Background: There is a growing interest in understanding the biological effects of timetested folk medicinal plants including the green leafy vegetables, which supply minerals and vitamins to the diet. Trigonella foenum-graecum L (fenugreek) is a dietary vegetable and there are reports concerning its antinociceptive effects in Iranian traditional medicine. Its seeds are also known for their carminative, tonic, antidiabetic, antineoplastic and restorative properties. These reports and the hypoglycemic effect of fenugreek leaf extract encouraged us to assay fenugreek aqueous extract for cytotoxicity on NIH3T3 mouse fibroblast cells.Methods: The NIH3T3 cell line was purchased from National Research Center for Genetic Engineering and Biotechnology of Iran. The cells were plated in 24-well microtiter plates with DMEM+F12 medium containing 10% fetal calf serum supplemented with 445 mg/L Lglutamine and maintained at 37°C with 5% CO2/95% air. Following a 24-hr incubation period, various concentrations (0.01-20 mg) of the extract to the culture wells. Cell viability was assessed using trypan blue and MTT assays after five days of incubation. Results: The results show that the IC50 of the fenugreek extract as calculated from the trypan blue and MTT assays were 1.25 and 2.5 mg/mL, respectively. Conclusions: Our findings, therefore, suggest that the aqueous extract of fenugreek is classified as nontoxic. This observed cytotoxicity is not specific and could be due to membrane disturbances.

Background Aging is one of the important factors in the development of age-related diseases. Acrylamide can be produced during carbohydrate-rich foods prepared at high temperatures. Recently, studies showed that acrylamide can induce cellular senescence. On the other hand, Rutin as a natural flavonoid, has a potent antioxidant activity. Objective This study aims to evaluate the ptotective effect of Rutin on oxidative-induced cellular senescence induced by acrylamide. Methods NIH3T3 mouse embryonic fibroblast cells were used in this study, which treated by different concentrations of acrylamide and Hydrogen Peroxide (H2O2). Oxidative stress was assessed by measuring the malondialdehyde and glutathion concentrations. To evaluate the senescence process, β,-galactosidase activity was measured by enzyme staining and β,-galactosidase activity assay kit. The MTT assay was used to evaluate the cell viability. Results Exposure of cells to acrylamide and H2O2 significantly reduced the cell viability, and Rutin significantly improved cell viability in cells treated by acrylamide (P<0. 05). Rutin also increased glutathion level in cells treated by acrylamide and H2O2 (P<0. 05). The rate of lipid peroxidation was significantly lower in the group treated by rutin and H2O2 than in the H2O2 group (P<0. 05). The β,-galactosidase activity was increased in the H2O2 and acrylamide groups. The use of rutin with acrylamide significantly reduced the activity of β,-galactosidase enzyme compared to the acrylamide group (P<0. 05). Conclusion Oxidative stress may be an important mechanism in acrylamide-induced senescence in NIH3T3 cells. On the other hand, Rutin, as a potent antioxidant, can reduce cellular senescence by inhibition of oxidative damage.

Objective: The purpose of regenerative medicine is to generate pluripotent cells with the genome of the patient and induce differentiation in these cells for transplantation. Stem cell therapy using induced Pluripotent Stem (iPS) cells provides hope for the treatment of diseases in the future. Several methods have been investigated to induce reprogramming of somatic cells. Using cell extract for reprogramming, seems to be very efficient and results in get the large quantities of reprogrammed cells. Materials and Methods: In this study we reprogrammed NIH3T3 cells to mouse Embryonic Stem Cells (mESCs) by mESC extract. Reprogrammed cells formed colonies and could form embryoid bodies. Expression of stem cell specific genes was corroborated by alkaline phosphatase assay, immunoflurescence staining for Nanog and Sox2 and also RT-PCR for Oct3/4 in colonies. In order to clarify the differentiation property of the reprogrammed cells, the cells were cultured in adipogenic media and then the differentiated cells were stained with oil red. Results: The reprogrammed cells showed lipid droplets in their cytoplasm after exposure to adipogenic media. Droplets of the lipids stained red that indicated cell differentiation towards adipocytes.Conclusion: These data provide evidence for the generation of ES like cells from differentiated somatic cells by mESC extract and the differentiation capacity of these cells.

Objective: Generation of patient specific stem cell is among the ultimate goals in regenerative medicine. On the other hand contact between matrix proteins and integrin receptors adjust many cell’s function such as migration during embryogenesis, immune responses and tumour invasion. In this study we de-differentiated NIH3T3 cells by mouse Embryonic Stem Cell (mESC) extract in order to determine the expression level of pluripotency markers as well as a2b1 and a5b1 integrins.Materials and Methods: Mouse embryonic stem cell extracts were prepared and NIH3T3 cells were reprogrammed by mESC extract. Generated iPS (induced Pluripotent Stem cells) colonies were picked up in the sterile conditions and checked for the pluripotency by alkaline phosphatase kit, Reverse Transcriptase PCR (RT-PCR) for oct3/4 and immunocytochemistry for oct3/4, sox2 and nanog. Then the expression level of oct3/4, a2b1 and a5b1 integrins was studied by RTPCR.Results: NIH3T3 cells treated with mESC extracts showed noticeable changes in cell morphology as early as day 3 post-treatment forming colonies similar to typical mESC morphology by day 10, after three passages. Alkaline phosphatase test and immunocytochemistry staining were performed for the iPS colonies. The results indicated that these colonies not only showed the alkaline phosphatase activity, but also the expression of sox2, oct3/4 and nanog by their specific antibodies in immunocytochemistry. Also RT-PCR results revealed the expression of oct3/4 a2b1 and a5b1 integrins in iPS cells.Conclusion: These data provide evidence for the generation of iPS cells from differentiated somatic cells by mESC extract also the expression of a2b1 and a5b1 integrins in iPS cells.

Objective: Nowadays, cell reprogramming is very fashionable in regenerative medicine. Therefore scientists try to do reprogramming in more efficient ways. There is evidence to show that cell reprogramming is controlled by epigenetic function, so cleaning of epigenetic may help to reprogramming. 5-Aza-2'- deoxycytidine (5-Aza-dc, methyltransferase inhibitor) and Trichostatin A (TSA, Histone deacetylase inhibitor) can partially clear epigenetic.Materials and Methods: In the present study, we de-differentiated NIH3T3 cells in the presence and absence of 5-Aza-dc and TSA by mouse Embryonic Stem Cell (mESC) extract. Then we checked cells morphology and alkaline phosphatase expression in the reprogrammed cells.Results: The cells without treatment showed morphologic changes in the day after reprogramming and formed colonies on the second day. Growth of these cells that were passaged every two days was similar to mESCs due to formation of embryoid like bodies. In pre-treated cells, colonies were formed four days after reprogramming and the number of colonies was remarkably less than the untreated cells. NIH3T3 cells reprogrammed by mESC extract showed alkaline phosphatase activity while NIH3T3 cells that were both treated with epigenetic eraser and reprogrammed by ESC extract didn’t show alkaline phosphatase expression. Conclusion: According to these data efficiency of dedifferentiation is better by using mESC extract and without epigenetic eraser materials.

Background: Ultraviolet B (UVB) irradiation induces photoaging and apoptosis in various cell types. Inhibition of UVB-induced apoptotic pathways has been explored in different apoptotic cascades. Conditioned media from human umbilical cord blood mesenchymal stem cells (CM-hUCB-MSC) contain important substances for cell regeneration. However, the potential of CM-hUCB-MSC in preventing UVB-induced apoptosis has not been clearly elucidated. Therefore, the current research was conducted to investigate the potential of CM-hUCB-MSC in inhibiting UVB-induced apoptosis and its role in the antiapoptotic signaling pathway.Methods: An experimental in vitro study was conducted at PT. Prodia StemCell Indonesia, Jakarta, Indonesia, 2019-2022. Initially, hUCB-MSCs were isolated and cultured to produce CM-hUCB-MSC. NIH3T3 cells were pretreated with/without 50 μM LY294002, treated with/without 10% CM-hUCB-MSC, and then irradiated with/without UVB. Subsequently, the cells were analyzed using sub-G1, immunofluorescence, and immunoblotting assays. One-way analysis of variance (ANOVA) was used for data analysis, followed by Tukey’s honest significant difference (HSD) test or the Kruskal-Wallis test, followed by the Dunn-Bonferroni test using IBM SPSS Statistics software version 21. Statistical significance was determined at P<0.05.Results: CM-hUCB-MSC significantly inhibited UVB-induced apoptosis in NIH3T3 cells (P=0.002, Dunn-Bonferroni test). CM-hUCB-MSC significantly induced Akt phosphorylation at Ser 473 in UVB-irradiated NIH3T3 cells (P<0.001, Tukey’s HSD test). The CM-hUCB-MSC-induced phosphorylation of Akt was significantly inhibited by LY294002 (P<0.001, Tukey’s HSD test).Conclusion: Taken together, it can be concluded that CM-hUCB-MSC inhibits UVB-induced NIH3T3 cell apoptosis via the activation of phosphatidylinositol-3-kinase (PI3K)/Akt signaling cascades.

Background: The aim of this investigation is to evaluate the histologic results of biopsy in women with atypical squamous cells of undetermined significance (ASCUS) cytologic diagnosis.Materials and Methods: We reviewed a series of cases with ASCUS pap smears from March 1999 to Feb 2002 in Imam Khomeini Hospital (n= 104), Who had cervical biopsy indirected colposcopy (103) and in Onec endocervical biopsy obtained without colposcopy. In 60 patients before colposcopy and biopsy repeat pap smear was tabled.Results: Biopsy revealed 28.8% SIL (14 LSIL and 16 HSIL),1 invasive carcinoma and 1 endometrial carcinoma. Pap smear repeated for 60 women before colposcopy examination, which 7 (11.7%) of them were normal. ASCUS persisted in 45 cases (75%) and 8 cases (13.3%) turned out to be SIL (6 LSIL, 2 HSIL) of 7 normal repeat smear, 2 marked as LSIL by biopsy. In colposcopic examination 22 of 103 (21.4%) had normal view which one of them was LSIL histologically.Conclusion: Based on these findings, it seems immediate colposcopy and directed biopsy are appropriate procedures for management of ASCUS and to detect underlying SIL.