Effect of icariin on the transformation efficiency of induced pluripotent stem cells into sperm cells in vitro / Estudio de los efectos de icariina en la eficiencia de la transformación de espermatozoides extrovertidos inducidos en cuerpos multicompetentes

Objective: To investigate the effect of icariin on the transformation efficiency of germ cell-like cells from mouse induced pluripotent stem cells into sperm cells in vitro. Methods: Firstly, mouse induced pluripotent stem cells were induced and cultured to transform into germ cell-like cells, and the primordial germ cell-like cells were identified by Western blot and RT-PCR. Then, different concentrations of icariin (0.1μg/mL, 1μg/mL, 10μg/mL and 100μg/mL) were added into the culture medium, and the obtained primitive germ cell-like cells were cultured, Western blot and RT-PCR were used to identify the obtained sperm cells, the transformation efficiency was compared. Results: The primordium germ cell-like cells obtained from mouse induced pluripotent stem cells in vitro specially expressed Oct-4 protein, C-kit protein, Mvh mRNA, Fragilis mRNA and Stella mRNA. The sperm cells were specially expressed VASA, SCP3 and γH2AX proteins. RT-PCR showed that the sperm cells were specially expressed Ddx4, Tp2 and Prm1 mRNA. Compared with the control group, the expression level of VASA protein (1.744±0.283, 2.882±0.373, 6.489±0.460), SCP3 protein (2.250±0.306, 7.058±0.521, 8.654±0.804), γH2AX protein (4.304±0.433, 5.713±0.339, 9.268±0.545), Ddx4 mRNA (1.374±0.145, 2.846±0.194, 4.021±0.154), Tp2 mRNA (1.358±0.130, 3.623±0.326, 5.811±0.390) and Prm1 mRNA (1.326±0.162, 3.487±0.237, 4.666±0.307) in 0.1μg/mL, 1μg/mL, 10μg/mL icariin experimental groups were all lower than that of VASA protein (10.560±0.413), SCP3 protein (13.804±0.642), γH2AX protein (11.874±0.464), Ddx4 mRNA (6.4005±0.361), Tp2 mRNA (7.314±0.256) and Prm1 mRNA (7.334±0.390) in 100μg/mL icariin experimental group. (AU)

Objetivo: Investigar el efecto de icariina en la eficiencia de la conversión in vitro inducida en espermatozoides de cultivos de células germinativas derivadas de la transformación de células madre pluripotentes inducidas de ratón. Métodos: Primero se indujeron y cultivaron células madre pluripotentes inducidas de ratón para transformarlas en células similares a las células germinales, y las células similares a las células germinales primordiales se identificaron mediante Western blot y RT-PCR. A continuación, se añadieron diferentes concentraciones de icariina (0,1μg/mL, 1μg/mL, 10μg/mL and 100μg/mL) al medio de cultivo, y se cultivaron las células primitivas similares a células germinales obtenidas, se utilizaron Western blot y RT-PCR para identificar las células espermáticas obtenidas, y se comparó la eficacia de la transformación. Resultados: Las células germinales primitivas obtenidas in vitro a partir de células madre pluripotentes inducidas de ratón expresaron especialmente la proteína Oct-4, la proteína C-kit, el ARNm de Mvh, el ARNm de Fragilis y el ARNm de Stella. Los espermatozoides expresaban especialmente las proteínas VASA, SCP3 y γH2AX. La RT-PCR mostró que los espermatozoides expresaban especialmente los ARNm Ddx4, Tp2 y Prm1. En comparación con el grupo de control, el nivel de expresión de la proteína VASA (1,744±0,283; 2,882±0,373; 6,489±0,460), la proteína SCP3 (2,250±0,306; 7,058± 0,521; 8,654±0,804), proteína γH2AX (4,304±0,433; 5,713±0,339; 9,268±0,545), ARNm Ddx4 (1,374±0,145; 2,846±0,194; 4,021±0,154), ARNm Tp2 (1,358±0,130; 3,623±0,326; 5,811±0,390) y ARNm Prm1 (1,326±0,162; 3,487±0,237; 4,666±0,307) en grupos experimentales de 0,1μg/mL, 1μg/mL, 10μg/mL de icariina fueron todos más bajos que los de la proteína VASA (10,560±0,413), proteína SCP3 (13,804±0,642), proteína γH2AX (11,874±0,464), ARNm Ddx4 (6,4005±0,361), ARNm Tp2 (7,314±0,256) y ARNm Prm1 (7,334±0,390) en 100μg/mL icariina grupo experimental. (AU)

Effect of icariin on the transformation efficiency of induced pluripotent stem cells into sperm cells in vitro.

OBJECTIVE: To investigate the effect of icariin on the transformation efficiency of germ cell-like cells from mouse induced pluripotent stem cells into sperm cells in vitro. METHODS: Firstly, mouse induced pluripotent stem cells were induced and cultured to transform into germ cell-like cells, and the primordial germ cell-like cells were identified by Western blot and RT-PCR. Then, different concentrations of icariin (0.1µg/mL, 1µg/mL, 10µg/mL and 100µg/mL) were added into the culture medium, and the obtained primitive germ cell-like cells were cultured, Western blot and RT-PCR were used to identify the obtained sperm cells, the transformation efficiency was compared. RESULTS: The primordium germ cell-like cells obtained from mouse induced pluripotent stem cells in vitro specially expressed Oct-4 protein, C-kit protein, Mvh mRNA, Fragilis mRNA and Stella mRNA. The sperm cells were specially expressed VASA, SCP3 and γH2AX proteins. RT-PCR showed that the sperm cells were specially expressed Ddx4, Tp2 and Prm1 mRNA. Compared with the control group, the expression level of VASA protein (1.744±0.283, 2.882±0.373, 6.489±0.460), SCP3 protein (2.250±0.306, 7.058±0.521, 8.654±0.804), γH2AX protein (4.304±0.433, 5.713±0.339, 9.268±0.545), Ddx4 mRNA (1.374±0.145, 2.846±0.194, 4.021±0.154), Tp2 mRNA (1.358±0.130, 3.623±0.326, 5.811±0.390) and Prm1 mRNA (1.326±0.162, 3.487±0.237, 4.666±0.307) in 0.1µg/mL, 1µg/mL, 10µg/mL icariin experimental groups were all lower than that of VASA protein (10.560±0.413), SCP3 protein (13.804±0.642), γH2AX protein (11.874±0.464), Ddx4 mRNA (6.4005±0.361), Tp2 mRNA (7.314±0.256) and Prm1 mRNA (7.334±0.390) in 100µg/mL icariin experimental group. CONCLUSIONS: Icariin can promote the transformation of mouse induced pluripotent stem cells into sperm cells in vitro, and it is concentration-dependent manner in a certain concentration range.

[Distribution Characteristics and Health Risk Assessment of Heavy Metals in a Soil-Rice System in an E-waste Dismantling Area].

This study selected Guiyu Town, Guangdong Province as the research area, the content of 15 kinds of metals (As, Be, Cd, Co, Cr, Cu, Hg, Li, Mn, Ni, Sb, Sn, Pb, V, and Zn) in the soil was determined, and the content of heavy metals (As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn in the rice of this research area was identified. Multivariate statistical analysis and a human health risk assessment model were used to investigate the distribution characteristics and health risk of heavy metals in a soil-rice system. The results showed that Hg, Sb, and Sn in the surface soil surrounding the electronic waste dismantling area have obvious accumulation effect. The average content of Cd and Hg exceeds the Ⅱ standard limit of the "Environmental Quality Standard for Soil" (GB 156182-1995), and that Guiyu Town is more seriously polluted than Chendian Town and Simapu Town. The multivariate statistical analysis showed that Cu, Sb, Ni, Zn, Sn, Pb, and Hg originated from the surrounding electronic waste dismantling activities, Cd and Be originated from other man-made sources of pollution, and V, Li, Cr, Co, As, and Mn originated from natural sources. Heavy metal evaluation concentration in the soil-rice system by heavy metal migration accumulated in rice are in compliance with national food hygiene standards, and the enrichment ability is Cd > Zn > Cu > Ni > As > Cr > Hg > Pb. Soil heavy metal health risk assessment results showed that children are more susceptible to heavy metal pollution, and handling-oral ingestion is the main way of soil exposure risk. The non-carcinogenic risk and carcinogenic risk of heavy metals in the soil of each town are acceptable. The health risk in Guiyu Town through ingestion of rice is mainly from the elements that include As, Cr, Cu, and Ni.

[Seasonal Characteristics and Ecological Risk Assessment of Heavy Metals in PM10 Around Electroplating Plants].

PM10 samples were collected from 45 sites around the electroplating factories in five towns in Dongguan at different times during all four seasons in 2015. The contents of 12 heavy metals (HMs) from the PM10 samples were analyzed by ICP-MS. The seasonal and spatial distribution characteristics and the ecological risk were analyzed to provide a scientific foundation for the relevant department to make decisions regarding the environmental hazard, risk assessment and, pollution control. The results showed that PM10 concentrations in the towns were lower than national standard level-Ⅱ, and the air pollution was heavier in winter than summer. The HM concentrations were higher in autumn and winter, and As, Cd, and Cr concentrations were higher than national standard (GB3095-2012). The concentrations of HMs in Humen, Shatian, and Dalingshan were much higher. The results for the enrichment factor and the geoaccumulation index indicated that Cd, Sb, Hg, and Co were in the extreme degree of pollution category, Pb and Zn were in the slight to extreme degree of pollution category, and Ni, Cr, Mn, and V were described as uncontaminated. The average potential ecological risk assessment (RI) of the HMs from the PM10 samples was more than 600, which suggested an extremely serious ecological risk in the study area.

[Distribution Characteristics and Health Risk Assessment of Heavy Metals in Surface Water Around Electroplating Factories].

To investigate the distribution characteristics and the human health risks of heavy metals in surface water samples, 30 samples were collected around electroplating factories of Machong, Shatian, Humen, Changan and Dalingshan towns in Dongguan city, 8 heavy metals(As, Cd, Cr, Cu, Hg, Ni, Pb and Zn) contents were measured and analyzed by using multivariate statistical analysis method and human health risk assessment model. The results showed that the maximum concentrations of Cr, Pb and the average concentration of Hg exceeded Environmental Quality Standards for Surface Water(GB 3838-2002, Grade Ⅲ), the concentrations of Cr, Cu, Hg, Ni, Zn and Pb during rainy season were all higher than that those during dry season. Multivariate statistical analysis indicated that Cd, Cr, Cu, Ni and Zn mainly originated from the contaminated electroplating factories, Pb and Hg were mainly affected by the traffic sources, and As was significantly correlated with natural sources. Health risk assessment result of surface water indicated that heavy metal pollution would lead to high health risks especially for children, and the health risks of heavy metals through drinking pathway were 2-3 orders of magnitude higher than the values caused by dermal contact pathway. Moreover, carcinogenic risks caused by Cr and As were higher than the maximum allowance levels (5×10-5 a-1), and non-carcinogenic risks of the heavy metals (10-10-10-7 a-1) decreased in the order of Pb > Ni > Cu > Hg > Zn, which were 4-5 orders of magnitude lower than the maximum allowance levels.

The aberrant expressions of nuclear matrix proteins during the apoptosis of human osteosarcoma cells.

The objective of this study was to investigate altered expressions of nuclear matrix proteins (NMPs) of human osteosarcoma (OS) MG-63 cells during curcumin-induced apoptosis of human OS MG-63 cells. MG-63 cells were cultured with curcumin (7.5 mg/L) for 72 hr. Morphological alterations of cells were captured using light microscopy and transmission electron microscopy, and cell cycle distribution was estimated by flow cytometry. NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis (2-DE) analysis. Western blots were performed to determine changes in the expression levels of specific NMPs. The results demonstrated that typical characteristics of apoptosis were observed. Cellular chromatin agglutinated, cell nuclei condensed, and apoptotic bodies were formed after treatment with curcumin. The 2-DE results displayed 27 NMPs, 21 of which were identified to have change in expression levels significantly during apoptosis. The altered expressions of three of these NMPs (nucleophosmin, prohibitin, and vimentin) were further confirmed by immunoblotting. These findings indicated that the apoptosis of MG-63 cells was accompanied by the expression alteration of NMPs. Our results might help to reveal the relationship between NMPs and the regulation of gene expression in the process of apoptosis, as well as provide the basic concepts for future studies on the mechanisms of apoptosis and the therapy for bone diseases.

Nuclear matrix protein, prohibitin, was down-regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG-63 cells induced by ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT).

Ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT) are effective anticancer and antioxidant constituents of traditional Chinese herbal medicines of Ginseng, Xuanseng, and Danseng. The molecular mechanisms of anticancer effects of those constituents and their targets are unknown. Prohibitin, an inner membrane-bound chaperone in mitochondrion involved in the regulation of cell growth, proliferation, differentiation, aging, and apoptosis, was chosen as a candidate molecular target because of its frequent up-regulation in various cancer cells. We demonstrated that prohibitin existed in the filaments of the nuclear matrix of the MG-63 cell and its expression was down-regulated by the treatment of RCT using proteomic methodologies and Western blot analysis. Immunogold electro-microscopy also found that prohibitin was localized on nuclear matrix intermediate filaments (NM-IF) that had undergone restorational changes after RCT treatment. Prohibitin may function as a molecular chaperone that might interact with multiple oncogenes and tumor suppressor genes. We found that oncogenes c-myc and c-fos and tumor suppressor genes P53 and Rb were regulated by RCT as well and that these gene products co-localized with prohibitin. Our study identified prohibitin as a molecular target of the effective anticancer constituents of Ginseng, Xuanseng, and Danseng that down-regulated prohibitin in nuclear matrix, changed prohibtin trafficking from nucleolus to cytoplasm, and regulated several oncogenes and tumor suppressor genes. Prohibitin downregulation and cellular trafficking from nucleolus to cytoplasm indicated RCT protective roles in cancer prevention and treatment.

[Changes of nuclear matrix proteins during apoptosis of human osteosarcoma MG-63 cells induced by curcumin].

Human osteosarcoma MG-63 cells were induced into apoptosis by curcumin (Cur). Its nuclear matrix proteins were selectively extracted, and subjected to two dimensional gel electrophoresis analysis. The resulted protein patterns were analyzed by Melanie software. There were 27 spots changed remarkably during the apoptosis induced by curcumin, 21 of which were identified. There were seven up-regulated proteins including DNA polymerase zeta and forteen down-regulated proteins including Prohibitin. This study suggests that the induced apoptosis of carcinoma cells is accompanied with changes of nuclear matrix proteins, and confirms the presence of some specific nuclear matrix proteins associated with carcinoma cell growth, apoptosis and the associated signal transduction pathways in induced apoptosis of carcinoma cells. It provides proofs and a new way to study the mechanisms of gene expression carcinogenesis which reveal the relationship between configuration and composition of nuclear matrix and cell apoptosis. Besides, this study provides several potential target proteins for cancer diagnosis and cancer therapy.