Jinyao Li

Enhanced contraceptive response by co‐immunization of DNA and protein vaccines encoding the mouse zona pellucida 3 with minimal oophoritis in mouse ovary

Zona pellucida 3 (ZP3) acts as the primary sperm receptor, induces autoantibody that can prevent oocyte fertilization and has been proposed as a vaccine candidate for contraception in humans. Due to the elicited autoreactive T cell inflammation that causes ovarian destruction, ZP3‐based vaccine with removed T epitopes from the ZP3 is considered as a preferred approach. We present here a new strategy to eliminate the T cell inflammation while retaining a high level of antibody by co‐immunization of mZP3 DNA and protein vaccines, which resulted in a higher reduction rate of fertility in this group. Histological analysis showed that there were normal follicular developments of infertile mice in the co‐immunized group; while other vaccine groups of the most infertile mice lacked mature follicles. There was a significant correlation between normal follicular development and the inhibition of T cell response in co‐immunized mice. At the same time, co‐immunization reduced the production of inflammatory cytokine, IFN‐γ, and increased the productions of IL‐10 and FoxP3 in CD4 T cells, suggesting the anti‐inflammation may be via a T regulatory function. The results indicate that co‐immunization of mZP3 DNA‐ and protein‐based vaccines can reduce fertility without interfering with the normal follicular development and present a novel strategy to develop a contraceptive vaccine in humans. Copyright

Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10 Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway.

Cistanche tubulosa phenylethanoid glycosides (CTPG) have been shown various biological activities including anti-allergy, hepatoprotective activity and bone regeneration. However, the anti-tumor activity of CTPG needs to be investigated. CTPG was used to treat B16-F10 cells both in vitro and in vivo. We found that CTPG dramatically changed the morphology of B16-F10 cells, and significantly reduced the viability of B16-F10 cells in a dose-dependent and time-dependent manner, which might be mediated by CTPG-induced apoptosis and cell cycle arrest. After CTPG treatment, the expressions of BAX and BCL-2 were up-regulated and down-regulated, respectively. Moreover, mitochondrial membrane potential was reduced and ROS generation was increased. Consequently, the levels of cytochrome c and cleaved-caspase-3 and -9 were up-regulated by CTPG treatment but not for cleaved-caspase-8. We further observed that CTPG significantly inhibited the tumor growth in vivo and improved the survival rate of tumor mice. We also observed that CTPG promoted the proliferation of splenocytes and increased the proportions of CD4+ and CD8+ T cells in spleens of tumor mice. The results showed that CTPG induced the apoptosis of B16-F10 cells through mitochondria-dependent pathway, suggesting that CTPG could be a potential candidate for treatment of cancer.

Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine

Licorice has been used as herbal medicine and natural sweetener. Here, we prepared Glycyrrhiza uralensis water extract (GUWE) and investigated the effect of GUWE on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We observed that GUWE dose-dependently promoted DC maturation and cytokine secretion through TLR4 signaling pathway. The capacity of DC to stimulate allogenic splenocyte proliferation was also enhanced by GUWE treatment. Compared with control group, GUWE treated DCs pulsed with human papillomavirus (HPV)-16 E6/E7 peptides significantly inhibited the tumor growth in both early and late therapeutic groups. In early therapeutic group, the frequencies of induced regulatory T cells (iTregs: CD4+CD25−Fopx3+) and CD4+ and CD8+ T cells were significantly decreased and increased, respectively. HPV-16-specific CD8+ T cell responses were significantly induced and negatively correlated with iTreg frequencies and tumor weight. These results indicated the immunoregulatory activities of licorice.

Capparis spinosa Fruit Ethanol Extracts Exert Different Effects on the Maturation of Dendritic Cells

Capparis spinosa L. (C. spinosa) has been used as food and traditional medicine and shows anti-inflammatory and anti-oxidant activities. Here, we prepared the C. spinosa fruit ethanol extracts (CSEs) using different procedures and investigated the effects of CSE on the maturation of mouse bone marrow-derived dendritic cells (DCs) in the absence or presence of lipopolysaccharide (LPS). DC maturation and cytokine production were detected by flow cytometry and ELISA, respectively. We obtained three different CSEs and dissolved in water or DMSO, named CSE2W, CSEMW, CSE3W, CSE2D, CSEMD, and CSE3D, respectively. These CSEs showed different effects on DC maturation. CSEMW and CSEMD significantly increased the expressions of CD40, CD80, and CD86, in a dose-dependent manner. CSE2W and CSE2D also showed a modest effect on DC maturation, which enhanced the expression of CD40. CSE3W and CSE3D did not change DC maturation but suppressed LPS-induced DC maturation characterized by the decreased levels of CD40 and CD80. CSE3W and CSE3D also significantly inhibited the secretions of IL-12p40, IL-6, IL-1β, and TNF-α induced by LPS. CSE3W further increased the level of IL-10 induced by LPS. Moreover, CSE3D suppressed LPS-induced DC maturation in vivo, which decreased the expressions of CD40 and CD80. These results suggested that CSE3W and CSE3D might be used to treat inflammatory diseases.

Enhanced contraception of canine zona pellucida 3 DNA vaccine via targeting DEC-205 in mice

Zona pellucida 3 (ZP3) is a potential antigen for the development of contraceptive vaccines to control animal population. In this study, we designed a canine ZP3 (CZP3) DNA vaccine through targeting DEC-205 (named as pcD-scFv-CZP3c) and investigated its contraceptive effect in mice. Female BALB/c mice were intramuscularly immunized 3 times at 2 weeks intervals. After immunization, humoral and cellular immune responses were detected by ELISA and flow cytometry. The results showed that pcD-CZP3 and pcD-scFv-CZP3c induced CZP3-specific antibody (Ab) responses both in serum and vaginal secretions compared to pcDNA3.1. Additionally, compared to pcD-CZP3, pcD-scFv-CZP3c increased the levels of CZP3-specific Abs after a third immunization. Abs induced by these two DNA vaccines could bind with mice and dogs oocytes. Moreover, pcD-scFv-CZP3c enhanced the activation of CD4+ T cells characterized by the increased frequencies of CD4+CD44+ T cells. Finally, the contraceptive effect was evaluated in the immunized mice. These two DNA vaccines significantly decreased a mean litter size of mice compared to pcDNA3.1, but pcD-scFv-CZP3c group showed the smallest mean litter size. The mean litter size of pcD-scFv-CZP3 were 3.2 ± 0.742 and 4.6 ± 1.118 in two mating tests, which were significantly lower than pcDNA3.1(P < 0.001 and P < 0.05). Our results suggest that the CZP3 DNA vaccine targeted with DEC-205 may be a potential strategy for developing a contraceptive DNA vaccine.

Cultivated and wild Pleurotus ferulae ethanol extracts inhibit hepatocellular carcinoma cell growth via inducing endoplasmic reticulum stress- and mitochondria-dependent apoptosis

Pleurotus ferulae is a kind of editable mushroom and has various biological functions such as antitumor, antioxidation and immunoregulation. Wild P. ferulae was successfully domesticated but the antitumor function and mechanisms of cultivated and wild P. ferulae need to be compared and explored. Here, we prepared cultivated and wild P. ferulae ethanol extracts (PFEE-C and PFEE-W) and compared their antitumor effect on hepatocellular carcinoma. Our data showed that PFEE-C and PFEE-W significantly inhibited the growth of H22 and HepG2 cells through induction of apoptosis. PFEE-W exhibited higher antitumor activity than PFEE-C. Both PFEE-C and PFEE-W induced endoplasmic reticulum (ER) stress characterized by the up-regulated levels of phosphorylated JNK, cleaved caspase-12 and HSP70, and mitochondrial dysfunction characterized by the reduction of mitochondrial membrane potential and the release of cytochrome c, which promoted the cleavage of caspase-3, -7, -9 and PARP. Moreover, PFEE-C and PFEE-W significantly increased ROS generation in H22 cells and suppressed H22 cell migration through reducing the levels of matrix metalloproteinase -2 and -9. Further, PFEE-C inhibited H22 tumor growth in mouse model and improved the survival of tumor mice. These results indicated that PFEE-C and PFEE-W could inhibit hepatocellular carcinoma cell growth through ER stress- and mitochondria-dependent apoptotic pathways.

N-Butanol Subfraction of Brassica Rapa L. Promotes Reactive Oxygen Species Production and Induces Apoptosis of A549 Lung Adenocarcinoma Cells via Mitochondria-Dependent Pathway

Brassica rapa L., an edible and medical vegetable, has been traditionally used in Uyghur folk medicine to treat coughs and asthma in the Xinjiang Uygur Autonomous Region, China. In this study, we prepared an n-butanol subfraction of B. rapa L. (BRBS) and investigated the anti-tumor effect on A549 lung adenocarcinoma cells. The proliferation of A549 cells was significantly inhibited by BRBS treatment in a dose- and time-dependent manner. BRBS significantly induced cell cycle arrest and apoptosis in A549 cells through increased reactive oxygen species (ROS) production and mitochondrial dysfunction characterized by a reduction in mitochondrial membrane potential and the release of cytochrome c, which promoted caspase-3 and poly(ADP-ribose) polymerase processing. Moreover, BRBS significantly suppressed the migration of A549 cells in vitro. These results suggest that BRBS inhibited A549 cell proliferation through increased ROS production and the mitochondria-dependent apoptosis pathway. Consequently, BRBS might be a potential candidate for the treatment of lung cancer.

Cistanche tubulosa phenylethanoid glycosides induce apoptosis in H22 hepatocellular carcinoma cells through both extrinsic and intrinsic signaling pathways

BackgroundCistanche tubulosa (Schenk) R. Wight is a traditional Chinese medicine that parasitizes the roots of the Tamarix plant and has been used to treat male impotence, sterility, body weakness, and as a tonic. However, its antitumor effect on hepatocellular carcinoma is still elusive. Here, we investigated the antitumor effect of C. tubulosa phenylethanoid glycosides (CTPG) on H22 hepatocellular carcinoma cells both in vitro and in vivo and its mechanisms.MethodsThe morphology, viability, apoptosis, cell cycle and mitochondrial membrane potential (Δψm) of H22 cells were analyzed by inverted microscopy, MTT assay and flow cytometry, respectively. The expression and activation of proteins in apoptosis pathway were detected by Western blot. The in vivo antitumor effect was evaluated in tumor mouse model established using male Kunming mice.ResultsCTPG treatment significantly suppressed H22 cell growth in a dose- and time-dependent manner, which was correlated with the increased apoptosis and cell cycle arrest at G0/G1 and G2/M phases. Moreover, the chromosomal condensation was observed in CTPG-treated H22 cells. CTPG treatment significantly increased Bax/Bcl-2 ratio, reduced Δψm and enhanced the release of cytochrome c. The levels of cleaved caspase-8 and caspase-9 in both extrinsic and intrinsic signaling pathways were significantly increased that sequentially activated caspase-7 and -3 to cleave PARP. Finally, CTPG inhibited the growth of H22 cells in mice and improved the survival rate of tumor mice.ConclusionsThese results suggested that CTPG suppressed H22 cell growth through both extrinsic and intrinsic apoptosis pathways.

λ-Carrageenan improves the antitumor effect of dendritic cellbased vaccine

In this study, we investigated the effect of λ-carrageenan on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We found that λ-carrageenan dose-dependently decreased the endocytosis of DCs, promoted DC maturation and increased cytokine production through TLR4 mediated signaling pathway. λ-carrageenan treatment also enhanced the ability of DCs in the stimulating allogenic splenocyte proliferation. In TC-1 tumor mouse model, HPV peptides pulsed λ-carrageenan-DC (HPV-CGN-DC) significantly inhibited tumor growth compared with control group. The frequencies of CD4+ and CD8+ T cells in spleens of tumor mice and their activation status were significantly increased in HPV-CGN-DC group, but the frequencies of natural regulatory T cells and CD11b+Gr-1+ cells were significantly decreased. Further, HPV-CGN-DC induced strong CD8+ T cell responses, which are negatively correlated with tumor volumes. The results suggested that λ-carrageenan promoted DC maturation through TLR4 signaling pathway and could be used as the adjuvant in DC-based vaccines.