| Chemotherapy is one of the three big major therapies for cancer.Since most chemo drags are targeted at DNA which is not highly specific,they damage normal cells(particularly active marrow hemopoiesis stem cells)while killing tumour cells.The damaged DNA protects its normal physiological function and the stable hereditary feature mainly through repair and apoptosis.In case of serious damage,the cell tends to die;in case of light damage,the cell cycle is blocked and suspended,which is continued after the DNA initiates and completes the repair.Since marrow is the hemopoietic tissue,once the cell cycle is disturbed,myelosuppression will arise,causing low hematopoietic function and falling circumference blood white cells with low immunologic function,and failure in chemotherapy or death from infection as well.Therefore,in this process,enzyme and protein which participate in the DNA repair are not only closely connected with the repair, affect the degree of suppression by acting on the cell cycle.Cyclophosphamide(CTX)is both an alkylation agent in the group of latent nitrogen mustard,and immunity inhibitor.It can interact with the macromolecules,and induce DNA damage.The cell purine may have N-methylation or O-methylation(inducing 67%phosphoric acid single groups and bases,33%N-7 guanine groups and bases).It may also damage the DNA complementary strand crosslinking and linking protein.Any DNA damage will first involve MGMT in direct fast repair.Meanwhile,nucleotide excision repair and basic group excision repair also play important role in DNA molecular (adducts)repair.Although it is generally accepted that acupuncture improves hemopoiesis and immunologic function post chemotherapy,very few is reported,on its mechanism, especially on micro mechanism.Based on previous researches on the mechanism of marrow injury by chemo drugs and progress of DNA repair studies as well as the research done over the past 20 years,the hypothesis is made that "promoting hemopoietic cell DNA repair is the key mechanism of acupuncture in relieving myelosuppresses,and protecting hemopoietic function." Starting with the CTX-injured sites and the key route of repair,the author observed the dynamic effect of acupuncture on DNA repair protein expressions in CTX-induced mice as well as changes of the cell cycle,analyzed the correlation between them and its variation so as to disclose the molecular mechanism of acupuncture in intervening(relieving)adverse reaction of chemotherapy,protecting marrow hemopoiesis cell,and improving myelosuppression,and providing theoretical evidence for acupuncture therapy in the treatmentof pathological hematopoietic injury as well.In addition,through comparative analysis,this paper aims to explore differences between acupuncture and moxibustion therapies in relieving myelosuppression.Methods:Grouping:Select 224 healthy male KM mice,divide by stratified random sampling every 4 mice into a block,56 blocks in total,according to body weight and numbers of white blood cell;divide the 4 mice in a block randomly into normal group,model group, acupuncture group and moxa group,namely 56 mice in each group(8mice/day×7days)×4groups =224mice.Duplication of models:duplicate models with myelosuppression as is stipulated in the Guidelines for Pre Clinical Study of New Drugs(Western Medicines)by abdominal cavity injection of endoxan,Injecting CTX 100mg/kg/d by abdominal cavity(According to does of 0.02ml/g for per mouse's injecting 5mg/ml CTX by abdominal cavity),followed by repeated injection 24h and 48h afterwards continuously for 3 days,the model is made 4h post repeated injections(in accordance with pharmacokinetics of CTX,endoxan tends to lose activity after 4h).Normal group is accordance with does of 0.02ml/g to inject 0.9%NS by abdominal cavity.The normal group is injected 0.9%NS ?ml by abdominal cavity.Acupuncture points selection and treatment:The mouse points were located in accordance with the guidelines in "Chinese Veterinary Acupuncture And Moxibustion,"and location of rat points:"Da Zhui" is between the seventh cervical vertebra and the first thoracic vertebra on the back;"Ge Shu" is on both sides under the seventh thoracic vertebra;"Shen Shu" is under the second lumbar vertebra on both sides between ribs, about each hole one side;"Zu San li(Hou San Li)" is about 1-2mm under fibula capitellum of the joint flank on each posterior leg.Acupuncture group:Apply acupuncture to the mice 4h post duplication.Select tubular needles of Hua Tuo brand with a vertically inserted depth of 0.3mm.Rotate the needles 5 times using tonic manipulation and retaine for 6min,once daily and continuously for 7 days.Moxa group:Apply moxa to the mice 4h post duplication.Light moxa stick and hold it over the respective point at a distance of 5mm for 7-8s until the mouse moves.Repeat this 3 times at the same point every 5s,once daily and continuously for 7 days.Normal group:Captured and fixed daily as is done in the acupuncture group and the moxa group,yet without any treatment.Model group:Captured and fixed daily as is done in the acupuncture group and the moxa group,yet without any treatment.Indicators and methods for observation:①Select mice every morning before injection of CTX through until after duplication of molds;cut the tails in the 4 groups in turn to obtain 10μl blood each;drip into test tubes of 390μl 3%glacial acetic acid dilution to be used for circumference white blood cell counting,continuously for 10 days.②Start from the 2nd day,take out the marrow karyocyte 4h post daily acupuncture and moxa treatment and make them into cellular suspension to determine the DNA volume of the marrow cells and marrow cell percentage at various cell cycles.Method:select 8 mice randomly from each group for execution,take humerus and thighbone from one sides. Place the humerus and thighbone on the ultra-clean work table,get rid of the muscles and tissues,rinse by 0.9%NS,keep in 75%ethyl alcohol for 2min and rinse by 0.9%NS again.Cut an opening at the upper extreme,use 5ml injector to take out 3.6ml 0.9%NS and inject them into medullary cavity of the thighbone,rinse all its karyocytes 2 or 3 times and disperse into separated cellular suspension.Inject the cellular suspension into the freeze test tube and preserve sample in the refrigerator at-80℃for determination of marrow cell DNA volume and marrow cell percentage at various cell cycles,continuously for 7 days.③From the 2nd day of duplication of models,take out marrow to determine the expression of Cyclin D1,MGMT,POLβand XPD proteins 4 hours after daily acupuncture and moxibustion treatment.Method:Takes humerus and thighbone marrow on the other side,fix the sample with 10%formalin,embedded by paraffin wax,slice it into 4μm thick. Observe the protein expression of Cyclin D1,MGMT,POLβand XPD with immunohistochernical method continuously for 7 days.Statistic analysisUse SPSS13.0 for Windows for data analysis.The data is expressed with mean value((?)) and standard deviation(s),test for equal variances is adopted in the comparison between different groups.In case of equal variances,one-way ANOVA is used;otherwise variances must be adjusted first before analysis,the test criteria isα=0.05.Results:1.Dynamic variation of circumference blood white blood cell counts post duplicationThere was no remarkable differences in white blood cell counts between different groups on the 1st day before duplication(P＞0.05),suggesting that mice are from the same origin, and conform to normal distribution,and can be used for experiment.White blood cell dropped gradually on the 2nd and 3rd day after injection of CTX.Compared with the normal group,WBC counts falls in the model,acupuncture and moxa groups with marked differences(P＜0.05);compared with the model group,there was no marked differences in the acupuncture and moxa groups(P＞0.05).This indicates that CTX inhibited the hemopoietic function of marrow,and markedly damaged stem cells.2.Acupuncture and moxibustion on the dynamic change of white blood cell counts in CTX-induced mice:Compared with the model group acupuncture and moxa could cause the level of WBC of CTX-induced mice to rise 1 day earlier and to surpass the basic level 1 day earlier. Especially between the 4thand 7th day,the cell count rose markedly in the acupuncture and moxa groups,proving that acupuncture does relieve marrow myelosuppression in CTX-induced mice,and raise the number of white blood cells.There was no remarkable difference between acupuncture and moxibustion(P＞0.05).3.Acupuncture and moxibustion on the dynamic change of DNA in CTX-induced mice marrow karyocyte:The results of the study show that G1 phase and G2 phase had generally the same tendency in DNA volume variations.After CTX chemotherapy,DNA volume reduced in the acupuncture,moxa,and model groups compared with the normal group,indicating CTX causes injury of the hematopoietic system.Compared with the model group,the DNA volume of the acupuncture group and the Moxa group on the 4th day has been higher, with marked difference on the 5th day,indicating that acupuncture and moxibustion can raise the marrow karyocyte DNA volume,promote the marrow karyocyte to mitose and proliferate.Acupuncture proves sooner effect than moxibustion in terms of time,the Moxa group is better than the acupuncture group,yet with no remarkable difference in terms of effect(P＞0.05).4.Acupuncture and moxibustion on the dynamic change of cell cycle of CTX-induced mice:4.1 Acupuncture and moxibustion on the dynamic,change of G1 phase in CTX-induced mice:The marrow cell percentage of G1 phase in the acupuncture group and moxa group between 2-7th day is lower than that of the model group,suggesting that acupuncture can relieve the G1 phase blocking,reduce the function of G1/S phase testing points,cause parts of the blocked cells to be repaired promptly at the G1 phase,help the marrow cells get through the G1/S phase testing point into the S phase to carry out DNA synthesis,continue splitting for the cycle.4.2 Acupuncture and moxibustion on the dynamic change of S phase in CTX-induced mice:The marrow cell percentage of S phase in the acupuncture group and moxa group between 2-7th day is higher than that of the model group,besides the remarkable difference on the 2nd,3rd and 4th day,suggesting that acupuncture and moxibustion are able reduce the injury of DNA caused by chemo therapy and promote marrow cell DNA repair and synthesis.4.3 Acupuncture and moxibustion on the dynamic change of G2 phase in CTX-induced mice:The marrow cell percentage of G2 phase in the acupuncture group and moxa group between 2-7th day is higher than that of the model group,besides the remarkable difference on the 2nd and 3rd day in acupuncture group and the remarkable difference on the 4th and 5th day in moxa group,suggesting that most cells are blocked at the G2 phase after duplication of models,the G2 phase is also the period when cell repairs the injured DNA before chromosome separation.A rise in the number of cells at the G2 phase indicates that the chance of DNA repair is increased,acupuncture and moxibustion may strengthen the function of G2/M testing points,promoting marrow cell DNA repair and accelerate enter the M phase to mitose and proliferate.Acupuncture has sooner effect than moxibustion in terms of time.Although the effect in the Moxa group is better than that in the acupuncture group in terms of effect,yet no remarkable difference is found.5.Acupuncture and moxibustion on DNA repair protein expression in CTX-induced mice marrow cell.5.1 Acupuncture and moxibustion on dynamic change of Cyclin D1 protein expression in CTX-induced mice marrow cell:After duplication of the molds with CTX,Cyclin D1 expression is reduced,affecting the smooth transformation of cell cycles,particularly the G1/S transformation,preventing cells entering the S phase from the G1 phase,causing reduced DNA synthesis.Starting from the 2nd day,figures in the acupuncture and Moxa groups are higher than those in the normal and model groups,with a peak on the 4th day,showing remarkable difference (p＞0.05).In terms of the average time cost to reach the peak,it is 1 day earlier in the acupuncture and moxa groups than in the model group,the Cyclin D1 protein expression elevation is consistent with S phase cell multiplication.After the 5th day,although the acupuncture group and Moxa group witnessed a gradual fall of Cyclin D1 expression,but it was still markedly higher than the model group and the normal group,showing periodic fluctuation.The above indicate that acupuncture and moxibustion can up-regulate the expression of Cyclin D1 in period. 5.2 Acupuncture and moxibustion on dynamic change of MGMT protein expression in CTX-induced mice marrow cell:The figure in the acupuncture group is obviously higher than that in the model group and the normal group from the 3rd day(p＜0.05),and also higher than that in the moxa group yet with no remarkable difference(p＞0.05);the figure in the moxa group on the 2nd day was remarkably higher than the normal group and the model group(p＜0.05).The peak comes on the 5th an 6th days in the acupuncture and moxa groups;the figure in the model group shows a rising tendency from the 4th day,but is lower than the acupuncture group, the Moxa group,and climbs to the maximum also to be lower than the acupuncture and moxa groups,the same case with the peak.On the 7th day each group shows a gradually falling tendency in terms of MGMT protein expression,with the acupuncture and moxa groups still higher than the model group.There is no significant difference between the acupuncture group and the moxa group.The above indicates that both acupuncture and moxibustion can elevate the expression of MGMT.5.3 Acupuncture and moxibustion on dynamic change of POL-βprotein expression in CTX-induced mice marrow cell:A rising tendency starts from the 2nd day in the acupuncture group,the Moxa group,and the model group,with the first two groups markedly higher than the model group and the normal group on the 3rd day(p＜0.05).The peak comes on the 4th day,gradual fall starts on the 5th day,but still obviously higher than the model group(p＜0.05).Although the model group shows a rising tendency from the 2nd day,but the figure is obviously lower than that in the acupuncture and moxa groups,with a peak on the 7th day yet without remarkable difference when compared with the acupuncture and moxa groups.In terms of the average time cost to reach the peak,it is 3 days earlier in the acupuncture and moxa groups than in the model group,and slower in the falling tendency.Acupuncture seems better than moxibustion,but with no remarkable difference(P＞0.05).The above indicates that both acupuncture and moxibustion can up-regulate the expression of POLβprotein.5.4 Acupuncture and moxibustion on dynamic change of XPD protein expression in CTX-induced mice marrow cell:It starts to rise gradually 3 days after the treatment in the acupuncture group,the moxa group,and the model group,with the acupuncture group obviously higher than the model group and the normal group(p＜0.05)between the 3rd and 5th day,the Moxa group higher than the model group and the normal group between the 3rd and 7th day(p＜0.05).Both groups reach its peak on the 5th day,and gradually fall from the 6th day,but still remarkably higher than the model group(p＜0.05).The model group shows a rise on the 4th day,but is obviously lower than the acupuncture group and the Moxa group. Acupuncture seems better than moxibustion,but with no remarkable difference(P＞0.05). The above indicate that both acupuncture and moxibustion can up-regulate the expression of XPD protein.Conclusion:1.Acupuncture and moxibustion definitely relieve myelosuppression post chemotherapy in CTX-induced mice,elevate white blood cells,the effect increases along with the times of treatment;The variation of circumference white blood cells has a close correlation with the ability of acupuncture and moxibustion to elevate expression of marrow cell DNA repair proteins,speed up transformation of cell cycles,promote DNA synthesis and cell proliferation.2.Acupuncture and moxibustion may increase marrow cell DNA volume at the G2 and G1 phases,raise the level of marrow cell proliferation.There is no significant difference between acupuncture and moxibustion.3.Acupuncture and moxibustion can help the marrow cells in G1 phase to get through G1/S testing points into the S phase for DNA synthesis and continue splitting on the cycle. There is no significant difference between acupuncture and moxibustion.4.Acupuncture and moxibustion are able to promote in CTX-induced mice marrow karyocyte repair and replication,thus reduce the influence of chemo drugs on S phase DNA synthesis,and elevate marrow karyocyte proliferative level.Although the moxa group is 1 day earlier in its action than the acupuncture group,but no significant difference was available when compared as a whole.5.CTX causes the majority of marrow karyocyte to retain in the G2 phase,acupuncture and moxibustion can take this opportunity to quickly repair those injured cell DNA,get marrow karyocyte DNA repaired at the G2/M phase and help them get through testing points in the G2/M phase and enter the M phase for splitting,have the function of promoting marrow cell repair and multiplication,thus reducing the influence of CTX on DNA synthesis at the S phase.Moxibustion seems more effective than acupuncture,but without any significant difference.6.Acupuncture and moxibustion raise the expression of Cyclin D1 in CTX-induced mice, promote cell cycle transformation,especially the G1/S transformation,raise the level of cell multiplication,thus relieving myelosuppression caused by chemotherapy,increase the marrow cell quantity.After the 5th day each group witnessed a gradual fall in Cyclin D1 expression,which conforms to pattern that "cell cycle protein is synthesized at each interphase,but degraded at the stage of mitosis,showing periodic fluctuation"The acupuncture group is 1 day earlier than the moxa group in terms of the time used for the elevation,and acupuncture proves more effective than moxibutstion with significant difference on 5th,6thand 7thday.7.Acupuncture and moxibustion can raise the expression of MGMT in CTX-induced mice marrow karyocyte,help to bring about direct and quick repair of marrow cell DNA injured by CTX,reduce myelosuppression,increase the number of white blood cells.The escalating trend of MGMT protein expression in the acupuncture group is consistent with the variation of white blood cells as well as with the change of cell percentage at various cell cycles.There is no significant difference between acupuncture and moxibustion.8.Acupuncture and moxibustion can markedly raise the polβprotein expression in CTX-induced mice,quickly complete base excision repair of the injured marrow cell DNA,combat CTX-induced cell injury,reduce myelosuppression,and increase the number of white blood cells.The change of polβprotein expression in the acupuncture and moxa groups is consistent with the variation of white blood cells as well as with the change of cell percentage at various cell cycles.Acupuncture seems better than moxibustion yet with no significant difference.9.Acupuncture and moxibustion can markedly,raise the XPD protein expression in CTX-induced mice marrow karyocyte and maintain such trend for a long time,which indicates that acupuncture may carry out nucleotide excision repair of the injured DNA supplementary chain,combat cytotoxicity caused by CTX,reduce myelosuppression,and increase the number of white blood cells.The change of XPD protein expression in the acupuncture and moxa groups is consistent with the variation of white blood cells as well as with the change of cell percentage at various cell cycles.The acupuncture group is 1 day earlier in action than the moxa group and with better effect,but no significant difference is available.In conclusion,the findings indicate that CTX causes decrease of the expression of DNA repair proteins MGMT,XPD,and polβas well as the decline of CyclinD1 expression,and leads to blocked cell cycle,a fall of DNA,and decrease of the peripheral leukocyte.Acupuncture and moxibustion can markedly elevate the expression of CTX-induced mice marrow karyocyte DNA repair protein,help with marrow cell DNA repair in the G2 phase and help it go through the G2/M phase testing points and enter the M phase for splitting;the rise of CyclinD1 in turn adjusts transformation from G1 to S phase,accelerating the transformation from G1 phase to the S phase,thus helping DNA synthesis,protecting marrow cells,and ensuring the maturing and renewal of blood cells constantly to be released into the circumference blood and increase the white blood cell counts.This indicates that the ability of acupuncture and moxibustion to accelerate the cell cycles in CTX-induced mice,promote marrow cell DNA repair,and protect hematopoietic cell from injury by chemo drugs is one of the essential mechanisms of acupuncture and moxibustion in relieving myelosuppression post chemotherapy,and protecting hematopoietic function,and increasing white blood cell counts as well.
|
PDF Full Text Request