Vitamin D and its binding protein in patients with leiomyomas.

AIM: This study examined the levels of VitD, VitD binding protein (DBP), and free VitD in leiomyomas patients and their association with the quantity, dimensions, and site of fibroid growths. Additionally, we evaluated the potentiality of employing these factors as a biomarker tool for the diagnosis and assessment of uterine fibroid progression. METHODS: This study involved the participation of 55 women with leiomyomas and 50 healthy women. We utilized commercial ELISA kits to measure the levels of total VitD and DBP in their serum. Additionally, we calculated the levels of free VitD and the ratio of VitD to DBP. Moreover, we determined the number, size, and location of the leiomyomas in the patients. RESULTS: There were no significant differences in the levels of total VitD between the groups. However, patients had significantly lower levels of free VitD and higher levels of DBP compared to the control group. The size of the largest leiomyomas showed a negative relationship with free VitD and a positive relationship with DBP. Receiver operating characteristic analyses, showed that the cut-off value for free VitD was 4.47 pg/mL, with a sensitivity of 75.6% and a specificity of 74.4%. The cut-off value for DBP was 256.2 µg/mL, with a sensitivity of 86% and a specificity of 70.3%. CONCLUSIONS: Free VitD and DBP potentially contribute to the development of leiomyomas and are linked to the size of these tumors. The measurement of serum levels of these factors could serve as additional biomarkers for the diagnosis of leiomyomas.

Biochanin-A has antidiabetic, antihyperlipidemic, antioxidant, and protective effects on diabetic nephropathy via suppression of TGF-ß1 and PAR-2 genes expression in kidney tissues of STZ-induced diabetic rats.

One of the major complications of diabetes is diabetic nephropathy, and often many patients suffer from diabetic nephropathy. That is why it is important to find the mechanisms that cause nephropathy and its treatment. This study was designed to examine the antidiabetic effects of biochanin A (BCA) and evaluate its effects on oxidative stress markers and the expression of transforming growth factor-ß1 (TGF-ß1) and protease-activated receptors-2 (PAR-2) genes in the kidney of type 1 diabetic rats. After induction of diabetes using streptozotocin (STZ), 55 mg/kg bw dose, rats were randomly divided into four groups with six rats in each group as follows: normal group: normal control receiving normal saline and a single dose of citrate buffer daily; diabetic control group: diabetic control receiving 0.5% dimethyl sulfoxide daily; diabetic+BCA (10 mg/kg) group: diabetic rats receiving biochanin A at a dose of 10 mg/kg bw daily; diabetic+BCA (15 mg/kg) group: diabetic rats receiving biochanin A at a dose of 15 mg/kg bw daily. TGF-ß1 and PAR-2 gene expression was assessed by real-time. Spectrophotometric methods were used to measure biochemical factors: fast blood glucose (FBG), urea, creatinine, albumin, lipids profiles malondialdehyde (MDA), and superoxide dismutase (SOD). The course of treatment in this study was 42 days. The results showed that in the diabetic control group, FBG, serum urea, creatinine, expression of TGF-ß1 and PAR-2 genes, and the levels of MDA in kidney tissue significantly increased and SOD activity in kidney tissue and serum albumin significantly decreased compared to the normal group (p < 0.001). The results showed that administration of biochanin A (10 and 15 mg/kg) after 42 days significantly reduced the expression of TGF-ß1 and PAR-2 genes and FBG, urea, creatinine in serum compared to the diabetic control group (p < 0.001), also significantly increased serum albumin compared to the diabetic control group (p < 0.001). The level of MDA and SOD activity in the tissues of diabetic rats that used biochanin A (10 and 15 mg/kg) was significantly reduced and increased, respectively, compared to the diabetic control group (p < 0.001). Also, the result showed that in the diabetic control group lipids profiles significantly is disturbed compared to the normal group (p < 0.001), the results also showed that biochanin A (10 and 15 mg/kg) administration could significantly improved the lipids profile compared to the control diabetic group (p < 0.001). It is noteworthy that it was found that the beneficial effects of the biochanin A were dose dependent. In conclusion, administration of biochanin A for 42 days has beneficial effect and improves diabetes and nephropathy in diabetic rats. So probably biochanin A can be used as an adjunct therapy in the treatment of diabetes.

Integrin α2ß1 inhibition attenuates prostate cancer cell proliferation by cell cycle arrest, promoting apoptosis and reducing epithelial-mesenchymal transition.

Integrin α2ß1 plays an important role in cellular migration and metastasis processes associated with prostate cancer. The aim of this study was to assess whether selective inhibition of integrin α2ß1 is an effective strategy to target metastatic prostate cancer cells. In this regard, we examined the effects of the inhibitor BTT-3033, which selectively interferes with the connection between integrin a2b1 and its ligand, on migration, epithelial-mesenchymal transition (EMT), cell cycle arrest, apoptosis, and specific intracellular signaling pathways using LNcap-FGC and DU-145 prostate cancer cell lines. Western blot analysis and immunocytochemistry assays showed that inhibition of integrin a2b1 inhibits EMT, through the increased expression of E-cadherin and decreased expression of N-cadherin and vimentin. Scratch wound healing assays revealed a direct effect on integrin α2ß1 in the migration capacity of cells. In addition, treatment with BTT-3033 induced a reduction in cell viability and proliferation, as assessed by MTT and BrdU assays. In addition, the results show that BTT-3033 inhibits cell proliferation by inducing G1 cell cycle arrest. Moreover, inhibition of integrin α2ß1 induces apoptosis through the activation of ROS, Bax protein upregulation, caspase-3 activation, and depletion of ΔΨm.  Molecular signaling studies showed that integrin α2ß1 was a positive regulator of MKK7 phosphorylation. In conclusion, our results reveal a critical role for integrin a2b1 in the proliferation of prostate cancer cells, as demonstrated by EMT inhibition, cell cycle arrest, and apoptosis induction in response to treatment with its specific inhibitor BT-3033.

Inhibition of didscoidin domain receptor 1 reduces epithelial-mesenchymal transition and induce cell-cycle arrest and apoptosis in prostate cancer cell lines.

Didscoidin domain receptor 1 (DDR1) is involved in the progression of prostate cancer metastasis through stimulation of epithelial-mesenchymal transition (EMT). So DDR1 inhibition can be a helpful target for cancer metastasis prevention. So, we studied the effects of DDR1 inhibition on EMT as well as induction of cell-cycle arrest and apoptosis in prostate cancer cell lines. DDR1 expression was evaluated using reverse-transcription polymerase chain reaction and western blot analysis. The EMT-associated protein expression was determined using the western blot analysis and immunocytochemistry following treatment with various concentrations of DDR1 inhibitor. The activation of DDR1 and also downstream-signaling molecules Pyk2 and MKK7 were determined using western blot analysis. Cell survival and proliferation after DDR1 inhibition were evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide, bromodeoxyuridine, and colony formation assays. Flow cytometry analysis was used to determine the effects of DDR1 inhibition on cell-cycle arrest and apoptosis using annexin V/propidium iodide-based flow cytometry. Results showed that the protein expression of N-cadherin and vimentin were decreased whereas protein expression of E-cadherin was increased after DDR1 inhibition. Results of our western blot analysis indicated that DDR1 inhibitor effectively downregulated P-DDR1, P-Pyk2, and P-MKK7 levels. This result also showed that DDR1 inhibition decreased cell survival and proliferation, induced G1 cell-cycle arrest, induced apoptosis by an increase in the Bax/Bcl-2 ratio and depletion of the mitochondrial membrane potential, and also by reactive oxygen species creation in prostate cancer cells. These data show that DDR1 inhibition can result in the EMT prevention via inhibition of Pyk2 and MKK7 signaling pathway and induces cell-cycle arrest and apoptosis in prostate cancer cell lines. Thus, this study identifies DDR1 as an important target for modulating EMT and induction of apoptosis in prostate cancer cells.

Antioxidant properties of Trifolium resupinatum and its therapeutic potential for Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is the most common cause of dementia and is characterized by a progressive deterioration in cognitive function, which typically begins with impairment in memory. Persian clover (Trifolium resupinatum) is an annual plant found in central Asia. Due to its contents (high flavonoid and isoflavones), extensive researches have been done on its therapeutic properties, such as multiple sclerosis (MS) treatment. In this study, we investigate the neuroprotective effects of this plant on Streptozotocin (STZ)-induced AD in rats. MATERIAL AND METHODS: This research aimed to evaluate the neuroprotective effect of Trifolium resupinatum on the spatial learning and memory, superoxide dismutase (SOD), expressions of ß amyloid 1-42 (Ab 1-42 ), and b amyloid 1-40 (Ab 1-40 ) in the hippocampus of STZ-induced Alzheimer rats. RESULTS: Our data showed that Trifolium resupinatum extract administration for two weeks before and one week after AD induction significantly improves maze escape latency ( p = 0.027, 0.001 and 0.02 in 100, 200, and 300 mg of the extract, respectively) and maze retention time ( p = 0.003, 0.04 and 0.001 in 100, 200, and 300 mg of the extract, respectively). Also, the administration of this extract significantly increases the SOD levels from 1.72+0.20 to 2.31+0.45 ( p = 0.009), 2.48+0.32 ( p = 0.001) and 2.33+0.32 ( p = 0.007) and decreases the expressions of Ab 1-42 ) ( p = 0.001 in all concentrations of the extract) and Ab 1-40 ) ( p = 0.001 in all concentrations of the extract) in the rat's hippocampus. CONCLUSIONS: This study suggests that the alcoholic extract of Trifolium resupinatum has anti-Alzheimer and neuroprotective effects on rats.

Allium jesdianum hydro alcoholic extract ameliorates diabetic nephropathy by suppressing connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) gene expression in diabetic rats with streptozotocin.

OBJECTIVES: Diabetic nephropathy is one of the major complications of diabetes, the use of medicinal plants is increasing due to fewer side effects. This study was designed to examine antidiabetic effects of Allium jesdianum (A. jesdianum) ethanolic extract and evaluate its effects on oxidative stress markers and the expression of connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) genes in the kidney of type 1 diabetic rats. METHODS: In this study, we randomly divided 24 rats into four groups with six rats in each group as follows: Cnt group: normal control receiving normal saline, Dibt group: diabetic control receiving normal saline daily, Dibt + A. jesdianum 250 group: diabetic rats receiving A. jesdianum at a dose of 250 mg/kg bw daily, Dibt + A. jesdianum 500 group: diabetic rats receiving A. jesdianum at a dose of 500 mg/kg bw daily. To induce diabetes, we used 55 mg/kg bw dose of streptozotocin intraperitoneally. The concentration of fasting blood glucose (FBG) and serum urea, creatinine and albumin, SOD, MDA (using spectrophotometric methods) and gene expression of CTGF and RAGE in kidney tissue (using real-time PCR methods) were quantified in the diabetic rats that received A. jesdianum for 42 days, and were compared to control rats. RESULTS: The results showed that in the diabetic group the FBG and serum urea, creatinine and expression of kidney CTGF and RAGE genes and the levels of SOD and MDA significantly increased and serum albumin significantly decreased compared to the Cnt group (p<0.001). Administration of A. jesdianum significantly improved the FBG and serum urea, creatinine and albumin compared to Dibt group (p<0.05). It was shown the A. jesdianum significantly decrease the kidney expression levels of CTGF and RAGE genes and improve oxidative stress (increased SOD and decreased MDA) in the kidney tissues when compared to Dibt group (p<0.001). Also, it was found that the beneficial effects of the A. jesdianum were dose-dependent. CONCLUSIONS: The results of this study showed that administration of A. jesdianum for 42 days has beneficial anti-diabetic and anti-nephropathic effects in diabetic rats and can be used as an adjunct therapy in the treatment of diabetes.

Serum transforming growth factor ß and leucine-rich α-2-glycoprotein 1 as potential biomarkers for diagnosis of uterine leiomyomas.

BACKGROUND AND AIM: Transforming growth factor ß (TGF-ß) and leucine-rich α-2-glycoprotein 1 (LRG1) play significant roles in the pathogenicity of uterine leiomyomas (ULMs). The current study aimed to assess the diagnostic values of serum TGF-ß and LRG1 in terms of the presence and severity of ULMs. METHODS: Premenopausal women with ULMs (n=44) together with age-adjusted ULM-free individuals (n=41) were incorporated into the study. ULMs were detected and evaluated using transvaginal ultrasonography. Serum levels of TGF-ß and LRG1 were quantified by enzyme-linked immunosorbent assay. RESULTS: Mean concentrations of serum TGF-ß and LRG1 were significantly higher in the group of patients with ULMs compared to the control group (p<0.05). The volume of the largest leiomyoma was positively correlated with the levels of TGF-ß (r = 0.414, p= 0.005) and LRG1 (r = 0.341, p= 0.023). The receiver-operating characteristics analysis demonstrated moderate and robust values of area under the curve for TGF-ß (0.755) and LRG1 (0.90), respectively. CONCLUSION: Increases in serum levels of TGF-ß and LRG1 is associated with the incidence and severity of ULMs. LRG1 in particular but also TGF-ß may be able to serve as reliable biomarkers for the diagnosis and monitoring of ULMs.

Efficient refolding of recombinant lipase from Escherichia coli inclusion bodies by response surface methodology.

An experimental design was employed to optimize the refolding conditions of a recombinant lipase from Pseudomonas sp. which expressed as inclusion bodies in Escherichia coli. The effects of several variables on the refolding and activation of the enzyme has been studied. Because of the complexity of the reaction with respect to the number of parameters that can affect the refolding efficiency, 2(6-1) half-fractional factorial design (H-FFD) was employed for initial screening of the factors, potentially influencing the response. Experiments were performed in triplicate at two levels. Subsequently, the selected factors were subjected to response surface methodology (RSM) with a four factor-five coded level central composite design (CCD), using Quadratic model for obtaining the optimum values for the factors. The adequacy of the calculated model was confirmed by the coefficient of determination (R(2)) and F value of 0.89 and 9.12, respectively. The optimized condition for the refolding was obtained in the refolding buffer containing unfolded lipase (10 microg/ml) and foldase (3 microg/ml) in combination with glycerol (10%), NaCl (1M) and sucrose (0.5M). Using chemicals in combination with foldase under the optimal condition exhibited a 50% increase in refolding yield over the conventional method.

Down-Regulation of DDR1 Induces Apoptosis and Inhibits EMT through Phosphorylation of Pyk2/MKK7 in DU-145 and Lncap-FGC Prostate Cancer Cell Lines.

BACKGROUND: In cancer cells, re-activation of Epithelial-Mesenchymal Transition (EMT) program through Discoidin Domain Receptor1 (DDR1) leads to metastasis. DDR1-targeted therapy with siRNA might be a promising strategy for EMT inhibition. Therefore, the aim of this study was to investigate the effect of DDR1 knockdown in the EMT, migration, and apoptosis of prostate cancer cells. For this purpose, the expression of DDR1 was down regulated by the siRNA approach in LNcap-FGC and DU-145 prostate cancer cells. METHODS: Immunocytochemistry was carried out for the assessment of EMT. E-cadherin, N-cadherin, Bax, Bcl2, and the phosphorylation level of Proline-rich tyrosine kinase 2 (Pyk2) and Map Kinase Kinase 7 (MKK7) was determined using the western blot. Wound healing assay was used to evaluate cell migration. Flow cytometry was employed to determine the apoptosis rate in siRNA-transfected cancer cells. RESULTS: Our findings showed that the stimulation of DDR1 with collagen-I caused increased phosphorylation of Pyk2 and MKK7 signaling molecules that led to the induction of EMT and migration in DU-145 and LNcap- FGC cells. In contrast, DDR1 knockdown led to significant attenuation of EMT, migration, and phosphorylation levels of Pyk2 and MKK7. Moreover, DDR1 knockdown via induction of Bax expression and suppression of Bcl-2 expression induces apoptosis. CONCLUSION: Collectively, our results indicate that the DDR1 targeting with siRNA may be beneficial for the inhibition of EMT and the induction of apoptosis in prostate cancer.

Histopathological and cytological analyses of endometrium in water buffaloes (Bubalus bubalis) to detect estrus and endometritis.

This study aimed to determine cytological, histopathological and cytomorphometrical characteristics of endometrium in healthy and endometritic uterus in the water buffalo. Fifty eight non-pregnant reproductive systems were collected from slaughterhouse. Efficiency of three methods of sampling including cotton swab, smear, and aspiration were compared for cytologic study. Concurrent histopathologic examination revealed endometritis in 38 uteri including 8 (21.00%) with mild endometritis, 7 (18.42%) with moderate endometritis, 6 (15.90%) with severe endometritis and 17 (44.73%) with chronic endometritis. Cyto-morphometrical results showed significant relationship between diameter and area of epithelial nuclei with phases of estrus cycle. Neutrophil and lymphocytes densities in swab and aspiration samples were significantly higher in severe endometritis than normal and chronic endometritis samples. Similarly, lymphocytes density in smear and aspiration methods was significant between normal and moderates, and also severe and chronic endometritis. Cytomorphometric analysis of epithelial nuclei characteristics (diameter and area) in buffalo were performed for the first time and it could be valuable to identify estrus cycle in this species. Aspiration had the most efficiency to identify endometritis in comparison with other methods.