New Wenshen Shengjing Decoction Improves Early Embryonic Development by Maintaining Low Levels of H3K4me3 in Sperm.

BACKGROUND: New Wenshen Shengjing Decoction (NWSSJD), a traditional Chinese compound medicine, has significant effect on spermatogenesis disorder and can significantly improve sperm quality. Many components in NWSSJD can induce epigenetic modifications of different types of cells. It is not yet known whether they can cause epigenetic modifications in sperm or early embryos. OBJECTIVE: This study investigated the effect of NWSSJD on mouse early embryonic development and its regulation of H3K4me3 in mouse sperm and early embryos. METHODS: Spermatogenesis disorder was induced in male mice with CPA (cyclophosphamide). NWSSJD was administrated for 30 days. Then, the male mice were mated with the female mice with superovulation, and the embryo degeneration rate of each stage was calculated. Immunofluorescence staining was used to detect the expression of H3K4me3 in sperm and embryos at various stages. Western blotting was performed to detect methyltransferase SETD1B expression. The expressions of development-related genes (OCT-4, NANOG, and CDX2) and apoptosis-related genes (BCL-2 and p53) were measured with qRT-PCR. RESULTS: Compared with the CPA group, NWSSJD significantly reduced the H3K4me3 level in sperms, significantly increased the number of normal early embryos (2-cell embryos, 3-4-cell embryos, 8-16-cell embryos, and blastocysts) per mouse, and reduced the degeneration rate of the embryos. The expression levels of H3K4me3 and methyltransferase SETD1B in early embryos were significantly elevated by NWSSJD. Additionally, NWSSJD significantly promoted BCL-2 expression, while reducing p53 expression, thus inhibiting embryonic cell apoptosis. Moreover, the expressions of development-related genes OCT-4 and CDX2 were significantly increased by NWSSJD, but NANOG expression had no significant difference. CONCLUSION: NWSSJD may promote early embryonic development possibly by maintaining low H3K4me3 levels in sperms and normal H3K4me3 modification in early embryos and by inhibiting embryonic cell apoptosis.

Protective effects of new Wenshen Shengjing Decoction on cyclosporine-induced impairment of testosterone synthesis and spermatogenic apoptosis.

The aim of the present study was to investigate the potential protective effects of new Wenshen Shengjing Decoction (new WSSJD; including Cornu Cervi Nippon Parvum, Panax ginseng, Cynomorium songaricum, Cistanche deserticola, Radix Astragali, Epimedium brevicornum and Angelica sinensis) on cyclosporine-induced impairment of testosterone synthesis and spermatogenic apoptosis in mice. A total of 90 adult male Kunming mice were divided into the following 6 groups: Control (no intervention), dimethylsulfoxide (DMSO; received only DMSO), cyclosporine A (CsA), clomifene citrate (CC; CsA + CC, 15 mg/kg/day), WSSJD (CsA + WSSJD, crude drug 12 g/kg/day) and new WSSJD (CsA + new WSSJD, crude drug 12 g/kg/day). All mice were treated for 30 days via oral gavage. The testes were subsequently fixed and stained with hematoxylin & eosin to assess the development of seminiferous epithelia. Immunohistochemical techniques were used to detect the expression of luteinizing hormone receptor (LHR) and P450 side chain cleavage (P450scc) in testicular Leydig cells. In addition, the apoptosis of spermatogenic cells in the testes was detected using a terminal dexynucleotidyl transferase-mediated dUTP nick-end labeling assay, and flow cytometry was used to analyze the survival rate and early apoptosis of sperm in the epididymis. Compared with the CsA and CC groups, new WSSJD administration significantly increased levels of serum testosterone and the expressions of LHR and P450scc in testicular Leydig cells (P<0.05), while the apoptosis of spermatogenic cells in the seminiferous tubules and early apoptosis of mature sperm were significantly decreased (P<0.05). These results suggest that new WSSJD may ameliorate CsA-induced spermatogenic damage in male mice by enhancing testosterone synthesis and the secretion of testicular Leydig cells, and by reducing the apoptosis of spermatogenic cells.

[Anti-angiogenesis of interferon-alpha2b in chronic myeloid leukemia in vitro].

This study was aimed to investigate the anti-angiogenesis of IFN-alpha2b in chronic myeloid leukemia (CML) in vitro by using K562 cell line and human umbilical vein endothelial cells (HUVEC). The levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in culture supernatant of K562 cells were determined by ELISA; the expressions of VEGF and bFGF mRNA after treating K562 cells with 10(3), 10(2) and 10 U/ml IFN-alpha2b for 24, 36, 48 hours were detected by real-time RT-PCR; the effects of K562 cell culture supernatant and IFN-alpha2b on proliferation, migration and differentiation of HUVEC in vitro were assayed by MTT, Transwell chamber and tubule formation assay respectively. The results showed that the K562 cells expressed and secreted VEGF and bFGF. The culture supernatant of K562 cells significantly promoted the proliferation, migration and tubule formation of HUVEC in vitro in a concentration-dependent manner. After treating K562 cells with IFN-alpha2b 10 U/ml for 24, 36 and 48 hours, the expression levels of VEGF and bFGF mRNA were 1.64+/-0.18, 1.49+/-0.14, 1.31+/-0.05 and 1.53+/-0.10, 1.29+/-0.15, 0.79+/-0.13 respectively (p=0.002), but the expression levels of VEGF and bFGF mRNA were not significantly different along with increasing of IFN-alpha2b concentration. It is concluded that the angiogenesis exists in CML. The K562 cell expresses and secrets VEGF and bFGF, which promotes the proliferation, migration and differentiation of HUVEC. The IFN-alpha2b displays anti-angiogenesis by inhibiting the proliferation, migration and tubule formation in vitro of HUVEC and down regulating the expression of VEGF and bFGF mRNA.