular ROSwould be eliminated by superoxide dis mutase.Butwhen accumulated ROS induced by ischemia-hypoxia exceed the compensatory ability of the body,excessive ROS can activate much apoptosisproteins such as apoptosis inducing factor,bcl-2/bax family proteins and tumor necrosis factor to induce energy metabolis m disorder which can decrease mitochondrial membrane potential and cell death[19,20].

After being treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxine B,we observed the pycnosised and heavily stained nucleis alleviated after hoechst stained than the negative control.We detected the intracellular ros level of oxidative injured cells decreased after treated with 7-deacetyl-taxine B and 5-cinnamoyloxy-taxine B.we get the conclusion that 7-Deacetyl-taxine B,5-Cinnamoyloxy-taxine B can get rid of intracellular ROS and protect cells from oxidative stress.The four taxines has the similar structure but different efficacy,maybe due to the subtle structure differences:5-Cinnamoyloxy-taxin B has an fat-soluble side chain at seventh cabon,and an acetyl group at the fifth and 7-Deacetyltaxine B has two adjacent hydroxyl groups at them.So, to further study theirmechnis ms and look formore taxanswith stronger effects will be an important research topic,and have broad application prospects.Itwill be a shortcut to search taxanes with two adjacent hydroxyl groups or fat-soluble side chain adjacent to acetyl group.

Acknowledgements The work discribed in this paper was financially supported from FRND of China(No: 2003AA2Z3527)and Scientific Research Foundation for the Returned Overseas Chinese Scholars from Hebei Province and State EducationMinistry of P.R.China to QWS is appreciated.

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