Despite having been formally defined over 150 years ago, the etiology of multiple sclerosis (MS) is still relatively unknown. However, it is now recognized as a multifactorial disease in which genetics, infection, immune function, and environment play a role. We propose an additional piece to the puzzle: milk. In this review, milk is highlighted as a potential risk factor for MS. We examine the overall correlation between bovine milk consumption and the incidence of MS. We then discuss possible mechanisms that may explain the positive association between milk consumption and the development of MS. For instance, butyrophilin (BTN), a milk glycoprotein, can provide molecular mimicry of myelin oligodendrocyte glycoprotein and induce an autoinflammatory response against myelin. Other milk components such as casein, gangliosides, xanthine oxidase, and saturated fats are also analyzed for their potential involvement in the pathophysiology of MS. Finally, we fit milk alongside other well known risk factors of MS: vitamin D levels, Epstein Barr virus infection, and gut dysbiosis. In conclusion, this review summarizes potential mechanisms linking milk as an underappreciated potential risk factor for the development of MS.

Metformin is an orally effective anti-hyperglycemic drug that despite being introduced over 60 years ago is still utilized by an estimated 120 to 150 million people worldwide for the treatment of type 2 diabetes (T2D). Metformin is used off-label for the treatment of polycystic ovary syndrome (PCOS) and for pre-diabetes and weight loss. Metformin is a safe, inexpensive drug with side effects mostly limited to gastrointestinal issues. Prospective clinical data from the United Kingdom Prospective Diabetes Study (UKPDS), completed in 1998, demonstrated that metformin not only has excellent therapeutic efficacy as an anti-diabetes drug but also that good glycemic control reduced the risk of micro- and macro-vascular complications, especially in obese patients and thereby reduced the risk of diabetes-associated cardiovascular disease (CVD). Based on a long history of clinical use and an excellent safety record metformin has been investigated to be repurposed for numerous other diseases including as an anti-aging agent, Alzheimer's disease and other dementias, cancer, COVID-19 and also atrial fibrillation (AF). AF is the most frequently diagnosed cardiac arrythmia and its prevalence is increasing globally as the population ages. The argument for repurposing metformin for AF is based on a combination of retrospective clinical data and in vivo and in vitro pre-clinical laboratory studies. In this review, we critically evaluate the evidence that metformin has cardioprotective actions and assess whether the clinical and pre-clinical evidence support the use of metformin to reduce the risk and treat AF.

Proteinase-activated receptor-2 (PAR2), which modulates inflammatory responses, is elevated in the central nervous system in multiple sclerosis (MS) and in its murine model, experimental autoimmune encephalomyelitis (EAE). In PAR2-null mice, disease severity of EAE is markedly diminished. We therefore tested whether inhibiting PAR2 activation in vivo might be a viable strategy for the treatment of MS. Using the EAE model, we show that a PAR2 antagonist, the pepducin palmitoyl-RSSAMDENSEKKRKSAIK-amide (P2pal-18S), attenuates EAE progression by affecting immune cell function. P2pal-18S treatment markedly diminishes disease severity and reduces demyelination, as well as the infiltration of T-cells and macrophages into the central nervous system. Moreover, P2pal-18S decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) production and T-cell activation in cultured splenocytes and prevents macrophage polarization in vitro. We conclude that PAR2 plays a key role in regulating neuroinflammation in EAE and that PAR2 antagonists represent promising therapeutic agents for treating MS and other neuroinflammatory diseases. SIGNIFICANCE STATEMENT: Proteinase-activated receptor-2 modulates inflammatory responses and is increased in multiple sclerosis lesions. We show that the proteinase-activated receptor-2 antagonist palmitoyl-RSSAMDENSEKKRKSAIK-amide reduces disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis by inhibiting T-cell and macrophage activation and infiltration into the central nervous system, making it a potential treatment for multiple sclerosis.

ABSTRACT Digital dermatitis (DD) is a skin disease in cattle characterized by painful inflammatory ulcerative lesions in the feet, mostly associated with local colonization by Treponema spp., including Treponema phagedenis . The reason why most DD lesions remain actively inflamed and progress to chronic conditions despite antibiotic treatment remains unknown. Herein, we show an abundant infiltration of proinflammatory (CD14 high CD16 low ) monocytes/macrophages in active DD lesions, a skin response that was not mitigated by topical treatment with oxytetracycline. The associated bacterium, T. phagedenis , isolated from DD lesions in cattle, when injected subcutaneously into mice, induced abscesses with a local recruitment of Ly6G + neutrophils and proinflammatory (Ly6C high CCR2 + ) monocytes/macrophages, which appeared at infection onset (4 days post challenge) and persisted for at least 7 days post challenge. When exploring the ability of macrophages to regulate inflammation, we showed that bovine blood-derived macrophages challenged with live T. phagedenis or its structural components secreted IL-1β via a mechanism dependent on the NLRP3 inflammasome. This study shows that proinflammatory characteristics of monocytes/macrophages and neutrophils dominate active non-healing ulcerative lesions in active DD, thus likely impeding wound healing after antibiotic treatment.

Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally-tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Biocompatible polymeric nanoparticles (NPs) as carriers for therapeutic agents with multifunctional activities have received unprecedented attention for a variety of bio-pharmaceutical applications. We describe the synthesis, the fluorescence properties, the bio-compatible nature and the alpha-1 adrenergic receptor bio-activity of engineered quantum dot-like polynorepinephrine (PNE) NPs. The spherical PNE NPs, which are internalized in smooth muscle cells via a receptor-selective mechanism, activate alpha-1-adrenoceptors in intact mouse aorta and aorta-derived cultured smooth muscle cells, leading to the activation of calcium signaling/contraction and stimulation of mitogen-activated protein kinase (MAPK), thereby displaying receptor-triggering biological activity, possibly acting both extracellularly and intracellularly. Our data indicate that NPs generated by the polymerization of pharmacologically active compounds like norepinephrine can retain receptor-selective biological activity coupled to inherent fluorescence.

Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2-biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE-independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non-protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen-presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease-activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro-Th2 cytokines (IL-6, IL-25, IL-1β, TSLP) and danger-associated molecular patterns (DAMPs; IL-33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL-33 to produce a super-active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at . G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Cathelicidin peptides secreted by leukocytes and epithelial cells are microbicidal but also regulate pathogen sensing via toll-like receptors (TLRs) in the colon by mechanisms that are not fully understood. Herein, analyses with the attaching/effacing pathogen Citrobacter rodentium model of colitis in cathelicidin-deficient (Camp−/−) mice, and colonic epithelia demonstrate that cathelicidins prevent apoptosis by sustaining post-transcriptional synthesis of a TLR adapter, toll-interacting protein (TOLLIP). Cathelicidins induced phosphorylation-activation of epidermal growth factor receptor (EGFR)-kinase, which phosphorylated-inactivated miRNA-activating enzyme Argonaute 2 (AGO2), thus reducing availability of the TOLLIP repressor miRNA-31. Cathelicidins promoted stability of TOLLIP protein via a proteosome-dependent pathway. This cathelicidin-induced TOLLIP upregulation prevented apoptosis in the colonic epithelium by reducing levels of caspase-3 and poly (ADP-ribose) polymerase (PARP)-1 in response to the proinflammatory cytokines, interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Further, Camp−/− colonic epithelial cells were more susceptible to apoptosis during C. rodentium infection than wild-type cells. This antiapoptotic effect of cathelicidins, maintaining epithelial TOLLIP protein in the gut, provides insight into cathelicidin’s ability to regulate TLR signaling and prevent exacerbated inflammation.

Proteinase-activated receptors (PARs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Proteinase-activated Receptors [39]) are unique members of the GPCR superfamily activated by proteolytic cleavage of their amino terminal exodomains. Agonist proteinase-induced hydrolysis unmasks a tethered ligand (TL) at the exposed amino terminus, which acts intramolecularly at the binding site in the body of the receptor to effect transmembrane signalling. TL sequences at human PAR1-4 are SFLLRN-NH2, SLIGKV-NH2, TFRGAP-NH2 and GYPGQV-NH2, respectively. With the exception of PAR3, synthetic peptides with these sequences (as carboxyl terminal amides) are able to act as agonists at their respective receptors. Several proteinases, including neutrophil elastase, cathepsin G and chymotrypsin can have inhibitory effects at PAR1 and PAR2 such that they cleave the exodomain of the receptor without inducing activation of Gαq-coupled calcium signalling, thereby preventing activation by activating proteinases but not by agonist peptides. Neutrophil elastase (NE) cleavage of PAR1 and PAR2 can however activate MAP kinase signaling by exposing a TL that is different from the one revealed by trypsin [87]. PAR2 activation by NE regulates inflammation and pain responses [115, 76] and triggers mucin secretion from airway epithelial cells [116].

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.

(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (−)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.

Protease‐activated receptor‐1 & ‐2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca 2+ signaling, but the mechanisms are incompletely understood. By using single‐cell Ca 2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR‐mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca 2+ waves that propagated across the endothelium. The PAR2‐evoked Ca 2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C‐coupled receptor that evokes Ca 2+ release from the IP 3 ‐sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium‐dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca 2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP 3 ‐mediated Ca 2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

Introduction Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Methods and Results Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10-expressing plasmids inhibited atherosclerosis in ApoE-/- mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10’s anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. Conclusion KLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Our results show that low concentrations of metformin (</=100 μM), in the presence of wild-type but not PPGG mutant NR4A1 which does not enable metformin interactions at the NR4A1 alternative ligand binding site, can both block TGF- β signaling and reduce cell-secreted MMP activity that in turn can drive tissue invasion and metastasis via PAR2 activation. Thus, NR4A1 may play a role in the antitumour actions of metformin.

To develop new degrader molecules from an existing protein ligand a linkage vector must be identified and then joined with a suitable E3 ligase without disrupting binding to the respective targets. This is typically achieved through empirically evaluating the degradation efficacy of a series of synthetic degraders. Our strategy for determining optimal linkage sites utilises biotinylated protein ligands, linked via potential conjugation sites of an inhibitor to confirm whether target protein is maintained after forming a conjugate. This method provides low-cost, qualitative evidence that the addition of a linker moiety at a specific position can be tolerated, guiding further optimisation. We demonstrate the application of this method through the exploration of linkage vectors on A-485, a known ligand of p300/CBP, and found a conjugation site through a urea moiety. Pomalidomide was then conjugated through this site with several different linkers and cell viability and degradation were assessed for this library using a myeloma cell line, MM1.S. Compound 18i, with a PEG4 linker, was found to be the most effective p300 degrader and linker length greater than 10 atoms afforded enhanced degradation.

The coronavirus disease 2019 (COVID-19) pandemic, causing considerable mortality and morbidity worldwide, has fully engaged the biomedical community in attempts to elucidate the pathophysiology of COVID-19 and develop robust therapeutic strategies. To this end, the predominant research focus has been on the adaptive immune response to COVID-19 infections stimulated by mRNA and protein vaccines and on the duration and persistence of immune protection. In contrast, the role of the innate immune response to the viral challenge has been underrepresented. This overview focuses on the innate immune response to COVID-19 infection, with an emphasis on the roles of extracellular proteases in the tissue microenvironment. Proteinase-mediated signaling caused by enzymes in the extracellular microenvironment occurs upstream of the increased production of inflammatory cytokines that mediate COVID-19 pathology. These enzymes include the coagulation cascade, kinin-generating plasma kallikrein, and the complement system, as well as angiotensin-generating proteinases of the renin-angiotensin system. Furthermore, in the context of several articles in this Supplement elucidating and detailing the trajectory of diverse profibrotic pathways, we extrapolate these insights to explore how fibrosis and profibrotic pathways participate importantly in the pathogenesis of COVID-19. We propose that the lessons garnered from understanding the roles of microenvironment proteinases in triggering the innate immune response to COVID-19 pathology will identify potential therapeutic targets and inform approaches to the clinical management of COVID-19. Furthermore, the information may also provide a template for understanding the determinants of COVID-19-induced tissue fibrosis that may follow resolution of acute infection (so-called "long COVID"), which represents a major new challenge to our healthcare systems.

Giardia duodenalis cysteine proteases have been identified as key virulence factors and have been implicated in alterations to intestinal goblet cell activity and mucus production during Giardia infection. The present findings demonstrate a novel mechanism by which Giardia cysteine proteases modulate goblet cell activity via cleavage and activation of protease-activated receptor 2. Giardia duodenalis (assemblage A) increased MUC2 mucin gene expression in human colonic epithelial cells in a manner dependent upon both protease-activated receptor 2 activation and Giardia cysteine protease activity. Protease-activated receptor 2 cleavage within the N-terminal activation domain by Giardia proteases was confirmed using a nano-luciferase tagged recombinant protease-activated receptor 2. In keeping with these observations, the synthetic protease-activated receptor 2-activating peptide 2fLIGRLO-amide increased Muc2 gene expression in a time-dependent manner. Calcium chelation and inhibition of the ERK1/2 mitogen activated protein kinase pathway inhibited Muc2 upregulation during Giardia infection, consistent with canonical protease-activated receptor 2 signaling pathways. Giardia cysteine proteases cleaved both recombinant protease-activated receptor 1 and protease-activated receptor 2 within their extracellular activation domains with isolate-dependent efficiency that correlated with the production of cysteine protease activity. Protease-activated receptors represent a novel target for Giardia cysteine proteases, and these findings demonstrate that protease-activated receptor 2 can regulate mucin gene expression in intestinal goblet cells.

Metformin was first used to treat type 2 diabetes in the late 1950s and in 2022 remains the first-choice drug used daily by approximately 150 million people. An accumulation of positive pre-clinical and clinical data has stimulated interest in re-purposing metformin to treat a variety of diseases including COVID-19. In polycystic ovary syndrome metformin improves insulin sensitivity. In type 1 diabetes metformin may help reduce the insulin dose. Meta-analysis and data from pre-clinical and clinical studies link metformin to a reduction in the incidence of cancer. Clinical trials, including MILES (Metformin In Longevity Study), and TAME (Targeting Aging with Metformin), have been designed to determine if metformin can offset aging and extend lifespan. Pre-clinical and clinical data suggest that metformin, via suppression of pro-inflammatory pathways, protection of mitochondria and vascular function, and direct actions on neuronal stem cells, may protect against neurodegenerative diseases. Metformin has also been studied for its anti-bacterial, -viral, -malaria efficacy. Collectively, these data raise the question: Is metformin a drug for all diseases? It remains unclear as to whether all of these putative beneficial effects are secondary to its actions as an anti-hyperglycemic and insulin-sensitizing drug, or result from other cellular actions, including inhibition of mTOR (mammalian target for rapamycin), or direct anti-viral actions. Clarification is also sought as to whether data from ex vivo studies based on the use of high concentrations of metformin can be translated into clinical benefits, or whether they reflect a 'Paracelsus' effect. The environmental impact of metformin, a drug with no known metabolites, is another emerging issue that has been linked to endocrine disruption in fish, and extensive use in T2D has also raised concerns over effects on human reproduction. The objectives for this review are to: 1) evaluate the putative mechanism(s) of action of metformin; 2) analyze the controversial evidence for metformin's effectiveness in the treatment of diseases other than type 2 diabetes; 3) assess the reproducibility of the data, and finally 4) reach an informed conclusion as to whether metformin is a drug for all diseases and reasons. We conclude that the primary clinical benefits of metformin result from its insulin-sensitizing and antihyperglycaemic effects that secondarily contribute to a reduced risk of a number of diseases and thereby enhancing healthspan. However, benefits like improving vascular endothelial function that are independent of effects on glucose homeostasis add to metformin's therapeutic actions.

Objectives: Dihydromyricetin (DHM), a natural flavonoid isolated from vine tea with anti-inflammatory activity was evaluated for its ability to prevent vascular endothelial dysfunction caused by hyperglycaemia. Methods: Vasoconstrictor (phenylephrine-PE) and vasodilator (acetylcholine-ACh) responses were monitored for female rat aorta rings maintained in a bioassay organ bath for 3 hat 37 degrees C in either low (LG: 10 mM) or high (HG: 40 mM, to mimic hyperglycaemia) glucose-Krebs buffer in the absence or presence of 50 mu M DHM. Tissues recovered from the organ bath at 3 h were fixed and analyzed for morphological changes and their expression of eNOS, iNOS, HIF-1 alpha, GLUT1, ROR2 tyrosine kinase, NF-kappa B, TNF-alpha, Bax, Bcl2, caspase-3, and forindices of increased oxidative stress. Key Findings: HG-incubated tissues showed increased PE-stimulated contractile response and decreased ACh-mediated endothelial vasodilation. DHM prevented both of these changes. Besides, HG incubation increased the immunoreactivity to iNOS, HIF-1 alpha, GLUT1, ROR2, NE-kappa B, TNF-alpha, Bax, and active caspase-3, and decreased the expression of eNOS and Bcl2. Hyperglycaemia-like conditions also increased the indices of oxidative/nitrosative stress. These HG-induced changes, which were accompanied by an increase in tissue adventitial thickness and inflammatory cell infiltration, were all prevented by DHM. Conclusion: Our data demonstrate an anti-inflammatory protective action of DHM to preserve vascular function in the setting of hyperglycaemia.