Trichostatin A Affect T-Cell Reaction In Mice And Suppress Rejection Response To Renal Transplantation In Rat

ObjectivesWith the development of organ transplantation technology,the organ transplantation recipients have been gradually increased in China for decades. However,the recipients need to be administered some kinds of immuno-suppressants to suppress rejection after transplantation for a long time even for a life-time.These immunosuppressants present various benefits for recipients through different pharmacological mechanisms and pathways,but unavoidably,they have lots of untoward effects in the clinical.The therapeutic windows of some immunosuppressants,especially,are narrow and the individual differences in pharmacokinetics are remarkable.Therefore,the therapeutic drug monitor(TDM)is necessary for the transplantation recipients,which impose a heavily financial burden on the recipients.Thus,it is urgent to research and develop new immunosuppressants with high efficacy and potency and no untoward effects.Histone deacetylase inhibitors(HDACIs)play a pivotal role in regulating gene expression,DNA duplication and reparation,participating a variety of gene transcription through maintaining the equilibrium between histone acetylation and histone deacetylation.The present paper is aimed at investigating the pharmacological mechanisms of Trichostatin A,a HDACI,on T-cell reaction in mice and on renal rejection in rat.It has been know that T-cell reaction is the central link in immune response of organ transplantation.Consequently,it is sure that the results in this paper would provide some usefully experimental and theoretical data for immunotherapy and immune tolerance induction of organ transplantation.Methods1.The mice were administered with different concentrations of TSA and the amounts of T cells in peripheral blood and spleen were determinedThe mice were randomly allocated to 5 groups(8 mice in each group):group A was control treated with normal saline(0.5 ml/kg·d);Group B was treated with CTX(20 mg/kg·d);Group C,D,E was administered TSA with TSA 0.4mg/kg·d, 0.8 mg/kg·d and 1.6 mg/kg·d,respectively.All reagents were injected subcutaneously for consecutive 7 days.The samples of blood and spleen of mice were drawn after 1 week treatment. The spleen tissues were made to be cell suspension right away.And then the lymphocytes in blood and spleen suspension were isolated.The numbers of T cells were counting by Acidα-naphthyl acetate esterase staining.2.The subgroup of T cells and ratio of CD4⁺ and CD8⁺ T cells in peripheral blood and spleen suspension of mice were detected after treating by TSAThe samples of blood and spleen suspension of mice were obtained.The lymphocyte of the blood and spleen cell suspension were isolated by applying the monoclonal antibody labeled by fluorescence(Rat anti-mouse Lyt2⁺ and Rat anti-mouse L3T4⁺),respectively.The cytometry was employed to observe the counting CD4⁺ and CD8⁺ T cells and their relative ratio.3.The effect of TSA on T cells proliferation was observed in mixed lymphocyte culture(MLC) of miceThe spleen of BALB/c and C₅₇BL mice were drawn and made to be single cell suspension.The lymphocytes were isolated by lymphocytes separating solution and adjusted the number to be 1×10⁶/ml.The lymphocyte were mixed by 1:1 and seeded to 96-well microplate with 100μL cell suspension per well.And then,100μL TSA with different concentrations of 0.16,0.8,4,20,100,500nmol/L was added into wells with RPMI-1640 for control well.After co-incubated 2 h,24 h,48 h.the co-incubating system was added 0.5%concentration MTT 20μL into each well for 4 h. After centrifuged for 10min,1500 rpm,the supernatant was abandoned,100μL DMSO was added.The micro-plate reader was used to detect the optical density at 570nm.4.The effect of TSA on IL-2 expression was determined in activated lymphocyteThe C₅₇BL mice spleen lymphocyte cell suspension was prepared and adjusted the cell numbers to be 1×10⁶/ml used as reactive cells,meantime,BALB/c mice spleen lymphocyte cell suspension was prepared,added 75umol/L mitomycin, incubated 30min and residual mitomycin was removed,then adjusted the cell numbers by 1×10⁶/ml used as stimulating cells later.The cell suspension was divided into 3 groups,including treatment group, positive control group and negative control group.In the treatment group,1ml reactive cell was taken to mix with 1ml stimulating cell and seed into 96-well microplate with 6 well duplicated.1μmol monensin was added to each well and incubated at 37℃,5%CO₂ for 48 h.50μL TSA was added by the final concentration of 0.16,0.8,4,20,100,500nmol/L,respectively.In the positive control group,TSA was instead of CTX.And in the negative control group, TSA was replaced by RPMI-1640 culture media.After incubated for 4 d,the wells were added IL-2 monoclonal antibody labelled by fluorescence and the fluorescence values were measured. 5.The renal orthotopic transplantation model was established in ratWistar rats,male,adult and inbred,were used in this study.The rats were anesthetized by intraperitoneal injection of 2%pentobarbital sodium.A T shape middle abdominal operative incision was made.Left kidney,abdominal aorta and inferior caval vein were revealed thoroughly.Left kidney and the vessel were isolated totally,ligated and then disconnected all branches of renal artery and renal veins, respectively,blocked abdominal aorta and inferior cava vein above the left renal vessels,punctured the abdominal aorta under the left renal artery and retained the cannulation,then cut the inferior cava vein under the left renal vein.The perfusion of kidney was carried out with 4℃hypertonic citrate adenine solution through the punctured cannulation until the kidney turned to be grey and perfusion fluid from the inferior cava vein was transparent.The ureter was separated till bladder with a piece of urinary bladder wall.The left renal vessel was disconnected with a little section of abdominal aorta and inferior cava vein.The whole donor kidney was taken out from the body and put into the ice normal saline.Kidney adipose capsule and the fascia of the vessels were removed,renal artery and renal vein was disconnected from the root of abdominal aorta and inferior cava vein separately,chipping the bladder wall and keep the section around the ureteric orifice.The kidney was put into the ice bag to be reserved for use later.The Wistar rats,male and inbred were used as recipients.The same surgical procedure was carried out.The left renal artery and vein were dissociated enough at length and blocked at the site near to abdominal aorta and inferior cava vein,then was cut off at one-third of the length to the root.The left ureter was ligated and disconnected and the left kidney was removed.The vein was sutured by 9-0 nylon stitch and the artery was sutured by 11-0.The donor bladder section was sutured with the recipient bladder after liberating the blood,. 6.To observe the histopathology change of the grafting renalNine rats orthotopic renal transplantation were successfully made and randomly divided into 3 groups,including control group treated by subcutaneous injection of normal saline(10 ml/kg·d);CTX group treated with CTX 20 mg/kg·d and TSA group treated by TSA of 1mg/kg·d once a day.After 3 d,the rats were killed and the renal graft were taken,fixed by 10%formaldehyde solution,then sliced and stained by HE.The histopathology change of the renal graft was observed.Results1.Effect of TSA on T-cell count,CD4⁺,CD8⁺ subgroup in peripheral blood in miceCompared to control,TSA significantly decreased T-cell counts in peripheral blood at the dosage of 0.4,0.8,1.6 mg/kg/day,respectively.At the same time,TSA 0.4～1.6 mg/kg/day also significantly decreased the counts and percentage of CD4⁺ T-cell subgroup.However,the counts and percentage of CD8⁺ T-cell subgroup were markedly different compared with control group only at the concentration of 0.8～1.6 mg/kg/day.Meantime,TSA significantly decreased the ratio of CD4⁺/CD8⁺ T-cell subgroup at more than 0.8mg/kg/day.The results suggested that TSA affected more effectively on CD4⁺ than on CD8⁺.2.Effect of TSA on T-cell count,CD4⁺,CD8⁺ subgroup in spleen in miceCompared to control,TSA significantly decreased the counts of T cells in spleen at the dosage of 0.8～1.6 mg/kg/day.On the other hand,TSA also significantly suppressed counts,percentage and ratio of CD4⁺,CD8⁺ T-cell subgroup in spleen in mice at the same dose.The results showed that TSA was obviously less effective to spleen than to peripheral blood compared with control.3.Inhibitory effect of TSA on lymphocytic proliferationTSA obviously inhibited lymphocytic proliferation time-dependently and concentration-dependently in MLC.The inhibition ratio reaches by 59.9%at the concentration of TSA 500nmol/L for 48 h.Compared with control,TSA 0.16nmol/L has no significant difference for 12 h.All other groups have statistically significant difference compared to control at different time points.4.Inhibitory effect of TSA on IL-2 expression of activated lymphocyte in one-way MLCTSA remarkably inhibit IL-2 expression of activated lymphocyte of mice in one-way MLC with the pattern of concentration-dependence.Compared with control group,TSA significantly suppressed IL-2 expression at the concentration of more than 0.8nmol/L.5.The establishment of orthotopic renal transplantation model in ratsThe orthotopic renal transplantation model was successfully established.One rat died after surgery because of anastomotic stoma hemorrhage,1 rat died on the first day post-operation because of thrombosis,1 rat was urine leakage but alive.The average duration of operation took about 150 min,which the transplantation operation spent about 100 min whereas the operation for gaining donor kidney need about 45 min.About 30 sec was necessary for warm ischemia and 90 min or so was for cold ischemia.The successful cases could be living over 3 ds.6.Inhibitory effect of TSA on acute rejectionSevere acute rejection characteristics,including interstitial edema,lymphocyte infiltrating,glomerulus hyperaemia,intumescentia of glomerular capillary, periglomerular lymphocyte infiltration glomerular capsule wall,nephric tubule necrosis and apomorphosis,occurred in the control group. In TSA group,no incidence of acute rejection happened,although lymphocyte infiltration around renal arteriole,punctiform lymphocytic infiltration in nephric tubule interstitium and periglomcrulus could be observed,the pathological change was inferior.In the meantime,glomerular blood vessels were gently expanded to less extent. Although interstitial edema,lymphocyte infiltration,lymphocyte infiltration in nephric tubule interstitium also can be observed,they were mild all.The morphology of nephric tubule was intact,which suggested that acute rejection was inhibited.Conclusions:1.TSA significantly decreased the counts of T-cell in peripheral blood and spleen in mice time-dependently and concentration-dependently.2.TSA significantly decreased the counts and ratio of CD4⁺,CD8⁺ T-cell subgroups in both peripheral blood and spleen in mice.Furthermore,TSA was less effectively in spleen than in peripheral blood and more effectively to CD4⁺ than to CD8⁺ T-cell subgroup..3.TSA inhibited lymphocyte proliferation with the pattern of concentration-dependence and time-dependence.4.TSA inhibited IL-2 expression on activated lymphocyte in one-way MLC.5.TSA suppressed rejection in orthotopic renal transplantation model in rats through inhibiting immune response to the graft.