Antioxidant and Anti-Inflammatory Effects of 3-Dehydroxyceanothetric Acid 2-Methyl Ester Isolated from Ziziphus jujuba Mill. against Cisplatin-Induced Kidney Epithelial Cell Death

Cisplatin is a platinum-based chemotherapeutic agent for treating solid tumors; however, it presents a risk factor for nephropathy. In the present study, we investigated the antioxidant and anti-inflammatory effects of 3-dehydroxyceanothetric acid 2-methyl ester (3DC2ME) isolated from Ziziphus jujuba Mill. in LLC-PK1 cells following cisplatin-induced cytotoxicity. These cells were exposed to 3DC2ME for 2 h, followed by treatment with cisplatin for 24 h. The treated cells were subjected to cell viability analysis using the Ez-Cytox assay. Reactive oxygen species (ROS) were detected via 2′, 7′- dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In addition, western blotting and fluorescent immunostaining were performed to evaluate protein expressions related to oxidative stress and inflammation pathways. Pretreatment with 3DC2ME protected LLC-PK1 cells from cisplatin-induced cytotoxicity and oxidative stress. In addition, pretreatment with 3DC2ME upregulated heme oxygenase 1 (HO-1) via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the cisplatin-treated LLC-PK1 cells. Furthermore, the increase in the expressions of IκB kinase α/β (IKKα/β), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in these cells was inhibited. These results provide basic scientific evidence for understanding the antioxidant and anti-inflammatory effects of 3DC2ME isolated from Z. jujuba against cisplatin-induced kidney epithelial cell death.

In vitro evaluation of antiviral efficacy of a Siddha Formulation against SARS-CoV-2

Introduction: SARS-CoV-2 virus caused COVID-19 pandemic with 218 million cases and 45 million deaths world over. It has challenged the already overburdened healthcare systems and created an urgent need to investigate solutions present in other healthcare systems. In this study Kabasura Kudineer is investigated as an intervention to influence the immune response which is beneficial for the host and stop the viral replication. Methods: Kabasura Kudineer is a polyherbal formulation containing 15 herbal drugs mixed in equal quantity. It is an official Siddha formulation, used for phlegmatic fevers and flu-like symptoms. To conduct this study Vero E6 (CL1008), the African monkey kidney epithelial cell line was taken and infected with SARS-CoV-2 viral isolate. The Kabasura Kudineer was added in different concentrations; 0.5 mg/mL, 0.25 mg/mL, 0.12 mg/mL, 0.06 mg/mL and 0.03 mg/mL to the infected cells respectively. These cell plates were incubated for 3 days in 5% CO2 incubator. Remdesivir was used as a positive control. The cells were fixed with formaldehyde, stained with crystal violet and plaques were visualised. Plaques were counted as PFU/ml. Result: Kabasura Kudineer was found to exhibit good antiviral activity against SARS-CoV-2. The highest antiviral activity was 81.5% at a concentration of 0.5 mg/ml. The IC-50 value was found to be 0.2 mg/mL. Conclusion: The antiviral efficacy of Kabasura Kudineer in our study showed reduction in the viral load which supports the results of clinical studies. Kabasura Kudineer can be used widely in a clinical setting as a treatment for COVID-19.

An In-vitro evaluation of a polyherbal formulation, against SARS-CoV-2

Background: COVID-19, caused by SARS-CoV-2 is one of the major health crisis that has affected the world in the past century. With the emergence of new strains of viruses and antimicrobial resistance, the world is looking for an alternate therapeutic option to fight infectious disease.Objective: The present study evaluated the efficacy of a novel polyherbal formulation, named NOQ19, against SARS-CoV-2 in an in vitro setting. NOQ 19 is an unique blend of 13 Ayurvedic herbs.Methodology: Vero E6 (CL1008), the African green monkey kidney epithelial cell, were infected with SARS-CoV-2 virus (isolate USA-WA1/2020) in a 96 well-plate. NOQ19 test material was diluted in different concentration as follows 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml and 0.9mg/ml. These different concentrations of NOQ19 were added to infected cells respectively and incubated for 3 days in 5% CO2 incubator. Remdesivir was used as a positive control.The cells were finally fixed with formaldehyde, stained with crystal violet and plaques were visualized. The number of plaques were counted to determine the PFU(plaque forming units)/mL.Results: Results demonstrated 100% antiviral efficacy of NOQ19 at 0.9mg/ml concentration with complete elimination of the virus. The IC50 of the drug was found to be 0.2mg/ml. The results of the present study demonstrated viral load reduction in SARS- CoV-2 infected Vero E6 cell lines.Conclusion: The result along with clinical trials could propose NOQ19 as a potential therapeutic option in the fighting the COVID-19 challenge.

Application of a Biosurfactant Produced by Bacillus cereus UCP 1615 from Waste Frying Oil as an Emulsifier in a Cookie Formulation

Biosurfactants have attracted increasing interest from the food industry due to their emulsifying, foaming and solubilizing properties. However, the industrial use of microbial biosurfactants has been hampered by the high production costs related mainly to the use of expensive substrates. The search for low-cost alternative substrates is one of the strategies adopted to overcome this problem. In the present study, a biosurfactant produced by Bacillus cereus UCP1615 by fermentation in a medium supplemented with waste frying soybean oil as a low-cost substrate was evaluated as a bioemulsifier for the production of cookies. The biosurfactant was evaluated for its emulsifying capacity against different vegetable oils, antioxidant activity and toxicity, demonstrating favorable results for use in food. In particular, it showed satisfactory antioxidant activity at the tested concentrations and no cytotoxicity to the L929 (mouse fibroblast) and Vero (monkey kidney epithelial) cell lines using the MTT assay. The biosurfactant was then added at different concentrations (0.25, 0.5 and 1%) to a standard cookie dough formulation to evaluate the physicochemical characteristics of the product. Cookies formulated with the biosurfactant exhibited similar energy and physical characteristics to those obtained with the standard formulation but with a lower moisture content. The biosurfactant also ensured a good preservation of the cookie texture after 45 days of storage. These results suggest that the biosurfactant has a potential application as a green emulsifier in accordance with the demands of the current market for biocompatible products.

Nano-Second Laser Interference Photoembossed Microstructures for Enhanced Cell Alignment

Photoembossing is a powerful photolithographic technique to prepare surface relief structures relying on polymerization-induced diffusion in a solventless development step. Conveniently, surface patterns are formed by two or more interfering laser beams without the need for a lithographic mask. The use of nanosecond pulsed light-based interference lithography strengthens the pattern resolution through the absence of vibrational line pattern distortions. Typically, a conventional photoembossing protocol consists of an exposure step at room temperature that is followed by a thermal development step at high temperature. In this work, we explore the possibility to perform the pulsed holographic exposure directly at the development temperature. The surface relief structures generated using this modified photoembossing protocol are compared with those generated using the conventional one. Importantly, the enhancement of surface relief height has been observed by exposing the samples directly at the development temperature, reaching approximately double relief heights when compared to samples obtained using the conventional protocol. Advantageously, the light dose needed to reach the optimum height and the amount of photoinitiator can be substantially reduced in this modified protocol, demonstrating it to be a more efficient process for surface relief generation in photopolymers. Kidney epithelial cell alignment studies on substrates with relief-height optimized structures generated using the two described protocols demonstrate improved cell alignment in samples generated with exposure directly at the development temperature, highlighting the relevance of the height enhancement reached by this method. Although cell alignment is well-known to be enhanced by increasing the relief height of the polymeric grating, our work demonstrates nano-second laser interference photoembossing as a powerful tool to easily prepare polymeric gratings with tunable topography in the range of interest for fundamental cell alignment studies.

Increased expression of LCN2 formed a positive feedback loop with activation of the ERK pathway in human kidney cells during kidney stone formation

Kidney stones are a common threat to the health of elderly patients with a high incidence of disease. However, the specific molecular mechanism of the formation of kidney stones has not been elucidated. Here, we combined signalling molecules with signalling pathways in a double positive circulation regulation model. In addition, we found that LCN2 plays a role in promoting kidney stones through regulation of the ERK signalling pathway and expression of other kidney stone-related genes. LCN2 expression was upregulated upon oxalate stimulation. P-ERK1/2 inhibition by U0126 in kidney epithelial cells resulted in decreased expression of LCN2. Furthermore, the upregulation of LCN2 not only depended on the activation of the ERK signalling pathway but also regulated the activation of the ERK signalling pathway. Importantly, upregulation of LCN2 not only caused kidney epithelial cell damage but also promoted the expression of other kidney stone-related genes. Our findings improved the understanding of LCN2 and might lead to the development of new therapeutic and prognostic markers for kidney stones.

Effects of gamma-aminobutyric acid and piperine on gene regulation in pig kidney epithelial cell lines

Objective: Gamma-aminobutyric acid (GABA) and piperine (PIP) are both nutritional supplements with potential use in animal diets. The purpose of this study is to investigate the effect of GABA and/or PIP treatment on the gene expression pattern of a pig kidney epithelial cell line.Methods: LLCPK1 cells were treated with GABA, PIP, or both, and then the gene expression pattern was analyzed using microarray. Gene ontology analysis was done using GeneOntology (Geneontology.org), and validation was performed using quantitative real-time polymerase chain reaction.Results: Gene ontology enrichment analysis was used to identify key pathway(s) of genes whose expression levels were regulated by these treatments. Microarray results showed that GABA had a positive effect on the transcription of genes related to regulation of erythrocyte differentiation and that GABA and PIP in combination had a synergistic effect on genes related to immune systems and processes. Furthermore, we found that effects of GABA and/or PIP on these selected genes were controlled by JNK/p38 MAPK pathway.Conclusion: These results can improve our understanding of mechanisms involved in the effect of GABA and/or PIP treatment on pig kidney epithelial cells. They can also help us evaluate their potential as a clinical diagnosis and treatment.

Anti-Apoptotic and Antioxidant Effects of 3-Epi-Iso-Seco-Tanapartholide Isolated from Artemisia argyi against Iodixanol-Induced Kidney Epithelial Cell Death

Iodixanol is a non-ionic iso-osmolar contrast agent, but it is a risk factor for kidney damage and increases morbidity and mortality. In this study, we investigated the effect of 9 sesquiterpenes isolated from mugwort (Artemisia argyi) in contrast agent-induced cytotoxicity in LLC-PK1 cells. Cells were exposed to nine sesquiterpene compounds for 2 h, followed by incubation with iodixanol for 3 h. Cell viability was assessed using the Ez-Cytox assay. The level of reactive oxygen species was measured using 2′,7′-dichlorodihydrofluorescein diacetate staining. Apoptotic cell death was detected using annexin V/PI staining. In addition, immunofluorescence staining and western blotting were performed using antibodies against proteins related to apoptosis, oxidative stress, and MAPK pathways. The most effective 3-epi-iso-seco-tanapartholide (compound 8) among the 9 sesquiterpene compounds protected LLC-PK1 cells from iodixanol-induced cytotoxicity, oxidative stress, and apoptotic cell death. Pretreatment with compound 8 reversed iodixanol-induced increases in the expression of JNK, ERK, p38, Bax, caspase-3, and caspase-9. It also reversed the iodixanol-induced decrease in Bcl-2 expression. Furthermore, pretreatment with compound 8 caused nuclear translocation of Nrf2 and upregulated HO-1 via the Nrf2 pathway in iodixanol-treated LLC-PK1 cells. Thus, we demonstrated here that compound 8 isolated from A. argyi has the potential to effectively prevent iodixanol-induced kidney epithelial cell death via the caspase-3/MAPK pathways and HO-1 via the Nrf2 pathway.

Structural Insights into Escherichia coli Shiga Toxin (Stx) Glycosphingolipid Receptors of Porcine Renal Epithelial Cells and Inhibition of Stx-Mediated Cellular Injury Using Neoglycolipid-Spiked Glycovesicles

Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Galα1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.