Research On The Mechanism Of ShouTaiWan Preventing Acute Rejection Of Ovarian Allotransplantation In Rats

Objective:To observe the effect and mechanism of ShouTaiWan in treating acute rejection ofovarian allotransplantation in SD rats and Wistar rats. To observe the effect of ShouTaiWanon ovarian histology and morphology, serum endocrine hormone, T lymphocyte subsets,and cytokines interleukin-2(IL-2) and interferon-γ(IFN-γ) of these rats.Methods:238 adult female Wistar rats were randomly divided into seven groups:â‘ lowdose of ShouTaiWan group,â‘¡middle dose of ShouTaiWan group,â‘¢high dose ofShouTaiWan group,â‘£CsA group,⑤CsA+ShouTaiWan group,â‘¥normal group,⑦modelgroup. No surgery was performed on the rats in the normal group. Ovarianallotransplantation surgery was performed on the rats of other groups.After surgery, rats in groupâ‘ were treated with ShouTaiWan decoction at 6.7g/kg/din 28 days. Rats in groupâ‘¡were treated with ShouTaiWan decoction at 13.4g/kg/d in28 days. Rats in groupâ‘¢were treated with ShouTaiWan decoction at 26.8g/kg/d in 28days. Rats in groupâ‘£received a 14-day course of cyclosporine A (CsA) at 10mg/kg/d, andthen another 14-day course of CsA at 6mg/kg/d. Rats in group⑤were treated withequivalent CsA as groupâ‘£,and with equivalent ShouTaiWan decoction as groupâ‘¡.Normal group and model group were treated with 0.9% NaCl at 5ml/kg/d in 28 days.Serum estradiol (E₂), progestogen (P) and follicle stimulating hormone (FSH) ofrecipient rats were measured before Ovarian allotransplantation surgery and in the 5th-14th,15th-28th, 29th-35th days postoperative. The Wistar rats were killed and evaluated at d7,d14, d21, d28 and d35 after transplantation. Number of rat spleen T lymphocyte and itssubsets were determined by flow cytometry. ELISA measured serum IL-2 and IFN-γlevelin recipient rats. The histological and morphological alteration of ovarian grafs was studiedwith microscope. Ovary ultrastructure was dectected by electric microscope. Generalizedcase of rats in every group was observed to analyze the side effect of medicines. Results:1. Histology, morphology and ultrastructure of ovarian graftsRate of rats restoring estrous cycle from high to low was : CsA+ShouTaiWan group,CsA group, high dose of ShouTaiWan group, middle dose of ShouTaiWan group, low doseof ShouTaiWan group, model group.Weight and volume of ovarian grafts in low dose of ShouTaiWan group, middle doseof ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly higher than those of normal group and modelgroup(P＜0.01).Numbers of corpus luteum, primordial follicles, primary follicles and graafian folliclesin low dose of ShouTaiWan group, middle dose of ShouTaiWan group, high dose ofShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly higherthan those of model group(P＜0.01). Numbers of primordial follicles and primary folliclesin CsA+ShouTaiWan group was significantly higher than those of CsA group (P＜0.01).21 days after operation, ultrastructure of ovarian grafts in model group was in accordwith that of acute rejection, ultrastructure of ovarian grafts in low dose of ShouTaiWangroup, middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group was normal, and the structure of newborn capillary vesselwas normal too. 35 days after operation, ovary ultrastructure of CsA+ShouTaiWan groupwas degenerated more slightly than other groups.2. Serum endocrine hormone E₂, P, and FSHThe mean levels of serum E₂ and P in normal group were significantly higher thanthose of other six groups before transplantation (P＜0.01). 5-14 days after transplantation,the mean levels of serum E₂ and P in every group were significantly higher than thosebefore transplantation (P＜0.01).15-28 days after transplantation, The mean levels of serum E₂ and P in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly higher than those of modelgroup(P＜0.01). The levels of serum E₂ and P in low dose of ShouTaiWan group weresignificantly lower than those of high dose of ShouTaiWan group (P＜0.01).29-35 days after transplantation, The mean levels of serum E₂ and P in low dose ofShouTaiWan group, middle dosc of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly higher than those of modelgroup(P＜0.01). The mean levels of serum E₂ and P in CsA+ShouTaiWan group weresignificantly higher than those in low dose of ShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group and model group. Thelevel of serum P in low dose of ShouTaiWan group were significantly lower than those ofhigh dose of ShouTaiWan group (P＜0.05).The mean level of serum FSH in normal group was significantly lower than that ofother six groups before transplantation(P＜0.01). 5-14,15-18,29-35 days after transplantation,the mean levels of serum FSH in in low dose of ShouTaiWan group, middle dose ofShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWangroup were significantly lower than those before transplantation. The change of serum FSHin model group after operation was not significant.3. Rat spleen T lymphocyte and its subsets7 days after transplantation, The mean levels of spleen CD3⁺, CD4⁺ and CD8⁺ innormal group was significantly lower than those of other six groups.14 days after transplantation, The mean level of spleen CD3⁺ in CsA+ShouTaiWangroup was significantly lower than those in the 7th day after transplantation. The meanlevels of spleen CD3⁺ in middle dose of ShouTaiWan group, high dose of ShouTaiWangroup, CsA group, and CsA+ShouTaiWan group were significantly lower than that ofmodel group. The levels of spleen CD4⁺ in high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group were significantly lower than those in the 7th day aftertransplantation. The levels of spleen CD4⁺ in low dose of ShouTaiWan group, middle doseof ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly lower than that of model group. The mean levels ofspleen CD8⁺ in low dose of ShouTaiWan group, middle dose of ShouTaiWan group, andhigh dose of ShouTaiWan group were significantly higher than that of normal group(P＜0.05).21 days after transplantation, the mean levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of modelgroup (P＜0.01). The levels of spleen CD3⁺ in low dose of ShouTaiWan group and middledose of ShouTaiWan group were significantly higher than that of normal group(P＜0.01).The levels of CD4⁺ in low dose of ShouTaiWan group and middle dose of ShouTaiWangroup were significantly higher than those of normal group, high dose of ShouTaiWangroup, CsA group, and CsA+ ShouTaiWan group.28 days after transplantation, the levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of model group (P＜0.01). The levels of spleen CD3⁺ and CD4⁺ in low dose of ShouTaiWan groupwere significantly higher than those in high dose of ShouTaiWan group, CsA group, CsA+ShouTaiWan group and normal group.35 days after transplantation, the levels of spleen CD3⁺ and CD4⁺ in low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those of modelgroup (P＜0.01). The levels of spleen CD3⁺ and CD4⁺ in low dose of ShouTaiWan groupwere significantly higher than those of normal group. The difference of spleen CD8⁺between seven groups was not significant.4. Serum cytokines IL-2 and IFN-γ7 days after transplantation, the level of serum IL-2 in normal group was significantlylower than those of other six groups. 14 days after transplantation, the mean level of serumIL-2 in middle dose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group,and CsA+ShouTaiWan group were significantly lower than that of model group. 21 daysafter transplantation, the levels of serum IL-2 in low dose of ShouTaiWan group, middledose of ShouTaiWan group, high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group were significantly lower than that of model group. 28 days aftertransplantation, the level of serum IL-2 in CsA+ShouTaiWan group were significantlylower than that of model group (P＜0.05). 35 days after transplantation, the difference ofserum IL-2 between seven groups was not significant.7 days after transplantation, the level of serum IFN-γin normal group wassignificantly lower than those of other six groups. 14 days after transplantation, the level ofserum IFN-γin model group was significantly higher than that of normal group(P＜0.01).21 days after transplantation, The levels of serum IFN-γin CsA group and CsA+ShouTaiWan group were significantly lower than those in the 7th day after transplantation(P＜0.05). 28 days after transplantation, The levels of serum IFN-γin low dose ofShouTaiWan group, middle dose of ShouTaiWan group, high dose of ShouTaiWan group,CsA group, and CsA+ShouTaiWan group were significantly lower than those in the 7thday after transplantation. The levels of serum IFN-γin middle dose of ShouTaiWan group,high dose of ShouTaiWan group, CsA group, and CsA+ShouTaiWan group weresignificantly lower than that of model group. 35 days after transplantation, the levels ofserum IFN-γin CsA+ShouTaiWan group were significantly lower than that of modelgroup(P＜0.05).Conclusion:1. ShouTaiWan helps the recovery of rat estrous cycle, and has obvious synergy combined with CsA. ShouTaiWan raise the survival rate, weight and size of allograftingovaries. High-dose group is superior to middle-dose group and low-dose group.ShouTaiWan can reduce the ovarian interstitial damage because of acute rejection, and hasobvious synergy combined with CsA. ShouTaiWan promote the development of ovarianluteal and various stages of follicle, especially promoting the growth of primordial folliclesand primary follicle. And there is a dose-response relationship. ShouTaiWan, combinedwith CsA, can reduce abnormal changes in the ultrastructure of ovarian grafts.2. ShouTaiWan can restore the endocrine function of ovarian grafts, reduce the loss oftheir endocrine function, and has obvious synergy combined with CsA. There was a certainamount of high-dose-effect relationship. ShouTaiWan can promote the establishment ofnegative feedback response between ovarian grafts and pituitary gland.3. ShouTaiWan inhibits the proliferation of T lymphocytes in allotransplantationmodel, particularly inhibiting the proliferation of auxiliary T lymphocytes, high-dose groupis superior to middle-dose group and low-dose group, showing a dose-responserelationship.4. ShouTaiWan can inhibit secretion of Th1-type cytokines in rats after ovariantransplantation, decrease the levels of serum IL-2 and IFN-γ, and has obvious synergycombined with CsA.ShouTaiWan maybe play the role of preventing acute rejection of allotransplantationin rats through all these mechanisms.