Circulatory levels of B-cell activating factor of the TNF family in patients with diabetic foot ulcer: Association with disease progression.

Enhanced and prolonged expression of tumor necrosis factor alpha (TNF-α), a potent pro-inflammatory cytokine is evidenced during the chronic wound healing process of infected diabetic foot ulcer (IDFU). B-cell activating factor (BAFF) is the member of TNF-α family, which implicit in B-cell dysfunction. This study was aimed to evaluate the role of BAFF in diabetic foot ulcer (DFU) patients and to correlate its association with other family of inflammatory cytokines. Circulating levels of BAFF and other cytokines were measured in IDFU (n = 44) and non-IDFU patients (n = 40) using multiplexed bead-based cytokine immunoassay. A stepwise significant increase was observed in both circulatory BAFF and C-reactive protein (CRP) during the disease progression. The area under the receiver operating characteristic curve (AUCROC ) for BAFF was found to be high (0.89; [95% CI: 0.73-1.0]), when compared to CRP (0.68; [95% CI: 0.61-0.76]). Optimum diagnostic cutoff level for BAFF was found to be ≥2.35 pg/mL with 62.0% sensitivity and 85.7% specificity. Further, BAFF levels showed a significant positive correlation with CRP among IDFU patients. With respect to other family cytokines, BAFF levels were positively correlated with TNF-α, interferon family cytokines such as IFN-α2, IL-28A/IFN-λ2, IFN-γ, and IL-10 family cytokines such as IL-19, IL-22, and IL-26 and negatively correlated with IL-6 receptor family such as gp130/sIL-6Rß. Hence, our data suggest that devising therapeutic strategies to reduce the levels of BAFF may contribute in amelioration of IDFU.

Association of single-nucleotide polymorphisms of the KEAP1 gene with the risk of various human diseases and its functional impact using in silico analysis.

Keap1, Kelch-like erythroid derived Cap 'n' collar homology (ECH) associated protein 1 is a highly redox-sensitive member of the BTB-Kelch substrate adaptor protein which acts as a major upstream regulator of Nrf2 (Nuclear factor erythroid 2-related factor 2) by Cul3 ubiquitin E3 ligase complex, leading to its proteasomal degradation. Oxidative and electrophilic stresses impair the structural integrity of Keap1-Cul3 ubiquitin E3 ligase complex resulting in the dissociation of Nrf2-Keap1 binding and nuclear accumulation of Nrf2. Studies on tissue-specific Keap1 null mutation have demonstrated the important roles of Keap1 mediated Nrf2 degradation. An increasing body of evidence suggests that loss of functional mutation in Keap1 arbitrates constitutive activation and expression of Nrf2 which in turn provokes the chemotherapeutic resistance in various diseases. The current review addresses the genetic aspects of KEAP1 including somatic mutations and in silico functional profiling of human disease-associated and polymorphic amino acid substitutions.

Targeting Nrf2/Keap1 signaling pathway by bioactive natural agents: Possible therapeutic strategy to combat liver disease.

BACKGROUND: Nuclear factor erythroid 2-related factor (Nrf2), a stress-activated transcription factor, has been documented to induce a defense mechanism against oxidative stress damage, and growing evidence considers this signaling pathway a key pharmacological target for the treatment of liver diseases. PURPOSE: The present review highlights the role of phytochemical compounds in activating Nrf2 and mitigate toxicant-induced stress on liver injury. METHODS: A comprehensive search of published articles was carried out to focus on original publications related to Nrf2 activators against liver disease using various literature databases, including the scientific Databases of Science Direct, Web of Science, Pubmed, Google, EMBASE, and Scientific Information (SID). RESULTS: Nrf2 activators exhibited promising effects in resisting a variety of liver diseases induced by different toxicants in preclinical experiments and in vitro studies by regulating cell proliferation and apoptosis as well as an antioxidant defense mechanism. We found that the phytochemical compounds, such as curcumin, naringenin, sulforaphane, diallyl disulfide, mangiferin, oleanolic acid, umbelliferone, daphnetin, quercetin, isorhamnetin-3-O-galactoside, hesperidin, diammonium glycyrrhizinate, corilagin, shikonin, farrerol, and chenpi, had the potential to improve the Nrf2-ARE signaling thereby combat hepatotoxicity. CONCLUSION: Nrf2 activators may offer a novel potential strategy for the prevention and treatment of liver diseases. More extensive studies are essential to identify the underlying mechanisms and establish future therapeutic potentials of these signaling modulators. Further clinical trials are warranted to determine the safety and effectiveness of Nrf2 activators for hepatopathy.

Analysis of the Exonic Single Nucleotide Polymorphism rs182428269 of the NRF2 Gene in Patients with Diabetic Foot Ulcer.

BACKGROUND: The pivotal role of Nuclear factor erythroid-2-related factor 2 (NRF2) in redox homeostasis and wound healing has been well documented. However, the genetic mechanisms that regulate NRF2 in type 2 diabetes and diabetic foot ulcers remain unexplored. The present study investigated the association of single nucleotide polymorphism rs182428269 (-127 C/T) in subjects with type 2 diabetes and diabetic foot ulcers. METHODS: This cross-sectional study comprised 400 participants that included group I: normal glucose tolerant subjects (NGT, n = 150), group II: type 2 diabetes mellitus subjects (T2DM, n = 150) and group III: infected diabetic foot ulcer subjects (DFU, n = 100). The non-synonymous SNP rs182428269 was selected based on in silico analysis and genotyped by PCR-restriction fragment length polymorphism (RFLP) followed by bidirectional Sanger sequencing. In addition, the gene expression of NRF2 in patients with polymorphism was analyzed by qPCR to evaluate the functional impact of the SNP. RESULTS: NRF2 expression was significantly decreased among the T2DM and DFU subjects when compared to the NGT subjects. Of particular interest, the homozygous mutant (TT) genotype of rs182428269 polymorphism was significantly associated with an increased risk for the development of T2DM (OR = 1.95 (1.02-3.72), p = 0.04) and DFU (OR = 5.66 (2.98-10.76), p = 0.0001). Furthermore, a progressive decline in NRF2 expression was observed among the T2DM and DFU subjects with "TT" genotype compared to the "CC" and "CT" genotypes. CONCLUSION: NRF2 polymorphism rs182428269 is associated with the pathogenesis of T2DM and DFU.

Gene Expression Profiling of Multiple Histone Deacetylases (HDAC) and Its Correlation with NRF2-Mediated Redox Regulation in the Pathogenesis of Diabetic Foot Ulcers.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a protein of the leucine zipper family, which mitigates inflammation and employs cytoprotective effects. Attempting to unravel the epigenetic regulation of type 2 diabetes mellitus (T2DM) and diabetic foot ulcer (DFU), we profiled the expression of eleven isoform-specific histone deacetylases (HDACs) and correlated them with NRF2 and cytokines. This study recruited a total of 60 subjects and categorized into DFU patients (n = 20), T2DM patients (n = 20), and healthy controls (n = 20). The DFU patients were subcategorized into uninfected and infected DFU (n = 10 each). We observed a progressive decline in the expression of NRF2 and its downstream targets among T2DM and DFU subjects. The inflammatory markers IL-6 and TNF-α were significantly upregulated, whereas anti-inflammatory marker IL-10 was significantly downregulated in DFU. Of note, a significant upregulation of HDAC1, 3, 4, 11, SIRT3 and downregulation of HDAC2,8, SIRT1, SIRT2, SIRT3, SIRT7 among DFU patients were observed. The significant positive correlation between NRF2 and SIRT1 in DFU patients suggested the vital role of NRF2/SIRT1 in redox homeostasis and angiogenesis. In contrast, the significant negative correlation between NRF2 and HDAC1, 3 and 4, implied an imbalance in NRF2-HDAC1, 3, 4 circuit. Furthermore, a significant positive correlation was observed between HDAC4 and IL-6, and the negative correlation between SIRT1 and IL-6 suggested the pro-inflammatory role of HDAC4 and the anti-inflammatory role of SIRT1 in NRF2 signaling. In conclusion, the epigenetic changes such as upregulation of HDAC1, 3, 4, 11, SIRT3 and downregulation of HDAC2, 8, SIRT1, SIRT2, SIRT6, SIRT7 and their association with NRF2 as well as inflammatory markers are suggestive of their roles in pathophysiology of T2DM and DFU.

Role of Nrf2 in MALAT1/ HIF-1α loop on the regulation of angiogenesis in diabetic foot ulcer.

Diabetic non healing wounds often result in significant morbidity and mortality. The number of effective targets to detect these wounds are meagre. Slow lymphangiogenesis is one of the complex processes involved in impaired healing of wounds. Long non coding RNAs (lncRNAs) have been importantly recognized for their role in pathological conditions. Multiple studies highlighting the role of lncRNAs in the regulation of several biological processes and complex diseases. Herein, we investigated the role of lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of diabetic foot ulcer (DFU). We report a significant reduction in the expression of lncRNA MALAT1 in the infected DFU subjects which was positively correlated with the expression of angiogenic factors such as Nrf2, HIF-1α and VEGF. Further, expression of pro-inflammatory markers TNF-α and IL-6 were found to be increased while, the expression of anti-inflammatory marker IL-10 was decreased in infected DFU tissues. Involvement of lncRNA MALAT1 in angiogenesis in EA.hy926 cells was demonstrated by silencing the expression of Nrf2, HIF-1α, and VEGF through interference mediated by MALAT1. In addition, its inflammatory role was demonstrated by decreased expression of TNF-α, IL-6 and not affecting the expression of IL-10. Further, CRISPR-Cas9 knock out of Nrf2 decreased the expression of lncRNA MALAT1, HIF-1α and VEGF which revealed the association of Nrf2 in regulating MALAT1/HIF-1α loop through positive feedback mechanism. Collectively, our results suggested the role of Nrf2 on MALAT1/HIF-1α loop in the regulation of angiogenesis, which could act as a novel target in the treatment of diabetic wounds.

Genetic Polymorphism of the Nrf2 Promoter Region (rs35652124) Is Associated with the Risk of Diabetic Foot Ulcers.

The genetic polymorphism in the nuclear factor erythroid 2-related factor 2 (Nrf2) gene has been reported as one of the prognosis markers for various diseases, including cancer. Nrf2 is a key transcription factor involved in wound healing by regulating angiogenesis. We investigated the genetic association of NRF2 single-nucleotide polymorphism rs35652124 with T2DM and DFU and assessed its functional impact. A total of 400 subjects were recruited for the study and categorized into three groups: infected DFU patients (DFU, n = 100), T2DM patients without complications (T2DM, n = 150), and healthy adults with normal glucose tolerance (NGT, n = 150). The subjects were genotyped by PCR-RFLP, and the polymorphism was identified by bidirectional Sanger sequencing. The expression of NRF2, IL-10, TNF-α, and IL-6 was studied by qPCR to evaluate the functional impact of rs35652124. The "TT" genotype of rs35652124 was associated with a significant risk for T2DM [OR = 2.2 (1.2-4.2), p = 0.01] and DFU [OR = 7.9 (4-14.9), p < 0.0001]. A significant decrease in transcriptional levels of NRF2 and IL-10 and a remarkable increase in TNF-α and IL-6 were observed in subjects with TT genotype. In conclusion, rs35652124 (TT) is a harmful genetic variant that predisposes to insulin resistance and impaired angiogenesis. Hence, it may serve as a diagnostic genetic marker for T2DM and DFU in combination with different inflammatory markers.

Tissue-specific role of Nrf2 in the treatment of diabetic foot ulcers during hyperbaric oxygen therapy.

Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic foot ulcer (DFU) treatment due to its antimicrobial effect, increased angiogenesis and enhanced collagen synthesis. The molecular mechanism underlying HBO therapy particularly the involvement of Nrf2 in the wound healing process was investigated in the present study. In addition, we have studied the levels of angiogenic markers in ulcer tissues and their correlation with Nrf2 during HBO therapy compared with standard therapy (Non-HBO) for DFU. A total of 32 Patients were recruited and randomized to standard wound care procedure alone (n = 17) or HBO therapy in combination with standard wound care procedure (n = 15) for 20 days. Our results showed that the tissue levels of Nrf2 along with its downstream targets were significantly increased in patients who underwent HBO therapy when compared to Non-HBO therapy. Further, HBO therapy induced angiogenesis as assessed by increased levels of angiogenesis markers such as EGF, VEGF, PDGF, FGF-2 and CXCL10 in the tissue samples. The expressions of eNOS and nitrite concentrations were also significantly increased in HBO therapy when compared to Non-HBO therapy subjects. Moreover, HBO therapy sensitises the macrophages to release FGF-2 and EGF thereby promotes angiogenesis. Further, it increased the levels of neutrophil attractant CXCL-8 thereby promotes the release of chemokine CCL2, a well-known mediator of neovascularization. The Pearson correlation showed that Nrf2 has a positive correlation with EGF, VEGF and PDGF. In conclusion, the findings of the present study suggest that HBO therapy promotes wound healing by increasing oxygen supply and distribution to damaged tissues, stimulating angiogenesis, decreasing inflammation, and increasing the nitrite levels. Increased levels of Nrf2 transiently regulate the expression of angiogenic genes in wound biopsies, which may result in accelerated healing of chronic wounds.

BSMI single nucleotide polymorphism in vitamin D receptor gene is associated with decreased circulatory levels of serum 25-hydroxyvitamin D among micro and macrovascular complications of type 2 diabetes mellitus.

Vitamin D Receptor (VDR) gene single nucleotide polymorphism (SNP) in the intron 8 is a well characterized SNP which has previously been linked to various diseases including Type 2 Diabetes Mellitus (T2DM). However, the association of this SNP with micro and macrovascular complications of T2DM remains poorly studied. Hence, the present study was designed to investigate the association of VDR gene SNP with complications of T2DM (disease phenotype) and correlated it with the serum levels of (25[OH]D) (intermediate phenotype) along with other clinical risk factors of T2DM. Genotyping was carried out in a total of 866 subjects, in which Group-I had Control subjects (n = 252), Group-II with T2DM without complications (n = 200); Group-III and Group-IV had subjects with micro (n = 216) and macrovascular (n = 198) complications respectively by PCR -RFLP. The 'G' allele of the VDR gene SNP is associated with a significant risk for T2DM, Diabetic Neuropathy (DN) and Coronary Artery Disease (CAD). In addition, subjects with mutant GG genotype had lower levels of (25[OH]D) in both T2DM and micro-macrovascular complications than AA genotype. Thus, VDR (rs1544410) SNP was found to be associated with decreased serum (25[OH]D) levels in both micro-macrovascular complications of T2DM among South Indian Population.

Single nucleotide polymorphisms in cytokine/chemokine genes are associated with severe infection, ulcer grade and amputation in diabetic foot ulcer.

Compared to other complications the genetics of diabetic foot ulcer is poorly studied. The Interleukin (IL)-6 (-174G > C/rs1800795), Tumor Necrosis Factor (TNF)-α (-308G > A/rs1800629) and (-238G > A/rs361525) and Stromal cell Derived Factor (SDF)-1 (+801G > A/rs1801157) are well characterized single nucleotide polymorphisms (SNPs) which were previously shown to be associated with Diabetic Foot Ulcer (DFU). In the present study, we looked at the association of these SNPs with foot microbial infection, Wagner's ulcer grade and treatment procedure, along with serum levels of these cytokines (intermediate phenotype) and other serum biomarkers (adiponectin, leptin, CRP and HOMA-IR) in subjects with DFU. Subjects with DFU (n = 270) were genotyped by PCR-RFLP and the serum levels of IL-6, TNF-α and SDF-1 were determined by ELISA. Microbial infections were determined by standard microbiological methods. Ulcer grade and treatment procedures were recorded. IL-6 (-174G > C), TNF-α (-308G > A) and SDF-1 (+801G > A) SNPs were associated with severe microbial infections. TNF-α (-308G > A) and (-238G > A) SNPs were associated with severe ulcer grades. SDF-1 (+801G > A) SNP was associated with major amputation even after adjusting for confounding variables. Identification of these SNPs in DFU subjects would help in identifying high risk individuals who need better treatment care.

Association of NF-E2 Related Factor 2 (Nrf2) and inflammatory cytokines in recent onset Type 2 Diabetes Mellitus.

We investigated the association of redox regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and inflammatory cytokines as well as clinical remission in patients with recent onset type 2 diabetes (DM). Blood was collected from 180 DM patients (105 males/75 females) and 150 control subjects (86 males/64 females). Blood glucose, HbA1c, lipid profile and Nrf2 levels were determined along with circulatory cytokines in study subjects. The data were adjusted with confounding factors such as age and sex using multiple logistic regression analysis. We found that Th1/Th2 and oxidative stress markers were significantly elevated, whereas Nrf2 and its downstream targets were decreased in peripheral blood mononuclear cells (PBMCs) of DM subjects when compared with control. The circulatory levels of Nrf2 showed a positive correlation with Th2 cytokines and negative correlation to Th1 cytokines. Further, the impaired insulin secretion in pancreatic ß-cells observed due to cytokine stress has been restored by activation of Nrf2 as assessed by glucose-stimulated insulin secretion (GSIS). This study identifies Nrf2 plays a central role in skewing Th1 and Th2 dominance in the progression of diabetes.

Association of A1538G and C2437T single nucleotide polymorphisms in heat shock protein-70 genes with diabetic nephropathy among South Indian population.

Diabetic Nephropathy (DN) is the leading cause of end-stage renal disease, characterized by progressive albuminuria and conferring additional risk of cardiovascular disease (CVD) and mortality. The crucial role of heat-shock proteins (HSPs) on renal function in patients with DN has been well documented. The present study was aimed to understand the association of HSP-70 gene variants on the susceptibility of Type 2 Diabetes Mellitus (T2DM) and DN. A total of 946 subjects (549 Males; 397 Females) were recruited and divided into four groups according to the levels of urinary albumin excretion (UAE): those with normoalbuminuria (UAE <30 mg/24 h; n=230), those with microalbuminuria (30≤ UAE ≤300 mg/24 h; n=230), and those with macroalbuminuria (UAE> 300 mg/24 h; n=230). The control group randomly enrolled a consecutive population of 256 healthy subjects who had a routine medical check-up in our hospital. Those subjects had no history or clinical symptoms of diabetes. Subjects were genotyped for HSP70-2 (+1538 A/G; rs2763979) and HSP70-hom (+2437 C/T; rs2227956) by PCR-restriction fragment length polymorphism (RFLP). The 'G' allele of HSP70-2 (+1538 A/G) single nucleotide polymorphism (SNP) showed relative risk for normoalbuminuria, microalbuminuria and macroalbuminuria subjects whereas the 'T' allele of HSP70-hom (+2437 C/T) SNP showed significant protection against macroalbuminuria subjects. In conclusion, our results indicate that the HSP70-2 (+1538 A/G) and HSP70-hom (+2437 C/T) SNPs are highly associated with renal complications in T2DM among the South Indian population.

Role of pterostilbene in attenuating immune mediated devastation of pancreatic beta cells via Nrf2 signaling cascade.

Nrf2 (nuclear factor erythroid 2-related factor-2) is a transcription factor that regulates oxidative/xenobiotic stress response and also suppress inflammation. Nrf2 signaling is associated with an increased susceptibility to various kinds of stress. Nrf2 has been shown as a promising therapeutic target in various human diseases including diabetes. Our earlier studies showed Pterostilbene (PTS) as a potent Nrf2 activator, and it protects the pancreatic ß-cells against oxidative stress. In this study, we investigated PTS confer protection against cytokine-induced ß-cell apoptosis and its role on insulin secretion in streptozotocin (STZ)-induced diabetic mice. The Nrf2 activation potential of PTS was assessed by dissociation of the Nrf2-Keap1 complex and by expression of ARE-driven downstream target genes in MIN6 cells. Further, the nuclear Nrf2 translocation and blockage of apoptotic signaling as demonstrated by the reduction of BAX/Bcl-2 ratio, Annexin-V positive cells and caspase-3 activity conferred the cyto-protection of PTS against cytokine-induced cellular damage. In addition, PTS treatment markedly improved glucose homeostasis and abated inflammatory response evidenced by the reduction of proinflammatory cytokines in diabetic mice. The inhibition of ß-cell apoptosis by PTS as assessed by BAX/Bcl-2 ratio and caspase-3 activity in the pancreas was associated with the activation of Nrf2 and the expression of its downstream target genes. PTS also inhibited the activation of iNOS and decreased nitric oxide (NO) formation in the pancreas of diabetic animals. The results obtained from both in vitro and in vivo experiments showed that PTS improves ß-cell function and survival against cytokine stress and also prevents STZ-induced diabetes.

Genetic association of IL-6, TNF-α and SDF-1 polymorphisms with serum cytokine levels in diabetic foot ulcer.

The IL-6 -174G/C (rs1800795), TNF-α -308G/A (rs1800629) and -238G/A (rs361525) and SDF-1 801G/A (rs1801157) are well characterized SNPs which have previously been linked to various diabetic complications. However, the involvement of these SNPs in DFU remains poorly studied. In the present study we looked at the association of these SNPs with DFU (disease phenotype) and correlated it with the serum levels of cytokines (intermediate phenotype) along with other clinical risk factors of DFU (adiponectin, leptin and hsCRP). Genotyping was carried out in Normal glucose tolerance ((NGT)/Control=106), T2DM without DFU (T2DM=139), T2DM with neuropathy (DFU-DN=191) and T2DM with PVD (DFU-PVD=79) subjects by PCR-RFLP and the serum cytokine levels were determined by ELISA. IL-6 -176 "C" allele conferred significant protection against T2DM but not against DFU. TNF-α -308 "A" allele (but not -238 SNP) conferred significant susceptibility towards both T2DM and DFU-DN. The SDF-1 "A" allele conferred significant protection against both DM and DFU-DN but not against DFU-PVD. Further, these alleles were shown to influence the serum cytokine/chemokine levels under diabetic conditions. Thus SNPs in cytokine/chemokine genes serve as valuable biomarkers for DFU.