Crosstalk between endoplasmic reticulum stress and oxidative stress in the progression of diabetic nephropathy.

Increasing evidence in substantiating the roles of endoplasmic reticulum stress, oxidative stress, and inflammatory responses and their interplay is evident in various diseases. However, an in-depth mechanistic understanding of the crosstalk between the intracellular stress signaling pathways and inflammatory responses and their participation in disease progression has not yet been explored. Progress has been made in our understanding of the cross talk and integrated stress signaling network between endoplasmic reticulum stress and oxidative stress towards the pathogenesis of diabetic nephropathy. In this present study, we studied the crosstalk between the endoplasmic reticulum stress and oxidative stress by understanding the role of protein disulfide isomerase and endoplasmic reticulum oxidase 1α, a key player in redox protein folding in the endoplasmic reticulum. We had recruited a total of 90 subjects and divided into three groups (control (n = 30), type 2 diabetes mellitus (n = 30), and diabetic nephropathy (n = 30)). We found that endoplasmic reticulum stress markers, activating transcription factor 6, inositol-requiring enzyme 1α, protein kinase RNA-like endoplasmic reticulum kinase, C/EBP homologous protein, and glucose-regulated protein-78; oxidative stress markers, thioredoxin-interacting protein and cytochrome b-245 light chain; and the crosstalk markers, protein disulfide isomerase and endoplasmic reticulum oxidase-1α, were progressively elevated in type 2 diabetes mellitus and diabetic nephropathy subjects. The association between the crosstalk markers showed a positive correlation with endoplasmic reticulum stress and oxidative stress markers. Further, the interplay between endoplasmic reticulum stress and oxidative stress was investigated in vitro using a human leukemic monocytic cell line under a hyperglycemic environment and examined the expression of protein disulfide isomerase and endoplasmic reticulum oxidase-1α. DCFH-DA assay and flow cytometry were performed to detect the production of free radicals. Further, phosphorylation of eIF2α in high glucose-exposed cells was studied using western blot. In conclusion, our results shed light on the crosstalk between endoplasmic reticulum stress and oxidative stress and significantly contribute to the onset and progression of diabetic nephropathy and therefore represent the major therapeutic targets for alleviating micro- and macrovascular complications associated with this metabolic disturbance. Graphical abstract.

Effect of yoga intervention on biochemical, oxidative stress markers, inflammatory markers and sleep quality among subjects with type 2 diabetes in South India: Results from the SATYAM project.

AIMS: The aim of this study was to investigate the effect of yoga intervention on the biochemical, oxidative stress markers and inflammatory markers and sleep quality among subjects with type 2 diabetes. METHODS: Subjects with type 2 diabetes attending a tertiary care centre for diabetes during Feb 2017 to Oct 2019 in Chennai, India were randomly assigned to two different groups. Group1(non-Yoga) (n = 150) was advised on simple physical exercises whereas group2(Yoga) (n = 150) was trained and advised to do yogasanas with static loosening exercises for 50 min for 5 days in a week. Both the groups were followed up for a period of 3 months. Anthropometric, biochemical, oxidative stress markers, inflammatory markers and sleep quality were assessed at baseline and after follow up. RESULTS: There was a significant reduction in BMI, blood glucose levels, HbA1c, lipid levels, IL6, TNFα and TBARS in Yoga group as compared to non-Yoga group. There was marked improvement in the levels of Adiponectin, PTGIS and sleep quality among subjects practising yogasanas. CONCLUSION: Regular practice of yogasanas improved glycaemic control, oxidative stress, inflammatory response and sleep quality among subjects with type 2 diabetes. Hence, Yogasanas can be used as an adjuvant therapy for managing type 2 diabetes.

LPS-induced Apoptosis is Partially Mediated by Hydrogen Sulphide in RAW 264.7 Murine Macrophages.

Lipopolysaccharide (LPS) induces apoptosis in murine macrophages through the autocrine secretion of tumor necrosis factor (TNF)-α and nitric oxide (NO). LPS-induced inflammation in murine macrophages is associated with hydrogen sulfide (H2S) production. In this present study, we reported the novel role of H2S in LPS-induced apoptosis and its underlying molecular mechanism specifically at late phases in murine macrophage cells. Stimulation of RAW 264.7 macrophages with LPS resulted in a time- and dose-dependent induction of apoptosis. We observed that the LPS-induced early apoptosis (associated with TNF-α secretion) in macrophages was not inhibited in the presence of H2S inhibitor (DL-propargylglycine), whereas early apoptosis was absent in the presence of TNF receptor antibody. Interestingly, LPS-induced late apoptosis paralleled with H2S production was reduced in the presence of H2S inhibitor but not with TNF receptor antibody. The late apoptotic events mediated by H2S and not the TNF-α induced early apoptosis correlated significantly with the induction of p53 and Bax expression in LPS-induced macrophages. Thus, it is possible that RAW 264.7 murine macrophages treated with LPS mediated early apoptosis through TNF-α and the late apoptotic events through the production of H2S.

A candidate gene identification strategy utilizing mouse to human big-data mining: "3R-tenet" in COPD genetic research.

BACKGROUND: Early life impairments leading to lower lung function by adulthood are considered as risk factors for chronic obstructive pulmonary disease (COPD). Recently, we compared the lung transcriptomic profile between two mouse strains with extreme total lung capacities to identify plausible pulmonary function determining genes using microarray analysis (GSE80078). Advancement of high-throughput techniques like deep sequencing (eg. RNA-seq) and microarray have resulted in an explosion of genomic data in the online public repositories which however remains under-exploited. Strategic curation of publicly available genomic data with a mouse-human translational approach can effectively implement "3R- Tenet" by reducing screening experiments with animals and performing mechanistic studies using physiologically relevant in vitro model systems. Therefore, we sought to analyze the association of functional variations within human orthologs of mouse lung function candidate genes in a publicly available COPD lung RNA-seq data-set. METHODS: Association of missense single nucleotide polymorphisms, insertions, deletions, and splice junction variants were analyzed for susceptibility to COPD using RNA-seq data of a Korean population (GSE57148). Expression of the associated genes were studied using the Gene Paint (mouse embryo) and Human Protein Atlas (normal adult human lung) databases. The genes were also assessed for replication of the associations and expression in COPD-/mouse cigarette smoke exposed lung tissues using other datasets. RESULTS: Significant association (p <  0.05) of variations in 20 genes to higher COPD susceptibility have been detected within the investigated cohort. Association of HJURP, MCRS1 and TLR8 are novel in relation to COPD. The associated ADAM19 and KIT loci have been reported earlier. The remaining 15 genes have also been previously associated to COPD. Differential transcript expression levels of the associated genes in COPD- and/ or mouse emphysematous lung tissues have been detected. CONCLUSION: Our findings suggest strategic mouse-human datamining approaches can identify novel COPD candidate genes using existing datasets in the online repositories. The candidates can be further evaluated for mechanistic role through in vitro studies using appropriate primary cells/cell lines. Functional studies can be limited to transgenic animal models of only well supported candidate genes. This approach will lead to a significant reduction of animal experimentation in respiratory research.

Putative Systemic Biomarkers of Biomass Smoke-Induced Chronic Obstructive Pulmonary Disease among Women in a Rural South Indian Population.

RATIONALE: Exposure to biomass smoke (BMS) has been implicated in chronic obstructive pulmonary disease (COPD). About 3 billion people worldwide use biomass fuel for cooking and heating. Women in rural communities of low- and lower-middle-income countries are disproportionately exposed to massive amounts of BMS during active cooking hours (4-6 h/day). Therefore, BMS exposure is considered as a risk factor for COPD in the same order of magnitude as tobacco smoke. In rural India, due to cultural reasons, women are the primary cook of the family and are mostly nonsmokers. Thus, BMS-induced COPD is predominant among rural Indian women. However, BMS-COPD remains a relatively unexplored health problem globally. Therefore, we investigated the serum chemokine and cytokine signatures of BMS-COPD and tobacco smoke-induced COPD (TS-COPD) patients compared to their control in a rural South Indian population for this field study. METHODS: Concentrations of 40 serum chemokines and cytokines were measured using a multiplexed immunoassay. The study cohort consisted of BMS-COPD (female; n = 29) and BMS-exposed subjects without COPD (BMS-CONTROL; female; n = 24). For comparison, data from TS-COPD patients (male, n = 23) and tobacco smokers without COPD (TS-CONTROL; male, n = 22) were investigated. Subjects were matched for age, sex, and biomass exposure. Tobacco consumption was slightly higher in TS-COPD subjects compared to TS-CONTROL. BMS-exposed and TS-exposed subjects (currently exposed) were from the same locality with similar dwelling habits and socioeconomic status. A validated structured questionnaire-based survey and spirometry was performed. An additional control group with no tobacco and BMS exposure (TS-BMS-CONTROL; n = 15) was included. Statistical significance was set at p ≤ 0.01. RESULTS: Serum median concentrations (pg/ml) of CCL15 [8799.35; 5977.22], CCL27 [1409.14; 1024.99], and CXCL13 [37.14; 26.03] were significantly higher in BMS-CONTROL compared to BMS-COPD subjects. Nine analytes exhibited higher concentrations in TS-CONTROL compared to TS-COPD subjects. Comparison of chemokine and cytokine concentrations among BMS-COPD versus TS-COPD and BMS-CONTROL versus TS-CONTROL subjects also revealed distinct molecular signatures. CONCLUSION: Our data identifies CCL27 and CXCL13 as putative, plausibly homeostatic/protective biomarkers for BMS-COPD within the investigated population that warrants validation in larger and multiple cohorts. The findings further indicate exposure-specific systemic response of chemokines and cytokines.

Transcriptomic analysis comparing mouse strains with extreme total lung capacities identifies novel candidate genes for pulmonary function.

BACKGROUND: Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity. METHODS: Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences. RESULTS: Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term "extracellular region" was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3. CONCLUSIONS: Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function.

Assessment of the Role of Noni (Morinda citrifolia) Juice for Inducing Osteoblast Differentiation in Isolated Rat Bone Marrow Derived Mesenchymal Stem Cells.

BACKGROUND AND OBJECTIVES: Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro. METHODS AND RESULTS: Treatment with 200 µg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo, which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation. CONCLUSIONS: These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis.

Novel phenanthridine (PHE-4i) derivative inhibits carrageenan-induced rat hind paw oedema through suppression of hydrogen sulfide.

This study was conducted to assess the anti-inflammatory effect of a novel synthesized phenanthridine alkaloid (PHE-4i) and to examine the possible involvement of hydrogen sulfide (H2S) in anti-inflammatory mechanism. The synthesized phenanthridine derivative PHE-4i (2, 5, and 10 mg/kg) was administered intraperitoneally to rats. One hour following treatment, inflammation was induced by intraplantar injection of carrageenan (1 %), in the hind paw. Paw volume as the index of inflammation was measured before and after carrageenan injection. Neutrophil sequestration into the hind paw was quantified by measuring tissue myeloperoxidase (MPO) activity and was compared for the inhibition of H2S production. Pretreatment with PHE-4i significantly reduced carrageenan-induced hind paw weight, MPO activity, leukocyte infiltration, and H2S production in a dose-dependent manner (p < 0.001). These results indicate that the anti-inflammatory effect of PHE-4i on carrageenan-induced rat paw oedema could be via the inhibition of the gaseous mediator H2S.

Ultrafine carbon particle mediated cardiovascular impairment of aged spontaneously hypertensive rats.

BACKGROUND: Studies provide compelling evidences for particulate matter (PM) associated cardiovascular health effects. Elderly individuals, particularly those with preexisting conditions like hypertension are regarded to be vulnerable. Experimental data are warranted to reveal the molecular pathomechanism of PM related cardiovascular impairments among aged/predisposed individuals. Thus we investigated the cardiovascular effects of ultrafine carbon particles (UfCP) on aged (12-13 months) spontaneously hypertensive rats (SHRs) and compared the findings with our pervious study on adult SHRs (6-7 months) to identify age related predisposition events in cardiovascular compromised elderly individuals. METHODS: Aged SHRs were inhalation exposed to UfCP for 24 h (~180 µg/m³) followed by radio-telemetric assessment for blood pressure (BP) and heart rate (HR). Bronchoalveolar lavage (BAL) fluid cell differentials, interleukin 6 (IL-6) and other proinflammatory cytokines; serum C-reactive protein (CRP) and haptoglobin (HPT); and plasma fibrinogen were measured. Transcript levels of hemeoxygenase 1 (HO-1), endothelin 1 (ET1), endothelin receptors A, B (ETA, ETB), tissue factor (TF), and plasminogen activator inhibitor-1 (PAI-1) were measured in the lung and heart to assess oxidative stress, endothelial dysfunction and coagulation cascade. RESULT: UfCP exposed aged SHRs exhibited increased BP (4.4%) and HR (6.3%) on 1(st) recovery day paralleled by a 58% increase of neutrophils and 25% increase of IL-6 in the BAL fluid. Simultaneously higher CRP, HPT and fibrinogen levels in exposed SHRs indicate systemic inflammation. HO-1, ET1, ET-A, ET-B, TF and PAI-1 were induced by 1.5-2.0 folds in lungs of aged SHRs on 1(st) recovery day. However, in UfCP exposed adult SHRs these markers were up-regulated (2.5-6 fold) on 3(rd) recovery day in lung without detectable pulmonary/systemic inflammation. CONCLUSIONS: The UfCP induced pulmonary and systemic inflammation in aged SHRs is associated with oxidative stress, endothelial dysfunction and disturbed coagulatory hemostasis. UfCP exposure increased BP and HR in aged SHRs rats which was associated with lung inflammation, and increased expression of inflammatory, vasoconstriction and coagulation markers as well as systemic changes in biomarkers of thrombosis in aged SHRs. Our study provides further evidence for potential molecular mechanisms explaining the increased risk of particle mediated cardiac health effects in cardiovascular compromised elderly individuals.

Secreted phosphoprotein 1 is a determinant of lung function development in mice.

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14-P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1((-/-)) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1((+/+)) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1((-/-)) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1((-/-)) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice.

Heterologous expression of oxalate decarboxylase in Lactobacillus plantarum NC8.

Lactic acid bacteria (LABs) are being used as a probiotic very often for various enteric problems. Many genetically modified LABs are created by different workers for various novel applications. In this study we examine the expression of heterologous oxalate decarboxylase (oxdc) in Lactobacillus plantarum NC8. Generally, this enzyme is not present in Lactobacillus spp. Oxdc gene from Bacillus subtilis was polymerase chain reaction-amplified and cloned in a shuttle vector pSIP400 series, downstream of the inducible promoter, P(orfx). In the presence of an inducing peptide, Sakacin-P, the expression of OxdC was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-free extract and the purified protein from the recombinant LABs showed the presence of OxdC activity. The above recombinant LABs, with desired modifications, can be used as a possible probiotic for the degradation of intestinal dietary oxalate for preventing enteric hyperoxaluria.