Nanobioelectrocatalysis Using Human Liver Microsomes and Cytochrome P450 Bactosomes: Pyrenyl-Nanocarbon Electrodes.

Human liver microsomes containing various drug-metabolizing cytochrome P450 (P450) enzymes, along with their NADPH-reductase bound to phospholipid membranes, were absorbed onto 1-pyrene butylamine pi-pi stacked with amine-functionalized multiwalled carbon nanotube-modified graphite electrodes. The interfaced microsomal biofilm demonstrated direct electrochemical communication with the underlying electrode surface and enhanced oxygen reduction electrocatalytic activity typical of heme enzymes such as P450s over the unmodified electrodes and nonenzymatic currents. Similar enhancements in currents were observed when the bioelectrodes were constructed with recombinant P450 2C9 (single isoform) expressed bactosomes. The designed liver microsomal and 2C9 bactosomal bioelectrodes successfully facilitated the electrocatalytic conversion of diclofenac, a drug candidate, into 4'-hydroxydiclofenac. The enzymatic electrocatalytic metabolite yield was several-fold greater on the modified electrodes than on the unmodified bulk graphite electrodes adsorbed with a microsomal or bactosomal film. The nonenzymatic metabolite production was less than the enzymatically catalyzed metabolite yield in the designed microsomal and bactosomal biofilm electrodes. To test the throughput potential of the designed biofilms, eight-electrode array configurations were tested with the microsomal and bactosomal biofilms toward electrochemical 4'-hydroxydiclofenac metabolite production from diclofenac. The stability of the designed microsomal bioelectrode was assessed using nonfaradaic impedance spectroscopy over 40 h, which indicated good stability.

Modeling dry eye with an air-liquid interface in corneal epithelium-on-a-chip.

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air-liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF), non-steroidal anti-inflammatory drug. Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell-cell junctions. They increased the expression of inflammatory genes (e.g., IL-6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the potential use of AL stimulus within the CEpOC to induce cellular characteristics relevant to DES.

Exploring the anti-inflammatory potential of topical hyaluronic acid for vocal fold injury in a rat model.

PURPOSE: Vocal fold injuries are associated with fibrosis and dysphonia, which is a major obstacle to surgical treatment. The aim of this study is to evaluate the effect of topical hyaluronic acid with or without diclofenac on the inflammatory phase of vocal fold wound healing. METHODS: Forty-one male Sprague-Dawley rats were randomly assigned to four groups: an uninjured control group, an injured control group without any treatment, and two intervention groups in which hyaluronic acid with or without diclofenac was applied to the injured vocal fold. Gene expression of inflammatory markers and ECM-related molecules were examined. RESULTS: Vocal fold injury resulted in a significant upregulation of inflammatory parameters [Ptgs2, Il1b and Il10] and Has1. Tgfb1, Has3 and Eln gene expression were significantly downregulated by the topical application of hyaluronic acid. The combination of hyaluronic acid and diclofenac did not result in any significant changes. CONCLUSIONS: Vocal fold wound healing was significantly improved by a single post-operative topical application of hyaluronic acid. The addition of diclofenac may provide no additional benefit.

Histopathological, physiological, and multi-omics insights into the hepatotoxicity mechanism of nanopolystyrene and/or diclofenac in Mylopharyngodon piceus.

Nanopolystyrene (NP) and diclofenac (DCF) are common environmental contaminants in the aquatic ecosystem; therefore, the present study aimed to investigate the hepatotoxicity of NP and/or DCF exposure on aquatic organisms and the underlying mechanisms. Juvenile Mylopharyngodon piceus were used as a model organism to study the effects of NP and/or DCF exposure at environmentally relevant concentrations for 21 days. Subchronic exposure to NP and/or DCF resulted in liver histological damage. In the NP group, the presence of large lipid droplets was observed, whereas the DCF group exhibited marked hepatic sinusoidal dilatation accompanied by inflammation. Additionally, this exposure induced liver oxidative stress, as evidenced by the changes in several physiological parameters, including catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), reactive oxygen species (ROS), and malondialdehyde (MDA). Integrated transcriptomic and metabolomic analysis was performed to further investigate the molecular mechanism underlying hepatotoxicity. Multi-omics analysis demonstrated, for the first time to our knowledge, that NP induced hepatic steatosis mainly through activating the glycerol-3-phosphate pathway and inhibiting VLDL assembly by targeting several key enzyme genes including GPAT, DGAT, ACSL, APOB, and MTTP. Furthermore, NP exposure disrupted arachidonic acid metabolism, which induced the release of inflammatory factors and inhibited the release of anti-inflammatory factors, ultimately causing liver inflammation in M. piceus. In contrast, DCF induced interleukin production and downregulated KLF2, causing hepatic sinusoidal dilatation with inflammation in juvenile M. piceus, which is consistent with the finding of JAK-STAT signaling pathway activation. In addition, the upregulated AMPK signaling pathway in the DCF group suggested perturbation of energy metabolism. Collectively, these findings provide novel insights into the molecular mechanism of the multiple hepatotoxicity endpoints of NP and/or DCF exposure in aquatic organisms.

A premature stop codon in the CYP2C19 gene may explain the unexpected sensitivity of vultures to diclofenac toxicity.

The unintended environmental exposure of vultures to diclofenac has resulted in the deaths of millions of old-world vultures on the Asian subcontinent. While toxicity has been since associated with a long half-life of elimination and zero order metabolism, the actual constraint in biotransformation is yet to be clarified. For this study we evaluated if the evident zero order metabolism could be due to defects in the CYP2C9/2C19 enzyme system. For this, using whole genome sequencing and de-novo transcriptome alignment, the vulture CYP2C19 open reading frame was identified through Splign analysis. The result sequence analysis revealed the presence of a premature stop codon on intron 7 of the identified open reading frame. Even if the stop codon was not present, amino acid residue analysis tended to suggest that the enzyme would be lower in activity than the equivalent human enzyme, with differences present at sites 105, 286 and 289. The defect was also conserved across the eight non-related vultures tested. From these results, we conclude that the sensitivity of the old-world vultures to diclofenac is due to the non-expression of a viable CYP2C19 enzyme system. This is not too dissimilar to the effects seen in certain people with a similar defective enzyme.

Evaluating the protective effect of metformin against diclofenac-induced oxidative stress and hepatic damage: In vitro and in vivo studies.

Diclofenac (DIC) is one of the most commonly prescribed non-steroidal anti-inflammatory drugs and has been shown to cause oxidative stress and liver injury. The current study investigated protective effects of metformin against DIC-induced hepatic toxicity in both in vitro and in vivo models. For the in vitro study, HepG2 cells were exposed to DIC in the presence or absence of metformin. The effect of metformin on cell viability was evaluated by MTT assay. Oxidative stress parameters (malondialdehyde (MDA), total thiol molecules (TTM), and total antioxidant capacity (TAC)) were assessed. For the in vivo study, thirty-six male Wistar rats were randomly divided into 6 groups. These groups were normal saline, metformin (200 mg/kg), DIC (50 mg/kg/day), DIC + metformin (50, 100, and 200 mg/kg/day). Histopathological studies and serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), albumin, direct and total bilirubin were measured. Also, oxidative stress parameters were assessed in liver tissue. Furthermore, expression of glutathione peroxidase (GPX)-1, -3, and -4, catalase (CAT), superoxide dismutase (SOD)-1, and -3 was examined using the real-time PCR method in hepatic tissue. In the in vitro study, metformin significantly prevented DIC-induced loss in cell viability in HepG2 cells. Metformin markedly reduced DIC-induced elevation of MDA levels and increased the TAC and TTM levels. In the in vivo study, metformin significantly prevented DIC-induced changes in hematological and histological markers. Administration of metformin significantly improved oxidative stress parameters in liver tissue. In addition, metformin increased the expression of antioxidant enzymes. Our results suggest that metformin exerts a significant protective effect against DIC-induced hepatic toxicity.

Tubuloside A, a phenylethanoid glycoside, alleviates diclofenac induced hepato-nephro oxidative injury via Nrf2/HO-1.

The most prominent adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DF) are hepato-renal damage. Natural antioxidants can be preferred as an alternative and/or combination to improve this damage. This present study was conducted to evaluate the protective effect of Tubuloside A (TA) against diclofenac (DF)-induced hepato-renal damage. TA (1 mg/kg, ip) was administered to male Sprague-Dawley rats for 5 days, and DF (50 mg/kg, ip) was administered on Days 4 and 5. Plasma aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine were measured to evaluate liver and kidney functions. Additionally, oxidative stress parameters (malondialdehyde, glutathione, superoxide dismutase, catalase, and 8-oxo-7,8-dihydro-2'-deoxyguanosine) in blood, liver, and kidney tissues, changes in mRNA expression of genes involved in the Nrf2/HO-1 signalling pathway (Nrf2, HO-1, NQO-1, IL-6, iNOS, Cox-2, TNF-α, IL1-ß and NFκB) and apoptotic process (Bcl-2, Cas-3 and Bax) in liver and kidney tissues were determined. Additionally, tissue sections were evaluated histopathologically. Biochemical, histopathological, and molecular results demonstrated the hepato-renal toxic effects of DF, and TA treatment protected the liver and kidney from DF-induced damage. This provides an explanation for the hepato-nephro damage caused by DF and offers new ideas and drug targets together with TA for the prevention and treatment of DF injury.

Pathological study of proximal tubule mitochondria in diclofenac-induced acute kidney injury model mice.

Diclofenac, a non-steroidal anti-inflammatory drug, reportedly targets mitochondria and induces nephrotoxicity via reactive oxygen species. However, there are few detailed reports of pathological analyses of mitochondria and the factors that cause acute kidney injury (AKI) as a result of nephrotoxicity. In this study, we investigated mitochondrial damage in the proximal tubule in AKI mice at 6, 12, and 24 h after administration of diclofenac. Statistical analysis of immunohistochemistry results confirmed that expression of p62 and LC3, which is associated with autophagy, reached a maximum level in the degenerated proximal renal tubule 12 h after diclofenac treatment, with high autophagy activity. Electron microscopy images provided clear evidence that confirmed mitochondrial degeneration and injury as well as autophagy (mitophagy) in mitochondria treated with diclofenac. The purpose of this study was to pathologically characterize both mitochondrial damage in the proximal renal tubules induced by diclofenac and the course of mitophagy to remove the damaged mitochondria. This report provides important information regarding mitochondrial damage in the proximal tubules in diclofenac-induced nephropathy.

A multibiomarker approach to assess the ecotoxicological effects of diclofenac on Asian clam Corbicula fluminea (O. F. Müller, 1774).

Diclofenac (DCF), one of the most current and widely used nonsteroidal anti-inflammatory drugs (NSAIDs), has been frequently detected in aquatic environments worldwide. However, the ecotoxicological effects of DCF on freshwater invertebrates remain largely unknown. In the present study, Corbicula fluminea were exposed to environmentally relevant concentrations of DCF (0, 2, 20, and 200 µg/L) for 28 days, and the potential adverse effects of DCF on siphoning behavior, antioxidant responses, and apoptosis were investigated. Our results showed that the siphon efficiencies of clams were significantly suppressed under DCF stress. DCF exerted neurotoxicity via reducing the activity of acetylcholinesterase (AChE) in gills and digestive gland of C. fluminea. Exposure to DCF induced antioxidant stress and increased malondialdehyde (MDA) levels in both gills and digestive gland of C. fluminea. Transcriptional alterations of apoptosis-related genes indicated that DCF might induce apoptosis by triggering mitochondrial apoptotic pathway. These findings can improve our understanding of the ecological risk of DCF in freshwater ecosystems.

Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine.

The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates thein vitrobioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. Thisin vitroapproach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation with and without limonene, giving a kinetic constant of the reaction,KM, of 103µM and 94.1µM, respectively, and a maximum reaction rate,Vmax, of 46.8 pmol min-1106cells-1and 56.0 pmol min-1106cells-1with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half-maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413µM.

Antioxidant and Hepatoprotective Potential of Echinops ritro L. Extracts on Induced Oxidative Stress In Vitro/In Vivo.

Echinops ritro L. (Asteraceae) is traditionally used in the treatment of bacterial/fungal infections and respiratory and heart ailments. The aim of this study was to evaluate the potential of extracts from E. ritro leaves (ERLE) and flowering heads (ERFE) as antioxidant and hepatoprotective agents on diclofenac-induced lipid peroxidation and oxidative stress under in vitro and in vivo conditions. In isolated rat microsomes and hepatocytes, the extracts significantly alleviated oxidative stress by increasing cell viability and GSH levels and reducing LDH efflux and MDA production. During in vivo experiments, the administration of the ERFE alone or in combination with diclofenac resulted in a significant increase in cellular antioxidant protection and a decrease in lipid peroxidation witnessed by key markers and enzymes. A beneficial influence on the activity of the drug-metabolizing enzymes ethylmorphine-N-demetylase and aniline hydroxylase in liver tissue was found. In the acute toxicity test evaluation, the ERFE showed no toxicity. In the ultrahigh-performance liquid chromatography-high-resolution mass spectrometry analysis, 95 secondary metabolites were reported for the first time, including acylquinic acids, flavonoids, and coumarins. Protocatechuic acid O-hexoside, quinic, chlorogenic and 3, 5-dicaffeoylquinic acid, apigenin; apigenin 7-O-glucoside, hyperoside, jaceosidene, and cirsiliol dominated the profiles. The results suggest that both extracts should be designed for functional applications with antioxidant and hepatoprotective capacity.

Green alga Chlamydomonas reinhardtii can effectively remove diclofenac from the water environment - A new perspective on biotransformation.

The use of unicellular algae to remove xenobiotics (including drugs) from wastewaters is one of the rapidly developing areas of environmental protection. Numerous data indicate that for efficient phycoremediation three processes are important, i.e. biosorption, bioaccumulation, and biotransformation. Although biosorption and bioaccumulation do not raise any serious doubts, biotransformation is more problematic since its products can be potentially more toxic than the parent compounds posing a threat to organisms living in a given environment, including organisms that made this transformation. Thus, two questions need to be answered before the proper algae strain is chosen for phycoremediation, namely what metabolites are produced during biotransformation, and how resistant is the analyzed strain to a mixture of parent compound and metabolites that appear over the course of culture? In this work, we evaluated the remediation potential of the model green alga Chlamydomonas reinhardtii in relation to non-steroidal anti-inflammatory drugs (NSAIDs), as exemplified by diclofenac. To achieve this, we analysed the susceptibility of C. reinhardtii to diclofenac as well as its capability to biosorption, bioaccumulation, and biotransformation of the drug. We have found that even at a relatively high concentration of diclofenac the algae maintained their vitality and were able to remove (37.7%) DCF from the environment. A wide range of phase I and II metabolites of diclofenac (38 transformation products) was discovered, with many of them characteristic rather for animal and bacterial biochemical pathways than for plant metabolism. Due to such a large number of detected products, 18 of which were not previously reported, the proposed scheme of diclofenac transformation by C. reinhardtii not only significantly contributes to broadening the knowledge in this field, but also allows to suggest possible pathways of degradation of xenobiotics with a similar structure. It is worth pointing out that a decrease in the level of diclofenac in the media observed in this study cannot be fully explained by biotransformation (8.4%). The mass balance analysis indicates that other processes (total 22%), such as biosorption, a non-extractable residue formation, or complete decomposition in metabolic cycles can be involved in the diclofenac disappearance, and those findings open the prospects of further research.

Effective bioremediation of clarithromycin and diclofenac in wastewater by microbes and Arundo donax L.

Bioremediation of pharmaceuticals has gained large research efforts, but there is still a need to improve the performance of bioremediation systems by selecting effective organisms. In this study, we characterized the capability to remove clarithromycin (CLA) and diclofenac (DCF) by the bacterium Streptomyces rochei, and the fungi Phanerochaete chrysosporium and Trametes versicolor. The macrolide antibiotic CLA and the non-steroid anti-inflammatory DCF were selected because these are two of the most frequently detected drugs in water bodies. Growth and content of the PhCs and a DCF metabolite (MET) by the energy crop Arundo donax L. were also evaluated under hydroponic conditions. The removal rate (RR) by S. rochei increased from 24 to 40% at 10 and 100 µg CLA L-1, respectively, averaged over incubation times. At 144 h, the RR by P. chrysosporium was 84%, while by T. versicolor was 70 and 45% at 10 and 100 CLA µg L-1. The RR by S. rochei did not exceed 30% at 1 mg DCF L-1 and reached 60% at 10 mg DCF L-1, whereas approached 95% and 63% by P. chrysosporium and T. versicolor, respectively, at both doses. Root biomass and length of A. donax were strongly affected at 100 µg CLA L-1. CLA concentration in roots and shoots increased with the increase of the dose and translocation factor (TF) was about 1. DCF severely affected both shoot fresh weight and root length at the highest dose and concentration in roots and shoots increased with the increase of the dose. DCF concentrations were 16-19 times higher in roots than in shoots, and TF was about 0.1. MET was detected only in roots and its proportion over the parent compound decreased with the increase of the DCF dose. This study highlights the potential contribution of A. donax and the tested microbial inoculants for improving the effectiveness of bioremediation systems for CLA and DCF removal.

Intestinal Metabolism of Diclofenac by Polymorphic UGT2B17 Correlates with its Highly Variable Pharmacokinetics and Safety across Populations.

Although the United States and Europe have shifted to the prescription use of oral diclofenac due to several serious incidences of cardiotoxicity, it is one of the most commonly used over-the-counter (OTC) pain medicines in major parts of the world. We elucidated the quantitative and tissue-specific contribution of uridine diphosphate-glucuronosyltransferases 17 (UGT2B17) in diclofenac metabolism and pharmacokinetics (PK). UGT2B17 is one of most deleted genes in humans with the gene deletion frequency ranging from ~ 20% in White population to 90% in Japanese population. The human intestinal and liver microsomes isolated from the high-UGT2B17 expressing individuals showed 21- and 4-fold greater rate of diclofenac glucuronide (DG) formation than in the null-UGT2B17 carriers, respectively. The greater contribution of intestinal UGT2B17 was confirmed by a strong correlation (R = 0.78, P < 0.001) between UGT2B17 abundance and DG formation in individual intestinal microsomes (n = 14). However, because UGT2B17 is a minor UGT isoform in the liver, DG formation rate correlated better with the expression of UGT2B7. The proteomics-informed physiologically-based pharmacokinetic (PBPK) model explains the reported higher exposure of diclofenac in women consistent with ~ 3-fold lower expression of UGT2B17. Similarly, our in silico predictions also corroborate with the reported higher exposure and lower standard clinical dose of diclofenac in Japanese population. Therefore, variable UGT2B17 mediated metabolism of oral diclofenac is a cause of concern, especially in the developing countries where it is still used as an OTC drug. The ontogeny data of UGTs in human hepatocytes can be utilized in developing PBPK models for predicting PK in the pediatric population.

Prediction of pharmaceuticals removal in activated sludge system under different operational parameters using an extended ASM-PhACs model.

Removal of pharmaceuticals is essential in wastewater treatment systems due to their release and accumulation in the environment, which are raising issues for the environment and human health. A mathematical model could be used to predict pharmaceuticals removal under various operational parameters and assess the contributions of different removal pathways to pharmaceuticals removal. Here an ASM-PhACs model was established to describe pharmaceuticals removal including diclofenac (DCF), erythromycin (ERY), gemfibrozil (GEM) and carbamazepine (CBZ) removal in activated sludge system. The pharmaceuticals removal processes linked to co-metabolic biodegradation through the growth of ammonia oxidizing bacteria (AOB), metabolic biodegradation through AOB, metabolic biodegradation through heterotrophic bacteria (HB) and sludge adsorption were incorporated into activated sludge model (ASM1) framework. The kinetic equations were established for each pharmaceuticals removal process. To provide the experimental data for model calibration and validation, two sets of batch tests were designed and conducted in the laboratory scale using SBR technology. According to the batch test data and results of sensitivity analysis, the newly added parameters and some original default parameters affecting pharmaceuticals removal processes were screened and calibrated. The model could accurately simulate all the dynamics of chemical oxygen demand, nitrogen and pharmaceuticals under various conditions. To explore the effect of operational parameters on pharmaceuticals removal efficiency, the wide range of operational parameters was analyzed during model simulation. According to the simulation results, both influent NH4+-N concentration and DO were found to be the significant parameters that impact the removal of DCF, ERY and GEM. AOB biodegradation played an important role in DCF, ERY and GEM removal. The developed model framework helps to investigate the removal mechanisms and key influencing factors of pharmaceuticals removal, thus providing guidelines for reactor design, operation and optimization aiming at pharmaceuticals removal.

Metabolomic response of microalgae towards diclofenac sodium during its removal from water and concomitant recovery of pigments and lipids.

The aim of this work was to assess the efficiency of freshwater green microalga, Chlorella sorokiniana for diclofenac sodium (DFS) removal, and metabolic response of alga to comprehend the metabolic pathways involved/affected during DFS decontamination. Results showed 91.51 % removal of DFS could be achieved within 9 days of algal treatment along with recovery of enhanced value-added bioresources i.e. chlorophyll, carotenoids, and lipids from the spent biomass. DFS also had an effect on enzyme activity including superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation (MDA). Furthermore, metabolomics profiling provided an in-depth insight into changes in the metabolic response of C. sorokiniana wherein DFS induced 32 metabolites in microalgae compared to unexposed-control. This study offers microalgae as a green option for DFS removal, and the metabolomics study complemented with DFS could be an approach to understand the stress-induced strategies of C. sorokiniana for concomitant value-added products recovery in presence of DFS.

[Research of diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity for promoting in vivo cartilage regeneration].

Objective: To develop a diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity and provide a new avenue for alleviating the inflammatory response and enhancing cartilage regeneration in vivo. Methods: Diclofenac sodium was homogeneously mixed with gelatin to prepare a diclofenac sodium-loaded porous gelatin scaffold by freeze-drying method as the experimental group, and a pristine porous gelatin scaffold was served as a control group. The general morphology of the scaffold was observed, the pore size of the scaffold was measured by scanning electron microscopy, the porosity of the scaffold was calculated by drainage method, the loading of diclofenac sodium into the gelatin scaffold was detected by fourier transform infrared spectrometer and X-ray diffraction examinations, and the release kinetics of diclofenac sodium from gelatin scaffold was tested using an in vitro release assay. The two scaffolds were co-cultured with lipopolysaccharide-predisposed RAW264.7 in vitro, and the expressions of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immuno sorbent assay, and Western blot, to detect the in vitro anti-inflammatory effect of the drug-loaded scaffold. Thereafter, the second generation chondrocytes of New Zealand white rabbits were inoculated on the two groups of scaffolds for in vitro culture, and the cytocompatibility of the scaffold was tested by live/dead staining and cell counting kit 8 assay, the feasibility of in vitro cartilage regeneration of the scaffold was evaluated via gross observation, HE staining, Safranin-O staining, and immunohistochemical collagen type Ⅱ staining, as well as biochemical quantitative analyses. Finally, the two groups of chondrocyte-scaffolds were implanted subcutaneously into New Zealand white rabbits, and after 4 weeks, the general observation, HE staining, safranin O staining, immunohistochemical collagen type Ⅱ staining, and biochemical quantitative analyses were performed to verify the cartilage regeneration in vivo, and the expression of inflammation-related genes CD3 and CD68 was detected by RT-PCR to comprehensively evaluate the anti-inflammatory performance of the scaffolds in vivo. Results: The two scaffolds exhibited similar gross, microporous structure, pore size, and porosity, showing no significant difference (P>0.05). Diclofenac sodium was successfully loaded into gelatin scaffold. Data from in vitro anti-inflammatory assay suggested that diclofenac sodium-loaded gelatin scaffold showed alleviated gene and protein expressions of IL-1ß and TNF-α when compared with gelatin scaffold (P<0.05). The evaluation of cartilage regeneration in vitro showed that the number of living cells increased significantly with the extension of culture time, and there was no significant difference between the two groups at each time point (P>0.05). White cartilage-like tissue was regenerated from the scaffolds in both groups, histological observation showed typical cartilage lacuna structure and specific cartilage extracellular matrix secretion. There was no significant difference in the content of cartilage-specific glycosaminoglycan (GAG) and collagen type Ⅱ between the two groups (P>0.05). In vivo experiments showed that the samples in the experimental group had porcelain white cartilage like morphology, histologic staining showed obvious cartilage lacuna structure and cartilage specific extracellular matrix, the contents of GAG and collagen type Ⅱ were significantly higher than those in the control group, and the protein and mRNA expressions of CD3 and CD68 were significantly lower than those in the control group, with significant differences (P<0.05). Conclusion: The diclofenac sodium-loaded gelatin scaffold presents suitable pore size, porosity, and cytocompatibility, as well as exhibited satisfactory anti-inflammatory ability, providing a reliable scheme for alleviating the inflammatory reaction of regenerated cartilage tissue after in vivo implantation and promoting cartilage regeneration in vivo.

Chronic Diclofenac Exposure Increases Mitochondrial Oxidative Stress, Inflammatory Mediators, and Cardiac Dysfunction.

PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly prescribed medications for pain and inflammation. Diclofenac (DIC) is a commonly prescribed NSAID that is known to increase the risk of cardiovascular diseases. However, the mechanisms underlying its cardiotoxic effects remain largely unknown. In this study, we tested the hypothesis that chronic exposure to DIC increases oxidative stress, which ultimately impairs cardiovascular function. METHODS AND RESULTS: Mice were treated with DIC for 4 weeks and subsequently subjected to in vivo and in vitro functional assessments. Chronic DIC exposure resulted in not only systolic but also diastolic dysfunction. DIC treatment, however, did not alter blood pressure or electrocardiographic recordings. Importantly, treatment with DIC significantly increased inflammatory cytokines and chemokines as well as cardiac fibroblast activation and proliferation. There was increased reactive oxygen species (ROS) production in cardiomyocytes from DIC-treated mice, which may contribute to the more depolarized mitochondrial membrane potential and reduced energy production, leading to a significant decrease in sarcoplasmic reticulum (SR) Ca2+ load, Ca2+ transients, and sarcomere shortening. Using unbiased metabolomic analyses, we demonstrated significant alterations in oxylipin profiles towards inflammatory features in chronic DIC treatment. CONCLUSIONS: Together, chronic treatment with DIC resulted in severe cardiotoxicity, which was mediated, in part, by an increase in mitochondrial oxidative stress.

Antioxidant and Antithrombotic Activities of Kenaf Seed (Hibiscus cannabinus) Coat Ethanol Extract in Sprague Dawley Rats.

Oxidative stress has been implicated in deadly lifestyle diseases, and antioxidants from plant sources are the primary option in the treatment regime. Kenaf seeds are the storehouse of potential natural antioxidant phytoconstituents. Perhaps, none of the studies documented the phytoconstituents and their antioxidant potential from Kenaf seed coat so far. Thus, the current study focuses on exploring the protective effect of Kenaf Seed Coat Ethanol Extract (KSCEE) against sodium nitrite and diclofenac-induced oxidative stress in vitro (red blood cell and platelets model) and in vivo (female Sprague Dawely rat's model) along with the antithrombotic activity. The infrared spectrophotometry data showed the heterogeneous functional groups (CH, OH, C = C, C = C-C) and aromatic rings. Reverse phase high-performance liquid chromatography and gas chromatography-mass spectrometry chromatogram of KSCEE also evidenced the presence of several phytochemicals. KSCEE displayed about 76% of DPPH scavenging activity with an IC50 value of 34.94 µg/ml. KSCEE significantly (***p < 0.001) normalized the stress markers such as lipid peroxidation, protein carbonyl content, superoxide dismutase, and catalase in sodium nitrite and diclofenac-induced oxidative stress in RBC, platelets, liver, kidney, and small intestine, respectively. Furthermore, KSCEE was found to protect the diclofenac-induced tissue destruction of the liver, kidney, and small intestine obtained from seven groups of female Sprague Dawely rats. KSCEE delayed the clotting time of platelet-rich plasma and platelet-poor plasma and activated partial thromboplastin time, suggesting its anticoagulant property. In addition, KSCEE also exhibited antiplatelet activity by inhibiting both adenosine diphosphate and epinephrine-induced platelet aggregation. In conclusion, KSCEE ameliorates the sodium nitrite and diclofenac-induced oxidative stress in red blood cells, platelets, and experimental animals along with antithrombotic properties.

Pennisetum glaucum Protein Extract Protects RBC, Liver, Kidney, Small Intestine from Oxidative Damage and Exhibits Anticoagulant, Antiplatelet Activity.

High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis.In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation.PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation.Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. ABBREVIATIONS: PGEPennisetum Glaucum protein ExtractAPPTActivated Partial Thromboplastin TimePTProthrombin TimeROSReactive Oxygen SpeciesPRPPlatelet Rich PlasmaPPPPlatelet Poor PlasmaSDS-PAGESodium Dodecyl Sulfate-Polyacrylamide Gel ElectrophoresisPASPeriodic Acid-schiff StainingODOptical DensityINRInternational Normalized RatioPBSPhosphate Buffered SalineSODSuperoxide DismutaseTCATrichloro Acetatic AcidDTNBDi-Thio-bis-NitroBenzoic acidSGOTSerum Glutamate Oxaloacetate TransaminaseSGPTSerum Glutamate Pyruvate TransaminaseALPAlkaline PhosphataseDFCDiclofenacSylSilymarinMEDMinimum Edema DoseMHDMinimum Hemorrhagic Dose.