Folic acid and melatonin mitigate diabetic nephropathy in rats via inhibition of oxidative stress.

BACKGROUND: Diabetes mellitus is a global epidemic leads to multiple serious health complications, including nephropathy. Diabetic nephropathy is a serious kidney-related complication of type 1 or 2 diabetes that is prevalent in almost 40% of the people with diabetes. We examined whether folic acid and melatonin can reduce progression of nephropathy in rats of type 1 diabetes mellitus by controlling the level of oxidative stress, glucose, lipids, and cytokines. METHODS: Forty-two male albino rats were distributed into six groups, (n = 7 per group). Five of the groups were induced with diabetes by a single intraperitoneal injection of freshly prepared streptozotocin at a dose of 50 mg/kg body weight. After the induction of diabetes, the rats were treated with folic acid (100 mg/kg) and melatonin (10 mg/kg) separately and in combination daily for 6 weeks, whereas, the other diabetic group was treated with glibenclamide (5 mg/kg). One of the diabetic groups served as a positive control. One-way ANOVA was used to compare those five subfields ability followed by LSD multiple comparisons. RESULTS: The data indicated that diabetes significantly altered the body weight, lipids and kidney function. Diabetic rats exhibited a significant increase in plasma levels of urea, uric acid, creatinine, sodium, tumor necrosis factor alpha (TNF-α), interleukin-6(IL-6), cholesterol, triglycerides, and low-density lipoprotein (LDL). In contrast, plasma total protein, potassium, high-density lipoprotein (HDL) and interleukin-10 (IL-10) decreased significantly in diabetic rats compared to the control rats. Moreover, levels of renal malondialdehyde (MDA) and nitric oxide (NO) were significantly increased while the levels of renal glutathione(GSH), superoxide dismutase(SOD), and catalase (CAT) were significantly decreased in diabetic rats comparison to those in the control rats. Hence, diabetic rats treated with folic acid and melatonin alone as well as in combination showed improvements with respect to the indices in addition to a significant recovery observed via histopathology when compared to the diabetic group. CONCLUSIONS: These results revealed that treatment with folic acid in combination with melatonin in diabetic rats was more effective than treatment with either of folic acid or melatonin alone to alleviate the symptoms of diabetic nephropathy.

Copper Mediates Anti-Inflammatory and Antifibrotic Activity of Gleevec in Hepatocellular Carcinoma-Induced Male Rats.

The elevated level of copper is one of the hallmark features of cancer cells in most of the types of cancer. In the present study, this feature has been targeted to investigate if coadministration of exogenous copper (Cu+) and its chelating agent like disulfiram (DSF+) influence the antineoplastic activity of the anticancer drug, Gleevec (GLV+), in hepatocellular carcinoma (HCC)-induced rats via immunomodulation. After the treatment, the level of proinflammatory interleukins (IL-1, 2, 6, and 7), anti-inflammatory interleukin (IL-10) concomitant with transcription factors (NF-kB and TNF-a), and the apoptotic marker (cleaved PARP) was estimated. The cancer-induced group without treatment (CN+) demonstrated abnormally elevated level of all proinflammatory cytokines and transcription factors concomitant with a compromised level of cleaved PARP as compared to the control normal (CN-). The detailed histological analysis also supported the results exhibiting extensive inflammation and tissue fibrosis confirming the second stage of HCC. Cu+, DSF+, and GLV+ displayed mild improvement in most of the parameters, but the combination group GLV + Cu+ demonstrated remarkable recovery in histology and most of the parameters tended towards the CN- followed by GLV + DSF+. Therefore, the management of copper level is critical in realizing the antineoplastic activity of GLV up to its full potential in cancer treatment. These findings will help in improving chemoimmunotherapy and personalized cancer treatment.

Modulatory roles of proinflammatory cytokines on the expression of cathelicidins in the lower regions of the oviduct of laying hens.

The current study aimed to confirm and examine the physiological roles of the proinflammatory cytokines IL1B and IL6 on the immune functions which mediated by cathelicidins (CATHs) in the uterus and vagina of laying hens. Snaps of the mucosal tissues of uterus and vagina were incubated in culture medium or chicken recombinant IL1B and IL6 for 1.5h or 3h before extraction of total RNA to be used for examination of IL1B and IL6, their receptors, and cathelicidins by semi-quantitative PCR; and to examine the changes in cathelicidins expressions by real-time PCR. PCR analysis confirmed that IL1B and IL6, their receptors, and CATH1-3 were expressed in the mucosal tissues of the uterus and vagina. In uterus tissue, IL1B did not affect the expression of CATH1 and -2 at different doses and incubation time, whereas CATH3 was significantly downregulated by incubation with IL1B for 1.5h. In the vaginal tissue, the expressed CATH1, -2 and -3 were significantly upregulated by incubation with IL1B for 1.5h in a dose-dependent manner. In uterus tissue, CATH1 expression was down-regulate by IL6 incubation for 1.5h, but not by 3h however, CATH3 expression was significantly increased by incubation with IL6 for 1.5h, but not for 3h. In the vaginal tissues, all CATHs expression was not affected significantly by incubation with IL6. These current observations suggest that CATH1, -2 and -3 in the vagina are upregulated by IL1B, and CATH3 in the uterus is also upregulated by IL6. IL1B and IL6 synthesized in response to infection by the microbes may enhance the defense system in the oviduct mucosal tissues by increasing the synthesis of CATHs.

Effects of Virus-associated Molecular Patterns on the Expression of Cathelicidins in the Hen Vagina.

The aim of this study was to examine the expression profiles of the cathelicidins (CATHs) in the oviduct and the effects of Toll-like receptor (TLR) ligands of virus-associated molecular patterns on CATHs expression in the vagina of hens. The mRNA expression of cathelicidins (CATH1, -2, -3 and -B1) in the oviductal mucosa was analyzed by RT-PCR. The effects of viral moleculs on the CATHs expression in the vagina was examined by incubating the mucosal tissue with virus molecular patterns, including poly I:C (dsRNA virus, TLR3 ligand), R848 (ssRNA virus, TLR7 ligand) and CpG-ODN (DNA virus, TLR21 ligand), followed by real-time PCR analysis. The expression of CATH1, CATH2 and CATH3 was identified in all oviductal segments, except for CATH2 which was lacked in the magnum. The expression of CATHB1 was not identified at any segments of the oviduct. Poly I:C down-regulated the expression of CATH1, -2 and -3, whereas R848 up-regulated the expression of CATH1 and CATH3 but down-regulated the expression of CATH2. CpG-ODN did not affect the CATHs expression. These results suggest that mucosal tissues of the oviduct express CATHs to provide the defense mechanism against microbes, and the expression of CATH1 and CATH3 is up-regulated against ssRNA viruses, whereas, dsRNA virus may suppress the expression of CATH1, -2 and -3.

Effects of different TLR ligands on the expression of proinflammatory cytokines and avian ß-defensins in the uterine and vaginal tissues of laying hens.

The immune response in the lower part of the hen oviduct is of crucial importance to protect the oviductal tissues from infection by microorganisms colonizing the cloaca. The aim of this study was to examine whether different TLRs can recognize their ligands to induce expression of proinflammatory cytokines and avian ß-defensins (AvBDs) in the uterus and vagina of laying hens. The mucosal tissues of the uterus and vagina were collected, cultured in TCM-199 medium and stimulated with or without different ligands of TLRs, namely Pam3CSK4 (TLR2), poly I:C (TLR3), flagellin (TLR5), R848 (TLR7), and CpG-ODN (TLR21) and incubated for 3h. The expression of IL1B in the uterus and vagina was upregulated by all TLR ligands tested. The expression of IL6 in the uterus and vagina was upregulated by poly I:C and CpG-ODN, and it was also upregulated by Pam3CSK4 in the uterus and by R848 in the vagina. The expression of AvBD10 was upregulated by poly I:C in the uterus and by flagellin in the vagina. On the other hand, the AvBD10 expression was downregulated by CpG-ODN in the uterus and by R848 in the vagina, whereas its expression was not affected by Pam3CSK4 in both tissues. The expression of AvBD12 in the uterus and vagina was not affected by any TLR ligands except for CpG-ODN, which downregulated its expression in the vagina. These results suggest that TLR2, 3, 5, 7, and 21 in the uterine and vaginal tissues are functionally active in inducing proinflammatory cytokines in response to their specific ligands, although the effect on the expression of AvBDs is limited. Proinflammatory cytokines induced by interaction of TLRs with their ligands may play roles in the defense against infectious microorganisms.

Induction of avian ß-defensins by CpG oligodeoxynucleotides and proinflammatory cytokines in hen vaginal cells in vitro.

Immune function in the vagina of hen oviduct is essential to prevent infection by microorganisms colonizing in the cloaca. The aim of this study was to determine whether CpG oligodeoxynucleotides (CpG-ODN) stimulate the expression of avian ß-defensins (AvBDs) in hen vaginal cells. Specific questions were whether CpG-ODN affects the expression of AvBDs and proinflammatory cytokines and whether the cytokines affect AvBDs expression in vaginal cells. The dispersed vaginal cells of White Leghorn laying hens were cultured and stimulated by different doses of lipopolysaccharide (LPS), CpG-ODN, interleukin 1ß (IL1B), or IL6. The cultured cell population contained epithelial cells, fibroblast-like cells, and CD45-positive leukocytes. The immunoreactive AvBD3, -10, and -12 were localized in the mucosal epithelium in the section of the vagina. The expression of AvBDs, IL1B, and IL6 was analyzed by quantitative RT-PCR. RT-PCR analysis showed the expression of AvBD1, -3, -4, -5, -10, and -12 in the cultured vaginal cells without stimulation. Toll-like receptors (TLRs) 4 and 21, which recognize LPS and CpG-ODN respectively and IL1 and IL6 receptors (IL1R1 and IL6R) were also expressed in them. The expression of IL1B, IL6, and AvBD10 and -12 was upregulated by LPS, whereas only IL1B and IL6 were upregulated by CpG-ODN. IL1B stimulation upregulated AvBD1 and -3 expression, whereas IL6 stimulation did not cause changes in AvBDs expression. These results suggest that CpG-ODN derived from microbes upregulates the expression of IL1B and IL6 by interaction with TLR21 and then IL1B induces AvBD1 and -3 to prevent infection in the vagina.