MYELOID-SPECIFIC FERRITIN LIGHT CHAIN DELETION DOES NOT EXACERBATE SEPSIS-ASSOCIATED AKI.

Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life threatening sequalae of sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of ferritin, an iron storage protein comprised of ferritin light (FtL) and heavy chain (FtH). Previous work revealed myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, myeloid FtL and serum ferritin levels were significantly lower in the knockout mice. Interestingly, while sepsis led to production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function and injury, identified by expression of kidney injury molecule-1 and neutrophil gelatinase associated lipocalin. RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy post-sepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. FtL deletion did not impact activation of NFkB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective, loss of FtL did not influence sepsis pathogenesis.

Downregulation of ATF-4 Attenuates the Endoplasmic Reticulum Stress-Mediated Neuroinflammation and Cognitive Impairment in Experimentally Induced Alzheimer's Disease Model.

Protein aggregation is invariably associated with the inflammation as a factor in Alzheimer's disease (AD). We investigated the interaction between downstream factors of endoplasmic reticulum (ER) stress pathway and inflammation, with implications in cognitive impairment in AD. Amyloid-ß (Aß)(1-42) was administered by bilateral intracerebroventricular (icv) injection in the brain of adult male Wistar rats to experimentally develop AD. The cognitive impairment was assessed by measuring behavioral parameters such as Morris water maze and novel object recognition tests. Levels of pro-inflammatory cytokines such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α and anti-inflammatory cytokines IL-4 and IL-10 were measured by the enzyme-linked immunosorbent assay (ELISA) in different rat brain regions. Inflammatory marker proteins such as cyclo-oxygenase (COX)-2 and phosphorylation of nuclear factor kappa B (NF-КB) (p65) were measured by the western blotting. Gene expression of ER stress downstream factors such as ATF-4, CHOP, and GADD-34 was analyzed by qRT-PCR. Histological studies were performed to check Aß accumulation and neuronal degeneration. Integrated stress response inhibitor (ISRIB) was used to confirm the specific role of ER stress-mediated inflammation in cognitive impairment. Administration of Aß(1-42) resulted in alteration in levels of inflammatory cytokines, inflammatory proteins, and mRNA levels of ER stress downstream factors. ISRIB treatment resulted in attenuation of Aß(1-42)-induced ER stress, inflammation, neurodegeneration, and cognitive impairment in rats. These results indicate that ER stress-mediated inflammation potentiates the cognitive impairment in AD. An understanding of cascade of events, interaction of ER stress which was a hallmark of the present investigation together with inflammation and modulation of downstream signalling factors could serve as potent biomarkers to study AD progression. Schematic representation of interaction between ER stress and inflammation. Administration of Aß(1-42) resulted in ER stress which caused the activation of factors of PERK pathway, inflammation, neuronal degeneration, and cognitive impairment. ISRIB treatment caused downregulation of ATF-4 and attenuation of inflammation indicating a role of ER stress-mediated inflammation in the cognitive impairment in AD. The site of action of ISRIB is shown in blue color.

Modulatory role of BV6 and chloroquine on the regulation of apoptosis and autophagy in non-small cell lung cancer cells.

Aims: Non-small cell lung cancer (NSCLC) is one of the aggressive tumors mostly diagnosed in the advanced stage. Therapeutic failure and drug resistance pose a major problem in NSCLC treatment primarily due to alterations in autophagy and loss of apoptosis. Therefore, the present study aimed to investigate the importance of the second mitochondria-derived activator of caspase mimetic BV6 and autophagy inhibitor chloroquine (CQ) on the regulation of apoptosis and autophagy, respectively. Subjects and Methods: Study was conducted on NCI-H23 and NCI-H522 cell lines to evaluate the effect of BV6 and CQ on the transcription and translation level of LC3-II, caspase-3, and caspase-9 genes by quantitative real-time-polymerase chain reaction and western blotting techniques. Results: In NCI-H23 cell line, BV6 and CQ treatments showed increased mRNA and protein expression of caspase-3, and caspase-9 compared to its untreated counterpart. BV6 and CQ treatments also caused downregulation of LC3-II protein expression compared to its counterpart. In NCI-H522 cell line, BV6 treatment showed a significantly increased expression of caspase-3 and caspase-9 mRNA and protein expression levels whereas BV6 treatment downregulated the expression level of LC3-II protein. A similar pattern was also observed in CQ treatment when compared with the respective controls. Both BV6 and CQ modulated in vitro expression of caspases and LC3-II which have critical regulatory roles in apoptosis and autophagy, respectively. Conclusions: Our findings suggest that BV6 and CQ could be promising candidates in NSCLC treatment and there is a need to explore them in vivo and in clinical applications.

Protective effect of rosmarinic acid on the transmembrane transporter Ctr1 expression in cisplatin-treated mice.

AIMS: Cisplatin (cis-diamminedichloroplatinum(II), CP) is a platinum-based anticancer drug widely used in the treatment of solid malignancies. However, its side effects, particularly nephrotoxicity, are limiting factors in its clinic use. Rosmarinic acid (RA), a natural antioxidant compound, is reported to attenuate oxidative stress and associated pathophysiological outcomes. Our study aimed to explore the protective effect of RA against CP-induced acute kidney injury (AKI). MATERIALS AND METHODS: We investigated the effect of RA at the dose of 100 mg/kg on AKI induced by CP (20 mg/kg) in mice. Various parameters of nephrotoxicity such as levels of serum electrolytes, albumin, and globulin were measured using standardized methods. Besides, a specific biomarker of damage to proximal tubular cells, kidney injury molecule-1 (Kim-1), was measured in the serum by ELISA. mRNA expression of Kim-1 and a transmembrane transporter, copper transporter 1 (Ctr1), was analyzed by quantitative reverse transcriptase-polymerase chain reaction. CTR1 expression was also analyzed by western blot technique. RESULTS: RA treatment restored the downregulated CTR1 , a renal transmembrane transporter in CP-treated mice. It was accompanied by a reduction in the level of serum albumin and globulin. Serum electrolytes such as Na+, K+, and Ca2+ in CP-treated mice were found to be restored with RA treatment. Moreover, RA also significantly downregulated the increased expression of nephrotoxicity biomarker KIM-1. CONCLUSIONS: Overall, RA proved to be an effective nephroprotective compound which afforded protection at cellular and subcellular levels with an appreciable modulatory effect on a transmembrane transporter.

Functional consequence of myeloid ferritin heavy chain on acute and chronic effects of rhabdomyolysis-induced kidney injury.

Acute kidney injury (AKI) is a serious complication of rhabdomyolysis that significantly impacts survival. Myoglobin released from the damaged muscle accumulates in the kidney, causing heme iron-mediated oxidative stress, tubular cell death, and inflammation. In response to injury, myeloid cells, specifically neutrophils and macrophages, infiltrate the kidneys, and mediate response to injury. Ferritin, comprised of ferritin light chain and ferritin heavy chain (FtH), is vital for intracellular iron handling. Given the dominant role of macrophages and heme-iron burden in the pathogenesis of rhabdomyolysis, we studied the functional role of myeloid FtH in rhabdomyolysis-induced AKI and subsequent fibrosis. Using two models of rhabdomyolysis induced AKI, we found that during the acute phase, myeloid FtH deletion did not impact rhabdomyolysis-induced kidney injury, cell death or cell proliferation, suggesting that tubular heme burden is the dominant injury mechanism. We also determined that, while the kidney architecture was markedly improved after 28 days, tubular casts persisted in the kidneys, suggesting sustained damage or incomplete recovery. We further showed that rhabdomyolysis resulted in an abundance of disparate intra-renal immune cell populations, such that myeloid populations dominated during the acute phase and lymphoid populations dominated in the chronic phase. Fibrotic remodeling was induced in both genotypes at 7 days post-injury but continued to progress only in wild-type mice. This was accompanied by an increase in expression of pro-fibrogenic and immunomodulatory proteins, such as transforming growth factor-ß, S100A8, and tumor necrosis factor-α. Taken together, we found that while the initial injury response to heme burden was similar, myeloid FtH deficiency was associated with lesser interstitial fibrosis. Future studies are warranted to determine whether this differential fibrotic remodeling will render these animals more susceptible to a second AKI insult or progress to chronic kidney disease at an accelerated pace.

Resveratrol-Loaded Glutathione-Coated Collagen Nanoparticles Attenuate Acute Seizures by Inhibiting HMGB1 and TLR-4 in the Hippocampus of Mice.

Epilepsy is a relatively complicated neurological disorder that results in seizures. The use of resveratrol in treating seizures has been reported in recent studies. However, the low bioavailability of resveratrol and the difficulty of reaching the targeted location in the brain reduce its efficacy considerably. The side effects due to the higher concentration of drugs are another matter of concern. The purpose of the present study is to enhance the antiepileptic potential of resveratrol by delivering it to the brain's targeted location by encapsulating it in glutathione-coated collagen nanoparticles. The collagen nanoparticles increase the bioavailability of resveratrol, while the transport of resveratrol to its target location in the brain is facilitated by glutathione. By encapsulating resveratrol in glutathione-coated collagen nanoparticles, the concentration also substantially decreases. Resveratrol encapsulated in synthesized nanoparticles is referred to as nanoresveratrol. In the present study, nanoresveratrol effectiveness was studied through PTZ-induced seizures (PTZ-IS) and the increasing current electroshock (ICES) test. The efficacy of nanoresveratrol was further established using biochemical analysis, histopathological examinations, ELISA and real-time-PCR tests, and immunohistochemistry examination of the hippocampus of mice. Hence, this study is unique in the sense that it synthesized nanoresveratrol by using glutathione-coated collagen nanoparticles, followed by its application to treating seizures. On the basis of the study results, nanoresveratrol was found to be effective in preventing cognitive impairment in the mice and controlling epilepsy seizures to a greater extent than resveratrol. The proposed nanoformulation also reduces the concentration of resveratrol considerably. The present study results show that even 0.4 mg/kg of nanoresveratrol outperforms 40 mg/kg of resveratrol.

NLRP3 inflammasome in rosmarinic acid-afforded attenuation of acute kidney injury in mice.

Cisplatin (CP) is a well-known anticancer drug used to effectively treat various kinds of solid tumors. CP causes acute kidney injury (AKI) and unfortunately, there is no therapeutic approach in hand to prevent AKI. Several signaling pathways are responsible for inducing AKI which leads to inflammation in proximal convoluted tubule cells in the kidney. Furthermore, the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome is involved in the CP-induced AKI. In this study, we investigated therapeutic effects of rosmarinic acid (RA) against inflammation-induced AKI. RA was orally administered at the dose of 100 mg/kg for two consecutive days after 24 h of a single injection of CP at the dose of 20 mg/kg administered intraperitoneally in Swiss albino male mice. Treatment of RA inhibited the activation of NLRP3 signaling pathway by blocking the activated caspase-1 and downstream signal molecules such as IL-1ß and IL18. CP activated HMGB1-TLR4/MyD88 axis was also found to be downregulated with the RA treatment. Activation of nuclear factor-κB and elevated protein expression of cyclooxygenase-2 (COX-2) were also found to be downregulated in RA-treated animals. Alteration of early tubular injury biomarker, kidney injury molecule-1 (KIM-1), was found to be subsided in RA-treated mice. RA has been earlier reported for antioxidant and anti-inflammatory properties. Our findings show that blocking a critical step of inflammasome signaling pathway by RA treatment can be a novel and beneficial approach to prevent the CP-induced AKI.

Tempol (4-hydroxy tempo) protects mice from cisplatin-induced acute kidney injury via modulation of expression of aquaporins and kidney injury molecule-1.

Tempol (4-hydroxy tempo), a pleiotropic antioxidant is reported to afford protection against cisplatin (CP)-induced nephrotoxicity. However, molecular mechanisms of action of tempol in improving the renal function in CP-induced nephrotoxicity are not fully understood. We investigated the attenuating effect of tempol against CP-induced alterations in kidney injury molecule-1 (KIM-1) and aquaporins (AQPs) in mice. Tempol (100 mg/kg, po) pretreatment with CP (20 mg/kg ip) showed restoration in renal function markers including electrolytes. CP treatment upregulated mRNA expression of KIM-1 and downregulated AQP and arginine vasopressin (AVP) expression which was attenuated by tempol. Immunoblotting analysis revealed that CP-induced alterations in KIM-1 and AQP expression were restored by tempol. Immunofluorocense study also showed restorative effect of tempol on the expression of AQP2 in CP-treated mice. In conclusion, this study provides experimental evidence that tempol resolved urinary concentration defect by the restoration of AQP, AVP and KIM-1 levels indicating a potential use of tempol in ameliorating the AKI in cancer patients under the treatment with CP.

mRNA expression and protein-protein interaction (PPI) network analysis of adrenal steroidogenesis in response to exposure to phthalates in rats.

Phthalate esters such as di-butyl phthalate (DBP) and di-ethyl hexyl phthalate (DEHP) used in personal care and consumer products and medical devices have potential to affect human health. We studied the effect of DBP and DEHP on critical enzymes of glucocorticoid biosynthesis pathway in the adrenal gland and pro-inflammatory cytokines in the serum in male Wistar rats. DEHP and DBP treatment altered the mRNA expression of enzymes of glucocorticoid biosynthesis pathway accompanied by a reduction in glucocorticoid production and elevation in the level of glucocorticoid regulated pro-inflammatory cytokines indicating a cascading effect of phthalates. The analysis of PPI (protein - protein interaction) network involving Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) of enzymes through STRING database revealed that all the proteins have the maximum level of interaction with the selected number of proteins. The STRING database analysis together with in vivo data indicates the potential effects of phthalates on various targets of steroidogenesis pathway with a global biological impact.

Resveratrol loaded nanoparticles attenuate cognitive impairment and inflammatory markers in PTZ-induced kindled mice.

Resveratrol has been found to exert protective effects in neurological disorders, including epilepsy. However, its poor bioavailability and difficulty in reaching the brain's targeted location reduce resveratrol's efficacy substantially. The side effects due to the higher concentration of drugs are another matter of concern. The objective of the present study is to propose solutions to these issues by encapsulating resveratrol in glutathione-coated collagen nanoparticles' core. The collagen nanoparticles increase the resveratrol's bioavailability, and glutathione helps in the passage of the encapsulated resveratrol to the target location in the brain. The concentration also substantially reduces due to resveratrol's encapsulation in glutathione-coated collagen nanoparticles. The encapsulated resveratrol is termed nanoresveratrol. The effectiveness of nanoresveratrol on epilepsy seizures was evaluated through histopathological examinations, ELISA tests, and qRT-PCR tests on the hippocampus of the kindled mice. The novelty of the present study thus lies in (i) the synthesis of nanoresveratrol using glutathione-coated collagen nanoparticles and (ii) the application of synthesized nanoresveratrol in the treatment of epilepsy. The study's outcome shows that nanoresveratrol has a favorable impact in reducing cognitive impairment in kindled mice, and it is more effective in controlling epilepsy seizures than resveratrol. The p-values of all the nanoresveratrol-given groups of mice (compared with the diseased group) were substantially smaller (∼10-4 to 10-2) than the significance level (0.05), indicating that the nanoresveratrol-given groups are significantly different from the diseased group, i.e., the nanoresveratrol has a significant effect on the mice. The concentration of resveratrol also decreases substantially in the proposed nanoformulation. It was observed that even 0.4 mg/kg of nanoformulation of resveratrol is performing better than 40 mg/kg of resveratrol.

Regulation of miR-126 and miR-122 Expression and Response of Imatinib Treatment on Its Expression in Chronic Myeloid Leukemia Patients.

BACKGROUND: MicroRNAs (miRNAs) have been observed to exhibit altered expression patterns in chronic myeloid leukemia (CML). Therefore, this study was aimed to evaluate the clinical importance of miR-126 and miR-122 expression in concert to imatinib response in CML patients. METHODS: The present study included 100 CML and 100 healthy subjects. The expression of the 2 miRNAs was performed using TaqMan probe chemistry, and snU6 was used as internal control. RESULTS: The expression of miR-126 and miR-122 was downregulated in CML patients, with a mean fold change ± SD 0.20 ± 0.33 and 0.22 ± 0.37, respectively. While the expression of both miRNAs was analysed before and after imatinib treatment, it was observed that the expression levels of both were increased after imatinib treatment by 26.25-fold (5.33 against 0.20) and 13.95-fold (3.07 against 0.22) and the increase was statistically significant (p < 0.0001 and p < 0.0001, respectively). The expression of miR-126 was not conclusive when compared in different clinical stages of the CML disease as it showed a decreased expression in patients with accelerated phase compared to chronic phase (mean fold change = 0.03 and 0.27, respectively), but patients with chronic phase and blastic phase had comparable expression (mean fold change = 0.27 and 0.24, respectively). We also observed an increased expression of both miRNAs after imatinib therapy in each clinical phase. CONCLUSION: The study concludes that expression of miR-126 and miR-122 increases after imatinib treatment in CML patients and that miR-126 defines the good responders of imatinib therapy.

Involvement of endoplasmic reticulum stress in amyloid ß (1-42)-induced Alzheimer's like neuropathological process in rat brain.

Amyloid-ß (Aß) accumulation in the brain is a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been implicated in aetiology of neurodegenerative disorders. We studied the involvement of ER stress in Aß-induced neuronal degeneration in rat brain to correlate it with cellular and molecular modifications in Aß-induced Alzheimer's like neuropathological process. Aß (1-42) (5 µg) was administered by bilateral intracerebroventricular (icv) injection in the brain of adult male Wistar rats. Acetylcholinesterase (AChE) activity and histological alterations were observed in different brain regions. ER stress-associated proteins- glucose regulated protein-78 (GRP78), eukaryotic translation initiation factor-2α (eIF2α) and growth arrest and DNA damage-inducible protein-153 (GADD153), neuronal marker- microtubule associated protein-2 (MAP-2) and microglial protein- ionized calcium binding adaptor molecule-1 (Iba-1) were measured by western blot. Reduced glutathione (GSH), nitrite level and levels of caspase-12 and caspase-3 were also measured. ER stress inhibitor, salubrinal (1 mg/kg, intraperitoneally, ip) was used to assess the specific role of ER stress. Aß (1-42)-induced increase in AChE activity, GRP78 and GADD protein levels, dephosphorylation of eIF2-α and caspase-12 and caspase-3 levels and decrease in GSH and MAP-2 levels were attenuated by salubrinal. Increase in Iba-1 protein and nitrite levels after Aß (1-42) administration were partially attenuated by salubrinal. Aß (1-42)-induced histological alterations were correlated with findings of ER stress. Results of present study implicate ER stress as a potential molecular mechanism in Aß-induced Alzheimer's like neuropathology which could serve as surrogate biomarker for study of AD progression and efficacy of therapeutic interventions for AD management.

Bisphenol A triggers axonal injury and myelin degeneration with concomitant neurobehavioral toxicity in C57BL/6J male mice.

Bisphenol A (BPA) is a ubiquitously distributed endocrine disrupting chemical (EDC). BPA exposure in humans has been a matter of concern due to its increased application in the products of day to day use. BPA has been reported to cause toxicity in almost all the vital organ systems even at a very low dose levels. It crosses the blood brain barrier and causes neurotoxicity. We studied the effect of BPA on the cerebral cortex of C57BL/6J mice and examined whether BPA exposure alters the expression of axonal and myelin structural proteins. Male mice were dosed orally to 40 µg and 400 µg BPA/kg body weight for 60 days. BPA exposure resulted in memory loss, muscle coordination deficits and allodynia. BPA exposure also caused degeneration of immature and mature oligodendrocytes as evaluated by decreased mRNA levels of 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), nestin, myelin basic protein (MBP) and myelin-associated glycoprotein-1 (MAG-1) genes revealing myelin related pathology. It was observed that subchronic BPA exposure caused neuroinflammation through deregulation of inflammatory cytokines mRNA and protein expression which further resulted into neurotoxicity through axonal as well as myelin degeneration in the brain. BPA also caused increased oxidative stress in the brain. Our study indicates long-term subchronic low dose exposure to BPA has the potential to cause axonal degeneration and demyelination in the oligodendrocytes and neurons which may have implications in neurological and neuropsychological disorders including multiple sclerosis (MS), neuromyelitis optica and others.

Expression Analysis of Serum microRNA-34a and microRNA-183 in Hepatocellular Carcinoma

Background/objective: HCC is a multistep process starting from chronic hepatitis that progress through cirrhosis to HCC. MicroRNA expression level was found to be deregulated in HCC. To find out whether the expression level of miR-34a and miR-183 was deregulated in HCC compared to controls without HCC. Methods: Real time quantitative PCR was done to find out the miRNA expression level in terms of Ct value followed by statistical analysis. Results: Over-expression of miR-183 and under-expression of miR-34a in HCC was detected. All changes in expression level of miR-34a and miR-183 were found to be due to HCC compared to controls without HCC. So both miR-34a and miR-183 were suitable to differentiate HCC from Cirrhosis and chronic hepatitis with an efficient diagnostic power of sensitivity, specificity and expression level. But they might not have any role in patients' survival. Conclusion: miR- 34a and miR-183 might be considered as potential markers of HCC screening molecule in addition to other approved panel of marker. Our study warrants further expression level study.