| Background and Purpose:In 1956,the British scholar,Harmna,first raised the aging theory associated with free radicals,it deemed that the free radicals,which attack the life macromolecules to cause tissues and cells injuries,are not only the primary cause of aging,but also the origin that induces malignant diseases,such as tumor.In 1990,Professor Sohal,a scholar in the aging study authority in America,pointed out many defects of the aging theory related to free radicals,and first put forward the concept of oxidative stress. Oxidative stress means the tissue injuries caused by the imbalance between the oxidative and antioxidative systems,which is induced by the body,overoxidation capacity in excess of antioxidant capacity because of the overproduction of overactive molecules in the body,such as active oxygen species and active nitrogen species. Almost all the human organs are vulnerable to the harmful effects of oxidative stress. When the level of oxidative stress raises,it can cause extensive damage to cells,and is closely related to inflammation,cancer,autoimmune diseases and aging,it can also cause diseases of multiple organs such as heart,brain,liver and kidney,which serve as serious hazards to human health.The generation of oxidative stress has both internal and external causes,and the external causes include exposure to pollution, petrochemical products or heavy metals;while the internal factors include chronic or acute infection,the degree of anemia,blood sugar levels and lifestyles.Free radicals,also known as the "liber radicals" on chemistry,are atomic groups which contain an unpaired electron.As the normal product of the body's metabolism, they have a strong oxidation capacity in vivo for easy damage to proteins,lipids and nucleic acids ultimately leading to body injury[1].Free radicals damage on the human body mainly lies in three aspects:First,inducing lipid peroxidation to cause membrane damage;secondly,inhibiting the activity of serum anti-protease to cause protein aggregation and cross-linking;thirdly,destroying the structure of nucleic acids and attacking the purine and pyrimidine base,which bring about the emergence and accumulation of cell variation.However,free radicals are also indispensable and active substances for the body,which can act as second messengers involved in cellular signal transduction[2].In fact,a certain degree of oxidation can provide the necessary energy for the body to maintan health and life,only overoxidation will be a strong risk for the life.Under norlmal physiological conditions,there is a dynamic balance between the production and clearance of oxygen free radicals[3].The substantial accumulation of oxygen free radicals in the body occurs only when this balance is broken,which causes the pathological damage of tissues by destroying the integrity of biofilm and both the structure and function of cells.The pathological damage of cells can also affect the metabolism of nucleic acid and destroy the structure of DNA,which causes the gene mutation that results in abnormalities of cell differentiatiation and tumorigenesis[4-5].The current study of free radicals and cancer:with the rapid development of life science and medical science,the study of major phenomenons of life and of many types of pathogenesis has been deep into the molecular and cell level.In foreign countries,study about free radicals in medicine and biology has proved to be a very active research field.With the development of the theory concerning free radicals as a cause of cancer,it has been fully realized that the free radicals have played an important role in the inducing,promoting and resisting tumorgenesis.Interational Cancer Research Center has raised that 80%-90%of human tumors are caused by chemical substances,most of which have undergone pilot process of generating free radicals during its metabolism process for the final course of cancer.Furthermore, study in foreign countries has found that tumor cells can generate superoxide anion, and the oxygen free radical scavenging systems of tumor cells from many patients are suppressed[6].Excessive free radicals can cause the expansion and chain reaction of carcinogens in the human body,and can attack DNA resulting in various forms of damage,which could induce many kinds of disease.Although it has been recognized that free radicals involve and play an important role in the occurrence and development of many diseases,the essence and details of the mechanism involved have not been known thoroughly yet.The mechanism of free radicals inducing and promoting tumorigenesis,the changes of free radicals in tumor occurrence and development,as well as the possibility of the level of free radicals as a supplementary target in disease diagnosis for a preliminary diagnosis are still needed to be further studied.Superoxide dismutase(superoxide dismutase,SOD),which is also called peroxide dismutase belong to metal enzyme,according to binding metal ion species,it has three kinds,CuZnSOD,Mn-SOD and Fe-SOD.They can catalysis superoxide anion radical anion disproportionate to hydrogen peroxide and oxygen.widely found in all living organisms in the nature.It is one of the important antioxidant enzyme.It has been deemed as the trash scavenger of the body for its capacity of removing excessive superoxide anion radicals,protecting structure and function of the biofilm and biological macromolecules,as well as inhibittng tumorigenesis.Endogenous changes in the level of SOD in body tissue may reflect the metabolism function of the body tissues at varying degrees[7].Under normal circumstances,SOD could specifically catalyze the disproportionation reaction of toxic oxygen free radicals,and remove them away from the body to maintain the dynamic balance between the production and clearance of free radicals.Any factors which enhance oxidation or reduce antioxidant can destroy this balance,resulting in a series of pathophysiological abnormalities[8].Levels of SOD synthesis in organisms are impacted by the concentration of oxygen free radicals.Under the induction of oxygen-derived free radicals,SOD strengthens its biosynthesis.The increase of local concentration of SOD in the body is a response from the endogenous defense system,which indirectly reflects the increased concentration of oxygen free radicals.As an important antioxidant enzyme,SOD activity has been reported in nervous system diseases and acute leukemia,such as Hujun et al.[9]who have found that in the course of cerebral ischemia reperfusion,oxygen free radical;oxyradical increased is the important patho-link,it make SOD dyssynthesis,initiate the biological membranelipid peroxidation,induce the nerves functional disturbance.it also initiate Ca2+ ATP enzymatic activity loss.Ca2+ inflow,pose vascular endothelial cell and parenchyma cell degeneration and necrosis.Meanwhile,it make the Na+-K+-ATP enzymolysis uncoupling and cause cerebral edema.Chen Huajian et al.[10]have checkouted the activity of SOD in 49 brain hemorrhage patients and 65 cerebral infarction patients,the results suggested that the activity of SOD participate the course of the patho- process of brain tissue damage,so,to check the activity of SOD conduce to judge the pathological changes degree and pathogenetic condition advancement.After detecting the level of serum SOD,glutathione peroxidase(glutathione peroxidase GSH-PX) in 42 cases of leukemia before and after chemotherapy.Li Xiumei et al.[11] found that serum SOD,GSH-PX levels in patients with leukemia before chemotherapy were significantly lower than the normal group,in addition,the SOD, GSH-PX levels of patients who relapse 6 months after chemotherapy keep abnormal,while they are nearly normal in patients who did not relapse,but DeviGS et al.[12] reported that,SOD activity in different types of acute leukemia showed no significant difference.The diagnosis of Central nervous system leukemia(certral nervous system leukemia,CNSL):Since many chemotherapeutic drugs are not easy to pass through the blood-brain barrier,the leukemia cells hidded in the central nervous system would hardly be effectively eliminated,giving rise to the CNSL,which has become one of the most important reasons of leukemia relapse.CNSL disease may emerge at any stage of disease,but often occurred in remission period after chemotherapy.Acute leukemia is rarely combined with CNSL at onset,but if no effective preventive measures adopted,it would not only cause a high incidence of CNSL,but also a bad prognosis.Chen Yi jian et al.[13]has reported the incidence of CNSL is about 10 - 20%, while acute lymphoblastic leukemia and acute non-lymphocytic leukemia combined with CNSL are 26 - 30%and 5 - 18%respectively.because of the adoption of preventive measures for CNSL,the CNS relapse rate of acute myeloid leukemia has dropped to 5 - 10%,while that of acute lymphoblastic leukemia has dropped to 2 - 10%[14].Therefore,timely detection and treatment of CNSL can significantly improve survival and curing rate of patients.At present,diagnostic criteria for the CNSL are as follows[15]:1.appearance of the central nervous system symptoms and signs(especially intracranial hypertension symptoms and signs). 2.changes in cerebrospinal fluid:(1) increased stress(＞0.02kPa or 200mmH2O).(2) WBC＞0.01×109 / L.(3)Leukemia cells seen on smear.(4) protein＞450mg / L,or PAN test positive.3.excluding the similar changes in central nervous system or cerebrospinal fluid cause by other diseases.Meeting the requirements of 3 plus(3) or any two of 2 woule confirm the diagnosis of CNSL.Any change in the cerebrospinal fluid coule also allow a diagnosis for CNSL,even Asymptomatic.If only the cerebrospinal fluid pressure kept rising,but dropped to normal after treatment by anti-CNSL,the diagnosis of CNSL can also be made.If no cerebrospinal fluid change,but there are symptoms and signs indicating brain,spinal cord or nerve roots infiltrated,other reasons can be ruled out,and significant improvement in symptoms emerges after anti-CNSL treatment,diagnosis of CNSL would be permitted too.CNSL usually started with occult onset,non-typical clinical manifestations,or even no positive symptoms and signs.The main clinical manifestations of CNSL are as follows:①intracranial hypertension;②cranial nerve damage;③symptoms of spinal cord damage;④symptoms of infiltration of brain parenchyma;⑤symptoms of nerve roots and peripheral nerves damage.According to research,under optical microscope,only one leukemia cell seen in one microlitre of cerebrospinal fluid(cerebrospinal fluid,CSF) indicates there are 105 leukemic cells existing in CSF[16].At this point,CNSL has actually developed to the severe extent, and lost the opportunities for early treatment.Therefore,early diagnosis and prevention for CNSL should be significantly emphasized,currently,there are various methods and criterias for early diagnosis for CNSL but no ideal diagnosis method concerning both of the specificity and sensitivity.Cerebrospinal fluid cytology test is valuable for early CNSL diagnosis,but only positive when leukemia cells infiltrating brain meninges.unfortunately,early infiltration of brain parenchyma and peripheral nerve always manifests inconspicuous changes in CSF,or even no change,which made simply CSF cytology for the early diagnosis of CNSL difficult.Thus,looking for a new early diagnosis criteria has significant clinical value for the prevention and treatment for CNSL,and for improving survival and cure rate of patients.Presently, experimental results indicated that the majority of patients with brain tumor showed increased serum SOD activity,therefore,the possibility of detecting SOD activity in cerebrospinal fluid as a supplemented diagnosis for CNSL needs to be investigated.The current treatments for CNSL:At present,the treatments for CNSL mainly include intracranial radiotherapy,intrathecal chemotherapy and systemic chemotherapy.Although these treatments can extend survival time of patients,defects raised,because of side effects and the difficuties for majority of drugs passing through the blood-brain barrier.In addition,repeated lumbar punctures woule aggravate the pain feeling of patients,resulting in inferior life quality and increased chances of puncture failure.Thus it is imperative for us to look for a new drug which can pass through the blood-brain barrier for the treatment of CNSL.The current research about Artesunate:Artesunate,an Artemisinin derivative and an anti-malarial ingredient in Abrotanum(a kind of Chiniese Traditional drug),is a sesquiterpene lactone compound with a peroxidized group extracted from Artemisinin(a kind of composite plant) and is the first internationally acknowledged and the frontline anti-malarial natural drug used worldwidely for its high efficiency, low toxicity,good liposolubility,high anti-malarialin activity in pharmacological properties.Inspired by the mechanism of Artemisinin eliminating plasmodiums, a number of laboratories both at home and abroad have studied the possibility of Artemisinin for anti-tumor use,results showed that the Artemisinin and its derivatives have the exact anti-tumor effects,and Artemisia-like drugs are selective anti-tumor inhibitors that can selectively inhibit the growth,induce the apoptosis,and inhibit the proliferation of tumor cells in vitro,such as leukemia cells,fibrosarcoma cells, ovarian tumor cells,breast tumor cells and cervix tumor cells.Its novel structure and its anti-tumor mechanisms with no cross-resistance to drugs make it different from traditional drugs.All of the above properties make it share a variety of advantages including killing multiple tumor cells,low toxicity,the possibility of reversing multiple drug resistance of tumor cells[20].Based on the anti-tumor effects of both the Artemisinin and its derivatives,and their pharmacokinetic characteristics of passing through the blood-brain barrier to penetrate into brain tissue[21],Artesunate may be speculated to bemome a new drug for the treatment of CNSL.In the previous study of our group,it had been verified that the artesunate-induced apoptosis of K562 cells was associated with iron metabolism,the mechanism of which may be artesunate and iron to form free radicals that kill tumor cells.Based on the capability of artemisinin-based drugs for passing through the blood-brain barrier,and on the convenience,economic cost as well as few toxic side effects of oral artemisinin-type drugs compared to chemotherapy drugs and radiotherapy,the use of artemisinin-based drugs may have significantly clinical value for treatment of CNSL, if it is confirmed that the incidence of CNSL,as well as artemisinin-based drug-induced tumor cell changes in apoptosis are related to free radicals.Based on the above,the purpose of this study is to investigate the value of SOD activity test in diagnosis of CNSL and the mechanism involved in artesunate interference,by detecting the changes of SOD activity in CSF and the markers such as SOD activity in the process of artesunate-induced apoptosis of K562 cells.Investigation methods1.Empirical materials①Collect the cerebrospinal fluid of 55 patients that come from deprtment of hematology of NanFang hospital.of the total,30 cases patients suffer with central nervous system leukemia,25 cases patients are suffer with acute leucemia but have no systema nervosum centrale impaired.②The K562 cell line is cultivated in the PRMI-1640 culture fluid which contain 10%FBS in the incubator with tempreture 37℃,5%CO2,saturation humidity,and passaged.once per 24-36 hours,take the cells in exponential phase of growth to experimentize.basis on the result of preliminary experiment in the invest groop, select the effect density of Artesunate with 4×10-5mol/L,2×10-4mol/L,1×10-3mol/L as experimentize groops,and control groups is not add artesunate.2.Empirical method①The analysis of the common practice,biochemistry,cells film preparation in cerebrospinal fluid.②The xanthine oxidase method detect the SOD activity in cerebrospinal fluid.③Apoptosis was mearsured by Annexin V-PI and Hochest 33258 stain.In the experiment,K562 cells were treated with different density of artesunate.④The xanthine oxidase method detect the SOD activity in the course of the k562 cells apoptosis with different density of artesunate treated,artesunate final concertration are 4×10-5mol/L,2×10-4mol/L and 1×10-3mol/L respectively.⑤The expression of MnSOD and SeGPX mRNA in K562 cells treated with three different concertration of artesunate for 48 hours was analyzed by Q-PCR.⑥Statistic methods:statistic software is SPSS13.0,statistic methods are K independent samples,One Way ANOVA,and multiple comparison(LSD).p value below 0.05 were considered significant.Results1.Compare with the blood corpuscle quantity,the protein level in cerebrospinal fluid,the CNSL groups has significant difference compared to the control groups(P=0.000,P=0.048).compare with the cerebrospinal fluid pressure,glucose, chlorine,the CNSL groups and the control groups has no significant difference (P＞0.05).the cerebrospinal fluid pressure of the the CNSL group are higher than the control groop,and the glucose,chlorine,of the CNSL group are lower than the control groop by the mean lenel. 2.the activity of SOD in CNSL groop and the control groop is 999.42±461.57, 662.17±354.38,compare with each other,two sets has significant difference (p=0.004).3.The blood corpuscle quantity and the protein level in cerebrospinal fluid each has direct correlation with the activity of SOD,the coefficient correlation is 0.871 and 0.518,p value is 0.000 and 0.003.4.In different age groups(＜45y,45-59y,＞60y),the activity of SOD in the cerebrospinal fluid from the detection is 755.64±345.77,1483.43±266.49, 1344.96±367.21.and the activity of SOD of the 45-59y groop is the highest.The＜45y groop has significant difference compared to theother two groups.(p＜0.05). between the 45-59y and＞60y groops,there has no significant difference(p＞0.05).5.Annexin V-PI double stain showed that K562 cells were treated with 4×10-5 mol/L,2×10-4 mol/L and 1×10-3 mol/L Artesunate for 48 hours,total apoptosis rate was in concentration dependent manner.6.The result of the activity of SOD on K562 cells treated with different density (4×10-5 mol/L,2×10-4mol/L,1×10-3mol/L) of Artesunate is 17.64±2.54,47.89±6.38 and 62.05±1.72.The 4×10-5 mol/L and 2×10-4mol/L groops has significant difference compare to the control groop(p＜0.05),the 4×10-5 mol/L and 2×10-4mol/L groops has significant difference compare to the 1×10-3 mol/L groop(p＜0.05).The 1×10-3 mol/L groop has no significant difference compare to the control groop,and between the 4×10-5 mol/L and 2×10-4mol/L groops there has no significant difference(p＞0.05). the activity of SOD in the 4×10-5 mol/L groop is the highest.7.Q-PCR showed that:The expression of MnSOD mRNA and SeGPX mRNA treated by different density Artesunate for 48 hours,the 4×10-5 mol/L and 2×10-4mol/L groups has significant difference compared to the control group.the 4×10-5 mol/L and 2×10-4mol/L groups has significant difference compared to the 1×10-3mol/L group(P＜0.05).between the 4×10-5mol/L groop and the 2×10-4mol/L group there were no significant difference.the 1×10-3mol/L group has no significant difference compared to the control group(P＞0.05).The expression of MnSOD mRNA and SeGPX mRNA in the 4×10-5 mol/L groop is the highest.Conclusion1.The blood corpuscle quantity and the protein level in cerebrospinal fluid of the CNSL groop is higher than that of the control groop,it suggest that The blood corpuscle quantity and the protein level in cerebrospinal fluid is important for the diagnose of CNSL.the cerebrospinal fluid pressure,glucose and chlorine in the two sets has no significant difference,it means that the cerebrospinal fluid pressure, glucose and chlorine are possible non- idio for the diagnose of CNSL.2.the activity of SOD of the CNSL groop is higher than that of the control groop. The blood corpuscle quantity and the protein level in cerebrospinal fluid each has direct correlation with the activity of SOD,it suggest that oxidative damage participate the course of the invasion of CNSL.Weather if the activity of SOD can be used for detecting the disease advancement,observing curative effect,criterion prognosis needs further research.3.The activity of SOD in the 45-59y groop is the highest Of the threet age groops, The activity of SOD in the＜45y groop has significant difference compared to the other two groups,between the 45-59y and＞60y groops,there has no significant difference,it suggest that The change of the activity of SOD in the cerebrospinal fluid is possible concerned with age.4.Artesunate can induce K562 cells apoptosis in different density(4×10-5 mol/L,2×10-4mol/L,1×10-3 mol/L),and the effect submit by density dependence,the more the density is the higher the aoptosis rate is.In the course of the K562 cells apoptosis by Artesunate has relationg with the activity of SOD and the expression of MnSOD mRNA and SeGPX mRNA.Of all the density groops,the aoptosis rate in the 4×10-5 mol/L groop is the highest.follow the density go on,the aoptosis rate go higher,but the activity of SOD and the expression of MnSOD mRNA,SeGPX mRNA down regulation,these demonstrate that oxidative damage participate the course of the K562 cells apoptosis by Artesunate,but it's possible not the main mechanism for the K562 cells apoptosis by Artesunate.
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