Honokiol: A polyphenol neolignan ameliorates pulmonary fibrosis by inhibiting TGF-ß/Smad signaling, matrix proteins and IL-6/CD44/STAT3 axis both in vitro and in vivo.

Pulmonary fibrosis (PF) is an epithelial/fibroblastic crosstalk disorder of the lungs with highly complex etiopathogenesis. Limited treatment possibilities are responsible for poor prognosis and mean survival rate of 3 to 5 years of PF patients after definite diagnosis. Once thought to be an irreversible disorder, recent evidences have brought into existence the concept of organ fibrosis reversibility due to plastic nature of fibrotic tissues. These findings have kindled interest among the scientific community and given a new direction for research in the arena of fibrosis for developing new anti-fibrotic therapies. The current study is designed to evaluate the anti-fibrotic effects of Honokiol (HNK), a neolignan active constituent from Magnolia officinalis. This study has been conducted in TGF-ß1 induced in vitro model and 21 day in vivo murine model of Bleomycin induced PF. The findings of our study suggest that HNK was able to inhibit fundamental pathways of epithelial to mesenchymal transition (EMT) and TGF-ß/Smad signaling both in vitro and in vivo. Additionally, HNK also attenuated collagen deposition and inflammation associated with fibrosis. We also hypothesized that HNK interfered with IL-6/CD44/STAT3 axis. As hypothesized, HNK significantly mitigated IL-6/CD44/STAT3 axis both in vitro and in vivo as evident from outcomes of various protein expression studies like western blotting, immunohistochemistry and ELISA. Taken together, it can be concluded that HNK reversed pulmonary fibrotic changes in both in vitro and in vivo experimental models of PF and exerted anti-fibrotic effects majorly by attenuating EMT, TGF-ß/Smad signaling and partly by inhibiting IL-6/CD44/STAT3 signaling axis.

Nimbolide ameliorates fibrosis and inflammation in experimental murine model of bleomycin-induced scleroderma.

BACKGROUND: Clinical manifestations of skin fibrosis are very variable and ambiguous, making its management quite critical and challenging. The lack of appropriate established pharmacological interventions make its treatment even more complicated. Intricate details of the underlying pathogenesis are thus imperative to further explore different treatment possibilities. Of note, the TGF-ß/Smad signaling axis and epithelial to mesenchymal transition (EMT) are the principal offenders in this fibrotic disorder. OBJECTIVE: Our current study is aimed at demonstrating the antifibrotic and anti-inflammatory potential of nimbolide, a triterpene derived from Indian traditional plant neem, in a murine model of Bleomycin-induced scleroderma. METHODS: Male C57BL/6 mice were administered with Bleomycin injections subcutaneously, daily for 28 days, at a constant site on the dorsum of the mice. Treatment with nimbolide lasted from day 1 to day 28. At the time of study termination, the injected sites were collected and stored suitably to conduct further molecular experiments and protein expression studies. RESULTS AND CONCLUSION: The results of our study show that nimbolide can significantly intervene in the TGF-ß/Smad signaling axis and the consequent EMT process, thus attenuating deposition of extracellular matrix. Nimbolide also profoundly caused the regression of established inflammation-driven fibrosis, thus demonstrating both antifibrotic and anti-inflammatory activities. Another commendable finding of this study is that nimbolide was able to decrease the levels of LOXL2, a collagen cross-linker, which is aberrantly expressed in scleroderma. Although further mechanistic studies are required, our study displays nimbolide for the first time as a potent antifibrotic agent which can be used as a pharmacological intervention for the treatment of scleroderma.

Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents.

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC50 value of 7.22 ±â€¯0.47 µM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile.

Withaferin A, a novel compound of Indian ginseng (Withania somnifera), ameliorates Cerulein-induced acute pancreatitis: Possible role of oxidative stress and inflammation.

Acute pancreatitis is an inflammatory disorder of the pancreas that may precipitate due to various reasons such as chronic alcoholism, gall stone obstruction, and life style. Current treatment options offer limited efficacy, as they provide only symptomatic relief. This study is an attempt to study the effects of Withaferin A (WFA) against Cerulein-induced acute pancreatitis in mice. Animals were pretreated with WFA via intraperitoneal route, for 7 days. Plasma amylase and lipase, tissue malondialdehyde (MDA), and glutathione were evaluated for all groups. Western blot analysis; haematoxylin and eosin staining of the liver, lung, and pancreas; immunohistochemistry for nitrotyrosine; and myeloperoxidase activity were performed. Haematoxylin and eosin stained sections significantly revealed the altered architecture and thereby damage in the pancreas, lungs, and liver that has been low in treatment groups. Increased myeloperoxidase and nitrotyrosine have also been reduced upon treatment with WFA. Increased levels of MDA, NO, and expression of myeloperoxidase and nitrotyrosine in the parameters estimated add evidence to the role of oxidative stress and inflammation in acute pancreatitis. WFA evidently altered these conditions upon pretreatment. Our study shows that this novel steroidal compound has potent anti-inflammatory property. Natural compounds can therefore be good remedies against many diseases if incorporated in routine diet as dietary supplement.

A facile I2-catalyzed synthesis of imidazo[1,2-a]pyridines via sp3 C-H functionalization of azaarenes and evaluation of anticancer activity.

A facile and efficient metal-free, one-pot, three component synthetic protocol has been developed for the synthesis of medicinally important substituted imidazo[1,2-a]pyridines via the iodine-catalysed oxidative amination of benzylic C-H bonds of azaarenes with 2-aminoazines and condensation with isocyanides. The presented methodology offers the advantages of wide substrate scope, moderate reaction conditions, environment friendliness, clean and simple protocol with high atom economy apart from higher yields. The synthesized compounds were screened for their anti-cancer activity in selected human cancer cell lines. Compounds 4a, 4b, 4c, 4i, 7a, 7b and 7m have significant IC50 values ranging from 4.88 ± 0.28 to 14.55 ± 0.74 µM.

The Role of Plant-derived Products in Pancreatitis: Experimental and Clinical Evidence.

Pancreatitis is a gastrointestinal disease with a worldwide sharp surge during the past decade. Pancreatitis includes acute and chronic subtypes, which are graded based on the amount of pancreatic inflammation. Phytoconstituents represent a promising class of therapeutic agents with wide acceptability not only based on folk practices but sound presence of pharmacological and molecular evidences. Growing research evidence indicates that different molecular mechanisms are involved in their protective effect. Many phytoremedies have been tried for the treatment of pancreatic injuries and have shown success in preclinical animal models of pancreatitis. The literature was largely collected through PubMed and Google scholar database. A large proportion of phytochemicals targets the inflammatory cascade and modulates the overtly acting redox balance among which nuclear factor kappa-light-chain-enhancer of activated B cells is the key molecule. Inhibition of apoptosis (artemisinin, embelin), inflammasome (withaferin A), neutrophil rolling (fucoidan), Ca+2 release (caffeine), mitogen activated protein kinase (guggulsterone) and many other novel mechanisms apart from antioxidant effect have been postulated behind the protective effect of phytoconstituents. The present review deals to fill the gap of hitherto unavailable comprehensive review on various plant products screened for the treatment of pancreatitis. The possible mechanistic profile of these phytochemicals is summarized. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibiting Triggering Receptor Expressed on Myeloid Cells-1 signaling to ameliorate skin fibrosis.

Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. Transforming growth factor-beta (TGF-ß) is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used novel ligand-independent TREM-1 inhibitors in order to investigate the pathogenic role of TREM-1 in SSc, using preclinical models of fibrosis, and explanted SSc skin fibroblasts. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.

Extracellular Matrix-Derived Damage-Associated Molecular Patterns (DAMP): Implications in Systemic Sclerosis and Fibrosis.

Damage-associated molecular patterns (DAMPs) are intracellular molecules released under cellular stress or recurring tissue injury, which serve as endogenous ligands for toll-like receptors (TLRs). Such DAMPs are either actively secreted by immune cells or passively released into the extracellular environment from damaged cells or generated as alternatively spliced mRNA variants of extracellular matrix (ECM) glycoproteins. When recognized by pattern recognition receptors (PRRs) such as TLRs, DAMPs trigger innate immune responses. Currently, the best-characterized PRRs include, in addition to TLRs, nucleotide-binding oligomerization domain-like receptors, RIG-I-like RNA helicases, C-type lectin receptors, and many more. Systemic sclerosis (SSc) is a chronic autoimmune condition characterized by inflammation and progressive fibrosis in multiple organs. Using an unbiased survey for SSc-associated DAMPs, we have identified the ECM glycoproteins fibronectin-containing extra domain A and tenascin C as the most highly upregulated in SSc skin and lung biopsies. These DAMPs activate TLR4 on resident stromal cells to elicit profibrotic responses and sustained myofibroblasts activation resulting in progressive fibrosis. This review summarizes the current understanding of the complex functional roles of DAMPs in the progression and failure of resolution of fibrosis in general, with a particular focus on SSc, and considers viable therapeutic approaches targeting DAMPs.

Pharmacological inhibition of TAK1 prevents and induces regression of experimental organ fibrosis.

Multiorgan fibrosis in systemic sclerosis (SSc) accounts for substantial mortality and lacks effective therapies. Lying at the crossroad of TGF-ß and TLR signaling, TGF-ß-activated kinase 1 (TAK1) might have a pathogenic role in SSc. We therefore sought to evaluate the TAK1 signaling axis in patients with SSc and to investigate pharmacological TAK1 blockade using a potentially novel drug-like selective TAK1 inhibitor, HS-276. Inhibiting TAK1 abrogated TGF-ß1 stimulation of collagen synthesis and myofibroblasts differentiation in healthy skin fibroblasts, and it ameliorated constitutive activation of SSc skin fibroblasts. Moreover, treatment with HS-276 prevented dermal and pulmonary fibrosis and reduced the expression of profibrotic mediators in bleomycin-treated mice. Importantly, initiating HS-276 treatment even after fibrosis was already established prevented its progression in affected organs. Together, these findings implicate TAK1 in the pathogenesis of SSc and identify targeted TAK1 inhibition using a small molecule as a potential strategy for the treatment of SSc and other fibrotic diseases.

Deficiency of inhibitory TLR4 homolog RP105 exacerbates fibrosis.

Activation of TLR4 by its cognate damage-associated molecular patterns (DAMPs) elicits potent profibrotic effects and myofibroblast activation in systemic sclerosis (SSc), while genetic targeting of TLR4 or its DAMPs in mice accelerates fibrosis resolution. To prevent aberrant DAMP/TLR4 activity, a variety of negative regulators evolved to dampen the magnitude and duration of the signaling. These include radioprotective 105 kDa (RP105), a transmembrane TLR4 homolog that competitively inhibits DAMP recognition of TLR4, blocking TLR4 signaling in immune cells. The role of RP105 in TLR4-dependent fibrotic responses in SSc is unknown. Using unbiased transcriptome analysis of skin biopsies, we found that levels of both TLR4 and its adaptor protein MD2 were elevated in SSc skin and significantly correlated with each other. Expression of RP105 was negatively associated with myofibroblast differentiation in SSc. Importantly, RP105-TLR4 association was reduced, whereas TLR4-TLR4 showed strong association in fibroblasts from patients with SSc, as evidenced by PLA assays. Moreover, RP105 adaptor MD1 expression was significantly reduced in SSc skin biopsies and explanted SSc skin fibroblasts. Exogenous RP105-MD1 abrogated, while loss of RP105 exaggerated, fibrotic cellular responses. Importantly, ablation of RP105 in mice was associated with augmented TLR4 signaling and aggravated skin fibrosis in complementary disease models. Thus, we believe RP105-MD1 to be a novel cell-intrinsic negative regulator of TLR4-MD2-driven sustained fibroblast activation, representing a critical regulatory network governing the fibrotic process. Impaired RP105 function in SSc might contribute to persistence of progression of the disease.

PLG nanoparticles target fibroblasts and MARCO+ monocytes to reverse multiorgan fibrosis.

Systemic sclerosis (SSc) is a chronic, multisystem orphan disease with a highly variable clinical course, high mortality rate, and a poorly understood complex pathogenesis. We have identified an important role for a subpopulation of monocytes and macrophages characterized by surface expression of the scavenger receptor macrophage receptor with collagenous structure (MARCO) in chronic inflammation and fibrosis in SSc and in preclinical disease models. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in topographic proximity to activated myofibroblasts in patients with SSc and in the bleomycin-induced mouse model of SSc. Short-term treatment of mice with a potentially novel nanoparticle, poly(lactic-co-glycolic) acid (PLG), which is composed of a carboxylated, FDA-approved, biodegradable polymer and modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture, PLG nanoparticles inhibited TGF-dependent fibrotic responses in vitro. Thus, MARCO+ monocytes are potent effector cells of skin and lung fibrosis and can be therapeutically targeted in SSc using PLG nanoparticles.

Gut microbe-derived metabolite trimethylamine N-oxide activates PERK to drive fibrogenic mesenchymal differentiation.

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown how it contributes to microvascular injury and fibrosis that are hallmarks of this disease. Trimethylamine (TMA) is generated by the gut microbiome and in the host converted by flavin-containing monooxygenase (FMO3) into trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular and metabolic diseases. Using cell culture systems and patient biopsies, we now show that TMAO reprograms skin fibroblasts, vascular endothelial cells, and adipocytic progenitor cells into myofibroblasts via the putative TMAO receptor protein R-like endoplasmic reticulum kinase (PERK). Remarkably, FMO3 was detected in skin fibroblasts and its expression stimulated by TGF-ß1. Moreover, FMO3 was elevated in SSc skin biopsies and in SSc fibroblasts. A meta-organismal pathway thus might in SSc link gut microbiome to vascular remodeling and fibrosis via stromal cell reprogramming, implicating the FMO3-TMAO-PERK axis in pathogenesis, and as a promising target for therapy.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs.

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-ß induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

Natural product topical therapy in mitigating imiquimod-induced psoriasis-like skin inflammation-underscoring the anti-psoriatic potential of Nimbolide.

BACKGROUND: Psoriasis is a chronic inflammatory dermatological disorder having complex pathophysiology with autoimmune and genetic factors being the major players. Despite the availability of a gamut of therapeutic strategies, systemic toxicity, poor efficacy, and treatment tolerance due to genetic variability among patients remain the major challenges. This calls for effective intervention with the superior pharmacological profile. Nimbolide (NIM), a major limonoid is an active chemical constituent found in the leaves of the Indian Neem tree, Azadirachta indica. It has gained immense limelight in the past decades for the treatment of various diseases owing to its anti-proliferative, anti-inflammatory, and anti-cancer potentials. OBJECTIVE: The present study was centered around evaluating the anti-psoriatic effect of NIM in the experimental model of Imiquimod (IMQ)-induced psoriasis-like inflammation model. MATERIALS AND METHODS: Application of IMQ topically on the dorsum of Balb/c mice from day 0-6 prompted psoriasis-like inflammatory symptoms. Treatment groups included topical administration of NIM incorporated carbopol gel formulation and NIM free drug given through subcutaneous route. Protein expression studies such as immunohistochemistry, Western blotting, and ELISA were employed. RESULTS: It was clearly observed from our results that NIM significantly ameliorated the expression of inflammatory and proliferation mediators. Further, NIM in the treatment groups significantly improved classic Psoriasis Area Severity Index scoring when compared to IMQ administered group. CONCLUSION: It is noteworthy that NIM showed a predominant therapeutic effect as compared to other treatment group. To recapitulate, NIM has shown promising activity as an anti-psoriatic agent by remarkably ameliorating inflammation and associated proliferation.

Targeting CD38-dependent NAD+ metabolism to mitigate multiple organ fibrosis.

The processes underlying synchronous multiple organ fibrosis in systemic sclerosis (SSc) remain poorly understood. Age-related pathologies are associated with organismal decline in nicotinamide adenine dinucleotide (NAD+) that is due to dysregulation of NAD+ homeostasis and involves the NADase CD38. We now show that CD38 is upregulated in patients with diffuse cutaneous SSc, and CD38 levels in the skin associate with molecular fibrosis signatures, as well as clinical fibrosis scores, while expression of key NAD+-synthesizing enzymes is unaltered. Boosting NAD+ via genetic or pharmacological CD38 targeting or NAD+ precursor supplementation protected mice from skin, lung, and peritoneal fibrosis. In mechanistic experiments, CD38 was found to reduce NAD+ levels and sirtuin activity to augment cellular fibrotic responses, while inhibiting CD38 had the opposite effect. Thus, we identify CD38 upregulation and resulting disrupted NAD+ homeostasis as a fundamental mechanism driving fibrosis in SSc, suggesting that CD38 might represent a novel therapeutic target.

Inhalation of sustained release microparticles for the targeted treatment of respiratory diseases.

Delivering drugs through inhalation for systemic and local applications has been in practice since several decades to treat various diseases. In recent times, inhalation drug delivery is becoming one of the highly focused areas of research in the pharmaceutical industry. It is being considered as one of the major portals for delivering drugs because of its wide range of advantages like requirement of low concentrations of drug to reach therapeutic efficacy, surpassing first pass metabolism and a very low incidence of side effects as compared to conventional delivery of drugs. Owing to these favorable characteristics of pulmonary drug delivery, diverse pharmaceutical formulations like liposomes, nanoparticles, and microparticles are developed through consistent efforts for delivery drugs to lungs in suitable form. However, drug-loaded microparticles have displayed various advantages over the other pharmaceutical dosage forms which give a cutting edge over other inhalational drug delivery systems. Assuring results with respect to sustained release through inhalational delivery of drug-loaded microparticles from pre-clinical studies are anticipative of similar benefits in the clinical settings. This review centralizes partly on the advantages of inhalational microparticles over other inhalational dosage forms and largely on the therapeutic applications and future perspectives of inhalable microparticle drug delivery systems.

Protective effects of nanoceria in imiquimod induced psoriasis by inhibiting the inflammatory responses.

Aim: To investigate the effect of cerium oxide nanoparticles (nanoceria) on psoriasis. Materials & methods: Fourier transform infrared, powder x-ray diffraction and scanning electron microscopy were used to characterize nanoceria. Imiquimod (62.5 mg/mice) was used for the induction of psoriasis while nanoceria was administered/applied via multiple routes (topical gel, intraperitoneal and subcutaneous) as a therapeutic intervention once daily. Results: Nanoceria significantly attenuated splenic hypertrophy, psoriasis area severity index scoring, and lipid peroxidation. It also reduced the expression of various inflammatory and proliferation markers such as IL-17, IL-22, IL-23, Ki-67, NF-κB, COX-2 and GSK3. Conclusion: Nanoceria exerts an antipsoriatic effect by inhibiting major pathogenic immune axes namely the Th-cell mediated IL-17/IL-23 axis and by downregulating other crucial inflammatory proteins like NF-κB, COX-2 and GSK3.

Niclosamide alleviates pulmonary fibrosis in vitro and in vivo by attenuation of epithelial-to-mesenchymal transition, matrix proteins & Wnt/ß-catenin signaling: A drug repurposing study.

Drug repurposing off late has been emerging as an inspiring alternative approach to conventional, exhaustive and arduous process of drug discovery. It is a process of identifying new therapeutic values for a drug already established for the treatment of a certain condition. Our current study is aimed at repurposing the old anti-helimenthic drug Niclosamide as an anti-fibrotic drug against pulmonary fibrosis (PF). PF is most common lethal interstitial lung disease hallmarked by deposition of extracelluar matrix and scarring of lung. Heterogenous nature, untimely diagnosis and lack of appropriate treatment options make PF an inexorable lung disorder. Prevailing void in PF treatment and drug repositioning strategy of drugs kindled our interest to demonstrate the anti-fibrotic activity of Niclosamide. Our study is aimed at investigating the anti-fibrotic potential of Niclosamide in TGF-ß1 induced in vitro model of PF and 21-day model of Bleomycin induced PF in vivo respectively. Our study results showed that Niclosamide holds the potential to exert anti-fibrotic effect by hampering fibroblast migration, attenuating EMT, inhibiting fibrotic signaling and by regulating WNT/ß-catenin signaling as evident from protein expression studies. Our study findings can give new directions to development of Niclosamide as an anti-fibrotic agent for treatment of pulmonary fibrosis.

Therapeutic effects of Nimbolide, an autophagy regulator, in ameliorating pulmonary fibrosis through attenuation of TGF-ß1 driven epithelial-to-mesenchymal transition.

Pulmonary fibrosis is an irreversible lung disorder with predictable decline in lung function leading to respiratory insufficiency. Incidence of pulmonary fibrosis has been apparently increasing worldwide. Though aetiology of this disease remains unclear, potential roles of infection, disordered cell biology, genetic influence etc. have been proposed. Pirfenidone and nintedanib are the only two US FDA approved drugs to treat pulmonary fibrosis. Autophagy is a catabolic intracellular pathway that plays a crucial role in maintaining cellular homeostasis, which is involved in many disorders including fibrotic diseases. The present study investigated the role of Nimbolide, an important active constituent of Neem in TGF-ß1 induced in vitro and bleomycin induced in vivo model of pulmonary fibrosis, with a slight emphasis on regulation of fibrosis related autophagy. Protein expression studies showed significant reduction in mesenchymal, fibrotic markers and a substantial up regulation of epithelial markers upon treatment with Nimbolide. Nimbolide regulated autophagy signaling by dampening LC-3 and p-62 expression and increasing Beclin 1 expression as evidenced by immunohistochemistry and confocal microscopy. Our study demonstrates Nimbolide as a potent anti-fibrotic agent and its ability to regulate fibrosis associated autophagy.

Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation.

Acute respiratory distress syndrome (ARDS) is characterized by an excessive acute inflammatory response in lung parenchyma, which ultimately leads to refractory hypoxemia. One of the earliest abnormalities seen in lung injury is the elevated levels of inflammatory cytokines, among them, the soluble tumor necrosis factor (TNF-α) has a key role, which exerts cytotoxicity in epithelial and endothelial cells thus exacerbates edema. The bacterial lipopolysaccharide (LPS) was used both in vitro (RAW 264.7, THP-1, MLE-12, A549, and BEAS-2B) and in vivo (C57BL/6 mice), as it activates a plethora of overlapping inflammatory signaling pathways involved in ARDS. Nimbolide is a chemical constituent of Azadirachta indica, which contains multiple biological properties, while its role in ARDS is elusive. Herein, we have investigated the protective effects of nimbolide in abrogating the complications associated with ARDS. We showed that nimbolide markedly suppressed the nitrosative-oxidative stress, inflammatory cytokines, and chemokines expression by suppressing iNOS, myeloperoxidase, and nitrotyrosine expression. Moreover, nimbolide mitigated the migration of neutrophils and mast cells whilst normalizing the LPS-induced hypothermia. Also, nimbolide modulated the expression of epigenetic regulators with multiple HDAC inhibitory activity by suppressing the nuclear translocation of NF-κB and HDAC-3. We extended our studies using molecular docking studies, which demonstrated a strong interaction between nimbolide and TNF-α. Additionally, we showed that treatment with nimbolide increased GSH, Nrf-2, SOD-1, and HO-1 protein expression; concomitantly abrogated the LPS-triggered TNF-α, p38 MAPK, mTOR, and GSK-3ß protein expression. Collectively, these results indicate that TNF-α-regulated NF-κB and HDAC-3 crosstalk was ameliorated by nimbolide with promising anti-nitrosative, antioxidant, and anti-inflammatory properties in LPS-induced ARDS.