Experimental approaches for induction of diabetes mellitus and assessment of antidiabetic activity: An in vitro and in vivo methodological review.

BACKGROUND: Diabetes mellitus poses a global health challenge, driving the need for innovative therapeutic solutions. Experimental methods play a crucial role in evaluating the efficacy of potential antidiabetic drugs, both in vitro and in vivo. Yet concerns about reproducibility persist, necessitating comprehensive reviews. OBJECTIVES: This review aims to outline experimental approaches for inducing diabetes and evaluating antidiabetic activity, synthesizing data from authoritative sources and academic literature. METHODS: We conducted a systematic search of prominent databases, including PubMed, ScienceDirect, and Scopus, to identify relevant articles spanning from 1943 to the present. A total of 132 articles were selected for inclusion in this review, focusing on in vitro and in vivo experimental validations of antidiabetic treatments. RESULTS: Our review highlights the diverse array of experimental methods employed for inducing diabetes mellitus and evaluating antidiabetic interventions. From cell culture assays to animal models, researchers have employed various techniques to study the effectiveness of novel therapeutic agents. CONCLUSION: This review provides a comprehensive guide to experimental approaches for assessing antidiabetic activity. By synthesizing data from a range of sources, we offer valuable insights into the current methodologies used in diabetes research. Standardizing protocols and enhancing reproducibility are critical for advancing effective antidiabetic treatments.

The potential effect of melatonin on in vitro oocyte maturation and embryo development in animals.

Melatonin is a hormone mainly secreted by the pineal gland during the circadian cycle, with low levels during the daytime and prominent levels during the night. It is involved in numerous physiological functions including the immune system, circadian rhythm, reproduction, fertilization, and embryo development. In addition, melatonin exerts anti-inflammatory and antioxidant effects inside the body by scavenging reactive oxygen and reactive nitrogen species, increasing antioxidant defenses, and blocking the transcription factors of pro-inflammatory cytokines. Its protective activity has been reported to be effective in various reproductive biotechnological processes, including in vitro maturation, embryo development, and survival rates. In this comprehensive review, our objective is to summarize and debate the potential mechanism and impact of melatonin on oocyte maturation and embryo development through various developmental routes in different mammalian species.

Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/ß-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/ß-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24 h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/ß-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

Arsenic trioxide-induced cytotoxicity in A549 cells: The role of necroptosis.

INTRODUCTION: Lung cancer is one of the deadliest cancers globally. Arsenic trioxide (ATO) is still present as a highly effective drug in treating acute promyelocytic leukemia (APL). Chemotherapy resistance is one of the major problems in cancer therapy. Necroptosis, can overcomes resistance to apoptosis, and can promote cancer treatment. This study examines the necroptosis pathway in A549 cancer cells exposed to ATO. METHODS: We used the MTT test to determine the ATO effects on the viability of A549 cells at three different time intervals. Also, the reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were performed in three-time intervals. The effect of ATO on apoptosis was evaluated by Annexin V / PI staining and, the RIPK1 and MLKL gene expression were measured by Real-Time PCR. RESULTS: The ATO has dose and time-dependent cytotoxic effects, so at 24, 48, and 72 h, the IC50 doses were 33.81 '11.44 '2.535 µM respectively. A 50 µM ATO is the most appropriate to increase the MMP loss significantly at all three times. At 24 and 48 h after exposure of cells to ATO, the ROS levels increased. The RIPK1 gene expression increased significantly compared to the control group at concentrations of 50 and 100 µM; however, MLKL gene expression decreased. CONCLUSIONS: The A549 cells, after 48 h exposure to ATO at 50 and 100 µM, induces apoptosis and necroptosis. Due to the reduced expression of MLKL, it can be concluded that ATO is probably effective in the metastatic stage of cancer cells.

Antibacterial Effect of Allium ampeloprasum and Allium porrum Extracts on Staphylococcus aureus and Pseudomonas aeruginosa.

Objectives: The most prominent microorganisms that cause hospital infections and acquire antibiotic resistance are Staphylococcus aureus and Pseudomonas aeruginosa. The present study aimed to compare the phenolic and flavonoid compounds of various Allium ampeloprasum and Allium porrum extracts and evaluate the antibacterial effects of these extracts against these two microorganisms. Methods: The total phenolic and flavonoid contents of the acetone, methanol, aqueous, and hexane leeks extracts from A. ampeloprasum and A. porrum were measured. The antibacterial activity of these extracts against S. aureus and P. aeruginosa was tested using the disk diffusion method for 24, 48, and 72 hours. Further, the minimum inhibitory concentrations and the minimum bactericidal concentrations of these extracts for these two bacteria were evaluated and compared with those of common antibiotics. Results: The aqueous extracts showed the highest phenolic and flavonoid contents and at concentrations of 35 and 40 mg per disk, showed the most antibacterial activity against S. aureus and P. aeruginosa; P. aeruginosa showed more sensitivity to the aqueous extracts than S. aureus. Conclusion: Aqueous A. ampeloprasum and A. porrum extracts may prevent the growth of hospital pathogens, especially P. aeruginosa; our findings will aid the discovery of new antimicrobial substances against antibiotic-resistant bacteria.

Histological and molecular evaluation of Mentha arvensis extract on a polycystic ovary syndrome rat model.

OBJECTIVE: This study aimed to investigate the impact of Mentha arvensis on a rat model of polycystic ovary syndrome (PCOS). METHODS: The PCOS rat model was made by the daily subcutaneous injection of testosterone enanthate (250mg/kg) for 21 days. Thirty rats were divided into five groups, including a healthy control group and four PCOS groups treated with various concentrations of hydroalcoholic extract of Mentha arvensis (0, 50, 100 and 200mg/kg). LH and FSH were measured in the blood. The ovaries were used for histological investigation, Cyp17 and Ptgs2 genes expression and total antioxidant capacity. RESULTS: Our results indicated that the level of LH and FSH hormones in treated PCOS rats with various concentrations of M. arvensis were reduced in comparison with the untreated PCOS group (p>0.01). Mentha arvensis in the highest concentration (200mg/kg) decreased the number of cysts in this group in comparison with the untreated PCOS group (p<0.01). The expression of Cyp17 and Ptgs2 genes in the treated group with the highest concentration of hydroalcoholic extract were decreased in comparison with the untreated PCOS group (p<0.05). Moreover, the antioxidant capacity in the rats receiving Mentha arvensis hydroalcoholic extract was significantly increased in comparison with that from the untreated PCOS rats (p<0.05). CONCLUSIONS: For the first time, Mentha arvensis hydroalcoholic extract proved to reduce some polycystic ovary syndrome symptoms. In the present experiment, a dose of 200mg/kg of Mentha arvensis hydroalcoholic extract was regarded as the most efficient dose.

Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review.

Since time immemorial, human beings have sought natural medications for treatment of various diseases. Weighty evidence demonstrates the use of chemical methodologies for sensitive evaluation of cytotoxic potentials of herbal agents. However, due to the ubiquitous use of cytotoxicity methods, there is a need for providing updated guidance for the design and development of in vitro assessment. The aim of this review is to provide practical guidance on common cell-based assays for suitable assessment of cytotoxicity potential of herbal medicines and discussing their advantages and disadvantages Relevant articles in authentic databases, including PubMed, Web of Science, Science Direct, Scopus, Google Scholar and SID, from 1950 to 2022 were collected according to selection criteria of in vitro cytotoxicity assays and protocols. In addition, the link between cytotoxicity assay selection and different factors such as the drug solvent, concentration and exposure duration were discussed.

Thymoquinone improves folliculogenesis, sexual hormones, gene expression of apoptotic markers and antioxidant enzymes in polycystic ovary syndrome rat model.

BACKGROUND: Nowadays, polycystic ovary syndrome (PCOS) is a prevalent cause of infertility affecting women of reproductive age around the world. Thymoquinone is a natural antioxidant, derived from Nigella sativa. OBJECTIVES: The current study aimed to evaluate the protective effects of thymoquinone on the detrimental effects of PCOS rats induced with letrozole. METHODS: Thirty-two female rats were randomly divided into four groups: (1) Control, (2) PCOS, (3) PCOS+5 mg/kg thymoquinone and (4) PCOS+10 mg/kg thymoquinone. Thymoquinone was administered every 3 days for 30 days. Ovaries were histopathologically and stereologically examined, and antioxidant and apoptotic enzymes gene expression in ovaries and sex hormones in serum were measured. RESULTS: The number of unilaminar, multilaminar, antral, and graffian follicles, volume density of corpus luteum (p < 0.01), and GPx1 gene expression in ovaries and level of FSH in the blood increased in both thymoquinone groups when compared to untreated PCOS (p < 0.05). Ovaries in thymoquinone groups showed a significant reduction in the number of atretic follicles, ovary weight and volume, volume density of cortex and ovarian cysts, Bax gene expression (p < 0.01) and Bax/Bcl2 ratio as well as levels of luteinizing hormone (LH), LH/FSH ratio and testosterone (p < 0.05) in the blood of female rats when compared to PCOS group. Administration of thymoquinone restored the most detrimental effects of PCOS on ovaries (p < 0.01) and sexual hormones (p < 0.05) in rats. CONCLUSIONS: These data suggest that thymoquinone has improved effects on ovarian function in the PCOS rat model. Therefore, thymoquinone might be useful as a protective agent and adjunct treatment in PCOS patients.

Protective effects of quercetin on uterine receptivity markers and blastocyst implantation rate in diabetic pregnant mice.

OBJECTIVE: Diabetic women have different reproductive problems. In pregnant diabetic women, high rates of perinatal mortality, spontaneous abortion and congenital anomalies are observed. We hypothesized that quercetin, as an antidiabetic and phytoestrogen, might have protective effects on the embryo implantation in pregnant diabetic mice. We investigated the ameliorative effects of quercetin on the levels of serum estrogen and progesterone, rate of blastocyst implantation, and uterine receptivity markers in diabetic mice. MATERIALS AND METHODS: Diabetic and healthy female mice were treated with quercetin (30 mg/kg/day) four weeks before pregnancy. Plasma sex-steroid levels were determined on day 4 of pregnancy. Also, uteri were harvested for investigation of protein and mRNA expression changes. In another set of our study, implantation rate was determined on day 5 of pregnancy. RESULTS: Our results indicated that quercetin was significantly reduced blood glucose levels in diabetic mice. The number of implantation sites as well as serum estradiol level was reduced in diabetic mice, and then treatment with quercetin significantly increased both. On the other hand, insulin like growth factor1, integrin αvß3, and cyclooxygenase2 mRNA expression in the uterus of diabetic mice were significantly reduced, and quercetin treatment augmented the expression level of these genes. Besides, the level of inactive ß-catenin protein level in the uterus of diabetic mice was higher than normal group; treatment with quercetin reduced the level of inactive ß-catenin protein as compared to diabetic mice. CONCLUSION: We conclude that administration of quercetin before pregnancy can probably alleviate reproductive problems in diabetic women likely via its estrogenic and antihyperglycemic effects.

Effects of Dextran-Coated Superparamagnetic Iron Oxide Nanoparticles on Mouse Embryo Development, Antioxidant Enzymes and Apoptosis Genes Expression, and Ultrastructure of Sperm, Oocytes and Granulosa Cells.

BACKGROUND: Although application of superparamagnetic iron oxide nanoparticles (SPIONs) in industry and medicine has increased, their potential toxicity in reproductive cells remains a controversial issue. This study was undertaken to address the response of sperm, oocyte, and resultant blastocyst to dextran-coated SPIONs (D-SPIONs) treatment during murine in vitro fertilization (IVF). MATERIALS AND METHODS: In this experimental study, murine mature oocytes were randomly divided into three groups: control, and low- and high-dose groups in which fertilization medium was mixed with 0, 50 and 250 µg/ml of DSPIONs, respectively. Sperm and/or cumulus oocyte complexes (COCs) were cultured for 4 h in this medium for electron microscopic analysis of sperm and COCs, and assessment of developmental competence and genes expression of Gpx1, Sod1, catalase, Bcl2l1 and Bax in the resultant blastocysts. RESULTS: Ultrastructural study of sperm, oocyte, and granulosa showed destructed mitochondria and membranes in spermatozoa, vacuolated mitochondria and distorted cristae in oocytes, and disrupted nuclei and disorganized cell membranes in granulosa in a dose-dependent manner. Data showed that cleavage and blastocyst rates in the 250 µg/ml of D-SPIONs were significantly lower than in the control group (P<0.05). Gene expression of GPx1, Sod1, catalase, Bcl2l1 and Bax in resultant blastocysts of the high-dose group and catalase and Bax in resultant blastocysts of the low-dose group, was higher than the controls. CONCLUSION: There is considerable concern regarding D-SPIONs toxic effects on IVF, and mitochondrial and cell membrane damage in mouse spermatozoa and oocytes, which may be related to oxidative stress and apoptotic events.

Comparison of Epigenetic Modifier Genes in Bovine Adipose Tissue-Derived Stem Cell Based Embryos, as Donors, with In Vitro and Parthenogenesis Embryos.

OBJECTIVE: Regarding that undifferentiated mesenchymal stem cells, as donor cells, require less epigenetic reprogramming, possibility of using bovine adipose tissue-derived stem cells (BASCs) with low level of DNMTs and HDACs expression was evaluated. MATERIALS AND METHODS: In this experimental study, we examined gene expression of epigenetic modifiers including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and histone deacetylases (HDAC1-3), as well as protein levels of histone H3 acetylation at lysine 9 (H3K9ac) and POU5F1 (also known as OCT4) at two stages of preimplantation development among in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) groups. RESULTS: The results revealed that developmental competence of IVF embryos was higher than SCNT embryos (P<0.05). In the PA and SCNT groups, DNMT1, HDAC2 and HDAC3 mRNA were overexpressed (P<0.05), and proteins levels of H3K9ac and POU5F1 were reduced at 6-8 cells and blastocyst stages compared to IVF (P<0.05). The mRNA expression of DNMT1 and HDAC1 and proteins levels of POU5F1 and H3K9ac were significantly different between SCNT and PA groups (P<0.05) in both developmental stages (except HDAC1 in blastocyst stage). CONCLUSION: The SCNT embryos derived from BASCs have endured considerable nuclear reprogramming during early embryo development. Comparison of PA and SCNT blastocysts demonstrated that HDAC1 and DNMT1 may attribute to developmental competence variability of bovine embryos.

Co-exposure to an Aryl Hydrocarbon Receptor Endogenous Ligand, 6-Formylindolo[3,2-b]carbazole (FICZ), and Cadmium Induces Cardiovascular Developmental Abnormalities in Mice.

6-Formylindolo[3,2-b]carbazole (FICZ) is a signal substance and an endogenous activator of aryl hydrocarbon receptor (AHR). Cadmium (Cd) is an environmental pollutant that can activate both AHR and Wnt/ß-catenin signaling pathways. We aimed to determine how dysregulated signaling through AHR-Wnt/ß-catenin cross-talk can influence mice heart development. Mice fetuses were exposed to Cd alone or in combination with FICZ in gestation day (GD) 0. In GD18, fetuses were harvested and randomly divided into two parts for stereological and molecular studies. Stereological and tessellation results revealed that when fetuses were co-exposed with FICZ and Cd, abnormalities were synergistically raised. In the presence of FICZ, mRNA expression levels of Wnt/ß-catenin target genes significantly enhanced, especially when animals co-treated with FICZ and Cd. Based on these findings, we propose that chemical pollutants can interfere with the normal function of AHR that has a physiological role in regulating Wnt/ß-catenin during cardiogenesis.

Comparative efficacy of herbal essences with amphotricin B and ketoconazole on Candida albicans in the in vitro condition.

BACKGROUND: The Candida species are the most important factors of fungal infections in humans and animals. It is necessary to prepare antifungal or antimicrobial drugs because of increasing drug resistance. The natural treatment of diseases of bacterial origin using medicinal plants is important. In this study the effect of antimicrobial medicinal herbal essential oils and conventional antifungal drugs were evaluated on Candida albicans in vitro. METHODS: Disc diffusion assay and the microbroth dilution method were used to investigate the anticandidal effects of Foeniculum vulgare Mill, Satureja hortensis L, Cuminum cyminum, and Zataria multiflora Boiss essential oils. The anticandidal effect of these essential oils was compared with that of amphotricin B and ketoconazole in vitro. We then measured the chemical composition of the studied essential oils using gas chromatography-mass spectroscopy. RESULTS: Z. multiflora Boiss essential oil at the minimum inhibitory concentration (MIC) of 34 µg/mL and minimal lethal concentration [i.e., minimal fungicidal concentration (MFC)] of 64 µg/mL had more powerful anti-Candida activity than the other essential oils. C. cyminum essential oil showed the least effect on the tested fungus. A comparison of the effect of the studied essential oils and antifungal drugs showed that the antifungal effect on the C. albicans fungus was better with the fungicides than with the essential oils. CONCLUSION: In the present study, essential oils with different components showed antifungal activity (especially Z. multiflora Boiss essential oil). They can therefore be used as new antifungal substances.

The interfering effects of superovulation and vitrification upon some important epigenetic biomarkers in mouse blastocyst.

Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers.

DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos.

Dppa3 has been described in mice as an important maternal factor contributed by the oocyte that participates in protecting the maternal genome from oxidation of methylated cytosines (5mC) to hydroxymethylated cytosines (5hmC). Dppa3 is also required for normal mouse preimplantation development. This gene is poorly conserved across mammalian species, with less than 32% of protein sequence shared between mouse, cow and human. RNA-seq analysis of bovine oocytes and preimplantation embryos revealed that DPPA3 transcripts are some of the most highly abundant mRNAs in the oocyte, and their levels gradually decrease toward the time of embryonic genome activation (EGA). Knockdown of DPPA3 by injection of siRNA in germinal vesicle (GV) stage oocytes was used to assess its role in epigenetic remodeling and embryo development. DPPA3 knockdown resulted in increased intensity of 5hmC staining in the maternal pronucleus (PN), demonstrating a role for this factor in the asymmetric remodeling of the maternal and paternal PN in bovine zygotes. Also, DPPA3 knockdown decreased the developmental competence of parthenogenetic and in vitro fertilized embryos. Finally, DPPA3 knockdown embryos that reached the blastocyst stage had significantly fewer ICM cells as compared with control embryos. We conclude that DPPA3 is a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, indicating that the role of DPPA3 during early development is conserved between species.

The effect of extract of Punica granatum var. pleniflora for treatment of minor recurrent aphthous stomatitis.

BACKGROUND: Herbal drugs are considered alternative agents and have been used for several years around the world. Recurrent aphthous stomatitis (RAS) is one of the most common problems recognized by dentists and skin specialists. This problem is characterized by recurring, painful, small oral mucosal ulcers with a round or oval aspect that mostly appear in keratinized mucosa, cheeks, and on the surface of the mouth under the tongue. METHODS: In our experiment, the alcoholic and water extracts of Punica granatum var. pleniflora, P. granatum var. Sweet Alak, and P. granatum var. Saveh Black were tested on minor RAS. The study was carried out using the double-blind method. The study population consisted of 210 participants, of whom 69 were females (32%) and 141 were males (68%). In addition to checking several factors, the pain and the degree of the participant's satisfaction had been determined based on visual analog scale. Data analysis was done in the form of a nonparametric method using Kruskal-Wallis test and SPSS version 20 software. RESULTS: The results show that the alcoholic and water extracts of P. granatum var. pleniflora have a meaningful therapeutic effect on minor RAS. Results from the antioxidant activity and its relation to total phenolics show that P. granatum var. pleniflora and P. granatum var. Sweet Alak are rich in phenols. CONCLUSION: The water and alcoholic extracts of P. granatum varpleniflora decreased the entire time of complete treatment, and the treatment was meaningfully satisfactory for patients who participated in this experiment.

Antimicrobial and cytotoxic evaluation of some herbal essential oils in comparison with common antibiotics in bioassay condition.

BACKGROUND: Since ancient times, various infectious diseases have been treated using herbal drugs. Today, efforts regarding the discovery of the effectual components of plants possessing antimicrobial properties are advanced. Herbal essential oils are widely used for treatment of various diseases, and they play an important role in health care considerations. METHODS: The antibacterial activity of Artemisia kermanensis, Lavandula officinalis, and Zataria multiflora Boiss essential oils against Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (PTCC 1310), and Klebsiella pneumonia (PTCC 1053) was evaluated using the disk diffusion method as well as determination of the minimal inhibitory concentration and minimal bactericidal concentration. The composition of the three essential oils was determined with gas chromatography-mass spectrometry. Variable amounts of different components (such as oxygenated monoterpenes, thymol, carvacrol, and 1,8-cineol) were found in all three oils. Among the tested bacteria, S. aureus was the most sensitive to the three essential oils. RESULTS: The obtained results showed that each of the three essential oils has an inhibitory effect on pathogenic strains. Of these three oils, Z. multiflora Boiss essential oil showed the highest inhibitory effect on microbial strains. Furthermore, comparison of the antibacterial effects of these three essential oils with ampicillin and tetracycline revealed that these antibiotics have a better effect in controlling pathogenic strains. CONCLUSION: The essential oils used in the present study with different components showed antibacterial activity (especially Z. multiflora Boiss essential oil), and therefore they can be used as a new antibacterial substance.