In previous studies, we have demonstrated that Tokishakuyakusan (TJ-23) can prolong the survival of allogeneic cardiac grafts and induce regulatory T cells. In this study we investigated the effects of Paeoniae radix and Cnidii rhizoma, two components of TJ-23, on alloimmune responses in a murine cardiac transplantation model and whether the two agents have synergistic effect. CBA mice underwent transplantation of a C57BL/6 heart and received oral administration of 2 g/kg/day of Paeoniae radix, Cnidii rhizoma, or the mixture of two agents from the day of transplantation until 7 days afterward. Naïve CBA mice rejected C57BL/6 cardiac graft acutely (median survival time (MST): 7 days). Paeoniae radix and Cnidii rhizoma prolonged C57BL/6 allograft survival (MSTs: 13.5 and 15.5 days, resp.). However, the mixture of two agents prolonged C57BL/6 allograft survival indefinitely (MST > 100 days). Secondary CBA recipients given whole splenocytes from primary combination-treated CBA recipients with B6 cardiac allografts 30 days after grafting had prolonged survival of B6 hearts (MST: 33 days). Flow cytometry studies showed that the CD4+CD25+Foxp3+ regulatory cell population was increased in combination-treated recipients. Combination of Paeoniae radix and Cnidii rhizoma induced hyporesponsiveness to fully allogeneic cardiac allografts and may generate CD4+CD25+Foxp3+ regulatory cells in our model.
Since Japanese government health officials officially recognized the therapeutic effects of Japanese herbal (Kampo) medicines about 30 years ago, these medicines have been widely used as alternative therapy for several diseases. In recent studies of our murine model, oral administration of the Japanese herbal medicines Tokishakuyakusan (TJ-23) [
Male C57BL/6 (B6,
All transplant procedures were performed on the mice under general anesthesia. Fully vascularized abdominal heterotopic hearts from B6 donors were transplanted into CBA mice by microsurgical techniques [
Naïve CBA recipients of a B6 heart were given no treatment, distilled water (control group), or oral administration of 2 g/kg/day of each component of TJ-23 (Poria sclerotium, Cnidii rhizoma, Paeoniae radix, Atractylodis lanceae rhizoma, Alismatis rhizoma, and Angelicae radix) or mixture of two components (Poria sclerotium, Cnidii rhizoma, and Paeoniae radix) from the day of transplantation to 7 days afterward. Other CBA recipients were also given mixture of 0.2 g/kg/day of Paeoniae radix and Cnidii rhizoma or mixture of 0.02 g/kg/day of Paeoniae radix and Cnidii rhizoma. All the agents were dissolved in distilled water and given orally by a metal tube (Thomas Scientific, Swedesboro, NJ). The agents were made by boiling water extraction, separating the effluents from the residuals, and spray-drying the effluents to produce the extract powder [
Adoptive transfer studies were conducted to determine whether regulatory cells were generated after treatment with mixture of Paeoniae radix and Cnidii rhizoma. Thus, 30 days after CBA recipients (primary recipients) underwent transplantation of a B6 cardiac allograft and were given mixture of Paeoniae radix and Cnidii rhizoma, splenocytes (5.0 × 107) from primary recipients with functioning allografts were adoptively transferred into naive CBA mice (secondary recipients). After the adoptive transfer, the secondary recipients underwent transplantation of a B6 heart immediately.
Cardiac allografts in untreated mice and mice given mixture of Paeoniae radix and Cnidii rhizoma were removed 14 days after transplantation and studied histologically and immunohistochemically. Histologic and immunohistochemical staining were performed as described previously [
The expression of CD4, CD25, and Foxp3 in splenocytes was determined by flow cytometry. After cardiac allograft transplantation for 14 days, splenocytes from CBA recipients given mixture of Paeoniae radix and Cnidii rhizoma, untreated recipients, and naïve CBA with/without mixture of Paeoniae radix and Cnidii rhizoma were stained with fluorochrome-conjugated anti-CD4 or anti-CD25 monoclonal antibody (mAb) (RM4-5 and PC61, resp.; BD Biosciences, San Jose, CA, USA) and anti-mouse Foxp3 mAb (FJK-16s; eBioscience, San Diego, CA), as well as their isotype controls (eBioscience). The stained cells were analyzed by using a FACS Canto2 system (BD Biosciences). The percentage of CD4+ CD25+ Foxp3+ in CD4+ cells was determined.
In mixed leukocyte culture (MLC) studies [
In some experiments, the MLC contained splenocytes from naïve CBA (responder cells; 2.5 × 106/mL) and MMC-treated splenocytes from B6 (stimulator cells; 5 × 106/mL). Two amounts of the mixture (0.5 and 5 mg/mL) were added to the MLC to assess the direct effects of these agents on cellular proliferation (direct MLC).
An ELISA was also performed to assess levels of IL-2, IL-4, IL-10, and interferon (IFN)-
Cardiac allograft survival in groups of mice was compared by using Mann-Whitney
CBA recipients without any treatment or given distilled water rejected B6 grafts acutely (median survival times (MSTs): 7 and 8 days, resp.; Figure
Graft survival of CBA mice given oral administration of agents and histologic studies. (a) Recipients with C57BL/6 hearts were either untreated, given distilled water, or given 2 g/kg/day of TJ-23 from the day of transplantation until 7 days afterward. MST: median survival time; #
When CBA recipients were given mixture of 0.2 g/kg of Paeoniae radix and Cnidii rhizoma or mixture of 0.02 g/kg/day of Paeoniae radix and Cnidii rhizoma, neither of them could prolong the survival of B6 cardiac allograft (Figure
Histologic examinations of cardiac allografts obtained 14 days after transplantation showed cell infiltrated but significantly preserved structure with a few myocardial injuries in transplant recipients given mixture of Paeoniae radix and Cnidii rhizoma, whereas allografts from untreated recipients showed severe myocyte damage and edema of the acute rejection process (Figure
In the adoptive transfer study, secondary CBA recipients given whole splenocytes from primary mixture of Paeoniae radix and Cnidii rhizoma-treated CBA recipients with B6 cardiac allografts 30 days after grafting had significantly prolonged survival of B6 hearts compared to secondary recipients which were adoptively transferred from naïve CBA splenocytes (MSTs: 33 days and 12 days, resp.;
Evidence of generation of regulatory cells in CBA allograft recipients treated with combination of Paeoniae radix and Cnidii rhizoma. (a) Cardiac allograft survival after adoptive transfer of whole splenocytes. MST: median survival time; *
In flow cytometry study, the percentage of population of CD4+CD25+Foxp3+ cells in CD4+ cells was increased in the spleens of recipients given mixture of Paeoniae radix and Cnidii rhizoma compared with those of untreated CBA recipients (
Proliferation of splenocytes from CBA recipients were significantly suppressed in treatment with Paeoniae radix, Cnidii rhizoma, and mixture of Paeoniae radix and Cnidii rhizoma compared with that of splenocytes from untreated mice (each
Evidence of induction of alloproliferative hyporesponsiveness in CBA recipients of allograft treated with combination of Paeoniae radix and Cnidii rhizoma. (a) Results of cell-proliferation assays in mixed leukocyte cultures (MLCs). The data shown are mean ± SD values derived from samples from 6 mice in each group. *
Level of IFN-
Moreover, the addition of mixture of Paeoniae radix and Cnidii rhizoma to an allogeneic MLC inhibited proliferation of CBA responder cells against B6 stimulator cells in a dose-dependent manner (Figure
There are several mechanisms by which treatment with combination of Paeoniae radix and Cnidii rhizoma has induced increased allograft survival in our model. The first possible mechanism of our results is the generation of regulatory cells. Active suppression by regulatory cells has been found to be one of the important mechanisms for induction and maintenance of self-tolerance [
A second possible mechanism for combination of Paeoniae radix and Cnidii rhizoma-induced hyporesponsiveness in our model is that the balance between Th-1 and Th-2 cytokines secretion may have a strong influence on the function of regulatory cells. Our previous studies have demonstrated that inducible regulatory T cells might enable the change of Th-1/Th-2 cytokines [
A third possible mechanism is the immunosuppressive effects by mixture of Paeoniae radix and Cnidii rhizoma. Actually, many reports demonstrated that some herbal medicines such as Kakkonto had the potentials of anti-inflammatory to allergy [
These findings demonstrated that treatment with Paeoniae radix and Cnidii rhizoma had an ability that induced prolonged allograft survival and generation of regulatory CD4+CD25+Foxp3+ cells in our
The authors declare that there is no conflict of interests regarding the publication of this paper.
Xiangyuan Jin and Lei Yu contributed equally to this work.
The authors thank Professor Toshio Fukusato and Mr. Masahito Watanabe, Department of Pathology, Teikyo University School of medicine, Tokyo, Japan, for technical assistance with the histologic studies.