Iranian Journal of Basic Medical Sciences
Volume:24 Issue: 11, Nov 2021

Metabolic syndrome (MetS), as a health-threatening factor, consists of various symptoms including insulin resistance, high blood sugar, hypertension, dyslipidemia, inflammation, and abdominal obesity that raise the risk of diabetes mellitus and cardiovascular disease. Cardiovascular diseases are important causes of mortality among the world population. Recently, there has been a growing interest in using phytomedicine and natural compounds in the prevention and treatment of various diseases. The data was gathered by searching various standard electronic databases (Google Scholar, Scopus, Web of Science, and PubMed) for English articles with no time limitations. All in vivo, in vitro, and clinical studies were included. Elettaria cardamomum (cardamom) is a rich source of phenolic compounds, volatile oils, and fixed oils. Cardamom and its pharmacologically effective substances have shown broad-spectrum activities including antihypertensive, anti-oxidant, lipid-modifying, anti-inflammatory, anti-atherosclerotic, anti-thrombotic, hepatoprotective, hypocholesterolemic, anti-obesity, and antidiabetic effects. This review aims to highlight the therapeutic effects of cardamom on MetS and its components including diabetes, hyperlipidemia, obesity, and high blood pressure as well as the underlying mechanisms in the management of MetS. Finally, it can be stated that cardamom has beneficial effects on the treatment of MetS and its complications.

Cervical cancer ranks as the fourth most common neoplasia in women worldwide in which epigenetic alterations play an important role. Several studies have reported pro-oncogenic role of the histone variant H2A.Z in different types of cancer; however, the role of H2A.Z in cervical cancer remains poorly studied. This study aimed to determine the potential role of H2A.Z in cervical cancer through a bioinformatic approach.

H2A.Z expression was analyzed in The Human Protein Atlas, The Cancer Genome Atlas, and Gene Expression Omnibus datasets. The promoter regions of H2AZ1 and H2AZ2 genes were downloaded from Expasy, and the prediction of transcription factor binding motifs was performed using CONSITE, Alibaba, and ALGGEN. ChIP-seq and RNA-seq data from HeLa-S3 cells were downloaded from ENCODE. The discovery motif was investigated using MEME-ChIP. The functional annotation was examined in Enrich.

The expression of H2A.Z is elevated in cervical cancer. Interestingly, DNA methylation, copy number, and transcription factors AP2α and ELK1 are involved in H2A.Z overexpression. Additionally, H2A.Z is enriched on promoter and enhancer regions of genes involved in pathways associated with cancer development. In these regions, H2A.Z enables the recruitment of transcription factors such as NRF1, NFYA, and RNA Pol II. Finally, H2A.Z allows the expression of genes associated with proliferation in patients with cervical cancer.

Our findings suggest that H2A.Z overexpression and its presence in promoters and enhancers could be regulating the transcription of genes involved in cervical carcinogenesis.

Vacuolar H+-ATPase is a highly conserved enzyme that plays an important role in maintaining an acidic environment for lysosomal function and accumulating neurotransmitters in synaptic vesicles. In the present study, we investigated the time-dependent changes in the expression of vacuolar H+-ATPase V1B2 (ATP6V1B2), a major neuronal subtype of vacuolar H+-ATPase located in the hippocampus, after 5 min of transient forebrain ischemia in gerbils. We also examined the pH and lactate levels in the hippocampus after ischemia to elucidate the correlation between ATP6V1B2 expression and acidosis.

Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 5 min and animals were sacrificed at various time points after ischemia for immunohistochemical staining of ATP6V1B2 and measurements of pH and lactate levels in the hippocampus.

ATP6V1B2 immunoreactivity was found to be transiently increased in the hippocampal CA1 region and dentate gyrus 12–24 hr after ischemia when the pH and lactate levels were decreased. In addition, ATP6V1B2 immunoreactivity significantly increased in the hippocampal CA3 and dentate gyrus, regions relatively resistant to ischemic damage, 4 days after ischemia, when the NeuN-positive, mature neuron numbers were significantly decreased in the hippocampal CA1 region. 

These results suggest that ATP6V1B2 expression is transiently increased in the hippocampus following ischemia, which may be intended to compensate for ischemia-related dysfunction of ATP6V1B2 in the hippocampus.

Myocardial infarction caused by ischemia of heart tissue is the main reason for death worldwide; therefore, early detection can reduce mortality and treatment costs. Erythropoietin (EPO) has protection effects on ischemic tissue due to nonhematopoietic peptide (pHBSP; ARA-290) which is derived from the B-subunit of EPO. 

We designed and synthesized a modified DOTA-(Lys-Dabcyl6, Phe7)-ARA-290 using Fmoc solid-phase peptide synthesis strategies. To improve serum stability, Fmoc-Lys-(Dabcyl)-OH as lipophilic amino acid was synthesized along with Fmoc-Phe-OH which then were substituted with Arg6 and Ala7, respectively; they were then investigated for the ability to detect ischemic cardiac imaging. DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 was labeled with technetium 99m, and its radiochemical purity (RCP), stability in the presence of human serum and, specific bind to hypoxic H9c2 cells were evaluated. In vivo studies for biodistribution and SPECT scintigraphy were checked in a normal and cardiac ischemia rat model. 

Radiolabeling purity was obtained more than 96% by ITLC, and in vitro stability of the radiopeptide up to 6 hr was 85%. The binding of 99mTc-ARA-290 to hypoxic cells was remarkably higher than normoxic cells (3 times higher than normoxic cells at 1 hr). Biodistribution and SPECT imaging on the cardiac ischemic model showed that radiopeptide considerably accumulated in the ischemic region (cardiac ischemic-to-lung rate = 3.65 ID/g % at 0.5 hr).

The results of studies, in vitro  and in vivo, indicated that 99mTc-DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 could be an appropriate candidate for early diagnosis of cardiac ischemia.

This study examines the impact of integrin-linked kinase (ILK), protein kinase B (AKT), glycogen synthase kinase-3β (GSK-3β), and β-catenin signal molecules in SKOV-3 ovarian cancer cells adhered to fibronectin.  Expression levels of α4, αv, β1, and β6 integrin subunits known as the fibronectin ligand were investigated with the flow cytometry technique. The effects of ILK, AKT, GSK-3β, and β-catenin on the binding of SKOV-3 cells to fibronectin were examined by using the Real-Time Cellular Analysis (RTCA) method. Additionally, the interaction of these proteins was investigated by using Western blot analysis.  The results show that the expression levels of integrin subunits were ranked as αv (67.8%), followed by α4 (48.55%), β6 (32.05%), and β1 (31%) on SKOV-3 cells. RTCA results showed that ILK (10 µM Cpd22), GSK-3β (50 μM GSK-3β inhibitor-XI), AKT (35 µM FPA 124), and β-catenin (50 μM cardamonin) inhibitors decreased significantly (P<0.01) binding to fibronectin at 24 hr. Western studies in SKOV-3 cells adhered to fibronectin have shown that in inhibition of ILK, AKT expression was strongly inhibited, whereas, in the inhibition of AKT, ILK expression was strongly inhibited. Furthermore, the expression of β-catenin is partially reduced in inhibition of these two molecules. In β-catenin inhibition, AKT and ILK expressions are also strongly inhibited. ILK, AKT, GSK-3β, and β-catenin were found to be fundamental molecules in binding of SKOV-3 cells to fibronectin. ILK and AKT affect strongly the level of expression of each other, and both also affect the signal path of β-catenin.

The present in vitro study aimed to evaluate whether Lactobacillus delbrueckii and Lactobacillus rhamnosus treatments can induce regulatory phenotype, together with modulating the expression of chemokine receptors (CRs) in dendritic cells (DCs). The CRs of DCs have been found to be involved in the pathogenesis of Systemic lupus erythematosus (SLE) through directing recruitment and migration of immune cells.

In brief, monocytes of patients with SLE and healthy donors were isolated and differentiated to regulatory or inflammatory mature DCs through treatment with L. delbrueckii, L. rhamnosus, mixed probiotics, and LPS. 

FACScan analysis showed that the expression of CRs including CXCR3, CCR5, CCR4, and CCR3, was significantly reduced in all probiotic-treated groups of SLE and healthy (control) donors when compared with the LPS treated group. 

The results demonstrated that tolerogenic probiotics could prevent or decrease the expression of inflammatory CRs on the surface of tolerogenic DCs during the maturation process.

Mouse breast cancer cell line 4T1 can accurately mimic the response to immune receptors and targeting therapeutic agents. Combined therapy has emerged as an important strategy with reduced side effects and maximum therapeutic effect. Mocetinostat (MGCD0103) is one of the members of Class I Histone Deacetylase Inhibitors (HDACi) and its mechanism of action has not been defined, yet. Capecitabine (Xeloda) is an antimetabolite and currently is widely utilized to treat a wide range of solid tumors. The aim of this study was to investigate the effects of the capecitabine, mocetinostat and their combined application on the 4T1 cell line.   The effects of combined administration of mocetinostat and capecitabine on 4T1 cells were investigated by cell viability and migration assays, apoptosis analysis, and Western blotting technique. The concentrations of drugs that give a half-maximal response (IC50) were detected for capecitabine (1700 µM), mocetinostat (3,125 µM), and 50 µM Capecitabine+1,5 µM Mocetinostat for 48 hr. In capecitabine+mocetinostat combine group, we observed that cell migration decreased, DNA fragmentation increased compared to the control group. capecitabine + mocetinostat group induced apoptosis by decreasing Bcl-2, PI3K, Akt, c-myc protein levels, while increasing Bax, Caspase-3, PTEN, cleaved-PARP, Caspase-7, Caspase-9, p53, cleaved-Cas-9 protein levels in 4T1 cells.  Capecitabine and mocetinostat played a toxic role through inducing apoptosis on 4T1 cancer cells in a time- and concentration-dependent manner. These results showed that combined therapy with low concentrations were detected to be more effective than that with high-concentration alone drug treatment.

In testis, the extracellular matrix (ECM) in addition to the supportive role for cells in the seminiferous epithelium, is also essential for the accurate functioning of these cells. Thus, using a decellularized testicular ECM (DTECM), as a scaffold for three-dimensional (3D) culture of testicular cells can mimic native ECM for studying in vitro spermatogenesis.  The rat testis was decellularized via perfusion of 0.5% sodium dodecyl sulfate (SDS) for 48 hr, followed by 1% Triton X-100 for 6 hr, and then 1% DNase I for 1 hr. The efficiency of decellularization was evaluated by histology, immunohistochemistry (IHC), scanning electron microscopy (SEM), and MTT test. The prepared scaffolds were recellularized with testicular cells and cultured and assessed with hematoxylin-eosin (H&E) staining after two weeks.  Based on the H&E image, no trace of cell components could be observed in DTECM. IHC images demonstrated collagen types I and IV, laminin, and fibronectin were preserved. Masson’s trichrome and alcian blue staining revealed that collagen and glycosaminoglycans (GAGs) were retained, and the SEM image indicated that 3D testicular architecture remained after the decellularization process. Based on the results of the MTT test, DTECM was cytocompatible, and H&E images represented that DTECM supports testicular cell arrangements in seminiferous tubule-like structures (STLSs) and organoid-like structures (OLSs).   The results showed that the applied protocol successfully decellularized the testis tissue of the rat. Therefore, these scaffolds may provide an appropriate vehicle for in vitro reconstruction of the seminiferous tubule.

Combination chemotherapy is a beneficial intervention for breast cancer, versus single therapy. We investigated the effect of Metformin (Met) on Lapatinib (Lap)-induced apoptosis in SK-BR3 cells.  

Toxic effect of Met and Lap on SK-BR3 cells was measured using MTT assay. Flow cytometry was used to measure the co-treatment effect of Met on lapatinib-induced apoptosis. The relative expression of Bax, Bcl2, and P21 was measured using a real-time PCR. The activity of caspase 3 and 9 was measured using an ELISA kit. The protein level of AMPK and Akt was determined using Western blot analysis.

Metformin and lapatinib alone and combined form showed significant time- and dose-dependent toxic effects on SK-BR3 cell viability. The greatest synergistic inhibitory effect on the cell viability [combination index (CI) = 0.51] was remarkable at Met 100 mM combined with Lap 100 nM. The combination has a stronger apoptotic death (46%) versus lapatinib alone. The combination considerably increased the mRNA expression of Bax and P21, and caspase 3 and 9 activity, while, decreasing the mRNA expression of Bcl2. Additionally, the combination significantly up-regulated and down-regulated the protein levels of AMPK and Akt, respectively.

The metformin-lapatinib combination can induce more potent apoptotic death versus each compound individually. The combination may be suggested as a valuable therapeutic intervention in patients with breast cancer. However, additional in vivo studies are necessary to evaluate the clinical use of the combination for induction of apoptosis and its antitumor effects.

To investigate the potential anti-breast cancer activity of zerumbone in regulating apoptotic mediators and cytokines in comparison with paclitaxel (positive control).  In this study, assays such as viability, apoptosis, reactive oxygen species, cell cycle, DNA fragmentation, and cytokines were carried out on MCF-7 cells after treatment with zerumbone and paclitaxel. The results showed that zerumbone demonstrated a higher (18-fold) IC50 value (126.7 µg/ml) than paclitaxel (7.29 µg/ml) in order to suppress proliferation and induce cell death of MCF-7. The cell cycle arrest at the G0/G1 phase and excessive intracellular ROS production during the in vitro zerumbone treatment indicated occurrence of apoptotic cell death although nuclear DNA fragmentation was not observed. The flow cytometer analysis of treated cells revealed secretion of proinflammatory cytokines suggesting the potential immunomodulatory activity of zerumbone.  Although, zerumbone exhibited a higher IC50 value compared with paclitaxel yet its anticancer activity against MCF-7 cells is still parallel to paclitaxel hence zerumbone has the potential to be an antineoplastic agent in the treatment of breast cancer especially the luminal type A.

Influenza is a highly contagious disease, which affects the respiratory system and seasonal influenza is common throughout the world. Influenza vaccination is an effective way to reduce the risk of death and hospitalization. This study aims at the expression of swine recombinant hemagglutinin protein in the baculovirus expression system and it offers a comparison of the immunologic parameters with the commercial vaccine.  The HA gene from the swine H1N1 strain of the Influenza virus was cloned into the Bac-To-Bac expression system in pFastBAC HTA vector and was transformed into Escherichia coli TOP10 strain. After the confirmation, the vector was transfected into the SF9 insect cell line. The recombinant HA was evaluated by SDS-PAGE and western blot. After formulation in Montanide ISA71 adjuvant, the immunization test was performed comparatively with Alum adjuvant, commercial vaccine in four groups of BALB/c mice, of which one group was control without any vaccination. Two weeks after the last immunization, the antibody response was assessed with HI assay, and experimental mice were challenged with mouse-adapted Influenza A/PR8/34 (H1N1) virus through nasal inhalation.  The immunoassay results revealed that the candidate vaccine induced the antibody response as the commercial one did but it did not significantly reduce the mortality rate, body loss, and severe fever.  To summarize, the results showed that the recombinant protein with the MontanideTM ISA- 71 adjuvant developed a more appropriate level of immunity than Alum adjuvant, so it might be used as a safe and reliable vaccine against H1N1 virus for further research.

One of the problems caused by infectious diseases is the decrease in sperm count and motility. Tannic acid is known as an anti-oxidant and anti-inflammatory agent. In this study, Cecal Ligation and Puncture (CLP) sepsis model was induced to investigate the effect of tannic acid on oxidative stress and inflammation in testicular and sperm structure and function. Twenty-four male Wistar rats (250–300 g) were randomly divided into 3 groups of 8: 1) sham, 2) sepsis, and 3) sepsis + tannic acid (20 mg/kg at 6, 12, and 24 hr after sepsis induction). Thirty hours after induction of sepsis, testicular samples were collected to measure SOD activity and MDA, IL-6, and TNF-α levels. Another part of the testis was fixed in 10% formalin for histological examinations.  In the sepsis group, testicular MDA, TNF-α, and IL-6 levels increased and SOD activity decreased compared with the sham group. In addition, the percentage of motile sperm and the survival rate of sperm decreased significantly in the sepsis group. Administration of tannic acid significantly decreased inflammatory markers (TNF-α and IL-6) and MDA levels and increased SOD activity. Furthermore tannic acid significantly improved sperm parameters and increased sperm and animal survival rates. The results of this study showed that the reproductive system may be strongly affected by the conditions created during sepsis. Tannic acid improved reproductive dysfunction in sepsis by reducing oxidative stress and inflammation.

Ovarian cancer is the most common gynecological malignancy, ranking as the fifth leading cause of cancer-related deaths among females in the United States. Homeobox D4 (HOXD4) is a transcription factor belonging to the homeobox protein family, which plays a critical role in morphogenesis during embryo development. Here we aimed to study the HOXD4 expression in ovarian serous carcinoma (OSC) and determine its clinical significance.  Real-time quantitative PCR and immunohistochemistry targeting human OSC tissues and adjacent ovarian tissues were performed to correlate the patterns of HOXD4 expression with clinical characteristics and survival outcomes. Cell lines and nude mice were used for verifying the role of HOXD4 in OSC. HOXD4 protein was predominantly expressed in OSC tissues compared with nontumorous tissues. The correlation test demonstrated a significant correlation between HOXD4 with tumor FIGO stage. Univariate and multivariate analyses found that HOXD4 expression was associated with poorer overall survival. Furthermore, high expression of HOXD4 protein was observed in OSC cell lines in vitro. Finally, the oncogenic effect of HOXD4 was confirmed by cellular and xenograft experiments. HOXD4 protein expression may be associated with a poorer prognosis in OSC. The unfavorable prognostic value of HOXD4 in malignancies and its underlying mechanism are worthy of further investigation.

This study aimed to evaluate the role of the linker histone (H1) in the binding interaction between ambochlorin (Amb), and calf thymus DNA (ctDNA) as binary and ternary systems. 

The project was accomplished through the means of absorbance, fluorescence, stopped-flow circular dichroism spectroscopy, viscosity, thermal melting, and molecular modeling techniques.

Spectroscopic analysis revealed that although Amb was strongly bound to both ctDNA and ctDNA-H1, it showed a greater tendency to ctDNA in the presence of the linker histone. The obtained thermodynamic parameters revealed that both Amb-ctDNA and Amb-ctDNA-H1 interactions were spontaneous, endothermic, and entropy-favored, and hydrophobic interactions played the main role in the formation and stabilization of complexes. Analysis of the stopped-flow circular dichroism results revealed that the binding process of Amb-ctDNA and Amb-ctDNA-H1 required a time of more than 150 milliseconds to complete. Moreover, Amb-ctDNA complex formation was marginally decelerated in the presence of the linker histone. The docking results suggested that the presence of the  linker histone may alter the binding sites of Amb from ctDNA minor grooves to major grooves. 

All quenching processes were governed by a dynamic mechanism. Additionally, Amb did not stabilize or induce considerable conformational changes in ctDNA and ctDNA-H1 complex upon binding. In silico molecular docking results confirmed that Amb was bound to the double-helical ctDNA and ctDNA-H1 via ctDNA grooves. In summary, some binding properties of the interactions between Amb and ctDNA change in the presence of the linker histone.

The use of mesenchymal stem cells in malignancies has attracted much attention due to their ability to deliver anticancer agents to tumors, including cytokines, chemokines, etc. This study aimed to investigate the effect of MSCs on the neuroblastoma SH-SY5Y cells through proliferation/apoptosis, senescence assessment, telomere length, and telomerase activity in vitro. BAX and BCL2 were also examined as potential signaling pathways in this process. For this reason, two cell populations (MSCs and SH-SY5Y cells) were co-cultured on trans-well plates for 7 days. In a subsequent step, SH-SY5Y cells were harvested from both control and experimental groups and subjected to flow cytometry, ELISA, real-time PCR, PCR-ELISA TRAP assay, and Western blotting assay for Ki67/Caspase3 investigation, β-Galactosidase assessment, telomere length, and telomerase activity assay. Also, expression of genes and proteins through real-time PCR and Western blotting demonstrated the involvement of the aforementioned signaling pathways in this process. It was found that MSCs contributed significantly to decrease and increase of Ki-67 and Caspase-3, respectively. Also, MSCs dramatically reduced the length of telomere and telomerase activity and increased the β-Galactosidase activity in a significant manner. In addition, significant increase and decrease were also seen in BAX and BCL2 gene and protein expressions, respectively.  These findings revealed that close interaction between MSCs and neuroblastoma cells causes inhibition of the SH-SY5Y cell proliferation and promotes cell senescence via BAX and caspase-3 cascade pathways.

Ischemic stroke is a disease with complex pathogenesis that requires timely and rational pharmacological intervention. One possible treatment for this condition may be to improve mitochondrial function as part of neuroprotective therapy. Cerebral ischemic damage was reproduced by middle cerebral artery permanent occlusion in Wistar male rats. 4-hydroxy-3,5-di-tretbutyl cinnamic acid was injected intraperitoneally in dose range of 25 mg/kg, 50 mg/kg, and 100 mg/kg. The time of administration was 3 days from the ischemia modeling. Further, changes in the rats’ cognitive functions in the Morris water maze test were evaluated, and the state of mitochondrial function in the hippocampal tissue was studied. The study showed that the use of the studied compound dose-dependently improved mitochondrial function in the rat hippocampus. At doses of 20 mg/kg and 50 mg/kg, administration of the test substance increased citrate synthase activity by 55.1% (P<0.05) and 43.4% (P<0.05), respectively and ATP content by 25.7% (P<0.05) and 23.9% (P<0.05). Also, the intensity of oxidative stress (activity of antioxidant enzymes increase whereas the concentration of TBARS reduces) and apoptosis (calcium content, concentration of apoptosis-inducing factor, and caspase-3 activity decrease; latent time of mitochondrial transition permeability pore opening increase) decreased against the background of administration of the test compound. At a dose of 100 mg/kg, the studied compound showed less effectiveness. Administration of 25 mg/kg and 50 mg/kg 4-hydroxy-3,5-di-tretbutyl cinnamic acid demonstrated neuroprotection action on hippocampal cells under the conditions of irreversible brain ischemia.

Delayed anti-inflammatory responses and scar-formation are the main causes for inability of injured body parts such as phalanges to regrow in mammals. Salamanders can regenerate fully scar-free body structures, followed by the appearance of anti-inflammatory responses at the injured site immediately after amputation. This study aimed to evaluate the local regenerative effects of direct amplified anti-inflammatory signals on regeneration of amputated mice digit tips using M2c-macrophages in a co-cultured organ system for the first time.  We used the amputated digits from the paws of 18.5E day old C57BL/6J mice. Monocytes were obtained from peripheral blood and co-cultured with amputated digits, which subsequently enhanced the M2c macrophage phenotype induced by IL-10. We also examined the regenerative effects of IL-10 and transcription growth factor-beta 1 (TGF-β1).  The regrowth of new tissue occurred 10 days post-amputation in all groups. This regrowth was related to enhanced Msh homeobox-1 (Msx1), Msh homeobox-2 (Msx2), and bone morphogenic protein-4 (Bmp4) genes. Increased expression of fibroblast growth factor-8 (Fgf-8) also increased the proliferation rate. Histological analyses indicated that epidermal-closure occurred at 3-dpa in all groups. We observed full digit tip regeneration in the co-cultured group. Particularly, there was new tissue regrowth observed with 40 µg/ml of IL-10 and 120 µg/ml of TGF-β. In contrast, the control group had no remarkable digit elongation. We propose that a direct amplified anti-inflammatory response at the digit injury site can regenerate epithelial and mesenchymal tissues, and might be useful for limb regeneration without scar formation in adult mammals.