Background: Chronic unpredictable stress triggers various pathological and metabolic alterations by modulating psychophysiological balance. Valeric acid (VA), a postbiotic material, has been reported to mitigate stress-induced behavioral changes in rodents. Objectives: To investigate the protective effect of valeric acid against chronic unpredictable stress in a rodent model by assessing neuro-physiological alterations along with changes in biochemical parameters to confirm the possible mechanism. Methods: A 14-day chronic unpredictable stress (CUS) model in albino Wistar rats was developed to check the stress-induced changes using forced swim test, tail suspension test and sexual behavior observation. Quantification of IL-6, TNF-α, IL-1β, plasma corticosterone level and oxidative stress parameters were also done. Results: Findings revealed the protective effects of valeric acid against CUS, which reversed the depression caused by a forced swim and tail suspension test in rats. Proinflammatory and oxidative stress markers were significantly (p < 0.05) restored in CUS rats treated with valeric acid as compared with the vehicle control, which was comparable to the standard drug, Panax ginseng. Conclusions: The present study concludes that valeric acid demonstrated significant (p < 0.05) anti-stress effect by modulating both behavioral responses and stress-related biochemical modifications.

BackgroundPathological NETosis contributes to immune dysregulation and organ damage in systemic lupus erythematosus (SLE), positioning it as a potential therapeutic target. Previous studies have shown suppressed NETosis in term low birth weight (tLBW) newborns. Several NETosis-suppressive molecules, including the α1-antitrypsin-derived C-terminal peptide and CRISPP have been reported in cord blood (CB) plasma and have shown suppressive effects on NET formation. Given the pathogenic role of excessive NETosis in SLE, these observations suggest that LBW newborns’ CB plasma (LP) may contain putative NETosis-inhibitory factors with therapeutic potential and support further investigations.ObjectivesTo evaluate the NETosis-inhibitory activity of LP and its effects on lupus-associated pathological features in neutrophils from patients with SLE and in the pristane-induced lupus (PIL) mouse model.MethodsEx vivo effects of LP on NETosis were evaluated using neutrophils from patients with SLE (n=35) and PIL mouse (n=18; n=6/group). NET formation was assessed by extracellular DNA release and expression of key markers, including citrullinated histone H3 (CitH3), neutrophil elastase (NE), and myeloperoxidase (MPO), along with cytoplasmic and mitochondrial ROS production. In vivo efficacy was determined by measuring reduction in NETosis, oxidative stress, anti-dsDNA autoantibody levels, proteinuria, and improvement of tissue oxygenation and vascularization in the heart, liver and kidneys of PIL mice.ResultsLP significantly reduced NET formation, as evidenced by reduced extracellular DNA release and expression of CitH3 and NE in SLE neutrophils. Extracellular DNA release decreased by 13% and 16.66% along with a reduction in netting neutrophils by 27.4% and 21.12% under basal and LPS-stimulated conditions, respectively. LP also suppressed ROS production in SLE neutrophils. In PIL mice, LP reduced NETosis (30.93%) and oxidative stress (15.34%mitochondrial ROS; 24.28% cytoplasmic ROS) in blood-derived neutrophils, decreased MPO and Sytox Orange signal intensities in the heart, liver, and kidneys. LP further decreased anti-dsDNA autoantibody levels (57.9%) and proteinuria (93.59%), preserved vascular integrity, and improved tissue oxygenation.ConclusionLP exhibits NETosis-inhibitory activity and ameliorates lupus-associated pathology in the PIL mouse and ex vivo SLE neutrophil system. These findings suggest the presence of as-yet-uncharacterized endogenous plasma components that modulate NET formation and support further investigation as potential therapeutics for SLE.

This study aimed to investigate the therapeutic potential of cell-free Dexamethasone (Dex) primed Wharton’s jelly Mesenchymal stem cells derived conditioned media (DW) in addressing complications associated with systemic lupus erythematosus (SLE), focusing on its immunomodulatory effects. Peripheral blood mononuclear cells from 74 SLE patients were stimulated and treated with Dex, DW and W. Culture supernatant were evaluated for autoantibody levels, IL-10 and TGF-β by ELISA, Treg subtypes, Breg subtypes, TH17 cells Double negative T cells and inflammatory neutrophils by flow cytometry, IL-10 and IL-17A by qPCR. In vivo studies were performed on 60 pristane induced female BALB/c mice. Dex and DW treatments were evaluated for autoantibody production, proteinuria, immunomodulation of immune cells, organ function, and histopathology. In vivo imaging of internal organs was done using VevoLAZR-X photoacoustic imaging system. DW treatment significantly expanded different Treg and Bregs subtypes. DW suppressed pathogenic TH17, Double negative T cells and inflammatory neutrophils. Comparative analyses with hydroxychloroquine showed similar effects, with combined treatment enhancing efficacy. Inhibition studies implicated the TGF-β pathway in DW's mechanism. In vivo studies using the PIL mouse model showed that DW treatment reduced mortality, prevented proteinuria, and ameliorated symptoms such as limb inflammation, seizures, and alopecia. Detailed organ-specific evaluations through live imaging and histopathological analyses revealed DW’s protective effects on kidneys, liver, lungs, heart, and spleen. DW shows promise as a cell-free biological therapy for SLE and related autoimmune disorders, capable of modulating immune responses effectively without the adverse effects of glucocorticoids.

BACKGROUND: Venous leg ulcers are responsible for more than half of all lower extremity ulcerations. Therefore, in this study we have proposed to understand the genetic changes associated with underlying disease mechanisms in venous ulcer by evaluating the gene expression of inflammatory gene/marker WAKMAR1, E2F1, RAD21 and NIPBL. METHODS: WAKMAR1, E2F1, RAD21 and NIPBL genes sequence was pulled out from NCBI database. The gene-specific primers were designed using Primer 3 software and were synthesized. Tissue sample collected before starting treatment and after 50% healing or after 2-6 months in non- healing ulcers. Samples stored at -80 degrees C, were thawed and dissected into small pieces. In all samples RNA was isolated and quantified using a Nano Drop. Standard 1% agarose gel under denaturing condition with ethidium bromide was used to assess the integrity of RNA. WAKMAR1, E2F1, RAD21 and NIBPL gene expression was directly analyzed by one step RT-PCR. Gene expression was made relative to expression of house-keeping beta actin gene. Statistical comparisons performed using Student's t-test with the help of GraphPad Prism version 8.0.2, NIH. RESULTS: RAD21 gene expression increased significantly in tissue samples from venous ulcer patients post treatment. NIPBL gene expression increased but not significantly in tissue samples from venous ulcer patients post treatment. Relative expression of RAD21 gene significantly increased in healing venous ulcer samples as compared to non-healing samples (P<0.05 in healing v/s non-healing group). Relative expression of NIPBL gene significantly increased in healing venous ulcer samples as compared to non-healing samples (P<0.0001 in healing v/s non-healing group). No expression for WAKMAR1 and E2F1 were noticed in venous ulcer tissue samples. CONCLUSIONS: NIPBL & RAD21 gene expression increased significantly in healed venous ulcers. This finding could be harnessed for the development of more effective wound therapy.

This study proposes a novel approach, utilizing cell-free dexamethasone (Dex) primed Whartons jelly mesenchymal stem cells derived conditioned media (DW), offering improved efficacy, simplicity, and alternative medicine for addressing complications associated with systemic lupus erythematosus (SLE). This study explores the immunomodulatory effects of DW treatment on immune cell populations in SLE patients and a pristane-induced lupus (PIL) mouse model. DW induces significant expansion of Tregs, Bregs, suppressing Th17, double-negative T cells, and inflammatory neutrophils through modulating IL-10 and IL-17A production. Comparisons with the standard drug hydroxychloroquine reveal similar effects, suggesting TGF beta; pathway mediation in DW's actions. Compared with the immunosuppressive drug Dex, DW better attenuated autoantibody production, increased anti-inflammatory cytokines and maintained a balanced Th17/Treg ratio. In the preclinical in vivo studies, DW exhibits therapeutic efficacy, reducing mortality, preventing proteinuria, and reversing limb inflammation, seizure and alopecia. Organ specific evaluations using advanced live imaging or histopathological analysis highlighted DW's protective effects on kidneys, liver, lungs, heart, and spleen. This provided insight into the immunomodulatory benefits of DW at various levels and suggested that it could be a potential therapeutic avenue for managing complications related to SLE. ### Competing Interest Statement The authors have declared no competing interest.

BackgroundSystemic Lupus Erythematosus (SLE) is characterized by dysregulated immune responses, with neutrophil extracellular traps (NETs) playing a significant role. NETs are recognized by autoantibodies in SLE patients, exacerbating pathology. Both excessive NET formation and impaired degradation contribute to SLE pathophysiology.ObjectiveTo investigate the immunomodulatory effects of Dexamethasone-primed Wharton’s jelly (WJ) derived MSCs CM (DW) and IFN-γ-primed WJ-MSCs-CM (IW) on NETosis and associated protein markers in SLE patients’ LPS or ribonucleoprotein immune complexes (RNP ICs) induced neutrophils and in pristane induced lupus (PIL) model. And to elucidate the mechanism involved therein.MethodsWe investigated the immunomodulatory effects of DW and IW on NETosis in SLE. Utilizing ex vivo and in vivo models, we assessed the impact of preconditioned media on NET formation and associated protein markers neutrophil elastase (NE), citrullinated histone (citH3), myeloperoxidase (MPO), cytoplasmic and mitochondrial ROS production. We also examined the involvement of key immunomodulatory factors present in DW and IW, including prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO), and transforming growth factor-beta (TGF-β).ResultsPreconditioned media effectively suppressed NETosis and reduced ROS generation in SLE neutrophils, indicating their immunomodulatory potential. Inhibition studies implicated IDO and PGE2 in mediating this effect. Combined treatment with DW or IW together with hydroxychloroquine (HCQ) demonstrated superior efficacy over HCQ alone, a standard SLE medication. In PIL mouse model, DW and IW treatments reduced NETosis, ROS generation, as evidenced by decreased NET-associated protein expression in vital organs.ConclusionOur study highlights the multifaceted impact of IW and DW on NETosis, ROS dynamics, and lupus severity in SLE. These findings underscore the potential of preconditioned media for the development of targeted, personalized approaches for SLE treatment.

BACKGROUND:Neutrophil extracellular traps (NETs) being one of the predominant activities of neutrophils has become its key defense mechanism owing to its extensive role in inflammation and infection. However, the mechanisms regulating NET formation or NETosis still remains to be better understood. Our earlier whole genome transcriptomic data revealed two G-protein couple receptors (GPCRs) - complement component 5a receptor 1 (C5aR1) and leukotriene B4 receptor 1 (LTB4R1) were downregulated in term low birth weight (tLBW) newborns with deficient NET formation abilities. Neutrophils employ C5aR1 and LTB4R1 for mediating their immune responses, inflammation and antimicrobial activity. Hence, this study was aimed to explore the role of two GPCRs, C5aR1 and LTB4R1 including their downstream signaling molecules in NETs induction and regulation. METHODS:The validation of the transcriptomic data for C5aR1 and LTB4R1 was done using quantitative real time PCR. Pharmacological inhibition of C5aR1 and LTB4R1 using W-54011 and LY223982 on neutrophils of adults and newborns' was done to study their impact on NETosis. Extracellular DNA release, Reactive oxygen species (ROS) generation, expression of NET proteins, and signaling molecules downstream to C5aR1 and LTB4R1 were quantified using plate reader based assay, immunofluorescence, and western blotting. Myeloperoxidase (MPO)-DNA quantified by flow cytometry. Knockdown studies using siRNA against C5aR1 and LTB4R1 were done in HL-60 cells derived surrogate neutrophils and expression of downstream molecules of the two GPCRs, C5aR1 and LTB4R1 signaling axis along with NET proteins was quantified by western blotting. RESULTS:The expression of C5aR1 and LTB4R1, extracellular DNA, ROS and NET associated proteins (NE, CitH3, PAD4 and MPO) was notably increased upon NET induction in healthy adults and normal birth weight (NBW) newborns' neutrophils. Pharmacological inhibition of these two GPCRs led to substantial reduction in NETosis, extracellular DNA, ROS generation, and expression of NET associated proteins like CitH3, NE, PAD4, MPO along with downstream signaling molecules Rap1a, B-Raf and pERK. Our observations suggest a precise role of C5aR1 and LTB4R1 on induction of NETs via Rap1a/B-Raf/ERK signaling axis. CONCLUSION:The C5aR1 and LTB4R1 signaling via Rap1a/B-Raf/ERK axis acts as a signal-relay mechanism to regulate NET formation in neutrophils. Further, C5aR1 and LTB4R1 signaling cascade along with NET-associated proteins are remarkably downregulated in tLBW newborns' neutrophils leading to impaired NETosis in them. Therefore, C5aR1 and LTB4R1 and their signaling molecules could provide an effective therapeutic target for compromised NETosis like tLBW newborns.

Tephrosia purpurea belongs to the family Fabaceae, is used for the treatment of inflammation, diabetes, cancer, chronic fever, boils, gingivitis etc. In the present study, three compounds (TP-1, TP-3 and TP-5) were isolated from ethyl acetate extract of the seeds of T. purpurea (EETP). Quantity of TP-3 (6.05%) in EETP was determined by HPLC. In-vitro anti-cancer activity of EETP and isolated compound TP-3 on SiHa cells as well as PBMCs (peripheral blood mononuclear cells) were evaluated by MTT (3-(4,5- di methyl thiazol -2-yl)-2,5-di phenyl tetrazolium bromide) assay. On the basis of spectroscopic analysis and physical properties, structure of isolated compounds TP-1, TP-3 and TP-5 were characterized as teclenone, pongamol and ss-sitosterol respectively. Isolated compound TP-3 showed cytotoxicity against SiHa cells with the IC50 33.06 mu gmL(-1). TP-3 showed significant cytotoxicity at the concentration range from 20-50 mu gmL(-1) as compared to control. Maximum cytotoxicity (72.75%) was observed at the concentration of 50 mu gmL(-1). TP-3 showed non-significant cytotoxicity against PBMCs cells at all the concentrations except at 50 mu gmL(-1) (cytotoxicity 24.19%). EETP showed significant cytotoxicity (IC50 value 113.63 mu gmL(-1)) against SiHa cells at the concentration range from 75-150 mu gmL(-1) as compared to control. Maximum cytotoxicity (75.34%) was observed at the concentration of 150 mu gmL(-1).

We report herein a one-step, biomimetic synthesis of silver nanoparticles (AgNPs) from Musa balbisiana leaf extract having a cytotoxic effect on SiHa and HL-60 cancer cell lines without affecting non-cancerous, healthy peripheral blood mononuclear cells (PBMCs). The EDX analysis of AgNP dispersion, with a peak of 3.2 keV, confirmed the presence of elemental silver with a weight percentage of 54.57%. As is revealed from both SEM and TEM, monodispersed AgNPs were spherical in morphology with a diameter ranging from 11.28 to 87.53 nm with an average size of 12 nm. The diffraction pattern revealed polycrystalline AgNPs with FCC crystal structure. We report increased cytotoxicity accompanied with characteristic morphological change in HL-60 and SiHa, significant reduction in proliferation, increased reactive oxygen species (ROS) generation, and decreased mitochondrial membrane potential (MMP) following AgNP treatment at various doses. No such detrimental cellular impact of AgNPs was observed on healthy PBMCs. Fluorescence microscopy tracked coumarin-loaded AgNP accumulation in lysosomes as well as the nucleus, suggesting that both lysosome and nucleus served as cellular targets of AgNPs. AgNPs were internalized through passive and active pathways and that energy-dependent, clathrin-mediated endocytosis was involved in the trafficking of AgNPs. Our in silico docking studies confirm the binding of AgNPs to adaptor protein 2 on the β2 subunit facilitating clathrin-mediated endocytosis.

Understanding of the genetic basis underlying inflammatory disorders has progressed in recent years. Contribution of proinflammatory cytokines, human leukocyte antigen (HLA), and non-HLA polymorphisms in the pathogenesis of several autoimmune and immune-mediated inflammatory disorder is critical. HLA plays a central role in disease pathology. Harmful stimuli triggering the signaling mechanisms including nuclear factor-kappa B pathway, Janus kinase-signal transducer and activator of transcription pathway, and mitogen-activated protein kinase pathway results in the release of inflammatory mediators. From acute to chronic inflammation, the etiology of various inflammatory disorders is poorly understood. Inflammatory disorder such as COVID 19 is a devastating havoc to the world. As we reach the end of 2020, >1 million people have succumbed to death worldwide. Disease-manifesting clinical features include mild to severe pneumonia, loss of respiratory function progressing to acute respiratory distress syndrome with occasional multiorgan failure. Cytokine storm, decreased T cell count, and insufficient immune response are conducive issues to COVID 19 pandemic. Varied immune responses to the same antigen across different individuals determine the genetic perspective of disease susceptibility. Through genome-wide association studies, next-generation sequencing and other genetic techniques, several genetic risk loci associated with various inflammatory diseases such as inflammatory bowel disease, psoriasis, sclerosis, and systemic lupus erythematosus (SLE) have been identified. Dysregulated inflammatory pathways, gene mutation, or elevated cytokine level may lead to the disease progression. However, the production of autoantibodies against the nuclear antigens is a hallmark of diseases like SLE and rheumatoid arthritis. Moreover, environmental factors like smoking also increase the risk of inflammatory disorders. Understanding the functional aspects of casual genetic factors underlying the disease pathogenesis greatly facilitates the ability to identify the therapeutic targets relevant to disease. The current chapter deals with the idea of genetic perspective associated with various inflammatory disorders and their potential therapeutic targets along with the factors contributing to disease susceptibility.

The pathological hallmarks of Alzheimer's disease (AD) are manifested as an increase in the level of oxidative stress and aggregation of the amyloid-β protein. In vitro, in vivo, and in silico experiments were designed and carried out with multifunctional cholinergic inhibitor, F24 (EJMC-7a) to explore its neuroprotective effects in AD models. The neuroprotection ability of F24 was tested in SH-SY5Y cells, a widely used neuronal cell line. The pretreatment and subsequent co-treatment of SH-SY5Y cells with different doses of F24 was effective in rescuing the cells from H2O2 induced neurotoxicity. F24 treated cells were found to be effective in the reduction of cellular reactive oxygen species, DNA damage, and Aβ1-42 induced neurotoxicity, which validated its neuroprotective effectiveness. F24 exhibited efficacy in an in vivo Drosophila model by rescuing eye phenotypes from degeneration caused by Aβ toxicity. Further, computational studies were carried out to monitor the interaction between F24 and Aβ1-42 aggregates. The computational studies corroborated our in vitro and in vivo studies suggesting Aβ1-42 aggregation modulation ability of F24. The brain entry ability of F24 was studied in the parallel artificial membrane permeability assay. Finally, F24 was tested at doses of 1 and 2.5 mg/kg in the Morris water maze AD model. The neuroprotective properties shown by F24 strongly suggest that multifunctional features of this molecule provide symptomatic relief and act as a disease-modifying agent in the treatment of AD. The results from our experiments strongly indicated that natural template-based F24 could serve as a lead molecule for further investigation to explore multifunctional therapeutic agents for AD management.

In our overall goal to overcome the limitations associated with natural products for the management of Alzheimer's disease and to develop in-vivo active multifunctional cholinergic inhibitors, we embarked on the development of ferulic acid analogs. A systematic SAR study to improve upon the cholinesterase inhibition of ferulic acid with analogs that also had lower logP was carried out. Enzyme inhibition and kinetic studies identified compound 7a as a lead molecule with preferential acetylcholinesterase inhibition (AChE IC50 = 5.74 ± 0.13 μM; BChE IC50 = 14.05 ± 0.10 μM) compared to the parent molecule ferulic acid (% inhibition of AChE and BChE at 20 μM, 15.19 ± 0.59 and 19.73 ± 0.91, respectively). Molecular docking and dynamics studies revealed that 7a fits well into the active sites of AChE and BChE, forming stable and strong interactions with key residues Asp74, Trp286, and Tyr337 in AChE and with Tyr128, Trp231, Leu286, Ala328, Phe329, and Tyr341 in BChE. Compound 7a was found to be an efficacious antioxidant in a DPPH assay (IC50 = 57.35 ± 0.27 μM), and it also was able to chelate iron. Data from atomic force microscopy images demonstrated that 7a was able to modulate aggregation of amyloid β1-42. Upon oral administration, 7a exhibited promising in-vivo activity in the scopolamine-induced AD animal model and was able to improve spatial memory in cognitive deficit mice in the Y-maze model. Analog 7a could effectively reverse the increased levels of AChE and BChE in scopolamine-treated animals and exhibited potent ex-vivo antioxidant properties. These findings suggest that 7a can act as a lead molecule for the development of naturally-inspired multifunctional molecules for the management of Alzheimer's and other neurodegenerative disorders.

Stem cells exist in many niches throughout the body and have the ability to self replicate and to differentiate to many lineages. As of a result of the advances in stem cell-based therapies, regenerative medicine is witnessing remarkable development. Encouraging positive outcomes from the use of stem cells in various diseases are extremely promising.. The popularity of stem cell-based therapy is due to its flexibility and potent approach in the treatment of numerous diseases. Treatment with genetically configured HSCs favors the engraftment of transplantation without rejection. MSCs hold an immunoregulatory capacity, elicit immuno- suppressive effects and are immune-privileged cells, due to the low expression of MHCII and costimulatory molecules on their cell surface. Encouraging, positive outcomes from the use of stem cells in immunodeficiency, cancer, hemoglobinopathy, bone, cartilage repair, autoimmune disorders, cardiac and neuronal diseases are extremely promising. Successful stem cells based clinical trials are the game changers in the progress of clinical use of stem cells. This review provides an up to date comprehensive overview of the clinical efficacy of stem cells.