Multifacted Role of TNF-α During the Pathogenesis of Rheumatoid Arthritis

Tumor necrosis factor alpha (TNF-α) a cytokine has been shown to be the key player during the pathogenesis of several autoimmune inflammatory disorders (presumably sterile inflammation) including rheumatoid arthritis (RA). Several studies have shown that TNF-α is mainly involved in the proinflammatory responses. However recent studies have reported multifunctional role of TNF-α during the development of RA. Therefore, in this article we have highlighted the distinct functions of TNF-α during pathogenesis of R

Novel Anti-Obesity Effects of Beer Hops Compound Xanthohumol: Role of AMPK Signaling Pathway

Background: Obesity alters adipose tissue metabolic and endocrine functioning, leading to an increased adiposity and release of pro-inflammatory cytokines. Various phytochemicals have been reported to contribute to the beiging of white adipose tissue in order to ameliorate obesity by increasing thermogenesis. Here, we show that the prenylated chalcone, xanthohumol (XN), induces beiging of white adipocytes, stimulates lipolysis, and inhibits adipogenesis of murine 3T3-L1 adipocytes and primary human subcutaneous preadipocytes and these effects are partly mediated by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. Methods: 3T3-L1 adipocytes and primary human subcutaneous preadipocytes were differentiated using a standard protocol and were treated with various concentrations of XN, dorsomorphin, an AMPK inhibitor, or AICAR, an AMPK activator, to investigate the effects on adipogenesis, beiging and lipolysis. Results: XN induced beiging of white adipocytes as witnessed by the increased expression of beige markers CIDE-A and TBX-1. XN increased mitochondrial biogenesis, as evidenced by increased mitochondrial content, enhanced expression of PGC-1α, and the thermogenic protein UCP1. Following 24 h of treatment, XN also increased oxygen consumption rate. XN stimulated lipolysis of mature 3T3-L1 and primary human subcutaneous adipocytes and inhibited adipogenesis of maturing adipocytes. XN activated AMPK and in turn, XN-induced upregulation of UCP1, p-ACC, HSL, and ATGL was downregulated in the presence of dorsomorphin. Likewise, an XN-induced decrease in adipogenesis was reversed in the presence of dorsomorphin. Conclusions: Taken together, XN demonstrates anti-obesity effects by not only inducing beiging but also decreasing adipogenesis and inducing lipolysis. The anti-obesity effects of XN are partly mediated by AMPK signaling pathway suggesting that XN may have potential therapeutic implications for obesity

Xanthohumol Stimulates the Secretion of Catecholamines and Induces M2 Polarization in Raw 264.7 Macrophages

Anti-inflammatory, anti-oxidant, and anti-cancer effects of xanthohumol (XN), a prenylated chalcone extracted from common hop plants, are gaining attention and research has been expanding on the beneficial effects of this compound. In this study, we have investigated the anti-inflammatory effects of XN using a mouse monocytic cell line, RAW 264.7. We hypothesized that the anti-inflammatory effects of XN are due to M2 polarization of macrophages which, in turn, is mediated partly through the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. RAW 264.7 cells were treated with either 0.1% DMSO or XN at varying concentrations for 24hrs. Cell culture supernatant was collected for ELISA and whole cell lysates were collected for Western blotting experiments. Our results suggest that XN upregulated the secretion of interleukin 10 (IL10), a signature cytokine for M2 polarization, in RAW 264.7 cells after 24 hours. We further demonstrated that XN increased arginase expression, a marker for M2 polarization, and failed to increase inducible nitric oxide synthase (iNOS) expression, a marker for M1 polarization. XN decreased interferon-γ (IFNγ) induced elevation of nitrite release, indicating the inhibitory effects of XN against M1 polarization. Additionally, XN at 25µM increased the secretion of catecholamines from macrophages comparable to interleukin 4 (IL4), an inducer of the M2 phenotype. Finally, XN upregulated the expression of phospho-AMPK in RAW 264.7 cells, indicating the role of AMPK signaling pathway in XN-induced effects. These results provide evidence for the anti-inflammatory properties of XN–mediated induction of M2 polarization. The M2 macrophage mediated anti-inflammatory effects, coupled with catecholamine secretion, and previously anti-adipogenic effects, makes XN an attractive molecule to study its beneficial effects on metabolic disease, like obesity and diabetes, that are associated with underlying chronic, low-grade inflammation

Direct and Indirect Effects of Guggulsterone on the Induction of Beiging in Mature 3T3-L1 Adipocytes

Phytochemicals have long demonstrated anti-obesity properties in adipocytes. Their ability, however, to induce browning in white adipose tissue is only beginning to emerge. We have recently established that the white adipocyte cell line, 3T3-L1, is capable of beiging under beta-adrenergic conditions. Using this information, we sought to investigate if the plant steroid guggulsterone (GS) can induce beiging in 3T3-L1s. 3T3-L1 preadipocytes were differentiated using established protocols supplemented with rosiglitazone and thyroid hormone. Direct effects of GS were measured by treating mature 3T3-L1s for 24 hours. Indirect effects were measured by treating mature 3T3-L1s with conditioned media from GS-treated RAW 264.7 macrophages. Direct treatment of 3T3-L1s with GS resulted in increased lipolysis, increased mitochondrial activity (11%), and increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) levels by 250% more than control. 3T3-L1s also demonstrated an increase in uncoupling protein 1 (UCP1) expression by 200% and beige marker, T-box protein 1 (TBX1) expression by 80% more than control. Furthermore, this was accompanied by increased levels of G protein-coupled bile acid receptor (TGR5) and its downstream target iodothyronine deiodinase 2 (DIO2). Treatment of RAW 264.7 macrophages with GS induced a 60% increase in catecholamine release into the media compared to control. Using this conditioned media from macrophages, 3T3-L1 adipocytes increased the expression of DIO2 and UCP1 following 24 hours of incubation. Results from this study demonstrate that GS can potentially induce beiging in white adipose tissue through two distinct mechanisms: (1) direct signaling through the TGR5-cAMP-DIO2 pathway and (2) indirectly through stimulating catecholamine release in macrophages. Thus, it is reasonable to conclude that GS may improve the metabolic capacity of adipose tissue thereby counteracting the effects of obesity

Lipocalin-2 deficiency may predispose to the progression of spontaneous age-related adiposity in mice

Lipocalin-2 (Lcn2) is an innate immune protein elevated by several orders of magnitude in various inflammatory conditions including aging and obesity. Recent studies have shown that Lcn2 is secreted by adipocytes in response to inflammation and is categorized as a new adipokine cross-linking innate immunity and metabolic disorders including obesity. However, the involvement of Lcn2 and its function during the progression of obesity is largely unknown. Recently, browning of white adipose tissue (WAT) has gained attention as a therapeutic strategy to combat obesity. Herein, we have shown that treatment of mature 3T3-L1 adipocytes with recombinant Lcn2 (rec-Lcn2) resulted in the up-regulation of thermogenic and beige/brown markers (UCP1, PRDM16, ZIC-1 and TBX1) and increased mitochondrial activity. Additionally, global Lcn2 genetic knockout (Lcn2KO) mice exhibited accelerated weight gain and visceral fat deposition with age, when compared to wild type (WT) mice. Taken together, both in vitro and in vivo studies suggest that Lcn2 is a naturally occurring adipokine, and may serve as an anti-obesity agent by upregulating the thermogenic markers resulting in the browning of WAT. Therefore, Lcn2 and its downstream signaling pathways could be a potential therapeutic target for obesity

Inhibitory effect of tanshinone derivatives against SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) enzyme

The lingering consequences of the COVID-19 pandemic continue to manifest as persistent effects on recovering patients. Recent research has identified RNA-dependent RNA polymerase (RdRp), 3-chymotrypsin-like protease (3CLpro), and papain-like protease (PLpro) as crucial components for SARS-CoV-2 replication and the regulation of the host\u27s immune response. Specifically, 3CLpro plays a pivotal role by cleaving 11 non-structural protein (NSPs) sites on the polyprotein of coronaviruses. Consequently, 3CLpro is an attractive target for potential antiviral drug development, with the potential to inhibit viral RNA production in SARS-CoV-2. Our study examined the inhibitory effects of 21 tanshinone derivatives on the SARS-CoV-2 3CLpro enzyme. Among these 21 compounds, seven tanshinone derivatives - namely 3alpha-hydroxytanshinone IIA, dihydroisotanshinone, dihydrotanshinone, 1,2-dihydrocryptotanshinone, 1,2 dihydrocryptotanshinone IIA, Dihydrotanshinone II, and tanshinone IIA - demonstrated an inhibitory effect on 3CLpro exceeding 50% at a concentration of 50uM. Moreover, our dose-response studies revealed IC50 values for these compounds ranging from 0.663 μM to 11.97 μM. Our data suggest that these seven tanshinone derivatives may be used to inhibit the replicative activity of the 3CLpro. We are actively investigating these derivatives\u27 inhibition mechanisms and cytotoxicity in vitro. Once evaluated, the pre-clinical studies must validate these derivatives\u27 therapeutic potential and safety before considering them for clinical trials

Inhibition of SARS-CoV-2 papain-like protease by bioactive compounds found in Salvia miltiorrhiza

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, remains a substantial global public health threat. Despite widespread vaccination efforts, reinfection rates are rising, necessitating a comprehensive examination of vaccine effectiveness against emerging variants. There is an immediate and pressing need to identify potential therapeutic interventions for combating resistant COVID-19 infections. Research has highlighted the significance of nonstructural proteins, including proteases and polymerases, in viral replication, making them attractive targets for drug development. Among these, the Papain-like protease (PLpro), 3-chymotrypsin-like protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) enzymes play pivotal roles in SARS-CoV-2 replication and are promising candidates for drug targeting. Currently, two antiviral drugs, LagevrioTM (an RdRp inhibitor) and Paxlovid® (a 3CLPro inhibitor), are available for COVID-19 treatment. However, the market lacks drugs targeting the PLP enzyme, which is essential for viral replication and suppression of the host\u27s antiviral immune response. Additionally, SARS-CoV-2 PLpro exhibits deubiquitination (DUB) activity. The DUB activity of SARS-CoV-2 PLpro disrupts the host\u27s antiviral immune response. Hence, our objective is to assess derivatives from Salvia miltiorrhiza against the SARS-CoV-2 PLpro enzyme, evaluating both its proteolytic and DUB activities. In this study, we tested twenty-one derivatives from Salvia miltiorrhiza against the PLpro enzyme, and among them, 19 were found to inhibit more than 50% of the proteolytic activity of SARS-CoV-2 PLpro at a concentration of 50uM. Furthermore, dose-response experiments revealed that the IC50 values of these 19 derivatives against PLpro proteolytic activity ranged from 0.25 to 31.02uM. We are actively investigating the inhibition of Salvia miltiorrhiza derivatives against DUB activity of PLpro. Additionally, we will evaluate the inhibition kinetics of these compounds against both proteolytic and DUB activities of PLpro, along with conducting in vitro cytotoxicity assessments. Our data suggest that the Salvia miltiorrhiza derivatives inhibit the SARS-CoV-2 viral replicative activity of the PLpro enzyme. However, pre-clinical studies need to validate the therapeutic potential and safety of these antibiotics before being considered for clinical trials

Effects of Anchor Structure and Glycosylation of Fcγ Receptor III on Ligand Binding Affinity

Isoforms of the Fcγ receptor III (FcγRIII or CD16) are cell surface receptors for the Fc portion of IgG and important regulators of humoral immune responses. Different ligand binding kinetics of FcγRIII isoforms are obtained in three dimensions by surface plasmon resonance and in two dimensions by a micropipette adhesion frequency assay. We show that the anchor structure of CD16 isoforms isolated from the cell membrane affects their binding affinities in a ligand-specific manner. Changing the receptor anchor structure from full to partial to none decreases the ligand binding affinity for human IgG1 (hIgG1) but increases it for murine IgG2a (mIgG2a). Removing N-glycosylation from the CD16 protein core by tunicamycin also increases the ligand binding affinity. Molecular dynamics simulations indicate that deglycosylation at Asn-163 of CD16 removes the steric hindrance for the CD16-hIgG1 Fc binding and thus increases the binding affinity. These results highlight an unexpected sensitivity of ligand binding to the receptor anchor structure and glycosylation and suggest their respective roles in controlling allosterically the conformation of the ligand binding pocket of CD16

Guggulsterone Activates Adipocyte Beiging through Direct Effects on 3T3-L1 Adipocytes and Indirect Effects Mediated through RAW264.7 Macrophages.

Background: Plant-derived phytochemicals have been of emerging interest as anti-obesity compounds due to their apparent effects on promoting reduced lipid accumulation in adipocytes. Despite such promising evidence, little is known about the potential mechanisms behind their anti-obesity effects. The aim of this study is to establish potential anti-obesity effects of the phytochemical guggulsterone (GS). Methods: Mature 3T3-L1 adipocytes were treated with GS, derived from the guggul plant native in northern India, to investigate its effects on mitochondrial biogenesis and adipocyte beiging. Further, to explore the relationship between macrophages and adipocytes, 3T3-L1s were treated with conditioned media from GS-treated RAW264.7 macrophages. Markers of mitochondrial biogenesis and beiging were measured by western blot. Results: GS treatment in adipocytes resulted in increased mitochondrial density, biogenesis (PGC1α and PPARγ), and increased markers of a beige adipocyte phenotype (UCP1, TBX1, and β-3AR). This upregulation in mitochondrial expression was accompanied by increases oxygen consumption. In GS-treated macrophages, markers of M2 polarization were elevated (e.g., arginase and IL-10), along with increased catecholamine release into the media. Lastly, 3T3-L1 adipocytes treated with conditioned media from macrophages induced a 167.8% increase in UCP1 expression, suggestive of a role of macrophages in eliciting an anti-adipogenic response to GS. Conclusions: Results from this study provide the first mechanistic understanding of the anti-obesity effects of GS and suggests a role for both direct GS-signaling and indirect stimulation of M2 macrophage polarization in this model