The KLF14 Variant is Associated with Type 2 Diabetes and HbA1C Level.

The purpose of this study was to scan variants in coding region of Krȕppel like factor14 (KLF14) locus and assess association related to type 2 diabetes (T2D) in Iranian population. We sequenced the coding region of KLF14 to scan variants in case-sibling study (92 individuals with T2D and 92 healthy older siblings). To confirm, we analyzed rs76603546 association with T2D in a larger unrelated case-control study by PCR-RFLP (475 cases and 512 controls). We analyzed the association of rs76603546 with HbA1C, BMI, fat mass, waist circumference, fasting glucose, cholesterol and HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) using one-way ANOVA analysis. Also, association of genotypes with T2D adjusted for confounding variables was analyzed using logistic regression. HaploReg v 4.1 was used to predict rs76603546 possible function. Sequencing results analysis revealed the association of C allele of rs76603546, synonymous variant C>T, [OR 2.10 (1.38-3.20), P value < 0.001] and CC genotype of rs76603546 [OR 4.3 (1.79-10.23), P value = 0.001] with susceptibility to T2D. PCR-Restriction Fragment Length Polymorphism (RFLP) results analysis confirmed the association of rs76603546 with T2D [C allele, OR 1.91 (1.59-2.29), P value = 0.002, CC genotype, OR 3.27 (2.26-4.73), P value = 0.002 and TC genotype, OR 1.74 (1.31-2.31), P value = 0.001]. The CC genotype of rs76603546 is associated with HbA1C level (P value < 0.001) and BMI (P value = 0.02). After adjustment with confounding variables, we observed association of CC genotype with T2D [OR 2.542 (1.25-3.77), P value = 0.03]. Among over 220 SNPs, rs76603546 was associated with T2D, HbA1C and BMI in our study.

Improvement of fertility parameters with Tribulus Terrestris and Anacyclus Pyrethrum treatment in male rats.

OBJECTIVE: Anacyclus Pyrethrum (AP) and Tribulus Terrestris (TT) have been reported as male infertility treatment in several studies; however, in Iranian traditional medicine these two plants are prescribed simultaneously. In this study, we aimed to determine the effects of AP and TT extracts both separately and simultaneously on the male Wistar rat fertility parameters. MATERIALS AND METHODS: 32 male Wistar rats were divided into 4 groups: Control, TT, AP, and AT treated groups. Treatment continued for 25 days and rats were weighed daily. Their testes were dissected for histological studies. Sperm analysis including sperm count, viability and motility were performed. Serum was obtained to evaluate testosterone, LH and FSH levels. Histological studies were conducted to study Leydig, and Sertoli cells, spermatogonia and spermatid cell numbers, and to measure seminiferous diameter and epithelium thickness. RESULTS: Sperm count increased in all the treatment groups. Sperm viability and motility in AT and AP groups were elevated. TT and AT groups showed signifi cantly increased testosterone level compared to control group (P=004, P=0.000, respectively) and TT, AP and AT treatment groups showed increased LH level (P=0.002, P=0.03 and P=0.000, respectively) compared to control, while only AT group showed increased FSH (p=0.006) compared to control. Histological studies showed signifi cant increase of spermatogonia, Leydig and Sertoli cell numbers and epithelial thickness in AT group compared to other groups. All the treatment groups had higher number of Leydig, spermatogonia and spermatid cells. CONCLUSION: TT and AP improved sexual parameters; however, their simultaneous administration had higher improving effects on studied parameters.

Improvement of fertility parameters with tribulus terrestris and anacyclus pyrethrum treatment in male rats

ABSTRACT Objective Anacyclus Pyrethrum (AP) and Tribulus Terrestris (TT) have been reported as male infertility treatment in several studies; however, in Iranian traditional medicine these two plants are prescribed simultaneously. In this study, we aimed to determine the effects of AP and TT extracts both separately and simultaneously on the male Wistar rat fertility parameters. Materials and Methods 32 male Wistar rats were divided into 4 groups: Control, TT, AP, and AT treated groups. Treatment continued for 25 days and rats were weighed daily. Their testes were dissected for histological studies. Sperm analysis including sperm count, viability and motility were performed. Serum was obtained to evaluate testosterone, LH and FSH levels. Histological studies were conducted to study Leydig, and Sertoli cells, spermatogonia and spermatid cell numbers, and to measure seminiferous diameter and epithelium thickness. Results Sperm count increased in all the treatment groups. Sperm viability and motility in AT and AP groups were elevated. TT and AT groups showed significantly increased testosterone level compared to control group (P=004, P=0.000, respectively) and TT, AP and AT treatment groups showed increased LH level (P=0.002, P=0.03 and P=0.000, respectively) compared to control, while only AT group showed increased FSH (p=0.006) compared to control. Histological studies showed significant increase of spermatogonia, Leydig and Sertoli cell numbers and epithelial thickness in AT group compared to other groups. All the treatment groups had higher number of Leydig, spermatogonia and spermatid cells. Conclusion TT and AP improved sexual parameters; however, their simultaneous administration had higher improving effects on studied parameters.

Prevention and treatment of experimental autoimmune encephalomyelitis induced mice with 1, 25-dihydroxyvitamin D3.

Multiple sclerosis (MS) is a complex inflammatory and demyelinating disease of the central nervous system (CNS) frequently starts in young adulthood. Demyelination, inflammatory and axonal damage in the CNS is the pathological hallmark of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. 1, 25-dihydroxyvitamin D3 (Vitamin D3) is involved in calcium regulation, phosphorus homeostasis, and bone mineralization. In addition, vitamin D3 has potential inhibitory effects on immune cells in various inflammatory and autoimmunity disease. C57BL/6 female mice were divided into prevention groups (low, middle and high doses) and treatment groups (middle and high doses). Prevention groups received vitamin D3 2 weeks before EAE induction, and treatment groups were treated with vitamin D3 simultaneous with EAE induction. Vitamin D3 inhibits the development of EAE in a dose-dependent manner. Histological studies revealed reduced demyelination and limited infiltration into CNS, moreover vitamin D3 increased the production of IL-4, IL-10, and TGF-ß, while a significant reduction in the production of IFN-γ, IL-6, TNF-α, and IL-17 was observed. Flow cytometry results for CD4+ T cell subsets in compliance with ELISA cytokine assay results showed a significant decrease in the percentage of Th1 and Th17, but also a significant increase in the percentage of Th2 and Treg for middle and high dose vitamin D3 treated mice. Real-time PCR results indicated that middle and high dose vitamin D3 treatment reduced T-bet and ROR-γt expression, but enhanced GATA3 and Foxp3 expression. Real-Time PCR results in CNS for T cell subsets related cytokines and transcription factors supported the results of flow cytometry and ELISA. This study indicated that middle and high doses of vitamin D3 deviate the balance between Th1/Th2 and Th17/Treg to Th2 and Treg. Moreover, vitamin D3 could reduce the incidence and severity of EAE clinical disease.

Downregulation of miR-542-3p Contributes to Apoptosis Resistance in Dermal Fibroblasts from Systemic Sclerosis Patients via Survivin Overexpression.

Systemic sclerosis (SSc) is characterized by excessive production of collagens by fibroblasts that leads to vast fibrosis. Resistance to apoptosis is one of the possible underlying mechanisms of fibrosis in these patients. Survivinis involved in inhibition of apoptosis and aberrantly functions in SSc. Since dysregulation of survivin-targeting microRNAs (miRNAs) has frequently been observed in cancer and some autoimmune disorders, this study aimed to investigate their expression status in dermal fibroblasts from SSc patients. DiffuseSSc patients were selected according to American College of Rheumatology criteria. Isolated fibroblasts from 10 SSc and 10 healthy skin biopsies were cultured. After examining purity of the cells, mRNA and miRNAs extraction was performed followed by complementary DNA (cDNA) synthesis. Relative expressions ofsurvivin mRNA, miR-16-5p, miR-320a, miR-218-5p, miR-708-5p and miR-542-3p were analyzed using real time PCR. Survivin mRNA expression was significantly 1.85-fold upregulated in fibroblasts from SSc patients compared with healthy controls (p=0.046). Among the studied miRNAs, miR-542-3p expression was significantly decreased (p=0.033), while enhanced expression of miR-708-5p was observed in SSc fibroblasts (p=0.05) in comparison to healthy subjects. Downregulation of miR-542-3p significantly correlated with survivin overexpression (r=-0.45, p=0.049). Downregulation of miR-542-3p that is correlated with higher surviving expression levels might be a possible cause of apoptosis resistance in SSc fibroblasts, hence providing a new understanding of the disease pathogenesis.

Altered expression patterns of complement factor H and miR-146a genes in acute-chronic phases in experimental autoimmune encephalomyelitis mouse.

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.

Overexpression of apoptosis-related protein, survivin, in fibroblasts from patients with systemic sclerosis.

BACKGROUND/OBJECTIVES: Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are resistant to apoptosis. Previous studies have shown that survivin, a member of inhibition of apoptosis (IAP) family, plays an important role in apoptosis resistance. Accordingly, we decided to study the expression of the most important members of IAP family in SSc fibroblasts, which can block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. METHOD: Skin biopsy samples were obtained from 19 patients with diffuse cutaneous SSc (DcSSc) and 16 healthy controls. Dermal fibroblasts were cultured and the total RNA was isolated from cells followed by cDNA synthesis. Real-time PCR was performed using SYBR Green PCR master mix and specific primers for cIAP1, cIAP2, XIAP, and Survivin mRNA quantification. RESULTS: A significantly increased expression level of Survivin was observed in fibroblasts from SSc patients compared to controls (2.26-fold, P = 0.04). However, mRNA expression of cIAP1, cIAP2, and XIAP did not change significantly between cases and controls. CONCLUSIONS: Our results showed that survivin is upregulated in SSc skin fibroblast which may lead to resistance to apoptosis. Further studies should be performed to reveal the role of survivin in apoptosis pathway of SSc fibroblasts.

Expressions of p53 and PUMA in fibroblasts of systemic sclerosis patients are normal at transcription level.

BACKGROUND: Systemic sclerosis (SSc) fibroblasts show resistance apoptosis mechanisms, which enhances the fibrosis stage of the disease. Impaired function of p53 upregulated modulator of apoptosis (PUMA) has been related to deficits in p53-dependant apoptosis pathway. This study aimed to evaluate the transcriptional levels of p53 and PUMA mRNAs in fibroblasts from SSc patients and compare it with healthy individuals. METHODS: In this case-control study, skin biopsy samples were obtained from 19 patients with diffuse cutaneous SSc (DcSSc) and 16 healthy controls. Afterward, dermal fibroblasts were isolated and cultured. After extraction of total RNA from cultured fibroblasts, complementary DNA (cDNA) was synthesized. mRNA quantification was carried out using real-time PCR, SYBR Green PCR master mix, and specific primers for p53 and PUMA. RESULTS: No significant alteration was observed in mRNA expression levels of p53 and PUMA (P = .99 and .23, respectively) in fibroblasts from SSc patients compared with controls. CONCLUSIONS: Apoptosis pathways are impaired in fibroblasts from patients with SSc, leading to chronic fibrosis. Nonetheless, PUMA/p53 pathway may not be involved in dysfunction of apoptosis mechanisms in fibroblasts of patients with SSc.

Hesperidin ameliorates immunological outcome and reduces neuroinflammation in the mouse model of multiple sclerosis.

Multiple sclerosis (MS) is the most abundant central nervous system (CNS) inflammatory disease, which is due to the reaction of auto reactive T cells with own myelin proteins, leading to physical disorder and paralysis among people suffering the disease. Hesperidin, a flavanone glycoside found abundantly in citrus fruits possesses a wide range of pharmacological properties including potential anti-inflammatory and anti-cancer effects. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of hesperidin on MS alleviation. Female C57BL/6 mice were immunized with MOG35-55. Clinical scores and other parameters were monitored daily for the 21days. At the end of the period, brain/spinal cord histology was performed to measure lymphocyte infiltration; T-cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed using real-time PCR; T cell differentiation was evaluated via flow cytometry. The results demonstrated that hesperidin inhibited development of EAE. Histological studies revealed limited leukocyte infiltration into the CNS. Hesperidin increased Treg cells production of interleukin IL-10 and transforming growth factor (TGF)-ß, but concurrently resulted in a significant reduction in production of IL-17 and IL-6. Flow cytometry revealed there were also significant decreases in the percentages of Th17 cells, as well as significant increase in percentages of Treg cells in the spleen and lymph nodes. Real-time PCR results indicated hesperidin treatment reduced ROR-γt factor expression, but enhanced Foxp3 expression. Collectively, these results demonstrated that hesperidin could reduce the incidence and severity of disease.

Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice.

Estrogen is a neuro-protective hormone in various central nervous system (CNS) disorders. The present study evaluated the role of estrogen during experimental autoimmune encephalomyelitis (EAE) at doses selected to mimic any suppressive potential from the hormone during pregnancy. Here, mice were ovariectomized and then 2 weeks later treated with MOG antigen to induce EAE. Concurrently, mice then received (subcutaneously) an implanted pellet to deliver varying estrogen amounts over a 21-day period. Clinical scores and other parameters were monitored daily for the 21 days. At the end of the period, brain/spinal cord histology was performed to measure lymphocyte infiltration; T-cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed using real-time PCR; T-cell differentiation was evaluated via flow cytometry. The results demonstrated that estrogen inhibited development of EAE. Histological studies revealed limited leukocyte infiltration into the CNS. High and medium dose of estrogen increased TH2 and Treg cell production of interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-ß, but concurrently resulted in a significant reduction in production of interferon (IFN)-γ, IL-17, and IL-6. Flow cytometry revealed there were also significant decreases in the percentages of TH1 and TH17 cells, as well as significant increase in percentages of Treg and TH2 cells in the spleen and lymph nodes. Real-time PCR results indicated that high- and medium-dose estrogen treatments reduced T-bet and ROR-γt factor expression, but enhanced Foxp3 and GATA3 expression. Collectively, these results demonstrated that a medium dose of estrogen - similar to a pregnancy level of estrogen - could potentially reduce the incidence and severity of autoimmune EAE and possibly other autoimmune pathologies.

Pregnancy level of estrogen attenuates experimental autoimmune encephalomyelitis in both ovariectomized and pregnant C57BL/6 mice through expansion of Treg and Th2 cells.

Pregnancy suppressive effect on autoimmune diseases including Multiple Sclerosis and Rheumatoid Arthritis may result from high levels of sex steroids such as estrogen and estriol. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of estrogen on MS alleviation. Female C57BL/6 mice were immunized with MOG35-55. Clinical scores and other relevant parameters were monitored daily. Brain and spinal cord histology was performed to measure lymphocyte infiltration and central nervous system demyelination. Th1/Th2/Th17 and Treg cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed by real-time PCR and T cell differentiation was explored through flow cytometry examination. Pregnancy and pregnancy level of estrogen alleviated clinical manifestations in EAE induced mice, reduced CNS demyelination and cell infiltration, suppressed spleen T cell proliferation, enhanced production of anti-inflammatory cytokines in splenocytes and increased the percentage of Th2 and Treg cells. Furthermore, the results of real-time PCR for transcription factors and related cytokines of Th1/Th2/Th17 and Treg cells in CNS showed reduced expression levels of Th1 and Th17 transcription factors, including T-bet and ROR-γt, and decreased Th1 and Th17 cytokines including IFN-γ, TNF-α, IL-17 and IL-23. These results are the first to indicate that pregnancy and pregnancy level of estrogen ameliorate the EAE condition by favoring Treg and Th2 differentiation through induced expression of Foxp3 and GATA3 in the CNS. Moreover, pregnancy and pregnancy level of estrogen decreased mRNA levels of T-bet and ROR-γt in the CNS.