A Long-time Study Of Side-effect, DNA Damage And Mechanism Of Cell Cycle In Combination Of Methotrexate With Cyclophosphamide On Rat

【Background and objectives】Rheumatoid arthritis(RA)is a highly disability disease.Now,disease modifying antirheumatic drugs(DMARDS)combination is still the main therapy.Traditional DMARDS combination include methotrexate(MTX)+leflunomide(LEF).Both of them belong to cell cycle specific agent(CCSA)and play a role on S stage of cell cycle.The side effect of them resemble each other,and the effect of combination of them is limited.The effect of Some combinations is more effective,such as cyclophosphamide(CTX)+ azathioprine(AZA)+ hydroxychloroquine(HCQ),but the side effect was so severe that most patients could not tolorate because CTX was given every day.RA patients need long-time treatment,so the therapeutic effect of new therapeutic alliance regiment is not only considered,but also low side effect and enhance patients dependence should be thought highly of,when exploring.There are many tumor features on RA patients,such as,loss of control on the cell cycle of synoviocyte and lymphocyte,abnormal apoptosis etc.For the past years,haemopoietic stem cell transplant and anti-CD20 monoclonal antibody were applied to treat refractory RA patients.During the clinical practice of RA therapy,according to tumor combination chemotherapy theory-cell cycle theory,low dosage methotrexate(MTX)combined with low dosage cyclophosphamide(CTX)were given RA patients.The remission rate improved 80%.Side effect is no obviously increased.Analyzing the cause of our good therapeutic effect and low side effect,firstly,according to the theory of cell cycle,we combined CCSA with CCNSA(cell cycle non-specific agent)to treat RA patients.Secondly,CTX was given intermittently in low dosage.MTX prevent G0/G1 stage cell changing to S stage cell.CTX is a difunctionality aldyl agent,killing every stage cell of cell cycle.We presume that MTX make most of cell be blocked in G0/G1 stage,then CTX will kill more G0/G1 stage cell by given every three week in low dosage.Side-effect will reduce.Retrieving the international and domestic literature,there was no theory of RA therapy according to Cell dynamic principle.Based on former clinical practice,we design this study.Because RA need long-dine therapia, survival time of CIA model is hard to be wanrranted.In ordered to prob the long-time study of side effect and pathology of cell cycle,DNA damage about methotrexate combined with cyclophosphamide on rat,we design empirical study on normal rat for 27 weeks.The objectives of our study is the following.Firstly,to investigate side effects of combination project through detecting blood routine,liver function,renal function and related organ pathology after given MTX and CTX.Secondly,to further investigate the side effect of combination project through detecting the DNA damage of liver and renal by Comet assay single cell gel electrophoresis(SCGE),simultaneously,to investigate the mechanism of unification of MTX and CTX through detecting the DNA damage of PBL,spleen,and bone marrow by SCGE.Thirdly,to explore the synergistic effect of MTX and CTX by detecting the cell cycle and cyclin D1 of PBL,spleen,and bone marrow cell and to provide scientific basis for the clinic medication.【Methods】Wistar rat was randomly divided to six groups to explore the routine side effects and DNA damage,including normal control,combination,low-dosage MTX,low-dosage CTX, high-dosage MTX,high-dosage CTX.Selecting the former four groups to explore the synergistic effect of MTX and CTX.MTX was given by intragastrically.Rats of High-dosage CTX group was given CTX by intragastrically.Low-dosage CTX group was given CTX by intraperitoneal injection.Respectively before and after using medcine 3 week,9 week,18 week,27 week, detecting blood routine,liver function,renal function and Using SCGE method to measure the DNA damage of PBL.At the same time,Using the flow cytometry with PI staining to detect the changes of cell cycle and cyclin D1 in the PBL.Dissecting 6 Wistar rats after using medcine 3 week,9 week,18 week,27 week,respectively,every group to detect pathology of liver,renal and spleen and to measure the DNA damage of liver,renal,PBL,spleen and bone marrow cell by SCGE At the same time,Using the flow cytometry with PI staining to detect the changes of cell cycle and cyclin D1 of PBL,spleen and bone marrow cell and using immunohistochemical stain to detect the changes of cyclin D1 of spleen.【Results】1.The influence of MTX and CTX on liver,renal and blood routine(1)The influence of general state①There are no statistic difference on weight before given medicine.The weight of high-dosage CTX group is lower than normal control after 3 weeks.There are no statistic difference on weight of other groups.②There are no obviouse difference on the quantity of unexpected death rats among low-dosage MTX,low-dosage CTX group and combination group.The quantity of unexpected death rats of CTX given every other day is higher than other groups,and rats of CTX given every other day unexpectedly died in 18 weeks. (2)The influence of MTX and CTX on liver function,renal function and blood routine①The count of white blood cell(WBC)and lymphocyte(LC)of normal control are higher than other groups.The count of WBC and LC of high-dosage CTX are higher than other groups. There are no statistic difference among other groups.There are no statistic difference among different dissect point.There are no statistic difference on red blood corpuscle platelet and among all the groups.②There are no statistic difference on ALT,AST,ALP,GGT and BUN,Cr among all the groups and dissect point.③MTX and CTX are synergistic effect on the count of PBL.MTX or CTX make the count of PBL decreased.MTX combinded with CTX make the count of PBL decreased more severe than MTX or CTX alone.(3)the influence of MTX and CTX on pathology of liver and renal①The cellularedema and degeneration of liver on high-dosage CTX group are more severe than other groups.There are no statistic difference among other groups.There are obvious difference on the angiectasis and congestion of liver,the severe degree is high-dosage CTX＞combination≈low-dosage CTX＞high-dosage MTX≈normal control≈low-dosage MTX in turn. There are no statistic difference on liver lymphocyte infiltrating among all the groups and dissect point.There are no statistic difference on the liver cellularedema,degeneration and angiectasis and congestion among dissect point.②The cellularedema and degeneration on renal tubular cell of high-dosage CTX group are nore severe than normal control and low-dosage MTX group.There are no statistic difference on the renal angiectasis,congestion,protein cast and lymphocyte infiltrating among all the groups and dissect point.③There are no statistic difference on the cellularedema and degeneration of liver between unexpected death and high-dosage CTX group.The cellularedema and degeneration of liver on rats of unexpected death are more severe than other groups.The angiectasis and congestion of liver on rats of unexpected death are more severe than other groups.There are no statistic difference on the pathology of renal between unexpected death and high-dosage CTX group.The renal pathology on rats of unexpected death are more severe than other groups.(4)The relationship of function and pathology①The cellularedema and degeneration of liver correlated with ALT and AST.The angiectasis and congestion of liver is not correlated with ALT and AST.There are relationship between cellularedema and degeneration of liver.ALT correlates with AST。②The pathology index of renal is not correlated with BUN and Cr.There are relationship between cellularedema and degeneration of renal.BUN correlates with Cr。The lymphocyte infiltrating is correlated with cellularedema,degeneration and angiectasis,congestion of renal. The protein cast is not correlated with cellularedema,degeneration and angiectasis,congestion of renal.2.The DNA damage of liver,renal,PBL,spleen and bone marrow cell by SCGE(1)The DNA damage of liver and renal①The comet tail length of liver cell on high-dosage CTX is longer than other groups. There are no statistic difference among these groups.There are obvious difference on the comet frequencies of liver cell,the severe degree is high-dosage CTX≈combination low-dosage CTX＞low-dosage MTX≈high-dosage MTX≈normal control in turn.There are no statistic difference on the comet tail length of liver cell among dissect point.The comet frequencies of liver cell are higher in given medicine 18weeks and 27weeks than that in 3weeks.②The comet tail length of renal cell on high-dosage CTX is longer than other groups. There are no statistic differences among these groups.There are obvious difference on the comet frequencies of renal cell,the severe degree is high-dosage CTX≈low-dosage CTX≈combination＞low-dosage MTX≈normal control in turn.There are no statistic difference between high-dosage MTX and combination groups.There are no obvious difference on the DNA damage of renal cell among high-dosage MTX,low-dosage MTX and normal control groups.There are no statistic difference on the DNA damage of renal cell among dissect point.(2)The DNA damage of PBL and spleen,bone marrow cell①There are obvious difference on the comet tail length of PBL,the severe degree is high-dosage CTX＞combination＞low-dosage CTX＞high-dosage MTX≈normal control≈low-dosage MTX in turn.There are statistic difference on the comet frequencies of PBL,the severe degree is high-dosage CTX＞combination≈low-dosage CTX＞high-dosage MTX≈normal control≈low-dosage MTX groups in turn.There are no statistic difference on the DNA damage of PBL among dissect point.②There are obvious difference on the comet tail length of spleen and bone marrow cell, the severe degree is high-dosage CTX＞combination≈low-dosage CTX＞high-dosage MTX＞normal control≈low-dosage MTX in turn.There are statistic difference on the comet frequencies of spleen and bone marrow cell,the severe degree is high-dosage CTX＞combination≈low-dosage CTX＞high-dosage MTX≈low-dosage MTX≈normal control groups in turn.There are no statistic difference on DNA damage of spleen and bone marrow cell among dissect point.(3)The relationship of DNA damage among different organ cell and the relationship of DNA damage of PBL and the count of PBLThe comet tail length correlates with the comet frequencies.The DNA damage of PBL correlates with liver,renal,spleen and bone marrow cell.There are inverse correlation between DNA damage of PBL and the count of PBL.The more severe of DNA damage of PBL,the fewer is the count of PBL.There are obvious correlation between DNA damage of PBL and cellularedema,degeneration and angiectasis,congestion of liver.There are obvious correlation between DNA damage of PBL and cellularedema,degeneration of renal.We presume that using SCGE to detect DNA damage of PBL is a sensitive index to monitor side effect of MTX and CTX.(4)Comparing the DNA damage of different organ cell.The DNA damage of PBL,spleen,bone marrow cell caused by MTX and CTX is more severe than that of liver and renal.3.The influence of MTX and CTX on cell cycle and cyclin D1of PBL,spleen and bone marrow cell(1)The changes of cell cycle of PBL,spleen and bone marrow cell①MTX cause cell percentage of S stage on PBL decreased obviously and cell percentage of G0/G1 stage increased slightly.CTX cause cell percentage slightly changed.There are no obvious differences on cell percentage of cell cycle among low-dosage CTX,combination and normal control groups.②The cell percentage of G0/G1 stage of combination is lower than normal control and the cell percentage of S stage of combination is higher than normal control.There are no obvious differences on cell percentage of spleen and bone marrow cell cycle among low-dosage CTX, combination and normal control groups.There are no obvious differences on cell percentage of G2/M stage on spleen and bone marrow cell among all groups.There are no obvious difference on cell percentage of spleen and bone marrow cell cycle among dissect point.③By the analysis of variance of factorial design,the interaction of MTX and CTX to cell percentage of G0/G1 stage and cell percentage of S stage appears antagonistic effect.MTX or CTX given alone cause cell percentage of G0/G1 stage increased and cell percentage of S stage decreased.When the two drugs are combined to use,cell percentage of G0/G1 stage decrease and cell percentage of S stage increased.We presume that most cell is blocked on G0/G1 stage by using MTX weekly,then CTX can kill more G0/G1 stage cell efficiently.There are no interaction to cell percentage of G2/M stage on spleen and bone marrow cell between MTX and CTX.(2)The changes of cyclin D1 of PBL,spleen and bone marrow cell①There are no statistic difference on cyclin D1 of PBL,spleen and bone marrow cell among all the groups and dissect point.There are no obvious correlation between the cyclin D1 results of FCM and immunohistochemical stain. ②There are no interaction effect to cyclin D1 on PBL,spleen and bone marrow cell between MTX and CTX.③The interaction effect on cell cycle caused by MTX and CTX is not correlated with the change of cyclin D1 of PBL,spleen and bone marrow cell.(3)The relationship between the cell percentage of G0/G1 stage and cyclin D1The cell percentage of G0/G1 stage are correlated among PBL,spleen and bone marrow cell.There are obvious correlation on cyclinD1 among PBL,spleen and bone marrow cell.There are inverse correlation between the cell percentage of G0/G1 stage and cyclinD1.The DNA damage of PBL correlates with liver,renal,spleen and bone marrow cell.The more severe of DNA damage of PBL,the fewer is the count of PBL.There are obvious correlation between DNA damage of PBL and cellularedema,degeneration and angiectasis,congestion of liver.There are obvious correlation between DNA damage of PBL and cellularedema, degeneration of renal.We presume that using SCGE to detect DNA damage of PBL is a sensitive index to monitor side effect of MTX and CTX.The count of PBL are positively correlated with the cell percentage of G0/G1 stage on spleen,and negatively correlated with the cell percentage of S stage on spleen.The comet frequencies of bone marrow cell are negatively correlated with the cell percentage of G0/G1 stage on spleen,and positively correlated with the cell percentage of S stage on spleen.【Conclusion】1.When MTX and CTX were used unitedly,the side effect is no more than they were used alone.The side effect of CTX given every other day is so higher that most rats can not tolerate.2.There are correlation between DNA damage of PBL and pathology of liver,renal,so we presume that using SCGE to detect DNA damage of PBL is a sensitive index to monitor side effect of MTX and CTX.3.The DNA damage of PBL,spleen and bone marrow cell is more slight caused by MTX than caused by CTX.The more severe of the DNA damage of PBL,the fewer is the count of PBL.Indicating that CTX inhibit cell by causing DNA damage of cell.4.MTX is the specific depressant of cell cycle and it can make the cell cycle of PBL, spleen and bone marrow cell change and a few occurs DNA damage.But CTX as the CCNSA, the inhibition to the cell manifest causing DNA damage of cell,then cell death.5.MTX or CTX given alone cause cell percentage of G0/G1 stage increased and cell percentage of S stage decreased.When the two drugs are combined to use,cell percentage of G0/G1 stage decrease and cell percentage of S stage increased.We presume that most cell is blocked on G0/G1 stage by using MTX weekly,then CTX can kill more G0/G1 stage cell efficiently.When the two drugs were combined to use,the synergistic effect is showed because they could act in different period and various site and target.6.The interaction effect on cell cycle caused by MTX and CTX is not correlated with the change of cyclin D1 of PBL,spleen and bone marrow cell.