The Effects of Polyphenol, Tannic Acid, or Tannic Acid in Combination with Pamidronate on Human Osteoblast Cell Line Metabolism

Background: This study investigates the effect of tannic acid (TA) combined with pamidronate (PAM) on a human osteoblast cell line. Methods: EC50 for TA, PAM, and different combination ratios of TA and PAM (25:75, 50:50, 75:25) were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The combination index value was utilized to analyze the degree of drug interaction, while trypan blue assay was applied to analyze the cells proliferation effect. The mineralization and detection of bone BSP and Osx genes were determined via histochemical staining and PCR test, respectively. Results: The EC50 of osteoblasts treated with TA and a 75:25 ratio of TA and PAM were more potent with lower EC50 at 0.56 µg/mL and 0.48 µg/mL, respectively. The combination of TA and PAM (75:25) was shown to have synergistic interaction. On Day 7, both TA and PAM groups showed significantly increased proliferation compared with control and combination groups. On Day 7, both the TA and combination-treated groups demonstrated a higher production of calcium deposits than the control and PAM-treated groups. Moreover, on Day 7, the combination-treated group showed a significantly higher expression of BSP and Osx genes than both the TA and PAM groups. Conclusion: Combination treatment of TA and PAM at 75:25 ameliorated the highest enhancement of osteoblast proliferation and mineralization as well as caused a high expression of BSP and Osx genes.

Synergistic Inhibitory Effect of Human Umbilical Cord Matrix Mesenchymal Stem Cells-Conditioned Medium and Atorvastatin on MCF7 Cancer Cells Viablity and Migration

Background: Tumor growth and metastasis eventuate from an interaction between cancer cells and the surrounding extracellular matrix. Recent studies demonstrated inhibitory effects of mesenchymal stem cells on breast tumors. Likewise, the emerging interest in statins as anticancer agents is based on their pleiotropic effects. In the present study, we investigated whether atorvastatin and umbilical cord matrix derived mesenchymal stem cells-conditioned medium alone and combined with each other affect the MCF7 cancer cells viability and interactions.Methods: We measured the viability, apoptosis, and necrosis of MCF7 by MTT assay, Annexin-V/PI staining by flow cytometry, and quantitative real-time PCR. Two-dimensional culture and hanging drop aggregation assay illustrated the morphological changes in single cancer cells and spheroid configuration respectively. We traced the MCF7 migration via scratch-wound healing and trans-well assays. Results: The results showed inhibition of cancer cell viability in all treated groups compared with the control group, but the effect of atorvastatin and conditioned medium combination was further than each one alone. Cancer cells shrinkage and chromatin condensation in the inverted light microscopy and fluorescent staining microscopy indicated apoptosis in treated cells. The annexin V/PI analysis especially in the combination-treated group displayed decreasing in DNA synthesis and cell cycle arrest. The mRNA expressions of caspases 3, 8, 9, and Bcl-2 genes were along with extrinsic and intrinsic apoptosis pathways. Conditioned medium disrupts the connections between cancer cells in the three-dimensional spheroids configuration. The migration of treated cells across the wound area and trans-well diminished, particularly in the combination-treated group. Conclusions: For the first time, the synergistic anti-proliferative and anti-motility effect of atorvastatin along with human umbilical cord mesenchymal stem cells-derived conditioned medium on MCF7 breast cancer cells have been proved. These findings point to some new therapeutic strategies with a focus on the crosstalk between breast cancer cells and microenvironment.

A NEW APPROACH IN TREATMENT ACUTE IVERMECTIN TOXICITY IN MALE BALB-C MICE

This study was conducted to investigate the potential therapeutic of flumazenil and neostigmine in single and combination in Balb-C mice subjected to ivermectin acute oral toxicity. Ivermectin-poisoning dose was considered the half dose of LD50 that computed in current study as 20.9 mg/kg BW. A total of 24 male Blub-C mice were divided randomly into four equal treatment groups and administered ivermectin-poisoning dose (10 mg/kg BW) and treated as follows: control group (C), treated orally with distilled water; neostigmine-treated group (NT), treated i.p. with 60 µg/kg BW of neostigmine; flumazenil-treated group (FT), treated i.p. with 7 µg/kg BW of flumazenil; and combination-treated group (CT), treated i.p. with 30 µg/kg BW of neostigmine plus 3.5 µg/kg BW of flumazenil. Oral administration of ivermectin at poisoning dose caused grooming, lethargy, depression, recumbency, with no mortalities recorded. The toxic signs of animals in FT and NT groups were disappeared within 1.23 and 1.98 h, respectively, comparing to animals in CT and C groups, in which the recovery time was prolonged to 40.62 and 4.52 h, respectively. Overall, both flumazenil and neostigmine medicines have the potential to overcome ivermectin acute poisoning in Bulb-C mice, with flumazenil being more efficient. However, the combination of both medicines can cause an adverse prognosis.

Malondialdehyde Level in Seminal Plasma of Cryopreserved Holstein Bull Semen after Addition of Zinc, Cysteine, Prostaglandin F2α and their Combination in vitro

The study was conducted to know the level of Malondialdehyde (MDA) in seminal plasma of cryopreserved semen of Holstein bulls after addition of zinc sulphate, cysteine, PGF2α and their combination in vitro. Semen was collected from 7 Holstein bulls, presented in Artificial insemination Center which belonged to the Directorate of Animal Resources/ Ministry of Agriculture at Abu-Graib at the west of Baghdad. Pooled semen were diluted with Tris- based extender and divided into five parts. The first part (T1) serve as a control (without addition). The 2nd part (T2) added to it zinc sulphate (0,576 mmol/ ml). The 3rd part (T3) added to it cysteine (5 mmol/ ml). The 4th part (T4) added to it PGF2α (37.5 pg/ ml). while the 5th part added to it a combination of previous substances at the same concentration. They packed in straws and cryopreserved in a liquid nitrogen and after 30, 60 and 90 days. Seminal plasma when isolated to measure the level of MDA. The results showed a significant decrease (P>0.01) in MDA level in the combination treated group (zinc, cysteine and PGF2α) 0.450 ± 0.11 (mmol/ ml) as compared with control group 1.025 ± 0.38 (mmol/ ml), zinc 0.867 ± 0.12 (mmol/ ml), cysteine 1.06 ± 0.12 (mmol/ ml) and PGF2α group 0.968 ± 0.17 (mmol/ ml) respectively. It was concluded from this study that addition of a combination of zinc, cysteine and PGF2α to the Holstein bull semen could decrease the level of MDA which might be due to the synergistic effect of these substances.