Journal of Cell and Molecular Research

Abstract Reactive oxygen species (ROS) are highly reactive molecules that play a key role in cellular physiology.in Teucrium persicum, an Iranian endemic plant native to the southern regions of Iran, particularly the Fars province. It has long been used in traditional medicine for the treatment of headaches, abdominal pain, diabetes, inflammation, and hyperlipidemia. Our previous findings demonstrated that treatment of PC-3 prostate cancer cells with T. persicum extract induces significant cytotoxicity and programmed cell death. In the present study, we aimed to further explore the effects of the methanolic extract of T. persicum on lactate dehydrogenase (LDH) activity, intracellular ROS species levels, mitochondrial membrane potential, and the expression of Matrix metalloproteinase-2 (MMP-2), Matrix metalloproteinase-9 (MMP-9), Tumor protein 53 (TP53), and β-catenin (CTNNB1) genes in PC-3 cells. For this purpose, PC-3 cells were treated with both sublethal concentrations and concentrations above the IC₅₀ value of the extract. The results revealed that T. persicum treatment caused marked mitochondrial membrane damage and increased LDH release. These effects were associated with decreased intracellular ROS levels and loss of mitochondrial membrane potential, which correlated with reduced cellular uptake of rhodamine 123 in a dose-dependent manner. Furthermore, gene expression analyses showed that exposure of PC-3 cells to the extract at the IC₅₀ concentration led to significant downregulation of MMP-2, MMP-9, and CTNNB1 genes, accompanied by upregulation of TP53. Collectively, these findings suggest that the methanolic extract of T. persicum can inhibit the proliferation and survival of PC-3 prostate cancer cells through multiple molecular pathways. Further comprehensive studies are warranted to elucidate the precise mechanisms underlying these anticancer effects.

Abstract Identification of genes affecting production is one of the important priorities in animal breeding strategies. The introgression of genes with a larger effect on quantitative traits into livestock can improve protein and economic resources. The present study aimed to evaluate high-resolution melting (HRM) analysis for genotyping bone morphogenetic protein receptor type 1B (Fecundity Booroola gene or FecB) in sheep and myostatin (MSTN) in cattle. This study was conducted on blood samples from 19 cattle and 170 sheep including: 19 control samples with approved FecB genotypes and 151 samples with unknown genotypes. After DNA extraction from blood and semen samples, a 176-bp fragment of the MSTN gene in cattle and a 140-bp fragment of the FecB gene in sheep were amplified. HRM was optimized and conducted in all samples then, the PCR-RFLP and sequencing methods were done to prove the HRM genotyping results. The results obtained using the HRM method matched those from sequencing and PCR-RFLP, confirming the effectiveness of HRM for genotyping and enabling rapid and accurate identification of individuals with FecB and MSTN mutations. Melting temperature range for MSTN genotypes was between 80.90 and 81.60 ºC and for FecB genotypes was between 79.6 and 80.50 ºC. Utilizing the HRM method for genotyping will provide researchers and commercial animal producers’ access to a low cost, and easier method to genotype for any SNP detection. In this study, we successfully established a genotyping method for two loci in cattle and sheep economically important genes by high-resolution melting method. This method could be used in a large sample size with a high level of confidence.
 

Abstract Peripheral blood mononuclear cells (PBMCs) are key components of the immune system and serve as a practical in vitro model in many research programs. Despite the widespread applications, popular standard methods for isolating and stimulating PBMCs in rats (rPBMCs) have remained limited. In this study, rPBMCs were isolated from rat peripheral blood using a modified Ficoll density gradient protocol, followed by a short-term adherence step to remove monocytes. A complete blood count (CBC) analysis and Giemsa staining confirmed that the isolated population consisted of approximately 98% lymphocytes, indicating high purity of the isolated cells. The cells were subsequently exposed to different concentrations of mitogens, concanavalin A (ConA; 1.25–10 µg/mL) and phytohemagglutinin (PHA; 1–2%) for 3 and 5 days. Cell proliferation and viability were assessed by MTT assay, and TNF-α secretion was quantified by ELISA. ConA treatment resulted in a concentration-dependent increase in rPBMCs proliferation, with the highest viability rate observed at 1.25–5 µg/mL on day 3. At a concentration of 5 μg/mL, ConA significantly enhanced rPBMCs viability. This elevated level remained constant between days 3 and 5, indicating a sustained cellular response. By comparison, the stimulatory effect of PHA proved either weaker or delayed, as it reached its peak only on day 5. Furthermore, TNF-α analysis revealed that ConA markedly enhanced cytokine secretion, whereas PHA triggered only a modest increase. These findings demonstrate that ConA is a more potent mitogen than PHA in rat PBMCs, promoting both proliferation and cytokine release in a dose-dependent manner. The optimized protocols for isolation and stimulation of rPBMCs presented here, providing a reliable framework for future in vitro studies on immune function and immunomodulation in rat models.

Abstract Collagen bioscaffolds show promising results in oral mucosa engineering due to the high biocompatibility and biodegradability of collagen. Because of the high collagen content of gingiva, it can be used to prepare a natural collagen scaffold. Hyaluronic acid (HA) is one of the major components of extracellular matrix that regulates cellular behavior. In the present study, the effects of HA on the behavior of adipose-derived mesenchymal stem cells (Ad-MSCs) cultured on a decellularized gingival matrix were investigated by histological methods. Human gingival tissue (5×5×5 mm) was decellularized using physical methods as well as treatment with sodium dodecyl sulfate and Triton X-100, followed by washing and sterilization procedures. Scaffolds were divided into two groups including soaked scaffolds in HA (0.3% solution) and scaffolds without HA, which served as controls. In the next step, 3×10⁵ Ad-MSCs were seeded on each scaffold in both groups, and histological studies were performed after 1, 2, 3 and 4 weeks of culture. Histological studies revealed effective cell removal and also preservation of collagen fibers in the decellularized gingival connective tissue. In vitro analysis of cellular behaviors showed adhesion and also migration of human Ad-MSCs towards connective tissue matrix, as well as formation of epithelial-like structures. Overall, statistical analyses indicated significant differences in cell density (P<0.01), migration (P<0.001) and nuclear size (P<0.01) at week one in HA-treated scaffolds versus the control group. The results of the present study demonstrated that the extracellular matrix of decellularized human gingival stroma can induce cellular behaviors such as adhesion, proliferation and migration of human Ad-MSCs.