Radioprotective Effects And Mechanism Of Mangiferin And Its Derivatives On Cells And Zebrafiish

From the earliest days of the nuclear era, the potential applications ofradioprotective chemicals in the event of nuclear accidents, as well as the possibility ofprotecting normal tissues but not tumors during radiotherapy, were considered. Animals,including humans, are exposed to ionizing radiation from natural and artificial sourcessuch as radon, cosmic rays, and nuclear medicine. The efficacy and toxicity of someclasses of radioprotective agents in animals or on cells has been extensively studied.Modulation of endogenous antioxidants, such as superoxide dismutase, may be useful inspecific radiotherapy protocols. Naturally occurring antioxidants, such as vitamin E andselenium, are less effective radioprotectors than synthetic thiols, such as WR-2721andWR-151327, but may provide a longer window of protection against lethality and othereffects of ionizing radiation. Some drugs that have antioxidant properties may have utilityas radioprotectors when administered alone or in combination with other radiomodifiers.As we all know, the damage of radiation mainly comes from the γ ray and neutron ray,and the biological effects are divided into two ways: the first is direct effect and the secondis indirect effect, which accounts for about70-80%of the damages. The indirect way cancause damages to tissues and organs through generating abundant free radicals fromradiolysis water. Cellular damage caused by oxidative stress after radiation is one of thekey factors responsible for many diseases, such as thyroid cancer, skin cancer, and breastcancer as well as leukemia. Studies on screening radioprotectors from antioxidants that canmodulate radiation-induced reactive oxygen species and parallel studies on otherpathologies involving radiation damage remain an active area of biomedical research.Mangiferin (MGN), a xanthone glucoside, is an active phytochemical isolated fromAnemarrhenae asphodeloidea, shows a wide variety of biological activities, such asantioxidant, antiinflammatory, antiviral analgesic and immunomodulatory properties.Professor Jagetia GC from India have studied the protective effect of mangiferin againstwhole body irradiation induced cellular damage in animal systems. It was reported thatmangiferin could increase the survival of animals exposed to different doses of γ-radiation and reduce radiation-induced micronuclei formation and initial DNA damage inthe HPBLs. The study screening a broad of derivatives of mangiferin and found thatpretreatment with most of the derivatives can increase the cell viability compared withirradiation-alone. Among these derivatives, mangiferin aglycone (norathyriol) has the mostsignificant protective effect, especially on the radiation-induced DNA damage. Ionizing radiation has unique characteristics as a genotoxic agent in terms of the DNA damage itproduces. This damage can cause mutation including activation of proto-oncogene,inactivation or mutation of anti-oncogene, DNA mismatch repair, loss control of cellproliferation, signal transduction change in specific gene or chromatosome. In this study,we mainly assessed the protective effects of mangiferin aglycone against radiation-inducedinjuries on normal human intestinal epithelial cells (HIEC), while using mangiferin as areference compound, and try to clarify the underlying mechanism.Recent studies have shown that mangiferin aglycone could reduce intestinal neoplasmsin rats, therefore, it may have special protective sensitivity on intestinal cells. Testis waschosen to study the underlying radioprotective mechanism of mangiferin aglycone andmangiferin on HIEC. Zebrafish (Danio rerio) embryos provide a unique vertebrate modelto screen therapeutic agents easily and rapidly because of their relatively close geneticrelationship to humans, ready abundance and accessibility, short embryonal development,and optical clarity. It allows the transparent visualization of the effects of ionizing radiationand the convenient testing of potential radioprotectors. What’s more, γ rays were shownto be an efficient mutagen in zebrafish and zebrafish embryogenesis. There are few reportsabout the effects of radioprotectors in zebrafish and there is a considerable gap in this areain the world. We conducted experiments to investigate the radioprotective effects ofmangiferin aglycone and mangiferin in zebrafish. Research on this area will fill the gap,which has great value.Collectively, these experiments showed that pretreatment of either mangiferin aglyconeor mangiferin could inhibit cytotoxic effects of ionizing irradiation (IR) on HIEC andattenuate radiation-induced damages in zebrafish tissues and embryos. In addition,mangiferin aglycone presents potential useful effects on IR-induced damage and may thusprovide great potential as a novel radioprotector without knowing toxic side effects.Contents of study:1. Preparation of drugs: Mangiferin and all the derivatives were synthesized by Collegeof Pharmaceutical Sciences, Second Military Medical University, China.2. Screening of mangiferin derivatives: we measured the viability of human intestinalepithelial cells (HIEC) and3T3cells under the influence of mangiferin derivatives onirradiation to find effective radioprotector.3. Radioprotective effects of mangiferin aglycone and mangiferin on HIEC: we observe the following subjects:①Radioprotective effects of different concentrations (0-100μg/mL) of the two drugs on HIEC irradiated with the same dose of IR.②Radioprotectiveeffects of70μg/mL of the two drugs irradiated with different doses of IR.③Theinfluence of mangiferin aglycone and mangiferin on apoptotic rate of irradiated cells.4. Radioprotective effects of mangiferin aglycone and mangiferin in zebrafish:①Radioprotective effects of the two drugs on zebrafish embryonic survival rate at differentperiods following fertilization with different doses of radiation.②Radioprotective effectsof the two drugs on hatching ratio and teratogeny rates of irradiated zebrafish embryos.③The influence of mangiferin aglycone and mangiferin on morphologic abnormalities ofirradiated fish.5. Preliminarily exploring the mechanism of radioprotective effects of mangiferinaglycone and mangiferin:①We assessed the hydroxyl radical (·OH) scavenging activityof mangiferin aglycone and mangiferin.②We monitored radiation-induced DNAdouble-strand breaks (DSBs) in HIEC to test whether mangiferin aglycone and mangiferincould modulate genotoxic effects of radiation.Method:1. Preparation of drugs: All the drugs were dissolved at a concentration of10mg/mL indimethyl sulfoxide (DMSO) when it was used. This stock was furtherly diluted withindouble distilled water (DDW) or diluted by DMEM medium as it was used.2. Cells: Human intestinal epithelial cells (HIEC) and Mouse swiss3T3cells (Long-timecultured in our laboratory) were maintained in RPMI1640(Invitrogen Corporation) with10%foetal bovine serum(Hyclone Corporation) and were incubated at37℃in ahumidified atmosphere of5%CO2in air.3. Animals: Wild-type adult zebrafish were obtained from local pet stores (Shanghai, CN)and embryos were obtained from laboratory of Cardiovascular Dept of Changhai Hospital(Shanghai, CN) and were maintained according to standard operating procedures.Zebrafish were kept at（28±1）℃on a14-hour day/10-hour night cycle.4. Irradiation: Cells and animals were treated with drugs prior exposure to60Co γ-rayswith a delivery rate of1.0Gy/min in the irradiation centre (Faculty of Naval Medicine,Second Military Medical University, China), and then exposed to different radiation doses.5. Determination of cell viability: We assessed the survival rate of different radiationgroups of cells by Cell Counting kit-8(CCK-8) and colony-forming assay. Formula of CCK-8: Viability=OD values of drug group-Background/control OD values-Background;Formula of colony-forming assay: Fraction=(number of colonies/number of cells plated)/(number of colonies for corresponding control/number of cells plated).6. Apoptosis analysis: We assessed the type of cell death induced by gamma radiation,cells were stained by Annexin V-FITC and propidium iodide (PI), and apoptotic rate wereassessed by flow cytometry.7. Zebrafish embryonic survival: We investigated the effects of ionizing radiation onzebrafish embryonic hatching ratio, survival rate and teratogeny rates with different dosesof irradiation.8. Histopathological changes: We screen hepatic, enteric and nephric responses inirradiated adult zebrafish by H&E histological analysis.9. Cell DNA Detection: We investigated the formation of DNA DSBs induced bygamma radiation and its subsequent repair in HIEC by using γH2AX foci formation.Olympus BX51fluorescence microscope (Olympus, Tokyo, Japan) was used to observe thefoci imagines and Image Pro Plus (Media Cybernetics) was used to count the number offoci in each cell.10.Identifies·OH: We assessed the hydroxyl radical (·OH) scavenging activity ofmangiferin aglycone and mangiferin by radiolysis of H2O using HPF.11. Statistical analyses: The mean and standard error of the mean (SEM) for eachtreatment group was calculated for all experiments. For multiple comparisons we used ananalysis of Variance (ANOVA). For pairwise comparison procedures including calculationsof P values were done using the Student’s t test method. A P value of less than0.05wasconsidered to be statistically significant.Results:1. Screening the radioprotective activity of mangiferin derivatives1.1Select the best protective concentration: Cells cultured with different concentrationsof mangiferin aglycone or mangiferin (0-100μg/ml) before exposure to IR and detectedcell viability at24,48and72h. The most significant difference between IR-alone groupand treatment group was observed at72h after exposure to gamma radiation. Cell survivalrate of IR-alone group at this time point was only33.03±0.036%. The survival rateresulted in a dose-dependent increase at each time point and the optimum protectiveconcentration of mangiferin aglycone was70μg/ml, which is higher than that ofmangiferin (50μg/ml). 1.2Radioprotective effects on HIEC and3T3cells: Pretreatment with mangiferin and itsderivatives can increased cell viability in varying degrees compared to IR-alone group. Thesurvival rates of HIEC cultured with mangiferin, mangiferin aglycone or No.10compoundare73.2±0.044%,78.3±0.029%,83.8±0.047%, respectively. The survival rates of3T3cells cultured with mangiferin, mangiferin aglycone or No.21compound are61.6±0.038%,69.4±0.042%,64.47±0.038%, respectively. Thus, we choose mangiferin aglycone to do thefollowing experiments.2. Radioprotective effects of mangiferin aglycone on HIEC2.1CCK-8assay detect cell viability: The radioprotective effects of mangiferin aglyconeand aglycone are dose-dependent. Pretreatment with either mangiferin aglycone ormangiferin significantly (P<0.05) increased cell viability compared to IR-alone group. Asignificant elevation of cell viability (1.52-,2.37-,2.41-and2.40-fold) was observed inmangiferin aglycone-pretreated groups in comparison with IR-alone groups(4,8,12and16Gy). This radioprotection effect of mangiferin aglycone was comparable with mangiferinat doses ranging from4to16Gy, while at the dose of8Gy mangiferin aglyconedemonstrated significantly (P<0.05) better protection on HIEC.2.2Clonogenic survival rate: Compared to irradiation alone group at a dose of8Gy,pretreatment with different concentrations of mangiferin aglycone or mangiferin (0-100μg/ml) for1.5h significantly (P<0.01) increased clonogenic survival of of HIEC. TreatingHIEC with70μg/ml mangiferin aglycone for1.5h before exposure to different doses ofgamma radiation resulted in2.11-,2.46-,3.71-and4.83-fold increase in clonogenicsurvival in comparison with4,8,12and16Gy IR-alone group, and closely reproduced bymangiferin.2.3FITC-Annexin V staining assess apoptosis: In the IR-alone group, the apoptotic cellsincreased to21.85%, while the AnnexinV-FITC-positive/PI-positive cells which representnecrosis and late apoptotic cells in upper right quadrant increased to2.64%. Pretreatmentwith70μg/ml mangiferin aglycone or mangiferin prior to radiation either the apoptotic orthe necrosis cells were reduced. Compared to IR-alone group, late apoptosis of the grouppretreated with mangiferin aglycone or mangiferin reduced by65.4%and57.39%respectively.3. Radioprotective effects of mangiferin aglycone on zebrafish3.1Zebrafish survival rate: Gama-irradiation has a great impact on morphology ofzebrafish embryonic development, and a clear dose-response relationship was found in a short period. With the increase of radiation dose, the mortality rate of embryos of larvalincreased, showing a significant positive correlation. Mangiferin aglycone or mangiferinwere injected into the yolk of zebrafishes’ gastrula stage embryos. We found that themortality rate of the injected embryos was significantly lower than that of the controls.Some juvenile fishes were feeded in the water with a certain concentration of mangiferinaglycone or mangiferin and did not find any observed influence on zebrafish death andteratogeny.3.2Zebrafish hatching ratio and teratogeny rate: IR exposure could lead to many kindsabnormal in zebrafishe，such as curve of the tail, expanding of pericardial cavity, spinaltwist and so on. With the radiation dose increase, the rates of malformation and severemalformation had an obvious increase, and all of these embryos eventually becamemalformation or even severe malformation. The hatching ratio was significantly (P<0.1)higher than that of the controls while the teratogeny rate of the injected embryos wassignificantly (P<0.05) lower.3.3Histopathology of irradiated zebrafish:①Intestinal: In the IR-alone group we cansee small intestinal epithelium cells are arranged irregular, with different degrees ofdegeneration and fall off and the margin of some of the cells disappear. The resultssuggested that pretreatment with mangiferin aglycone or mangiferin preserve better themorphology of the small intestinal than the IR-alone group, especially in maintaining theoriginal form of intestinal crypt and increasing the number of epithelial cells.②Liver: Thehepatocyte borders are obscure and cell vacuolar degeneration can be observed in theIR-alone group. Destruction of cellular structures, hepatocyte denaturation and necrosiscan also be observed. Some hepatocyte nuclei have become enlarged and pycnotic. Bothmangiferin aglycone and mangiferin can attenuates these hepatocyte damage induced byIR.③Kidney: Compared with the kidney of healthy fish, some of the renal tubules in thekidneys of irradiated fish displayed a lot of debris from necrotic epithelium and lost thenormal shape. The interstitial areas between the renal tubules, or glomeruli were alsoenlarged. Pretreatment with mangiferin aglycone and mangiferin can reduces thesedeleterious effect in irradiated fish kidney.4. The mechanism of radioprotective effects of mangiferin derivatives on HIEC4.1Hydroxyl-Radical scavenging activity: The fluorescence of mangiferin aglyconegroup and mangiferin group were both significantly less than that of control group. Thefluorescence signals of the oxidized HPF were reduced by these two compounds at all doses and a maximum reduction was observed at7μg/mL. However, there was nosignificant difference between them.4.2Attenuate DSBs caused by IR: Radiation damage to cellular DNA and radiosensitivityof normal cells under a variety of conditions can be evaluated using the γH2AX foci assay.This technique was used to evaluate the radioprotective effect of mangiferin aglycone inHIEC before irradiation. Mangiferin aglycone alone did not alter radiation-induced DNAdamage to irradiated HIEC in vitro. However, a significant radioprotective effect (P<0.05)was observed in vitro with mangiferin aglycone at concentrations between50and100μg/mL. γH2AX foci formation in HIEC provide a good measure of IR exposure with clearreproducibility and high sensitivity. Compared to IR-alone group, fluorescence signals ofcells pre-treated with mangiferin aglycone before exposed to1,2and4Gy irradiationreduced by41.3%,46.8%and43.4%relatively, while the fluorescence signals of cellspretreated with mangiferin reduced by36.2%,40%and37.5%. Within30min after IR,formation of γH2AX foci occurred and the formation of γH2AX was also dose-dependent.Discussion:Treatment of radiation damage has been a very difficult problem in biological andmedical domain. Many countries have invested much money to study new radiationprotection drugs. However, so far, only one kind of radiation protection drug (WR2721)approved by FBA was used in clinic for treating radiation damage from radiotherapy.There is no approved drug available for the prevention or treatment of Acute RadiationSyndrome at present. Thus, finding novel agents ultimately intended for human use in thecontext of therapeutic or accidental radiation exposure is important.It is generally accepted that radiation damage to the body can be divided into directdamage and indirect damage, the main point of radiation protection is against indirectdamage by clearing the radiation induced free radicals. Due to presence of a phenolicgroup with an aromatic conjugation in mangiferin, it has a potent activity for scavenginghydroxyl radicals (·OH) and it also has been shown to protect ROS-induced cell death,preventing mitochondrial dysfunctions and restoring the SOD activity. Therefore, its robustantioxidant effects make mangiferin an attractive candidate for radioprotection. Howevermangiferin itself is difficult to dissolve in water, which greatly affected its biologicaleffects. Twenty five brand new compounds were synthesized to find better radioprotectiveagents by testing the survival rate of irradiated cells. In this study, we measured the viability of human intestinal epithelial cells (HIEC) and3T3cells under the influence of mangiferin derivatives on irradiation to find effectiveradioprotector. Our results show that some of the mangiferin derivatives have greatradioprotective effects and among which mangiferin aglycone has the best protectiveeffects. What’s more, Mangiferin aglycone is a better water-soluble than mangiferin due toits small molecular. We also found that irradiated zebrafish embryos administered mangiferinaglycone or mangiferin significantly improved the survival rate as well as the hatching rateand decrease the teratogeny rates. Meantime, both of these two drugs can reduce radiationdamage of intestinal tissue, liver and kidney of zebrafish. They can inhibiting of small intestinalcrypt epithelial cell apoptosis and maintaining the integrity of the intestinal bacterial. At presentthe mechanism of therapeutic effect of mangiferin aglycone is still not entirely clear, thereforewe further studied it on HIEC. The results showed that both mangiferin aglycone andmangiferin can reduce radiation-induced hydroxyl radicals (·OH)，at the same time，theycan be very effective in reducing DNA damage of the HIEC. There are researches on theclinical use of mangiferin and it showed no obvious acute toxicity, which is the same in ourexperiments. Both mangiferin aglycone and mangiferin are classified as nontoxic product.Complementing earlier results from our group, it appears possible to conclude thatmangiferin aglycone presents potential useful effects on IR-induced damage and may be abetter radioprotective agent than mangiferin therapeutically.