Gene Expression Of Inflammatory Markers Research Articles

Gallic Acid Ameliorates Angiotensin II-Induced Atrial Fibrillation by Inhibiting Immunoproteasome- Mediated PTEN Degradation in Mice.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and is a major cause of stroke and heart failure. We and others have found that gallic acid (GA) plays a beneficial role in cardiac hypertrophic remodeling and hypertension. However, the effect of GA on angiotensin II (Ang II)-induced AF and atrial remodeling as well as the underlying mechanisms remain unknown. AF was induced in mice by Ang II infusion (2000 ng/kg/min) for 3 weeks. Blood pressure was measured using the tail-cuff method. Atrial volume was evaluated by echocardiography. Atrial remodeling was studied using hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining. Atrial oxidative stress was assessed by dihydroethidium staining. The gene expression of fibrotic and inflammatory markers and protein levels of signaling mediators were measured by quantitative real-time PCR and western blot analysis. In mice, GA administration significantly attenuated Ang II-induced elevation of blood pressure, AF incidence and duration, atrial dilation, fibrosis, inflammation, and oxidative stress compared with the vehicle control. Furthermore, GA downregulated Ang II-induced activity and expression of immunoproteasome subunits (β2i and β5i), which reduced PTEN degradation and led to the inactivation of AKT1 and downstream signaling mediators. Importantly, blocking PTEN activity by VO-Ohpic markedly reversed the GA-mediated protective effects on Ang II-induced AF and atrial remodeling. Therefore, our results provide novel evidence that GA exerts a cardioprotective role by inhibiting immunoproteasome activity, which attenuates PTEN degradation and activation of downstream signaling, and may represent a promising candidate for treating hypertensive AF.

Effect of camphor on biochemical factors and gene expression of antioxidant enzymes, inflammatory and apoptotic factors against gentamicin-induced nephrotoxicity in rats

Introduction: Camphor is a natural antioxidant with anti-inflammatory and tissue repair properties. Nephrotoxicity is the most important side effect of gentamicin (GEM) administration. Therefore, investigating the effect of natural antioxidants can resolve this complication. Objectives: We aimed to assay the effect of camphor on biochemical factors and gene expression of antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPX]) and inflammatory markers (tumor necrosis factor-alpha [TNF-α], nuclear factor kappa-B [NF-κB], interleukine-6 [IL-6]), and apoptotic indices (BCL2-associated X protein [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3)], against GEM-induced nephrotoxicity in rats. Materials and Methods: Thirty adult male Wistar rats were allocated to five groups. Positive control and treatment groups were given GEM to induce nephrotoxicity. Animal treatment groups were treated with camphor in olive oil for 12 days. Renal biopsies, serum, extraction of renal tissue and urine of rats were taken after the twelfth day. Biopsies were examined for structural changes using a light microscope, moreover, apoptosis, desired biochemical and inflammatory factors, were investigated by suitable methods. Results: Camphor had no effect on biochemical factors, including malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), urea, creatinine and urine protein. However, it reduced the gene expression of TNF-α, NF-κB, IL-6, Bax, and caspase-3 and increased the gene expression of GPX and CAT and Bcl-2. Moreover, camphor improved kidney histopathological changes in the camphor groups in comparison with the GEM group. Conclusion: Camphor can be useful in the attenuation of GEM-induced nephrotoxicity based on expression levels of examined enzymes and factors and improving kidney histopathological changes.

Spermidine, a caloric restriction mimetic, provides neuroprotection against normal and D-galactose-induced oxidative stress and apoptosis through activation of autophagy in male rats during aging.

Spermidine (SPD) is a natural polyamine present in all living organisms and is involved in the maintenance of cellular homeostasis by inducing autophagy in different model organisms. Its role as a caloric restriction mimetic (CRM) is still being investigated. We have undertaken this study to investigate whether SPD, acting as a CRM, can confer neuroprotection in D-galactose induced accelerated senescence model rat and naturally aged rats through modulation of autophagy and inflammation. Young male rats (4months), D-gal induced (500mg/kg b.w., subcutaneously) aging and naturally aged (22months) male rats were supplemented with SPD (10mg/kg b.w., orally) for 6weeks. Standard protocols were employed to measure prooxidants, antioxidants, apoptotic cell death and electron transport chain complexes in brain tissues. Gene expression analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to assess the expression of autophagy and inflammatory marker genes. Our data demonstrate that SPD significantly (p ≤ 0.05) decreased the level of pro-oxidants and increased the level of antioxidants. SPD supplementation also augmented the activities of electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria. RT-PCR data revealed that SPD up-regulated the expression of autophagy genes (ATG-3, Beclin-1, ULK-1 and LC3B) and down-regulated the expression of the inflammatory gene (IL-6) in aging brain. Our results provide first line of evidence that SPD provides neuroprotection against aging-induced oxidative stress by regulating autophagy, antioxidants level and also reduces neuroinflammation. These results suggest that SPD may be beneficial for neuroprotection during aging and age-related disorders.

Intermittent hypoxia induces premature adipose tissue senescence leading to cardiac remodeling

Obstructive sleep apnea is a growing worldwide health problem. The landmark feature of OSA is a chronic intermittent hypoxia (CIH) responsible for multiple organ damages including heart diseases. CIH profoundly alters both visceral adipose tissue (VAT) and heart structures and functions, but little is known regarding their interactions in the context of CIH. We recently showed that VAT senescence drives myocardial alterations through the release of profibrotic factors in aged mice. Thus, we aim at demonstrating that CIH induces a premature vWAT senescent phenotype, responsible for subsequent heart dysfunction. In a first series of animals, ten-week old C57BL6 male mice ( n = 10/group) were exposed to 14 days CIH protocol (8 hours daily, 5–21% cyclic inspired oxygen fraction, 60 seconds per cycle). In another series, mice were submitted to VAT surgical lipectomy or sham-surgery ( n = 10/group) and then exposed to the same CIH protocol. VAT and heart were assessed by histology for fibrosis (Sirius red), cell hypertrophy (wheat-germ agglutinin) and reactive oxygen species (ROS) (4-HNE), and markers of senescence (p16, p21, p53), inflammation (CD68, Tnfa, Serpine1), fibrosis (Col1a1) and hypertrophy were also evaluated by RT-qPCR and western blot. CIH induced an increased macrophage infiltration in VAT, along with an increased expression of senescence markers, ROS production and gene expression of inflammatory and fibrogenic markers. This was associated with CIH-induced myocardial interstitial fibrosis and upregulation of profibrotic gene expression in myocardium. Interestingly, VAT lipectomy prevented CIH-induced myocardial fibrosis. Short-term exposure to CIH is sufficient to induce both VAT remodelling, characterized by oxidative stress, inflammation and fibrosis, and cardiac interstitial fibrosis that was prevented by VAT lipectomy. This strongly suggest a causal crosstalk between CIH-induced VAT senescence and cardiac remodeling.

The Absence of Adiponectin Alters Niacin’s Effects on Adipose Tissue Inflammation in Mice

Obesity is an immunometabolic disease associated with chronic inflammation and the dysregulation of pro- and anti-inflammatory cytokines. One hallmark of obesity is reduced concentrations of the anti-inflammatory adipokine, adiponectin. Pharmacologic doses of niacin produce multiple metabolic benefits, including attenuating high-fat diet (HFD)-induced adipose tissue inflammation and increasing adiponectin concentrations. To determine if adiponectin mediates the anti-inflammatory effects of niacin, male C57BL/6J (WT) and adiponectin null (Adipoq-/-) mice were maintained on a low-fat diet (LFD) or HFD for 6 weeks, before being administered either vehicle or niacin (360 mg/kg/day) for 5 weeks. HFD-fed mice had increased expression of genes associated with macrophage recruitment (Ccl2) and number (Cd68), and increased crown-like structure (CLS) number in adipose tissue. While niacin attenuated Ccl2 expression, there were no effects on Cd68 or CLS number. The absence of adiponectin did not hinder the ability of niacin to reduce Ccl2 expression. HFD feeding increased gene expression of inflammatory markers in the adipose tissue of WT and Adipoq-/- mice. While niacin tended to decrease the expression of inflammatory markers in WT mice, niacin increased their expression in HFD-fed Adipoq-/- mice. Therefore, our results indicate that the absence of adiponectin alters the effects of niacin on markers of adipose tissue inflammation in HFD-fed mice, suggesting that the effects of niacin on tissue cytokines may involve adiponectin.

Angiotensin-(1-7) Improves Integrated Cardiometabolic Function in Aged Mice.

Angiotensin (Ang)-(1-7) is a beneficial renin–angiotensin system (RAS) hormone that elicits protective cardiometabolic effects in young animal models of hypertension, obesity, and metabolic syndrome. The impact of Ang-(1-7) on cardiovascular and metabolic outcomes during aging, however, remains unexplored. This study tested the hypothesis that Ang-(1-7) attenuates age-related elevations in blood pressure and insulin resistance in mice. Young adult (two-month-old) and aged (16-month-old) male C57BL/6J mice received Ang-(1-7) (400 ng/kg/min) or saline for six-weeks via a subcutaneous osmotic mini-pump. Arterial blood pressure and metabolic function indices (body composition, insulin sensitivity, and glucose tolerance) were measured at the end of treatment. Adipose and cardiac tissue masses and cardiac RAS, sympathetic and inflammatory marker gene expression were also measured. We found that chronic Ang-(1-7) treatment decreased systolic and mean blood pressure, with a similar trend for diastolic blood pressure. Ang-(1-7) also improved insulin sensitivity in aged mice to levels in young mice, without effects on glucose tolerance or body composition. The blood pressure–lowering effects of Ang-(1-7) in aged mice were associated with reduced sympathetic outflow to the heart. These findings suggest Ang-(1-7) may provide a novel pharmacological target to improve age-related cardiometabolic risk.

Physical Exercise Exacerbates Acute Kidney Injury Induced by LPS via Toll-Like Receptor 4.

Introduction: Lipopolysaccharide (LPS) is a systemic response-triggering endotoxin, which has the kidney as one of its first targets, thus causing acute injuries to this organ. Physical exercise is capable of promoting physiological alterations and modulating inflammatory responses in the infectious process through multiple parameters, including the toll-like receptor (TLR)-4 pathway, which is the main LPS signaling in sepsis. Additionally, previous studies have shown that physical exercise can be both a protector factor and an aggravating factor for some kidney diseases. This study aims at analyzing whether physical exercise before the induction of LPS endotoxemia can protect kidneys from acute kidney injury.Methods: C57BL/6J male mice, 12 weeks old, were distributed into four groups: (1) sedentary (control, N = 7); (2) sedentary + LPS (N = 7); (3) trained (N = 7); and (4) trained + LPS (N = 7). In the training groups, the animals exercised 5×/week in a treadmill, 60 min/day, for 4 weeks (60% of max. velocity). Sepsis was induced in the training group by the application of a single dose of LPS (5 mg/kg i.p.). Sedentary animals received LPS on the same day, and the non-LPS groups received a saline solution instead. All animals were euthanized 24 h after the administration of LPS or saline.Results: The groups receiving LPS presented a significant increase in serum urea (p < 0.0001) and creatinine (p < 0.001) concentration and renal gene expression of inflammatory markers, such as tumor necrosis factor alpha and interleukin-6, as well as TLRs. In addition, LPS promoted a decrease in reduced glutathione. Compared to the sedentary + LPS group, trained + LPS showed overexpression of a gene related to kidney injury (NGAL, p < 0.01) and the protein levels of LPS receptor TLR-4 (p < 0.01). Trained + LPS animals showed an expansion of the tubulointerstitial space in the kidney (p < 0.05) and a decrease in the gene expression of hepatic AOAH (p < 0.01), an enzyme involved in LPS clearance.Conclusion: In contrast to our hypothesis, training was unable to mitigate the renal inflammatory response caused by LPS. On the contrary, it seems to enhance injury by accentuating endotoxin-induced TLR-4 signaling. This effect could be partly due to the modulation of a hepatic enzyme that detoxifies LPS.

Autologous microfragmented adipose tissue reduces inflammatory and catabolic markers in supraspinatus tendon cells derived from patients affected by rotator cuff tears

Rotator cuff tears are common musculoskeletal disorders, and surgical repair is characterized by a high rate of re-tear. Regenerative medicine strategies, in particular mesenchymal stem cell-based therapies, have been proposed to enhance tendon healing and reduce the re-tear rate. Autologous microfragmented adipose tissue (μFAT) allows for the clinical application of cell therapies and showed the ability to improve tenocyte proliferation and viability in previous in vitro assessments. The hypothesis of this study is that μFAT paracrine action would reduce the catabolic and inflammatory marker expression in tendon cells (TCs) derived from injured supraspinatus tendon (SST). TCs derived from injured SST were co-cultured with autologous μFAT in transwell for 48h. Metabolic activity, DNA content, the content of soluble mediators in the media, and the gene expression of tendon-specific, inflammatory, and catabolic markers were analyzed. μFAT-treated TCs showed a reduced expression of PTGS2 and MMP-3 with respect to untreated controls. Increased IL-1Ra, VEGF, and IL-6 content were observed in the media of μFAT-treated samples, in comparison with untreated TCs. μFAT exerted an anti-inflammatory action on supraspinatus tendon cells in vitro through paracrine action, resulting in the reduction of catabolic and inflammatory marker expression. These observations potentially support the use of μFAT as adjuvant therapy in the treatment of rotator cuff disease.

Interleukin 1 Receptor 1 Knockout and Maternal High Fat Diet Exposure Induces Sex-Specific Effects on Adipose Tissue Adipogenic and Inflammatory Gene Expression in Adult Mouse Offspring.

Background: The global incidence of obesity continues to rise, increasing the prevalence of metabolic diseases such as insulin resistance, dyslipidemia, and type 2 diabetes mellitus. Low-grade chronic inflammation, associated with the obese state, also contributes to the development of these metabolic comorbidities. Interleukin-1-receptor-1 (IL-1R1), a pro-inflammatory mediator, bridges the metabolic and inflammatory systems. In young male mice, deficiency of IL-1R1 (IL-1R1−/−) paired with a high-fat diet (HFD) offered beneficial metabolic effects, however in female mice, the same pairing led to metabolic dysfunction. Therefore, we examined the contribution of maternal HFD in combination with IL1R1−/− to metabolic health in adult offspring.Methods: Female C57BL/6 and IL-1R1−/− mice were randomly assigned to a control diet (10% kcal from fat) or HFD (45% kcal from fat) 10 days prior to mating and throughout gestation and lactation. Male and female offspring were housed in same-sex pairs post-weaning and maintained on control diets until 16 weeks old. At 15 weeks, an oral glucose tolerance test (OGTT) was performed to assess glucose tolerance. Histological analysis was carried out to assess adipocyte size and gene expression of adipogenic and inflammatory markers were examined.Results: IL-1R1−/− contributed to increased body weight in male and female adult offspring, irrespective of maternal diet. IL-1R1−/− and maternal HFD increased adipocyte size in the gonadal fat depot of female, but not male offspring. In female offspring, there was reduced expression of genes involved in adipogenesis and lipid metabolism in response to IL1R1−/− and maternal HFD. While there was an increase in inflammatory gene expression in response to maternal HFD, this appeared to be reversed in IL1R1−/− female offspring. In male offspring, there was no significant impact on adipogenic or lipid metabolism pathways. There was an increase in inflammatory gene expression in IL1R1−/− male offspring from HFD-fed mothers.Conclusion: This study suggests that IL-1R1 plays a complex and important role in the metabolic health of offspring, impacting adipogenesis, lipogenesis, and inflammation in a sex-specific manner.

Effects of Berries on High-Fat Diet-Induced Inflammation

Abstract Objectives Studies suggest that inflammation mediates the link between obesity and its comorbidities including type 2 diabetes and cardiovascular disease. Hence, there is a demand for effective alternative or complementary approaches to treat obesity-associated inflammation. The objective of this study was to determine whether consumption of blackberries (BL) and raspberries (RB) alone or in combination reduce obesity-induced inflammation. Methods In Vitro Study: RAW 264.7 macrophages were pretreated with either BL, RB, or BL + RB, each at a final concentration of 200 µg/mL for 2 h. LPS (1 ng/mL) was then added to the media for 16 h. mRNA expression of inflammatory cytokines was measured. In Vivo Study: Five-week-old mice were acclimated to a low-fat low-sucrose (LFLS) diet for one week after which mice were randomized 10 per group to one of five groups: 1) LFLS, 2) high-fat high-sucrose (HFHS), 3) HFHS + 10% BL, 4) HFHS + 10% RB, or 5) HFHS + 5% BL + 5% RB. Expression of inflammatory markers was measured in the liver as well as epididymal and inguinal white adipose tissue. Results In Vitro Study: Each berry alone and in combination suppressed the LPS-induced increase in inflammatory markers, with the combination (BL + RB) having the greatest effect. The combination suppressed LPS-induced expression of Ccl2, Tnfa, F4/80, and Il6 by 3.7−, 5.3−, 5.3−, and 4.4-fold, respectively. In Vivo Study: Gene expression analysis indicated that berry consumption had no significant effect on proinflammatory (Ccl2, Il1b, Tnfa, Il6, Itgam) or anti-inflammatory (Adipoq, Arg1, Mgl1) markers in adipose tissue depots or liver. However, relatively low gene expression of inflammatory markers in the tissues indicates that the mice fed the HFHS diet failed to develop a robust inflammatory state. Conclusions BL and RB have direct anti-inflammatory effects on immune cells. Initial analysis indicates that consumption of BL and RB has no significant effects on markers of inflammation in a diet-induced mouse model of obesity. However, it is possible that the relatively low levels of inflammation in these mice masked the anti-inflammatory potential of BL and RB. Ongoing analysis will provide additional insights into the effects of BL and RB on inflammation in these tissues. Funding Sources Lewis Foundation Award.

Dietary Advanced Glycation End Products (AGEs) could alter ovarian function in mice

Nutrition is an important source of exogenous AGEs and thermally processed foods present in western-style diets contain a large amount of these pro-inflammatory AGEs. Additionally, the intake of dietary AGEs could upregulate ovarian gene expression of inflammatory macrophage markers. The objective of this study was to investigate the effect of diet rich in AGEs on estrous cyclicity and ovarian function in a mouse model. Six-week old C57BL/6 J female mice were randomly subjected to either a diet low in AGEs (L-AGE) or a diet high in AGEs (H-AGE) for a total of 13 weeks. Experiments performed included daily vaginal smears to assess estrous cyclicity, ovarian superovulation with gonadotropins to assess the number of oocytes released, whole ovarian tissue mRNA quantification by RT-PCR to quantify genes involved in folliculogenesis, steroidogenesis, and macrophage markers, and ovarian morphology for follicle count. Outcome measures included estrous cyclicity, number of oocytes following superovulation, expression of genes involved in folliculogenesis, steroidogenesis, and macrophage infiltration as well as the number of primordial, primary, secondary, antral follicles and corpora lutea. Compared to mice on L-AGE diet, mice on H-AGE spent significantly longer time in the diestrus phase, had similar number of oocytes released following ovarian superovulation, and showed significant alterations in genes involved in steroidogenesis (increase in Star mRNA expression levels) and folliculogenesis (increase in Gdf-9 and Fshr mRNA expression levels). Mouse macrophage marker F4/80 mRNA expression was upregulated in mice on H-AGE diet compared to mice on L-AGE diet. Finally, mice on H-AGE diet had significantly fewer corpora lutea in their ovaries. These results indicate that the ingestion of high amounts of dietary AGEs could disrupt folliculogenesis and steroidogenesis that might lead to abnormal estrous cyclicity. Intake of dietary AGEs could also upregulate ovarian gene expression of inflammatory macrophage markers.

Angiotensin‐(1‐7) Improves Integrated Cardiometabolic Function in Aged Mice

BackgroundAging is associated with impaired cardiometabolic function including insulin resistance, elevated blood pressure, and cardiac hypertrophy. Angiotensin (Ang)‐(1‐7) is a beneficial hormone of the renin‐angiotensin system that improves control of blood pressure and glucose homeostasis in models of cardiovascular and metabolic diseases in young mice. To our knowledge, no studies have evaluated the importance of Ang‐(1‐7) to integrated cardiometabolic function in aging. We hypothesized that Ang‐(1‐7) would improve insulin sensitivity as well as decrease blood pressure and cardiac hypertrophy in healthy aged mice.MethodsSixteen month‐old male C57BL/6J mice received 6‐week Ang‐(1‐7) [400 ng/kg/min, n=9] or saline infusion (n=9) via subcutaneous osmotic mini‐pumps. All mice were fed standard chow diet (18% kcal from fat). During the last week of treatment, intraperitoneal insulin and glucose tolerance tests were performed and blood pressure was measured via a carotid artery catheter. Heart weight was measured at the end of experiments. Gene expression of tyrosine hydroxylase (index of sympathetic activity), inflammatory markers (IL10, IL1β, IL6, TNFα) and Ang II AT1 and Ang‐(1‐7) Mas receptors were measured in myocardial tissue by quantitative real‐time PCR and quantified with 2‐ΔΔCT methods.ResultsWe found that Ang‐(1‐7) decreased systolic [Ang‐(1‐7): 105±2 mmHg, saline: 116±4 mmHg, P=0.031], diastolic [Ang‐(1‐7): 77±2 mmHg, saline: 83±2 mmHg, P=0.041], and mean [Ang‐(1‐7): 86±2 mmHg, saline: 94±3 mmHg, P = 0.023] blood pressure. Furthermore, Ang‐(1‐7) reduced heart weight [Ang‐(1‐7): 0.18±0.01 g; saline: 0.21±0.01 g, P=0.042]. The improvement in cardiac hypertrophy with Ang‐(1‐7) was associated with decreased tyrosine hydroxylase gene expression in the heart [Ang‐(1‐7): 0.4±0.1, saline: 0.9±0.1, p=0.014], with no effect on gene expression of inflammatory markers or angiotensin receptors (p&gt;0.3). Ang‐(1‐7) did not alter body mass [Ang‐(1‐7): 46.5±2.0 g, saline: 45.9±1.5 g, p=0.792] or body composition (p&gt;0.2). Ang‐(1‐7) also improved insulin sensitivity [Ang‐(1‐7): −2896±593 AUC, saline:‐1171 ± 624 AUC, p=0.033] but did not affect fasting glucose levels [Ang‐(1‐7): 180±5 mg/dL, saline: 169±8 mg/dL, p=0.225] or glucose tolerance (p=0.202).ConclusionsChronic Ang‐(1‐7) treatment decreases blood pressure, cardiac hypertrophy, and markers of cardiac sympathetic activity as well as increases insulin sensitivity in aged mice. These beneficial cardiometabolic effects of Ang‐(1‐7) occurred in the absence of significant changes in body weight or composition. Overall, these data suggest that Ang‐(1‐7) may provide a novel pharmacological target to improve age‐related cardiovascular and metabolic risk.Support or Funding InformationNIH Pathway to Independence Award: R00 HL122507 to A.C. ArnoldAHA Postdoctoral Fellowship: 18POST33960087 to A.J. Miller

Phenformin Attenuates Renal Injury in Unilateral Ureteral Obstructed Mice without Affecting Immune Cell Infiltration.

Phenformin and metformin are antihyperglycemic drugs that belong to the class of biguanides. Previously, we demonstrated that metformin elicits renoprotective effects in unilateral ureteral obstructed mice by reducing the infiltration of immune cells into the kidney. Since phenformin is a more potent drug as compared to metformin, we investigated the renoprotective properties of phenformin. We studied the efficacy of both drugs using mice that underwent unilateral ureteral obstruction. Renal damage was evaluated on RNA and protein level by qPCR, Western blotting, and immunohistochemistry. Moreover, we studied immune cell infiltration using flow cytometry. Both biguanides significantly reduced UUO-induced kidney injury, as illustrated by a reduction in KIM-1 protein expression. In addition, both metformin and phenformin impacted the gene expression of several inflammatory markers but to a different extent. Moreover, in contrast to metformin, phenformin did not impact immune cell infiltration into UUO kidneys. In conclusion, we demonstrated that phenformin has similar renoprotective effects as metformin, but the mechanism of action differs, and phenformin is more potent. The beneficial effects of phenformin are probably due to inhibition of the STAT3 pathway and mitochondrial complex I. Further research is needed to unveil the therapeutic potential of phenformin for the treatment of renal injury, either at low, non-toxic concentrations or as part of a combination therapy.

A Robust Model System for Retinal Hypoxia: Live Imaging of Calcium Dynamics and Gene Expression Studies in Primary Human Mixed Retinal Culture.

The detailed mechanisms underlying oxidative stress that leads to neuroinflammation and neurodegeneration in retinal vascular conditions, including diabetic retinopathy, retinopathy of prematurity etc., remain largely unexplored mainly due to a lack of suitable disease models that can simulate the inherent neuron–glia interactions in human retina. Specifically, establishment of a mixed retinal culture (MRC) containing both neuron and glial cell types remains a challenge due to different conditions required for their optimal growth and differentiation. Here, we establish a novel primary MRC model system containing neurons, astrocytes, Müller glia, and microglia from human donor retina that can be used to study the neuromodulatory effects of glial cells under the stress. The cell characterization based on immunostaining with individual cell type–specific markers and their presence in close vicinity to each other further underscores their utility for studying their cross talk. To the best of our knowledge, this is the first instance of an in vitro model obtained from human donor retina containing four major cell types. Next, we induce hypoxic stress to MRC to investigate if hypoxia activated neuroglia modulates altered gene expression for inflammatory, apoptotic, and angiogenic markers and Ca2+ transients by live cell imaging. Further, we performed k-means clustering of the Ca2+ responses to identify the modification of clustering pattern in stressed condition. Finally, we provide the evidence that the altered Ca2+ transient correlates to differential expression of genes shown to be involved in neuroinflammation, angiogenesis, and neurodegeneration under the hypoxic conditions as seen earlier in human cell lines and animal models of diabetic retinopathy. The major features of the hypoxic conditions in the proposed human MRC model included: increase in microglia activity, chemokine and cytokine expression, and percentage of cells having higher amplitude and frequency of Ca2+ transients. Thus, the proposed experimental system can potentially serve as an ideal in vitro model for studying the neuroinflammatory and neurodegenerative changes in the retina and identifying newer drug targets.

Ursolic acid induces the production of IL6 and chemokines in both adipocytes and adipose tissue

ABSTRACT Adipose tissue inflammation plays an important role in the regulation of glucose and lipids metabolism. It is unknown whether Ursolic acid (UA) could regulate adipose tissue inflammation, though it can regulate inflammation in many other tissues. In this study, 3T3-L1 adipocytes, DIO mice and lean mice were treated with UA or vehicle. Gene expression of inflammatory factors, chemokines and immune markers in adipocytes and adipose tissue, cytokines in cell culture medium and serum, and inflammation regulatory pathways in adipocytes were detected. Results showed that UA increased the expression of interleukins and chemokines, but not TNFα, in both adipocytes and adipose tissue. IL6 and MCP1 levels in the cell culture medium and mouse serum were induced by UA treatment. Cd14 expression level and number of CD14+ monocytes were higher in UA treated adipose tissue than those in the control group. Glucose tolerance test was impaired by UA treatment in DIO mice. Mechanistically, UA induced the expression of Tlr4 and the phosphorylation levels of ERK and NFκB in adipocytes. In conclusion, our study indicated that short-term UA administration could induce CD14+ monocytes infiltration by increasing the production of interleukins and chemokines in mouse adipose tissue, which might further impair glucose tolerance test.

Chromolaena odorata flavonoids attenuate experimental nephropathy: Involvement of pro-inflammatory genes downregulation

Nephropathy is a serious complication comorbid with a number of life-threatening diseases such as diabetes. Flavonoids are well known cytoprotective phytochemicals. Here, nephropathy associated with streptozotocin (STZ) treatment in experimental animals was challenged by flavonoids (CoF) isolated from Chromolaena odorata. Experimental animals were divided into control (n = 5), STZ (40 mg/kg b.w. i.p. n = 5) and STZ-CoF (CoF = 30 mg/kg b.w. oral, 60 days, n = 7) groups. Blood urea nitrogen (BUN) and serum creatinine (SC) levels were quantified using ELISA. Kidney function, inflammatory marker, and antioxidant gene expression levels were also evaluated using reverse-transcription and polymerase chain reaction protocols. Histological assessment was also performed using Haematoxylin and Eosin (H&E) staining protocols. CoF improved kidney function by restoring BUN/SC levels to pre-STZ treatment states. KIM-1, TNF-α, and MCP-1 but not TNF-R and IL-10 genes were significantly downregulated in STZ-CoF treated group in comparison with STZ-treated group (p < 0.05). Anti-oxidant genes (GPx-1, CAT) significantly (p < 0.05 vs. control) upregulated in STZ-treatment did not respond to CoF treatment. STZ treatment associated Bowman’s space enlargement, thickened basement membrane, and glomerulosclerosis were completely reversed in STZ-CoF group. Finally, CoF has demonstrable anti-nephropathic via downregulation of proinflammatory genes and may represent new management option in clinical nephropathy.

Topical application of Chinese herbal medicine DAEP relieves the osteoarthritic knee pain in rats

BackgroundThe potential adverse effects of conventional oral pharmacotherapy of osteoarthritis (OA) restrict their long-term use. Topical application of a Chinese herbal paste for relieving OA knee pain can be effective and safe. However, evidence-based scientific research is insufficient to support its application worldwide. The aim of this study was to investigate the in vivo efficacy of a topical Chinese herbal paste on relieving OA knee pain and its underlying mechanism.MethodsAn OA rat model was developed by anterior cruciate ligament transection (ACLT) followed by treadmill running. A herbal paste including Dipsaci Radix, Achyranthis Bidentatae Radix, Eucommiae Cortex and Psoraleae Fructus, named as DAEP, was applied topically on the knee joint of the rats (DAEP). The rats without DAEP treatment served as Control. Rats with surgery but without ACLT, treadmill running and DAEP treatment acted as Sham. The morphologic change of the knee joint was observed radiographically. Nociception from the knee of the rats was assessed using Incapacitent test and CatWalk gait system. The therapeutic mechanism was investigated by analyzing the gene and protein expression of inflammatory markers via qPCR and Western blot, respectively.ResultsRadiographic images showed less destruction at the posterior tibial plateau of the DAEP group compared with the Control after 2 weeks of treatment. The static weight ratio and the gait parameters of the Control were reduced significantly via Incapacitance test and CatWalk gait analysis, respectively. DAEP treatment increased the Print Area and Maximum Intensity significantly compared with the Control. DAEP significantly suppressed the upregulation of gene expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS).ConclusionsDAEP exhibited its effect via the nuclear factor (NF)-κB pathway by suppressing the phosphorylation of IκB kinase αβ (p-IKKαβ) and cyclooxygenase-2 (COX-2) protein expression. This study provides scientific evidence to support the clinical application of the Chinese herbal paste on reliving OA pain.

Increased inflammasome activity in markedly ill psychiatric patients: An explorative study

The aim of this study was to investigate inflammatory perturbations in 40 patients with severe and complex psychiatric disorders by studying the activity of the NLRP3 inflammasome, with a trans-diagnostic approach. Gene expression of CASP1, NLRP3, PYCARD, IL1B, IL1RN, TNF showed a significant increase in the patient group compared to a matched control group. Plasma levels of IL1Ra, IL-18, TNF, IL-6 and CRP were increased in the patient group. Within the patient group, increased gene expression of inflammatory markers correlated with increased disease severity. The findings support the inflammation hypothesis for markedly ill psychiatric patients across diagnostic groups.

Determinants of the lung microbiome in intubated premature infants at risk for bronchopulmonary dysplasia

Background Airway dysbiosis in premature infants may be associated with bronchopulmonary dysplasia (BPD). Early oropharyngeal colostrum (OPC) administration alters the oral microbiome, which may impact the lung microbiome. We aim to compare the oral and tracheal microbiota during the first week of life, and to determine whether early OPC administration affects microbial diversity or leukocyte inflammatory activity in the lung. Methods Intubated premature infants (n = 42) were evaluated. The oral microbiome was characterized on day of life (DOL) 3, and the tracheal microbiome on DOL 3 and DOL 7, using 16S ribosomal DNA sequencing. Gene expression for inflammatory markers was quantified in airway leukocytes by real-time q-PCR. Results The oral and tracheal microbiota were significantly different on DOL 3, but the tracheal microbiome on DOL 7 was more similar to the oral from DOL 3. Tracheal bacterial diversity decreased from DOL 3 to DOL 7. Longer time to first OPC administration tended to be associated with lower bacterial diversity in the airways. Conclusions The tracheal microbiome in intubated premature infants in the first week is likely determined, in part, by the composition of the oral microbiome. Bacterial diversity in intubated babies decreases during the first week of life, a pattern that could be consistent with risk for BPD. Decreased bacterial diversity and increased inflammatory activity in the lung may also be associated with delayed administration of OPC.

Colonic neuronal loss and delayed motility induced by high-fat diet occur independently of changes in the major groups of microbiota in Swiss mice.

Obesity has been linked to gastrointestinal disorders, and the loss of myenteric neurons in the intestine caused by high-fat diets (HFD) has been attributed to changes in microbiota and lipotoxicity. We investigated whether the prebiotic inulin modulates bacterial populations and alleviates neuronal loss in mice fed HFD. Swiss mice were fed purified rodent diet or HFD (59% kcal fat), or both diets supplemented with inulin for 17weeks. Intestinal motility was assessed and a metagenome analysis of the colonic microbiota was performed. The gene expression of inflammatory markers was evaluated, and immunofluorescence was performed for different types of myenteric neurons and glial cells in the distal colon. The HFD caused obesity and delayed colonic motility. The loss of myenteric neurons and glial cells in obese mice affected all of the studied neuronal populations, including neurons positive for myosin-V, neuronal nitric oxide synthase, vasoactive intestinal peptide, and calretinin. Although obese mice supplemented with inulin exhibited improvements in colonic motility, neuronal, and glial cell loss persisted. The HFD did not altered the expression levels of inflammatory cytokines in the intestine or the prevalence of the major groups in microbiota, but inulin increased the proportion of the genus Akkermansia in the obese mice. In Swiss mice, the HFD-induced neuronal loss but did not change the major groups in microbiota. This suggests that, despite the increase in the beneficial bacteria, other factors that are directly linked to excess dietary lipid intake affect the enteric nervous system.