Combined resveratrol and vitamin D treatment ameliorate inflammation-related liver fibrosis, ER stress, and apoptosis in a high-fructose diet/streptozotocin-induced T2DM model.

A high fructose diet is a major cause of diabetes and various metabolic disorders, including fatty liver. In this study, we investigated the effects of resveratrol and vitamin D (VitD) treatments on endoplasmic reticulum (ER) stress, oxidative stress, inflammation, apoptosis, and liver regeneration in a rat model of type 2 diabetes mellitus, namely, T2DM Sprague-Dawley rats. This T2DM rat model was created through a combination treatment of a 10% fructose diet and 40 mg/kg streptozotocin (STZ). Resveratrol (1 mg/kg/day) and VitD (170/IU/week) were administered alone and in combination to both the diabetic and control groups. Immunohistochemical staining was performed to evaluate PCNA, NF-κB, TNF-α, IL-6, IL-1ß, GRP78, and active caspase-3 in liver tissue. The TUNEL method and Sirius red staining were used to determine apoptosis and fibrosis, respectively. G6PD, 6-PGD, GR, and GST activities were measured to determine oxidative stress status. We found that the expressions of cytokines (TNF-α, IL-6, and IL-1ß) correlated with NF-κB activation and were significantly increased in the T2DM rats. Increased GRP78 expression, indicating ER stress, increased in apoptotic cells, enhanced caspase-3 activation, and collagen accumulation surrounding the central vein were observed in the T2DM group compared with the other groups. The combination VitD + resveratrol treatment improved antioxidant defense via increasing G6PD, 6-PGD, GR, and GST activities compared to the diabetic groups. We concluded that the combined administration of resveratrol with VitD ameliorates the adverse effects of T2DM by regulating blood glucose levels, increasing antioxidant defense mechanisms, controlling ER stress, enhancing tissue regeneration, improving inflammation, and reducing apoptosis in liver cells. In conclusion, this study indicates that the combination treatment of resveratrol + VitD can be a beneficial option for preventing liver damage in fructose-induced T2DM.

AR-A014418 as a glycogen synthase kinase-3 inhibitor: anti-apoptotic and therapeutic potential in experimental spinal cord injury.

OBJECTIVES: We aimed to investigate the effects of AR-A014418, a strong inhibitor specific to GSK-3beta, on neuronal apoptosis and neuroprotection in the traumatic SCI model. MATERIALS AND METHODS: In this study, three groups were generated from 36 Wistar rats; (1) control, (2) spinal cord trauma group created by clip compression technique after laminectomy, and (3) AR-A014418 (4mg/kg, i.p., DMSO) treatment group after laminectomy and spinal cord trauma. The TUNEL assay for apoptosis detection, immunohistochemical staining for bax and TGF-beta were applied in spinal cord tissues. For light microscopic examination, necrotic, and apoptotic cells were counted, and PMNL counting was applied to detect inflammation. Functional recovery was tested by field locomotor test in the 3rd and 7th days following surgery. RESULTS: In the trauma group, diffuse hemorrhage, cavitation, necrosis and edematous regions, degeneration in motor neurons and leukocyte infiltration were observed in gray matter. In the AR-A014418-treated groups, healthy cells were observed in more places compared to the trauma groups, however, cavitation, hemorrhagic, and edematous areas were seen in gray matter. In the AR-A014418-treatment groups, the number of apoptotic cells in the 3rd and 7th days (respectively; p<0.05, p<0.01), were significantly decreased compared to the trauma groups, as were the levels of bax (p<0.01) and TGF-beta 1 immunoreactivity. Results of the locomotor test were significantly increased in the treatment group (p<0.001) as compared to the trauma group. CONCLUSIONS: In this experimental spinal cord trauma model study neural apoptosis was significantly triggered in secondary damage developed after trauma, however, neurological healing was expedited by preventing mitochondrial apoptosis and reducing the inflammation by the potent inhibitor AR-A014418, which is GSK-3beta selective.

Therapeutic potential of the PI3K inhibitor LY294002 and PARP inhibitor Talazoparib combination in BRCA-deficient triple negative breast cancer cells.

Poly (ADP-ribose) polymerase (PARP) inhibitors provide a promising therapeutic strategy for triple-negative breast cancers (TNBCs) with BRCA1/2 mutation. However, acquire resistance mechanisms and genetic alterations limit the clinical efficacy of PARP inhibitors. The aberrant activation of phosphatidylinositol 3-kinase (PI3K) is a significant problem for cancer development and thus the inhibition of PI3K by PI3K inhibitors is a novel targeted therapy in advanced breast cancer. Here, we, for the first time, investigated that the combined inhibition of PARP by Talazoparib (TAL) and PI3K by LY294002 synergistically inhibited proliferation of BRCA1 mutant HCC1937 TNBC cells through apoptosis, G0/G1 arrest, oxidative stress and increased DNA damage compared to drug alone. Additionally, TAL and LY294002 combination could be a promising strategy for overcoming TAL resistance. Co-treatment of TAL with LY294002 considerably suppressed the activation of PI3K, Akt1 and mTOR expression and phosphorylated protein levels in TNBC cells and caused changes in the multiple kinase phosphorylation. Our findings revealed that the dual inhibition of PARP and PI3K might represent an effective therapeutic strategy for TNBC and potentially overcome TAL resistance.

Evaluation of trace elements and oxidative stress levels in the liver and kidney of streptozotocin-induced experimental diabetic rat model.

In this study, we aimed to investigate the relationship among trace elements (Cu, Fe, Zn and Mg) on oxidative and anti-oxidative substances in liver and kidneys tissues in streptozotocin (STZ) diabetic rat model. The mean levels of Fe and Cu were found significantly higher in the liver and kidneys of the diabetic rats, in comparison to the control rats. On the other hand, the mean levels of Zn and Mg in the liver and kidneys of the diabetic rats were significantly lower than in the control rats. The liver and kidneys malonaldehyde (MDA) levels of the experimental group were found to be higher than in the control group (p < 0.001; p < 0.01, respectively) after 4 weeks of the experimental period. Superoxide dismutase (SOD) activities and glutathione (GSH) levels in the liver tissue of STZ-induced diabetic rats were found to be lower in the experimental group than in the control group (p < 0.01). SOD activity and GSH concentration in kidneys of the diabetic rats were significantly diminished with respect to the control group (p < 0.01). In conclusion, the present results indicate that the increase of Fe and Cu together with decreas of Zn and Mg concentration in liver and kidney of STZ-induced diabetic rats may be involved in disturbances of oxidative balance in both the tissues. Therefore, these findings may contribute to explain the role of impaired ion metabolism of some elements in the progression of diabetic oxidative complications.

Synergistic Effect of Apigenin and Curcumin on Apoptosis, Paraptosis and Autophagy-related Cell Death in HeLa Cells.

BACKGROUND/AIM: We aimed to investigate the synergistic effects of apigenin and curcumin on the cross-talk between apoptosis and autophagic cell death, as well as on paraptosis in HeLa cells. MATERIALS AND METHODS: Cell viability was measured using the MTT assay. Synergistic effects were measured using the Bliss independence model. qRT-PCR was used to study the expression of genes related to apoptosis, autophagic cell death, and cross-talk. GRP78/BiP immunostaining was used to identify endoplasmic reticulum (ER) stress. RESULTS: Treatment with a combination of apigenin and curcumin increased the expression levels of genes related to cell death in HeLa cells 1.29- to 27.6-fold. The combination of curcumin and apigenin showed a synergistic anti-tumor effect via cross-talk between processes leading to apoptosis and autophagic cell death, as well as ER stress-associated paraptosis. GRP78 expression was down-regulated, and massive cytoplasmic vacuolization was observed in HeLa cells. CONCLUSION: The combination of curcumin and apigenin is an effective potential therapeutic for cervical cancers.

Association between ß arrestin 2 and filamin A gene variations with medical treatment response in acromegaly patients.

BACKGROUND: Acromegaly is a disease that occurs as a result of excessive growth hormone caused by pituitary adenomas. Some acromegaly patients show resistance to somatostatin analog (SSA) treatment. Filamin-A (FLNA) and ß-arrestins are thought to play a role in the response to SSAs. We aimed to investigate the relationship between FLNA-rs782079491 and ß-arrestin-2-rs34230287 single-nucleotide polymorphisms and disease risk, as well as treatment response in patients with acromegaly in the Turkish population. METHODS: The genotypes of 110 acromegaly patients and 99 controls were determined by realtime PCR. The genotype distributions were compared with clinical data on the disease. RESULTS: There was no association between the ß-arrestin-2 gene polymorphism and the response to SSA treatment in acromegaly patients. For responder patients to SSAs, the ß-arrestin-2-rs34230287 CT+TT genotype was associated with higher microadenoma as compared with the CC genotype (p = 0.017). The FLNA polymorphism was not observed in the study group. CONCLUSIONS: We showed that there was no association between the polymorphic genotypes of FLNA and ß-arrestin-2 genes with acromegaly disease and SSAs response in the Turkish population. However, there was a relationship between ß-arrestin-2 and some of the clinical characteristics. Furthermore, the CC genotype and the C allele are risk factors associated with tumor growth rate in acromegaly patients.

SNPs of miR-23b, miR-107 and HMGA2 and their Relations with the Response to Medical Treatment in Acromegaly Patients.

INTRODUCTION: Acromegaly is a chronic disease of increased growth hormone (GH) secretion and elevated insulin-like growth factor-I (IGF-I) levels induced by a pituitary adenoma. HMGA2 (high mobility group A2) and AIP (aryl hydrocarbon receptor-interacting protein) expression levels are related to GH-secreting adenomas, and also a response to Somatostatin Analogs (SSAs). We studied SNPs in miR-107 and miR-23b that related with AIP and HMGA2 genes respectively and control their expression, and also SNP in the 3'UTR of HMGA2 gene. Our aim was to investigate genotype distributions of the studied SNPs, as well as the possible relationship between disease and/or response to SSAs treatment in patients with acromegaly. MATERIAL AND METHODS: Genotypes were determined by qRT-PCR method from DNA materials obtained blood samples of acromegaly patients (141) and healthy individuals (99). The genotype distributions of patients and healthy groups, as well as the relationship between the clinical data of the disease and genotypes were statistically compared. RESULTS: In acromegaly patients with T-allele, p53 expression (p=0.049) was significantly higher. In patients with CT+TT genotype and T-allele who were responder to SSA-treatment Ki-67 index (respectively p=0.019, p=0.020 respectively) was higher. We did not observe the genotypes for miR-23b and miR-107 polymorphisms in the patients and control group of Turkish population. CONCLUSION: The genetic variations of the miRNAs genes related with HMGA2 and AIP genes were not seen in our study. Although there is no relationship between HMGA2-rs1351394 polymorphism and acromegaly disease, T allele was associated with some clinical features related to adenoma in patients with acromegaly.

Antiapoptotic effect of angiotensin-II type-1 receptor blockade in renal tubular cells of hyperoxaluric rats.

In this study, we investigated the protective effect of losartan as an AT1 receptor antagonist by evaluating the expression of apoptosis-regulatory genes that contribute to the progressive damage in the renal tubules of hyperoxaluric rats. Rats were divided into 4 groups of 10 each; control (C), ethylene glycol (EG), ethylene glycol + losartan (EG + L) and Losartan (L). For 4 weeks 0.8% EG, as a precursor for oxalate, was administered to EG and EG + L and losartan (300 mg/l) was administered to groups EG + L and L. Urine and blood samples were collected for biochemical determination. Bcl-2, bax, caspase-3 and TGF-beta 1 antibodies were used for immunohistochemistry. Apoptosis was determined by TUNEL method. A marked increase in urinary oxalate levels of the rats in EG and EG + L groups was found. In the EG group a diffuse amount of oxalate crystals into the tubular lumina and interstitium in the cortex was observed. In the EG group GBM thickening, interstitial fibrosis and tubular atrophy with infiltration of mononuclear cell findings reduced in the EG + L group were presented as well. In the EG group, immunoreactivity of TGF-beta 1 was increased in glomeruli and tubuli. In the EG + L group, immunoreactivity of TGF-beta 1 was decreased compared to the EG group. Bax expression increased in the renal tubules of EG group and reduced in the EG + L group comparing to the control. In the EG + L group, the immunoreactivity of bcl-2 was increased in glomeruli. In EG + L treated group, number of caspase-3 immunopositive cells were decreased compared to all groups (P < 0.01). Apoptotic cells were increased in the EG-treated group compared to the other groups. Decreased apoptotic cell number was observed in the EG + L compared to the EG group (P < 0.01). Our findings suggest that losartan may provide a beneficial effect against tubulointerstitial damage and decrease renal tubular apoptosis caused by hyperoxaluria.

Transdifferentiation of both intra- and extra-islet cells into beta cells in nicotinamide treated neonatal diabetic rats: An in situ hybridization and double immunohistochemical study.

We aimed to study the effect of nicotinamide (NA) on beta (ß)-cell regeneration and apoptosis in streptozotocin induced neonatal rats (n-STZ). Three groups were performed: Control group, n2-STZ group (100 mg/kg STZ on the second day-after birth), n2-STZ + NA group (STZ;100 mg/kg + NA;500 mg/kg/day for 5 days). The pancreatic tissue sections were immunostained with insulin, glucagon, somatostatin, Pdx1, Notch1 and active caspase-3 antibodies, and double immunostained with insulin/PCNA, insulin/glucagon and insulin/somatostatin antibodies. In situ hybridization carried out with insulin probe. Apoptotic ß-cell were shown by TUNEL assay, followed by immunostaining. The number of insulin/PCNA, insulin/glucagon and insulin/somatostatin double-positive cells significantly increased in n2-STZ + NA group compared with the other groups (p < 0.001). n2- STZ group had lower number of insulin and Pdx1 positive cells in islets, compared to NA treated diabetics. The insulin and Pdx1 immun positive cells were located in the small clusters or scattered through the exocrine tissue and around to ducts in n2-STZ + NA group. Notch1 positive cell numbers were increased, whereas caspase-3 and TUNEL positive ß-cell numbers were decreased in n2-STZ + NA group. NA treatment induces the neogenic insulin positive islets orginated from the differentiation of ductal progenitor cells, transdifferentiation of acinar cells into ß cells, and transformation of potent precursor cells and centroacinar cells via the activated Notch expression into ß-cells in n-STZ rats.

A Novel Expression Profile of Cell Cycle and DNA Repair Proteins in Nonfunctioning Pituitary Adenomas.

The molecular mechanisms underlying the formation of nonfunctioning pituitary adenomas (NFAs) are largely unknown. In this study, we aimed to understand the relationship between NFAs and functional pituitary adenomas and the possible role of proteins involved in cell cycle, senescence, and DNA damage control mechanisms in the etiology of NFA. We analyzed pATM-S1981, pRb-S608, Rb, pE2F1-S364, p16, E2F1, p73, cyclin D1, and CHEK2 protein expression (in a group of 20 patients with acromegaly, 18 patients with Cushing's disease (CD), and 29 NFA patients) by immunohistochemistry and their relevant mRNA expression by qRT-PCR (in a group of 7 patients with acromegaly, 7 patients with CD, and 7 NFA patients). The clinical and histopathological results on the patients were statistically evaluated. pE2F1-S364 protein expression in the CD group was significantly lower than that in the NFA and acromegaly groups (p = 0.025, p = 0.034, respectively). However, the expression of the p16 protein was lower than in the NFA group than in the CD and acromegaly groups (p = 0.030, p = 0.033, respectively), and E2F1 protein expression was significantly higher in the NFA group than in the CD group (p = 0.025). p73 protein expression in patients with acromegaly was significantly higher (p = 0.031) than that in the CD group. CHEK2 mRNA expression in the CD group was significantly higher than that in the acromegaly group (p = 0.012). The selective and tumor-specific associations between E2F1, pE2F1-S364, CHEK2, and p73 mRNA and protein levels indicate their involvement in pituitary adenoma formation in NFA, CD, and acromegaly patients.

The Detection Techniques for Autophagy-Associated Cell Death-Related Genes and Proteins: Gene Expression Assay and Immunohistochemistry.

Autophagy is important in cellular homeostasis for the cell survival mechanism. Deficiency or excess of autophagy is generally related to some of diseases such as cancer and neurodegeneration. Although autophagy is a cell survival mechanism, it can mediate programmed cell death in several conditions. Autophagy-related genes (ATGs) regulate the autophagy and also control the crosstalk with autophagy-associated cell death and apoptosis in some condition. Various methods have been used to detect the marker genes and the proteins involved in these processes. Quantitative real-time PCR (qRT-PCR) method for monitoring the expression of genes involved in autophagy or autophagic cell death is often preferred because of its sensitivity, high efficiency potential, accurate quantification, and high-grade potential automation. The detection of the markers for autophagy-related process by immunohistochemistry in paraffin sections of various patient tissues has become a reliable method for monitoring autophagy. Here, we introduce protocols for detecting autophagy and autophagy-associated cell death in HeLa cells by using gene expression assays qRT-PCR, and also in paraffin-embedded tissue section from human biopsy material by using immunohistochemistry.

DPP4 inhibitor induces beta cell regeneration and DDR-1 protein expression as an endocrine progenitor cell marker in neonatal STZ-diabetic rats.

BACKGROUND: We aim to investigate the effects of dipeptidyl-peptidase-4 inhibitor (Vildagliptin-VG) on DDR-1 as a marker for endocrine progenitor cells, ß-cell regeneration, and apoptosis in neonatal streptozotocin (n2-STZ) diabetics. METHODS: Neonatal rats were divided into two main groups as short- and long-term treatment, each consisted of four groups; (1) Control, (2) n2-STZ diabetic (single dose of 100 mg/kg STZ at 2nd day of birth), (3) n2-STZ + VG (60 mg/kg/day VG orally; for 8 and 28 days), (4) VG (60 mg/kg/day orally; for 8 and 28 days). Blood glucose levels and body weights were measured, and the tissue sections were immunostained using insulin, glucagon, somatostatin, PCNA, Pdx-1 and DDR-1 antibodies. The TUNEL method was used for apoptosis. RESULTS: The number of ß cells in islets of the n2-STZ + VG group increased compared to the n2-STZ group; insulin (+) cells were observed individually or as small clusters in exocrine tissue, between pancreatic duct epithelial cells, and around the ducts. The number of Pdx-1 and DDR-1 positive cells in islet and extra-islet pancreas tissue was elevated as a result of VG application compared to the STZ diabetic group; the number of double positive cells for DDR-1 and insulin increased in n2-STZ + VG rats. CONCLUSION: We showed that vildagliptin promotes ß cell neogenesis and regeneration, stimulates DDR-1 expression as an endocrine cell progenitor marker, suppresses apoptosis, induces islet cell proliferation and rearranges islet morphology in the n2-STZ diabetes model.

The effects of ACE inhibitor and angiotensin receptor blocker on clusterin and apoptosis in the kidney tissue of streptozotocin-diabetic rats.

Our first aim was to determine the effects of secreted clusterin (sCLU) and nuclear clusterin (nCLU) in diabetic nephropathy. We also aimed to investigate the post-effects of angiotensin II blockage treatment on clusterin expression and to compare these with apoptosis. Five groups of Wistar albino rats were used: First group consisted of healthy controls; the second group included the untreated STZ-diabetics; 30 days of irbesartan or perindopril treated STZ-diabetics formed the third and the fourth groups, respectively; while the subjects receiving a combined treatment with irbesartan and perindopril for 30 days consisted the fifth group. TUNEL method for apoptosis and immunohistochemical staining for TGF-beta1, alpha-SMA, clusterin-beta and clusterin-alpha/beta antibodies were performed. Apoptotic cells especially increased in the kidney tubuli of untreated diabetic group and on the contrary, a significant decrease was observed in the group that received a combined drug treatment. While sCLU was increased in the glomeruli and tubuli of the untreated diabetic group, it was decreased in all the treated groups. An increase in the nCLU immunoreactivity was observed in the podocytes, mesangial cells, and the injured tubule cells of the untreated diabetic group. nCLU immunopositive cells were decreased in all treated diabetic groups. In addition to this, the distribution of nCLU was similar to the distribution of apoptotic cells in the diabetic groups. Our results indicate that sCLU expression in diabetic nephropathy was induced due to renal tissue damage, and the nCLU expression increase in renal tubuli was related to apoptosis. Although irbesartan and perindopril prevented further renal injury in diabetes, a combined application of low-dose ACEI and AT1R blockers revealed more efficient measures, by means of renal damage prevention.

Okadaic acid-induced tau hyperphosphorylation and the downregulation of Pin1 expression in primary cortical neurons.

Hyperphosphorylation of tau leading to neurofibrillary tangles (NFT) is one of the key pathological hallmarks in neurodegenerative disorders such as Alzheimer disease (AD). Peptidyl-prolyl cis-trans isomerase (Pin1) regulates the phosphorylation of Ser/Thr sites of tau protein, and promotes microtubule assembly. In this study, we aimed to determine the effect of tau hyperphosphorylation on Pin1 expression in primary cortical neurons in order to investigate the results of the pathological process on Pin1, an important enzyme involved in various cellular mechanisms. Primary cortical neurons were prepared from embryonic day 16 -Sprague Dawley rat embryos. The cultures were treated with 25 nM okadaic acid (OKA) on day 7 in order to promote tau hyperphosphorylation. The cytotoxicity was determined with LDH release and measured by ELISA. Tau phosphorylation was confirmed by western blot using anti-tau antibodies Thr231 and Tau-1. Pin1 mRNA expression level was determined by qRT-PCR at 8 and 24 h. Pin1 protein expression was analyzed with immunofluorescent labeling at 8 and 24 h. Tau phosphorylation on Thr231 was increased and non-phosphorylated Tau-1 was decreased in OKA treated group compared with the untreated control at 8 h of treatment. While Pin1 mRNA expression levels at 8 h post-OKA treatment were lower than that of control groups, there were no differences between OKA-treated group and control groups in Pin1 protein expression. Whereas no significant differences for Pin1 mRNA expression, protein expression levels were decreased OKA-treated group compared to control groups at 24 h of treatment. The LDH release of OKA-treated group was significantly increased at 24 h. Our study indicates that although OKA treatment suppressed Pin1 mRNA expression and induced tau phosphorylation at 8 h of treatment, its influence on Pin1 protein expression has 16 h phase delay. Given the important role of Pin1 in many cellular mechanisms these results might indicate that tau hyperphosphorylation involved in many neurodegenerative disorders may cause some alterations in brain microenvironment via Pin1.This is the first demonstration of the alteration of the Pin1 mRNA and protein expression in OKA induced model in primary cortical neurons.

Both secreted and the cellular levels of BDNF attenuated due to tau hyperphosphorylation in primary cultures of cortical neurons.

Intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a major neuropathological hallmark of taupathies such as Alzheimer's disease. Okadaic acid (OKA) is a potent inhibitor of PP2A, leading to abnormal tau phosphorylation. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that is selectively downregulated in AD. In this study, we investigated the effects of OKA induced tau hyperphosphorylation on secreted and cellular levels of BDNF in primary cortical neurons that were treated with 25nM OKA. Tau phosphorylation at threonine 231 (Thr231) sites was assessed by Western blot using antibodies against phospho-Thr231. Non-phosphorylated tau protein was detected with the Tau-1 antibody. Levels of BDNF secreted to the culture medium were determined by ELISA at the 8th and 24th hours of treatment. Cellular localization and protein expression of BDNF and tau were assessed by immunofluorescent labeling and fluorescent intensity measurements at 24h of treatment. Tau hyperphosphorylation was confirmed with increase in Thr231 and the decrease in Tau-1 signals after 8h of OKA treatment, compared with the control groups, secreted BDNF levels in the OKA-treated group were significantly lower after 24h of treatment but were not significantly different at 8h of treatment. BDNF immunoreactivity was seen in cytoplasm and neurites of the neurons in control group. BDNF immunoreactivity significantly decreased in the OKA treated group and this attenuation was significant especially at neurites. Our results suggest that the decrease in BDNF secretion and the BDNF expression might depend on the disruption of microtubule structure caused by tau hyperphosphorylation.

Regulation of the Ku70 and apoptosis-related proteins in experimental diabetic nephropathy.

BACKGROUND: Apoptosis contributes nephropathy pathogenesis in diabetes. However, its mechanisms still remain unclear. We examined the extent to which the angiotensin-II type 1 receptor blocker (AT1RB) irbesartan and the angiotensin converting enzyme inhibitor (ACEI) perindopril affected the apoptosis-related proteins Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 in streptozotocin (STZ)-diabetic rats. MATERIALS AND METHODS: Animals were divided into five groups of eight each, four of which received STZ (60mg/kg in a single dose, i.p.) to induce diabetes. The groups were performed as untreated diabetic; non-diabetic control; daily irbesartan (15mg/kg/day) or perindopril (6mg/kg/day) and also combined irbesartan and perindopril (respectively, 5mg/kg/day, 3mg/kg/day) were applied by gavage for 30days to STZ-diabetic rats. The kidney tissue analysis was performed by using immunohistochemical staining with Bcl-2, Bax, caspase-3, cytochrome-c and Ku70 antibodies and by using Western blot analysis with caspase-3 and cytochrome-c antibodies. RESULTS: Immunoreactivity of Bax, caspase-3, cytochrome-c and Ku70 was increased in the tubuli and glomeruli of the untreated diabetic group, but decreased in all treated diabetic groups. In the irbesartan and perindopril treated diabetic groups Bcl-2 immunoreactivity was higher than that of the untreated diabetic group. Caspase-3 and cytoplasmic cytochrome-c protein levels increased in the untreated diabetic group. CONCLUSIONS: We conclude that the increased expression of Bax and caspase-3, and the increased level of cytoplasmic cytochrome-c relate to renal tissue injury. This case is also seen in the early stages of diabetes as a result of the damage caused by local increased expression of renin angiotensin system (RAS) in the renal tissue, which is induced by hyperglycemia. The increase of the cytosolic cytochrome-c, caspase-3 and Ku70 expression in the tubuli is suggestive of apoptosis. Overall, our results show that treatments of irbesartan and perindopril are effective and efficient in preventing renal tissue injury and apoptosis by blocking the RAS in experimental diabetic nephropathy and reducing the expression of proteins associated with apoptosis.

The Effects of Colchicum baytopiorum on Regulatory Genes of Apoptotic and Autophagic Cell Death in HeLa Cells.

BACKGROUND: The natural products have increasing important for the development of anticancer agents. Colchicum baytopiorum C.D. Brickell (C. baytopiorum), an endemic species for Turkey, contains colchicine and its derivatives. Stimulation of apoptotic and autophagy-mediated cell deaths are effective strategy for anticancer therapies. AIM: The aim of the study is to determine the role of the extract on both apoptotic and autophagic cell death in HeLa cell line. METHODS: The cell viability of C. baytopiorum (0.1 mg/ml) was determined by MTT assay. Active caspase-3 and t-Bid expressions were evaluated by immunohistochemical method. The mRNA expression of apoptotic regulatory genes (Bcl-xL, Bid, Bad, PUMA, NOXA, Caspase-3, -8, -9, Fas, FADD, TRADD, TRAF2, TNF, TNFR1), autophagic cell death related genes (Atg5-12, Beclin-1, DAPK), and also both autophagic and apoptotic cell death regulatory genes (Bif-1 and BNIP-3) were investigated by qRT-PCR. RESULTS: We determined that the expressions of both apoptotic and autophagic regulatory genes were significantly increased in the treatment group compared to control group. Also, we showed that C. baytopiorum crude extract induces the cross-connection between apoptotic and autophagic cell deaths in HeLa cells. CONCLUSION: We suggested that this endemic plant extract seems to be a new promising therapeutic approach in cancer.

Protective effects of a calcium channel blocker on apoptosis in thymus of neonatal STZ-diabetic rats.

Streptozotocin (STZ) is known to induce insulin-dependent diabetes in experimental animals. In STZ-induced diabetes, atrophy of the thymus is caused by elevated intracellular calcium levels leading to apoptosis. Hyperglycemia is known to result in a decrease in numbers of T cells in the thymus and circulation. Intracellular calcium levels increase in diabetic animals after induction by STZ. Hyperglycemia inhibits Ca2+-ATPase and increases intracellular calcium levels. We have investigated apoptosis in thymus tissue of neonatal STZ (n-STZ)-diabetic rats and the effects of isradipine as a calcium channel blocker (CCB) on apoptosis. Five groups of newborn Wistar rats were used. On the second day after birth, 100 mg/kg STZ was given i.p. to the first two groups. The first group was n-STZ diabetic. To the second group, starting from the 12th week, 5 mg/kg/day isradipine (i.p) was given for 6 weeks. To the third group, the same dose of isradipine was given on the second day, followed by STZ treatment. The fourth group was non-diabetic and treated with 5 mg/kg/day isradipine for six weeks. The fifth group consisted of non-diabetic rats. To the sixth group, dexamethasone (5 mg/kg i.p.) was given to adult rats. For detection of apoptotic cells in paraffin-embedded thymus sections, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay was used. The DNA ladder method was performed for analysis of DNA fragmentation. In the isradipine-treated non-diabetic group, typical apoptotic banding patterns were found, whereas thick bands between 123 and 246 bp length were found in the n-STZ- and n-STZ+isradipine-treated groups. More apoptotic cells were observed in the thymus of isradipine-treated, n-STZ-treated and n-STZ+isradipine-treated groups when compared with the non-diabetic control and isradipine+n-STZ-treated groups. In conclusion, we observed that long-term STZ diabetes results in apoptosis in the thymus. We also found that isradipine administered before STZ has protective effects against apoptosis, whereas isradipine alone induces apoptosis.

The neuroprotective effects of z-DEVD.fmk, a caspase-3 inhibitor, on traumatic spinal cord injury in rats.

BACKGROUND: Apoptosis is one of the most important forms of cell death seen in a variety of physiological and pathological conditions, including traumatic injuries. This type of cell death occurs via mediators known as caspases. Previous studies have investigated the roles that apoptosis and different caspases play in the pathogenesis of secondary damage after spinal cord injury (SCI). The aim of this research was to assess the neuroprotective effect of z-DEVD.fmk, a caspase-3 inhibitor, in a rat model of SCI. METHODS: Forty-five Wistar albino rats were studied in 3 groups of 15 animals: sham-operated control animals (group 1); trauma-only control animals (group 2); and rats subjected to trauma + z-DEVD.fmk treatment (group 3). Spinal cord injury was produced at the thoracic level using the weight-drop technique. Responses to injury and the efficacy of z-DEVD.fmk were assessed by light microscopy and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in cord tissues collected at 4 and 24 hours posttrauma. Five rats from each group were used to assess functional recovery at 7 days after SCI. The functional evaluations were done using the inclined-plane technique and a modified Tarlov motor grading scale. RESULTS: At 4 hours postinjury, the mean apoptotic index in groups 1, 2, and 3 was 0, 33.01+/-6.62, and 16.40+/-4.91, respectively. The group 3 count was significantly lower than the group 2 count (P<.01). At 24 hours postinjury, light microscopic examination of group 2 tissues showed widespread hemorrhage, necrosis, polymorphonuclear leukocyte infiltration, and vascular thrombi. The group 3 tissues showed similar features. The prominent findings in group 2 were hemorrhage and necrosis, whereas the prominent findings in group 3 were focal hemorrhage and leukocyte infiltration. The mean inclined-plane angles in groups 1, 2, and 3 were 64.5 degrees+/-1.0 degrees, 41.5 degrees+/-1.3 degrees, and 47 degrees+/-2.0 degrees, respectively. Motor scale results in all groups showed a similar trend. CONCLUSION: Local application of z-DEVD.fmk after SCI in rats reduces secondary tissue injury and helps preserve motor function. These effects can be explained by inhibition of apoptotic death in all cell types in the spinal cord.

Effects of ACE inhibition on the expression of type IV collagen and laminin in renal glomeruli in experimental diabetes.

In the present study, we examined electron microscopically and immunohistochemically the effects of perindopril, an angiotensin-converting enzyme inhibitor, on renal microangiopathy in streptozotocin-induced diabetes in rats. To investigate changes in glomerular basement membrane (GBM) and tubular basement membrane components, we immunohistochemically localized type IV collagen and laminin. Animals have been divided into three groups of eight adult male rats each. The first group was the non-diabetic control group. The second group consisted of untreated diabetic rats. The third group consisted of diabetic rats that were treated with perindopril for 6 weeks. Blood glucose levels and body weight were measured. Morphometric analysis of kidney tissue was performed using light and electron microscopy to quantify glomerular size and thickness of the GBM. Blood glucose levels in diabetic rats were significantly increased when compared with non-diabetic controls. Blood glucose levels were not affected by perindopril treatment. Untreated diabetic rats showed increased glomerular size, thickening of the GBM and an increase in mesangial matrix as compared with controls. Treatment with perindopril prevented effectively glomerular hypertrophy and thickening of the GBM. Significant increase in type IV collagen and laminin was found in thickened GBM and mesangial matrix in kidneys of untreated diabetic rats. In perindopril-treated diabetic rats, staining of type IV collagen and laminin was less strong when compared with untreated diabetic rats. In conclusion, our data suggest that perindopril treatment is effective in preventing renal lesions possibly by ameliorating the diabetes-induced increase in expression of type IV collagen and laminin.