Role of olmesartan in ameliorating diabetic nephropathy in rats by targeting the AGE/PKC, TLR4/P38-MAPK and SIRT-1 autophagic signaling pathways.

Diabetic nephropathy (DN) is one of the most serious consequences of diabetes and the most common reason for end-stage renal disease. The current study was set out to investigate the ability of olmesartan medoxomil (OM) to treat DN by evaluating the reno-protective effects of this drug on fat/fructose/streptozotocin (F/Fr/STZ)-induced diabetic rat model. This model was induced by feeding rats high F/Fr diet for 7 weeks followed by injection of a single sub-diabetogenic dose of STZ (35mg/kg; i.p). The F/Fr/STZ-induced diabetic rats were orally treated with either OM (10 mg/kg) or pioglitazone (10 mg/kg); as a standard drug daily for four consecutive weeks. F/Fr/STZ-induced diabetic rats propagated inflammatory, oxidative, and fibrotic events. OM was able to oppose the injurious effects of diabetes; it significantly reduced the elevated levels of advanced glycated end products (AGEs) and downregulated PKC gene expression, therefore, indicating its antioxidant capacity evidenced by mitigation in GSH, MDA renal content. Moreover, OM impaired the inflammatory cascade by suppressing the elevated level of renal TLR4 as well as diminished the inflammatory profibrotic cytokine TGF-ß1. Additionally, OM was able to turn off the MAPK cascade mediated by an upsurge in renal angiotensin 1-7 content and decrease the level of renal tubular injury marker, KIM-1. Furthermore, OM enhanced the autophagic activity pathway by upregulating of gene expression of SIRT-1. The histopathological examination confirmed these results. Finally, OM protected against type 2 diabetes-related nephropathy complications by altering inflammatory pathways, oxidative, fibrotic, and autophagic processes triggered by renal glucose overload. This study shows that OM has a reno-protective effect against DN in rats by inhibiting the AGE/PKC, TLR4/P38-MAPK, and SIRT-1 autophagic signaling pathways.

Contribution of glutathione peroxidase 1 (Pro200Leu) single nucleotide polymorphism and serum homocysteine levels in the risk of acute myocardial infarction in Egyptians.

BACKGROUND: Oxidative stress is among the most common risk factors in the pathogenesis of acute myocardial infarction (AMI). Glutathione peroxidase 1 enzyme coded by the GPX1 gene plays an essential role in reducing oxidative stress. Previous studies correlated the GPX1 (Pro200Leu) single nucleotide polymorphism (SNP) with AMI incidence. Elevated homocysteine (Hcy) levels induce oxidative stress and are considered an independent risk factor for AMI. Evidence showed a complex relationship between Hcy and GPx-1 activity. This study examined the association of the common (Pro200Leu) SNP in GPX1 with AMI incidence in an Egyptian population. This study is the first to check this association in an Egyptian population. Moreover, the association between serum Hcy and the incidence of AMI was checked, and the novelty was to statistically correlate GPX1 Pro200Leu genotypes with serum Hcy levels in patients and control subjects. Hundred control subjects and hundred and twenty AMI patients were genotyped using PCR-RFLP analysis. An ELISA was used to measure serum Hcy levels. RESULTS: The GPX1 (Pro200Leu) genotype distribution and allele frequency were not significantly different between patients and control subjects (P = 0.60 and P = 0.62, respectively). Serum levels of Hcy were significantly elevated in patients compared to control subjects (P ≤ 0.0001). However, no significant difference was observed in serum Hcy levels among different GPX1 genotypes in neither patients nor control subjects. CONCLUSIONS: The minor T allele of GPX1 Pro200Leu is not associated with AMI risk in this Egyptian population. However, high homocysteine serum levels might contribute independently to the risk of AMI. Finally, Hcy levels were not significantly different in homozygous minor TT compared to homozygous wild CC.

Is there a correlation between -174(G/C) polymorphism of IL-6 gene and the incidence of acute myocardial infarction?

BACKGROUND: Cardiovascular disease (CVD) remains the major cause of death worldwide. Most CVD can be prevented by addressing risk factors. Acute myocardial infarction (AMI) is an inflammatory disorder characterized by changes in several cytokines including the interleukins (ILs). Studies are running to evaluate the genetic variation in the inflammatory system and their influence on the risk factors for CVD aiming for future prevention of this global disease. The aim of the current study was too investigate the association of -174 (G/C) IL-6 polymorphism with the incidence of AMI in a representative sector of the Egyptian population and to examine the contribution of IL-6, as a biomarker, in the pathogenesis of AMI. Genotyping of -174 (G/C) IL-6 polymorphism was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) while IL-6 levels were assayed by ELISA. RESULTS: The genotype distribution of -174 (G/C) IL-6 gene was not significantly different between the control subjects (GG 81.7%, GC 16.3%, CC 1.9%) and the AMI patients (GG 79%, GC 19%, CC 2%).The serum levels of IL-6 were significantly elevated in the AMI patients in comparison to the control subjects (P < 0.0001). CONCLUSIONS: There is no significant association of -174(G/C) polymorphism in the promoter sequence of IL-6 and the incidence of AMI in the examined sample of Egyptian population. Elevated levels of serum IL-6 confirmed the relationship between inflammation and the incidence of AMI.

Ginkgo biloba extract (EGb 761) mitigates methotrexate-induced testicular insult in rats: Targeting oxidative stress, energy deficit and spermatogenesis.

Methotrexate (MTX) is commonly used as a therapeutic agent in the treatment of malignancies and autoimmune disorders. Risk of subsequent infertility following MTX administration has been reported as a significant side effect due to testicular toxicity. The aim of the study was to evaluate the modulatory effects of Ginkgo biloba (standardized extract, EGb 761) on MTX-induced testicular oxidative stress, energy deficits and spermatogenic status in rats. All groups received intraperitoneal injection of MTX (0.5 mg/kg) twice weekly for four weeks except the control group that received the corresponding vehicles. Other groups received oral EGb761, at doses 25, 50 or 100 mg/kg/day, for four weeks, concurrently with MTX. Blood and semen sampling followed by testis dissection were performed 24 h after last EGb 761 treatment. Semen was examined for sperm progressive motility, percent of normal spermatozoa and sperm cell concentration as well as seminal plasma essential and non-essential amino acids. Serum LH, FSH and testosterone were detected as well as testicular MDA, GSH, GSSG, TNF-α, IL-1ß, IL-6, NF-κB and the nuclear, cytoplasmic and mRNA expression levels of Nrf-2 besides the testicular cell energy; AMP, ADP and ATP. Histopathological studies of interstitial fibrosis and the severity of germ cell degeneration were also conducted. MTX induced significant decline in sperm quality along with decreased essential and non-essential amino acids in seminal plasma. MTX reduced serum FSH, LH and testosterone as well as testicular ATP, GSH and Nrf2, while increased levels of testicular ADP, AMP, MDA, GSSG and TNF-α. Results were confirmed by prominent interstitial fibrosis and severe germ cell degeneration in MTX group. Concurrent treatment with EGb 761 alleviated MTX-induced testicular insult evidenced by amelioration of oxidative stress biomarkers, energy functions, seminal sperms abnormalities and spermatogenesis status. The present study suggests a beneficial role of EGb 761 in MTX-induced testicular injury and subsequent distortion of spermatogenesis.

Effects of sulforaphane on D-galactose-induced liver aging in rats: Role of keap-1/nrf-2 pathway.

Aging; a biological phenomenon characterized by progressive decline in cellular functions, is considered as a major risk factor of various liver diseases that plays as an adverse prognostic role, thus increasing mortality rate. However, diet is the main environmental factor that has a major impact on the aging process whereas; sulforaphane (SFN), an isothiocyanate organosulfur compound in cruciferous vegetables, has been reported with myriad biological effects. In the present study, SFN antiaging properties were evaluated on D-galactose (D-Gal)-induced liver aging in rats. For this purpose, forty adult male Wistar rats were divided into five groups. All animals, except the normal control, were intraperitoneally injected with D-Gal (300 mg/kg/day for 5 days a week) for six consecutive weeks. In the hepatoprotective groups, animals received oral SFN (0.5, 1.0 and 2.0 mg/kg) for 6 weeks concurrently with D-GAL. SFN administration improved liver biomarkers through decreasing serum levels of AST, ALT, total and direct bilirubin when compared to D-Gal-aging group. SFN significantly increased hepatic GSH level as well as catalase and glutathione-S-transferase activities while counteracted the elevation in hepatic oxidative stress markers; MDA, NO and protein carbonyl in aged rats. SFN abrogated the dysregulation in hepatic Keap-1, Nrf-2 and HO-1and limited the elevation of TNF-α and TGF-ß concentrations in aging liver. Histopathologically, SFN decreased the intensity of hepatic fibrous proliferation in D-Gal-induced aging. In conclusion, SFN has shown hepatic anti-aging potential through promoting the antioxidant machinery via regulating Keap-1, Nrf-2 and HO-1 and antioxidant enzyme activities as well as ameliorating oxidative stress, hampering the inflammatory cytokines; TNF-ɑ and TGF-ß, and limiting hepatic fibrosis in a dose dependent manner.

The Association of Fractalkine Receptor (T280M) Polymorphism in the Pathogenesis of Acute Coronary Syndrome in the Egyptian Population.

BACKGROUND: Fractalkine (FKN) in its free and membrane bound-forms and its receptor CX3CR1are reported to have an atherosclerotic effect. The relationship of Single Nucleotide Polymorphisms (SNPs) in FKN and CX3CR1genes with the Coronary Artery Disease (CAD) risk showed conflicting results in different populations. The aim of this study was to investigate the influence of CX3CR1 threonine 280 methionine (T280M) polymorphism in the predisposition of Acute Coronary Syndrome (ACS) in Egyptians. METHODS: 200 Egyptian subjects were recruited for the study. They were divided into 100 ACS patients and 100 healthy controls. Genotyping of CX3CR1 T280M was performed using a Polymerase Chain Reaction-restriction Fragment Length Polymorphism (PCR-RFLP). Serum FKN was assayed by Enzyme - Linked - Immuno- Sorbent-Assay (ELISA). RESULTS: T and M allele frequencies for CX3CR1gene were not significantly different between ACS and Controls (p=0.76). Moreover, none of the genotypes had an atheroprotective effect. Serum analysis showed higher levels of FKN in ACS patients (p=0.041). FKN levels were not significantly different among genotypes of control and ACS groups (p=0.34) and (p=0.38) respectively. CONCLUSION: This study shows that CX3CR1 T280M polymorphism does not affect the incidence of ACS the Egyptian population. Moreover, none of the genotypes were associated with higher FKN levels.

C(-260)T Polymorphism in CD14 Receptor Gene of Egyptians with Acute Myocardial Infarction.

BACKGROUND: Despite the significance of the traditional risk factors, recently published studies have suggested that inflammatory processes and variations in the genetics of the inflammatory system may participate in the initiation of atherosclerosis and its complications. OBJECTIVE: To investigate the possible association between CD14 C(-260)T (rs2569190) gene polymorphism and the risk of acute myocardial infarction in the Egyptian population. METHODS: We enrolled 100 acute myocardial infarction patients in addition to 107 healthy controls. Deoxyribonucleic acid was extracted, purified and used for the genotype assay of C(-260)T polymorphism in promoter region of CD14 gene. Genotyping was conducted using polymerase chain reactionrestriction fragment length polymorphism. Polymerase chain reaction product was digested using a restriction enzyme and the digestion products were specified. Serum CD14 levels were determined by Enzyme Linked Immunosorbent Assay. RESULTS: CD14 genotypic distribution (CC: 15.9% vs. 16%, CT: 62.6% vs. 58%, TT: 21.5% vs. 26% in controls versus acute myocardial infarction patients, p > 0.05 for all variables) and allele frequencies (C allele: 47% vs., 45%, T allele: 52% vs. 55% in controls versus acute myocardial infarction patients, p > 0.05 for all variables) did not show a statistical significant difference. Serum CD14 levels were elevated in acute myocardial infarction patients (5.73±0.62 vs. 4.48±0.28 pg/ml, p < 0.05). However, there was no statistically significant difference in serum CD14 levels among different CD14 genotypes. CONCLUSION: CD14 C-(260)T polymorphism is not associated with incidence of acute myocardial infarction in Egyptians who showed elevated serum CD14 levels in comparison to healthy individuals.

Polymorphisms in Gap Junction Proteins and their Role in Predisposition of Acute Myocardial Infarction in Egyptians.

BACKGROUND: Connexin (Cx) proteins are the building blocks of gap junctions. Among these, Cx37 and Cx40 are expressed on vascular system and reported to have cardioprotective role. Linking polymorphisms in genes coding for Cx and coronary artery disease (CAD) risk showed conflicting results in different populations. None has been studied before in Egyptians. Therefore, the aims of this study were to investigate the influence of Cx37 C1019T and Cx40 A71G polymorphisms on the predisposition of acute myocardial infarction (AMI) in Egyptians, to study linkage disequilibrium (LD) and combined effects of single nucleotide polymorphisms (SNPs) and to correlate the genotypes with sVCAM-1 serum levels. METHODS: Total of 201 Egyptian subjects were recruited for the study. They were divided into 104 AMI patients and 97 healthy controls. Genotypes for each participant were determined using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Serum sVCAM-1was measured by ELISA. RESULTS: Allele frequencies for both Cx37 and Cx40 were not significantly different between AMI and Controls (p=0.93 and p=0.26 respectively). Moreover, studying the dominant and recessive models concluded that none of the genotypes was a risk factor. Both SNPs were not in LD (R2=0.0027). Serum analysis showed higher levels of sVCAM-1 in AMI patients (p<0.0001). sVCAM-1 levels were not significantly different among SNPs (Cx37; p=0.244 and Cx40; p=0.266). CONCLUSION: This study shows that Cx37 C1019T and Cx40 A71G polymorphisms are not associated with cardioprotective role in Egyptians. Moreover, both SNPs are inherited separately and none of the genotypes were associated with higher sVCAM-1 levels.

Association of Thrombospondin-1 (N700S) and Thrombospondin-4 (A387P) Gene Polymorphisms with the Incidence of Acute Myocardial Infarction in Egyptians

BACKGROUND: Thrombospondin (TSP) 1 and 4 are extracellular matrix glycoproteins that me- diate cell proliferation, platelet aggregation and inflammatory response. Conflicting data addressed the possible contribution of TSP-1 and TSP-4 gene polymorphisms to acute myocardial infarction (AMI). OBJECTIVE: Our study aimed to examine the association of TSP-1 (N700S) and TSP-4 (A387P) genetic variants with the incidence of AMI in Egyptians. It also correlated TSP-1 variants to TSP-1 and TNF-α serum concentrations while TSP-4 variants to IL-8 concentration identifying TSPs' contribution to vascular inflammation. METHODS: Genotyping was done in 214 subjects; 114 AMI patients and 100 controls using PCR-RFLP analysis. Serum Tsp-1, TNF-α and IL-8 levels were measured by ELISA assay. RESULTS: For TSP-4, (GC and CC) genotype distribution and the (C) allele frequency were significantly higher in AMI patients than controls (p = 0.0186), (p = 0.0117) respectively. In contrast, TSP-1 genotypes and allele frequencies showed no significant difference between AMI and controls (p = 0.7124 and p = 0.7201, respectively). Serum TSP-1, TNF-α and IL-8 concentrations were significantly elevated in AMI compared to controls (p = 0.0146, p < 0.0001 and p = 0.0057) respectively. Serum IL-8 levels had a significant difference among TSP-4 genotypes (p= 0.0368), being highest in the mutant C allele. Serum TSP-1 and TNF-α concentrations showed no significant difference among TSP-1 genotypes, but there was a positive correlation between both concentrations in AMI patients (p = 0.0014), (r = 0.4125). CONCLUSION: TSP-4 A387P polymorphism, but not TSP-1 polymorphism, is an independent risk factor for AMI in the Egyptians.

Association of manganese superoxide dismutase Ala16Val polymorphism in the incidence of acute myocardial infarction in the Egyptians.

Background: Oxidative stress has been implicated in various diseases including atherosclerosis; the most common pathologic process underlying acute myocardial infarction (AMI). The manganese superoxide dismutase (MnSOD) antioxidant enzyme affords the major defense against reactive oxygen species (ROS) within the mitochondria. MnSOD Alanine16Valine (A16V) single nucleotide polymorphism (SNP) has been shown to decrease MnSOD detoxification activity. Aim: A case-control study was conducted to investigate the association between MnSOD A16V polymorphism and the incidence of AMI in the Egyptians, investigate the contribution of oxidative stress represented by hexanoyl lysine adduct (HEL), an oxidative stress biomarker, in the pathogenesis of AMI and finally correlate the MnSOD genotypes with HEL serum levels. Methods: A total of 200 Egyptian subjects were recruited for the study; 100 AMI patients and 100 control subjects. Genotypes of the MnSOD A16V polymorphism were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum HEL was measured by ELISA. Results: A significant difference in the distribution of the MnSOD A16V genotypes was observed; VV genotype was significantly higher in AMI than controls (p ≤ 0.0001). Also, studying the allele frequencies revealed that Val allele was significantly higher in AMI than controls (p ≤ 0.0001). Serum analysis showed higher levels of HEL in AMI patients (p = 0.0142). Furthermore, HEL levels were found to be significantly higher in VV genotype in AMI (p = 0.0273). Conclusions: Our study suggests that MnSOD A16V polymorphism is associated with increased risk of developing AMI in the Egyptians. Moreover, the VV genotype is associated with higher HEL levels.

Assessment of the link between endothelin K198n Snp, endothelin concentration and acute myocardial infarction in Egyptians.

The aim of the current study was to assess the link between EDN K198N SNP, ET-1 serum concentration and acute myocardial infarction (AMI) in Egyptians. The study cohort consisted of 84 patients at AMI onset and 84 age-matched healthy controls. Endothelin genotypes and concentrations were determined by sequencing and ELISA, respectively. Genotype distribution was not significantly different between AMI patients and controls (P=.8341). The mean serum ET-1 concentration of patients (13.83±0.7 pg/mL) was significantly higher than controls (7.26±0.2 pg/mL) (P<.0001). ET-1 serum concentrations did not vary significantly among various EDN genotypes in patients (P=.378) and controls (P=.6164). Hence, we conclude that EDN K198N genotypes were not related to either ET-1 concentration or incidence of early-onset AMI in Egyptians. But, AMI patients had higher ET-1 concentrations than controls.

Addressing the link between paraoxonase-1 gene variants and the incidence of early onset myocardial infarction.

INTRODUCTION: The enzyme paraoxonase-1 (PON1) represents an endogenous defense mechanism against vascular oxidative stress, thereby contributing to the prevention of atherosclerosis. Several polymorphisms have been reported in the PON1 gene, including Q192R. PON1 phenotype is commonly expressed as the paraoxonase/arylesterase ratio (PON/ARE). The major aim of this study was to investigate the association between PON1 Q192R polymorphism, PON1 phenotypes and the incidence of early-onset acute myocardial infarction (AMI) in Egyptians. MATERIAL AND METHODS: The study subjects consisted of 102 AMI patients and 72 age-matched healthy controls. Genotyping and enzyme activities were determined using PCR-RFLP and kinetic spectrophotometric assays, respectively. RESULTS: The genotype distribution for the PON1 gene was significantly different between AMI patients (QQ = 38.24%, QR = 49.02%, RR = 12.75%) and controls (QQ = 66.67%, QR = 25%, RR = 8.33%). Allele frequencies were also significantly different between patients (Q = 62.75%, R = 37.25%) and controls (Q = 79.17%, R = 20.83%). The genotypes QR and RR showed higher risk for AMI compared to the homozygous QQ (odds ratio (OR) = 3.231, p < 0.001). The average PON/ARE ratio in MI patients (1.187 ±0.1) did not differ significantly from controls (1.118 ±0.26). However, it showed a significant difference among different genotypes in both AMI patients (QQ = 0.91 ±0.11, QR = 1.09 ±0.11 and RR = 2.65 ±0.4) (p = 0.0002) and controls (QQ = 0.68 ±0.1, QR = 1.07 ±0.11 and RR = 4.89 ±2.84) (p < 0.0001). CONCLUSIONS: PON1 192R allele represents an independent risk factor for early-onset AMI in Egyptians, and PON1 Q192R polymorphism modulates the paraoxonase phenotype.

C242T polymorphism of NADPH oxidase p22phox gene reduces the risk of coronary artery disease in a random sample of Egyptian population.

The p22phox protein subunit is essential for NADPH oxidase activity. The prevalence of C242T variants of p22phox gene was studied in 101 healthy Egyptian controls and 104 acute myocardial infarction (AMI) Egyptian patients. Contribution of oxidative stress, represented by serum oxidized-LDL (ox-LDL), in development of AMI was also examined and correlated with C242T gene variants. Genotyping and ox-LDL were assessed by PCR-RFLP and ELISA. Results showed that wild type CC genotype is prevalent in 27 % of controls; CT and TT are in 72 and 1 %. In patients, the distribution was 40.2, 59.8 and 0 % for CC, CT and TT; respectively, showing a significant difference (p = 0.0259). Serum ox-LDL levels were higher in patients than controls (p ≤ 0.0001). Subjects having CT genotype had lower levels of ox-LDL than CC genotype (p ≤ 0.005). C242T polymorphism of p22phox gene of NADPH oxidase is a novel genetic marker associated with reduced susceptibility to AMI.

Endothelial nitric oxide synthase (G894T) gene polymorphism in a random sample of the Egyptian population: comparison with myocardial infarction patients.

AIM: The aim of this study was to detect endothelial nitric oxide synthase (eNOS) Glu298Asp gene variants in a random sample of the Egyptian population, compare it with those from other populations, and attempt to correlate these variants with serum levels of nitric oxide (NO). The association of eNOS genotypes or serum NO levels with the incidence of acute myocardial infarction (AMI) was also examined. METHODS: One hundred one unrelated healthy subjects and 104 unrelated AMI patients were recruited randomly from the 57357 Hospital and intensive care units of El Demerdash Hospital and National Heart Institute, Cairo, Egypt. eNOS genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Serum NO was determined spectrophotometrically. RESULTS: The genotype distribution of eNOS Glu298Asp polymorphism determined for our sample was 58.42% GG (wild type), 33.66% GT, and 7.92% TT genotypes while allele frequencies were 75.25% and 24.75% for G and T alleles, respectively. No significant association between serum NO and specific eNOS genotype could be detected. No significant correlation between eNOS genotype distribution or allele frequencies and the incidence of AMI was observed. CONCLUSION: The present study demonstrated the predominance of the homozygous genotype GG over the heterozygous GT and homozygous TT in random samples of Egyptian population. It also showed the lack of association between eNOS genotypes and mean serum levels of NO, as well as the incidence of AMI.