Leishmania spp. amastigotes surrounding sensory nerve fibers in human painless skin ulcers: Evidence of pathogen-neuron proximity and absence of neuronal apoptosis.

In this present study, carried out between November 2020 and July 2023 at Londrina's University Hospital, patients with active lesions of cutaneous leishmaniasis (CL) were analyzed regarding pain perception and anatomopathological aspects of the ulcers. Pain was assessed using a numerical rating scale (NRS) to compare five patients diagnosed with CL with four control patients diagnosed with vascular skin ulcers. Histopathological evaluations were used to investigate the nociceptor neuron-Leishmania interface. Patients with CL ulcers reported less pain compared to patients with vascular ulcers (2.60 ± 2.30 and 7.25 ± 0.95, respectively, p = 0.0072). Histopathology evidenced Leishmania spp. amastigote forms nearby sensory nerve fibers in profound dermis. Schwann cells marker (S100 protein) was detected, and caspase-3 activation was not evidenced in the in the nerve fibers of CL patients' samples, suggesting absence of apoptotic activity in nerve endings. Additionally, samples taken from the active edge of the lesion were negative for bacilli acid-alcohol resistant (BAAR), which excludes concomitant leprosy, in which painless lesions are also observed. Thus, the present data unveil for the first time anatomopathological and microbiological details of painless ulcers in CL patients, which has important clinical implications for a better understanding on the intriguing painless clinical characteristic of CL.

Aspirin-Triggered Resolvin D1 (AT-RvD1) Protects Mouse Skin against UVB-Induced Inflammation and Oxidative Stress.

Intense exposure to UVB radiation incites excessive production of reactive oxygen species (ROS) and inflammation. The resolution of inflammation is an active process orchestrated by a family of lipid molecules that includes AT-RvD1, a specialized proresolving lipid mediator (SPM). AT-RvD1 is derived from omega-3, which presents anti-inflammatory activity and reduces oxidative stress markers. The present work aims to investigate the protective effect of AT-RvD1 on UVB-induced inflammation and oxidative stress in hairless mice. Animals were first treated with 30, 100, and 300 pg/animal AT-RvD1 (i.v.) and then exposed to UVB (4.14 J/cm2). The results showed that 300 pg/animal of AT-RvD1 could restrict skin edema, neutrophil and mast cell infiltration, COX-2 mRNA expression, cytokine release, and MMP-9 activity and restore skin antioxidant capacity as per FRAP and ABTS assays and control O2•- production, lipoperoxidation, epidermal thickening, and sunburn cells development. AT-RvD1 could reverse the UVB-induced downregulation of Nrf2 and its downstream targets GSH, catalase, and NOQ-1. Our results suggest that by upregulating the Nrf2 pathway, AT-RvD1 promotes the expression of ARE genes, restoring the skin's natural antioxidant defense against UVB exposition to avoid oxidative stress, inflammation, and tissue damage.

The Flavonoid Hesperidin Methyl Chalcone Targets Cytokines and Oxidative Stress to Reduce Diclofenac-Induced Acute Renal Injury: Contribution of the Nrf2 Redox-Sensitive Pathway.

Hesperidin is derived from citrus fruits among other plants. Hesperidin was methylated to increase its solubility, generating hesperidin methyl chalcone (HMC), an emerging flavonoid that possess anti-inflammatory and antioxidant properties. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a powerful regulator of cellular resistance to oxidant products. Previous data evidenced HMC can activate Nrf2 signaling, providing antioxidant protection against diverse pathological conditions. However, its effects on kidney damage caused by non-steroidal anti-inflammatory drugs (NSAIDs) have not been evaluated so far. Mice received a nephrotoxic dose of diclofenac (200 mg/kg) orally followed by intra-peritoneal (i.p.) administration of HMC (0.03-3 mg/kg) or vehicle. Plasmatic levels of urea, creatinine, oxidative stress, and cytokines were assessed. Regarding the kidneys, oxidative parameters, cytokine production, kidney swelling, urine NGAL, histopathology, and Nrf2 mRNA expression and downstream targets were evaluated. HMC dose-dependently targeted diclofenac systemic alterations by decreasing urea and creatinine levels, and lipid peroxidation, as well as IL-6, IFN-γ, and IL-33 production, and restored antioxidant properties in plasma samples. In kidney samples, HMC re-established antioxidant defenses, inhibited lipid peroxidation and pro-inflammatory cytokines and upregulated IL-10, reduced kidney swelling, urine NGAL, and histopathological alterations. Additionally, HMC induced mRNA expression of Nrf2 and its downstream effectors HO-1 and Nqo1, as well as reduced the levels of Keap1 protein detected in renal tissue. The present data demonstrate HMC is a potential compound for the treatment of acute renal damage caused by diclofenac, a routinely prescribed non-steroidal anti-inflammatory drug.

Association between IL-10 systemic low level and highest pain score in patients during symptomatic SARS-CoV-2 infection.

BACKGROUND: Despite the wide variety of Covid-19 symptoms, pain and the related mechanisms underlying unsettled nociceptive status are still under-prioritized. Understanding the complex network of Covid-19-related pain may result in new lines of study. It is unknown whether patient's immunological background influences pain in the acute phase of Covid-19, including musculoskeletal pain. Thus, we evaluated the blood levels of selected molecules that are upregulated in SARS-CoV-2 infection and analyzed a possible correlation with pain during Covid-19. METHODS: A cohort of 20 hospitalized patients with confirmed diagnoses for Covid-19 were evaluated in the context of pain. Visual analogic scale (VAS) was applied to quantitate pain level. Blood tests were used to determine the systemic levels of cytokines (IL-10 and IL-1ß), substance P, and leptin. The data were correlated when appropriate to determine the association between pain-related markers and assessed pain intensity. RESULTS: Our findings show that systemic levels of IL-10 have strong negative correlation with pain intensity on Covid-19 patients. Additionally, we also show that leptin systemic levels were increased in Covid-19 patients with pain, however, with moderate positive correlation between these events. IL-1ß and SP levels did not differ between Covid-19 patients with or without pain. Men reported less pain compared to women. No differences were found between genders in the levels of the molecules evaluated in patients with pain. CONCLUSION: IL-10 has been described over the years as an anti-inflammatory and analgesic cytokine. The present data support that low IL-10 levels might contribute to Covid-19-associated pain.

Sphagneticola trilobata (L.) Pruski-derived kaurenoic acid prevents ovalbumin-induced asthma in mice: Effect on Th2 cytokines, STAT6/GATA-3 signaling, NFκB/Nrf2 redox sensitive pathways, and regulatory T cell phenotype markers.

ETHNOPHARMACOLOGICAL RELEVANCE: Sphagneticola trilobata (L.) Pruski is used in traditional medicine in Brazil for inflammatory diseases treatment including asthma. The diterpene kaurenoic acid (KA) is one of its active compounds, but whether KA activity could explain the traditional use of S. trilobata in asthma is unknown. AIM: Investigate KA effect and mechanisms in asthma. METHODS: Experimental asthma was induced by ovalbumin immunization and challenge in male Swiss mice. KA (0.1-10 mg/kg, gavage) was administered 1 h before the ovalbumin challenge. Total leukocytes, eosinophil, and mast cell were counted in bronchoalveolar lavage fluid (BALF), and lung histopathology was performed. Lung mRNA expression of Th2 and regulatory T cells markers, and BALF type 2 cytokine production were quantitated. NFκB activation and oxidative stress-related components in pulmonary tissue were measured. RESULTS: KA inhibited the migration of total leukocytes and eosinophils to BALF, reduced lung histopathology (inflammatory cells and mast cells), mRNA expression of IL-33/ST2, STAT6/GATA-3 and NFκB activation in the lung, and reduced IL-33, IL-4, IL-5 production in the BALF. KA also reduced the mRNA expression of iNOS and gp91phox, and superoxide anion production accompanied by the induction of Nrf2, HO-1 and NQO1 mRNA expression, thus, exerting an antioxidant effect. Finally, KA induced nTreg-like and Tr1-like, but not Th3-like markers of suppressive T cell phenotypes in the lung tissue. CONCLUSION: KA prevents antigen-induced asthma by down-regulating Th2 and NFκB/cytokine-related pathways, and up-regulating Nrf2 and regulatory T cells' markers. Thus, explaining the ethnopharmacological use of S. trilobata for the treatment of lung inflammatory diseases.

The Lipoxin Receptor/FPR2 Agonist BML-111 Protects Mouse Skin Against Ultraviolet B Radiation.

Excessive exposure to UV, especially UVB, is the most important risk factor for skin cancer and premature skin aging. The identification of the specialized pro-resolving lipid mediators (SPMs) challenged the preexisting paradigm of how inflammation ends. Rather than a passive process, the resolution of inflammation relies on the active production of SPMs, such as Lipoxins (Lx), Maresins, protectins, and Resolvins. LXA4 is an SPM that exerts its action through ALX/FPR2 receptor. Stable ALX/FPR2 agonists are required because SPMs can be quickly metabolized within tissues near the site of formation. BML-111 is a commercially available synthetic ALX/FPR2 receptor agonist with analgesic, antioxidant, and anti-inflammatory properties. Based on that, we aimed to determine the effect of BML-111 in a model of UVB-induced skin inflammation in hairless mice. We demonstrated that BML-111 ameliorates the signs of UVB-induced skin inflammation by reducing neutrophil recruitment and mast cell activation. Reduction of these cells by BML-111 led to lower number of sunburn cells formation, decrease in epidermal thickness, collagen degradation, cytokine production (TNF-α, IL-1ß, IL-6, TGF, and IL-10), and oxidative stress (observed by an increase in total antioxidant capacity and Nrf2 signaling pathway), indicating that BML-111 might be a promising drug to treat skin disorders.

[Ru(bpy)2(NO)SO3](PF6), a Nitric Oxide Donating Ruthenium Complex, Reduces Gout Arthritis in Mice.

Monosodium urate crystals (MSU) deposition induces articular inflammation known as gout. This disease is characterized by intense articular inflammation and pain by mechanisms involving the activation of the transcription factor NFκB and inflammasome resulting in the production of cytokines and oxidative stress. Despite evidence that MSU induces iNOS expression, there is no evidence on the effect of nitric oxide (NO) donors in gout. Thus, the present study evaluated the effect of the ruthenium complex donor of NO {[Ru(bpy)2(NO)SO3](PF6)} (complex I) in gout arthritis. Complex I inhibited in a dose-dependent manner MSU-induced hypersensitivity to mechanical stimulation, edema and leukocyte recruitment. These effects were corroborated by a decrease of histological inflammation score and recruitment of Lysm-eGFP+ cells. Mechanistically, complex I inhibited MSU-induced mechanical hypersensitivity and joint edema by triggering the cGMP/PKG/ATP-sensitive K (+) channels signaling pathway. Complex I inhibited MSU-induced oxidative stress and pro-inflammatory cytokine production in the knee joint. These data were supported by the observation that complex I inhibited MSU-induced NFκB activation, and IL-1ß expression and production. Complex I also inhibited MSU-induced activation of pro-IL-1ß processing. Concluding, the present data, to our knowledge, is the first evidence that a NO donating ruthenium complex inhibits MSU-induced articular inflammation and pain. Further, complex I targets the main physiopathological mechanisms of gout arthritis. Therefore, it is envisaged that complex I and other NO donors have therapeutic potential that deserves further investigation.

The citrus flavanone naringenin attenuates zymosan-induced mouse joint inflammation: induction of Nrf2 expression in recruited CD45+ hematopoietic cells.

BACKGROUND: Naringenin is a biologically active analgesic, anti-inflammatory, and antioxidant flavonoid. Naringenin targets in inflammation-induced articular pain remain poorly explored. METHODS: The present study investigated the cellular and molecular mechanisms involved in the analgesic/anti-inflammatory effects of naringenin in zymosan-induced arthritis. Mice were pre-treated orally with naringenin (16.7-150 mg/kg), followed by intra-articular injection of zymosan. Articular mechanical hyperalgesia and oedema, leucocyte recruitment to synovial cavity, histopathology, expression/production of pro- and anti-inflammatory mediators and NFκB activation, inflammasome component expression, and oxidative stress were evaluated. RESULTS: Naringenin inhibited articular pain and oedema in a dose-dependent manner. The dose of 50 mg/kg inhibited leucocyte recruitment, histopathological alterations, NFκB activation, and NFκB-dependent pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-33), and preproET-1 mRNA expression, but increased anti-inflammatory IL-10. Naringenin also inhibited inflammasome upregulation (reduced Nlrp3, ASC, caspase-1, and pro-IL-1ß mRNA expression) and oxidative stress (reduced gp91phox mRNA expression and superoxide anion production, increased GSH levels, induced Nrf2 protein in CD45+ hematopoietic recruited cells, and induced Nrf2 and HO-1 mRNA expression). CONCLUSIONS: Naringenin presents analgesic and anti-inflammatory effects in zymosan-induced arthritis by targeting its main physiopathological mechanisms. These data highlight this flavonoid as an interesting therapeutic compound to treat joint inflammation, deserving additional pre-clinical and clinical studies.

Kaurenoic acid extracted from Sphagneticola trilobata reduces acetaminophen-induced hepatotoxicity through inhibition of oxidative stress and pro-inflammatory cytokine production in mice.

Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, acetaminophen overdose can be fatal. Currently, the only treatment available is the N-acetyl cysteine. The diterpene kaurenoic acid (ent-kaur-16-en-19-oic acid, KA) is the major constituent of Sphagneticola trilobata (L.) Pruski. KA presents anti-inflammatory, anti-nociceptive and antioxidant properties. In this study, we evaluated the efficacy of KA in a model of acetaminophen-induced hepatotoxicity. KA increased, in a dose-dependent manner, the survival rate after acetaminophen overdose. KA reduced acetaminophen-induced hepatic necrosis and ALT and AST levels. KA decreased acetaminophen-induced neutrophil and macrophage recruitment, oxidative stress and the production of IL-33, TNF-α and IL-1ß, alongside with normalisation of IL-10 levels in the liver. Therefore, KA showed preclinical efficacy in acetaminophen-induced hepatotoxicity and lethality.

The Lipid Mediator Resolvin D1 Reduces the Skin Inflammation and Oxidative Stress Induced by UV Irradiation in Hairless Mice.

UV irradiation-induced oxidative stress and inflammation contribute to the development of skin diseases. Therefore, targeting oxidative stress and inflammation might contribute to reduce skin diseases. Resolvin D1 (RvD1) is a bioactive metabolite generated during inflammation to actively orchestrate the resolution of inflammation. However, the therapeutic potential of RvD1 in UVB skin inflammation remains undetermined, which was, therefore, the aim of the present study. The intraperitoneal treatment with RvD1 (3-100 ng/mouse) reduced UVB irradiation-induced skin edema, myeloperoxidase activity, matrix metalloproteinase 9 activity, and reduced glutathione depletion with consistent effects observed with the dose of 30 ng/mouse, which was selected to the following experiments. RvD1 inhibited UVB reduction of catalase activity, and hydroperoxide formation, superoxide anion production, and gp91phox mRNA expression. RvD1 also increased the Nrf2 and its downstream targets NQO1 and HO-1 mRNA expression. Regarding cytokines, RvD1 inhibited UVB-induced production of IL-1ß, IL-6, IL-33, TNF-α, TGF-ß, and IL-10. These immuno-biochemical alterations by RvD1 treatment had as consequence the reduction of UVB-induced epidermal thickness, sunburn and mast cell counts, and collagen degradation. Therefore, RvD1 inhibited UVB-induced skin oxidative stress and inflammation, rendering this resolving lipid mediator as a promising therapeutic agent.

Pyrrolidine dithiocarbamate inhibits mouse acute kidney injury induced by diclofenac by targeting oxidative damage, cytokines and NF-κB activity.

AIMS: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used and effective anti-inflammatories despite the well-known side effects such as gastrointestinal damage, acute kidney injury (AKI), and cardiovascular dysfunctions. Diclofenac is among the most prescribed NSAIDs due to its efficient analgesic and anti-inflammatory properties. Patients using diclofenac possess 77% risk increase to develop AKI. Activation of NF-κB contributes to diclofenac-induced AKI, which is in line with the use of glucocorticoids as one of the management choices to treat AKI patients. MAIN METHODS: In this work, we investigate the efficacy of pyrrolidine dithiocarbamate (PDTC) in diclofenac-induced AKI in mice given it is a known NF-κB inhibitor. KEY FINDINGS: We observed that diclofenac increased proteinuria and urine neutrophil gelatinase-associated lipocalin (NGAL), blood levels of urea, creatinine, oxidative stress, C-reactive protein (CRP), and pro-inflammatory cytokine after 24 h of a bolus administration. In renal tissue, diclofenac also induced morphological changes consistent with kidney damage, modulated cytokine production, increased oxidative stress and reduced antioxidant defenses. These alterations induced by diclofenac were accompanied by activation of NF-κB in the kidney. Treatment with PDTC dose-dependently reduced diclofenac-induced blood urea, creatinine, and oxidative stress. In addition, PDTC reduced proteinuria and urine NGAL levels and blood CRP and pro-inflammatory cytokines. In the kidney, PDTC inhibited diclofenac-induced morphological changes, pro-inflammatory cytokine production, oxidative stress, and NF-κB activation, and increased antioxidant defenses and anti-inflammatory cytokine IL-10. SIGNIFICANCE: Our data demonstrate that PDTC ameliorates diclofenac-induced AKI and that targeting NF-κB signaling pathway is a promising therapeutic approach for the treatment of diclofenac-induced AKI.

Vinpocetine Ameliorates Acetic Acid-Induced Colitis by Inhibiting NF-κB Activation in Mice.

The idiopathic inflammatory bowel diseases (IBD) comprise two types of chronic intestinal disorders: Crohn's disease and ulcerative colitis. Recruited neutrophils and macrophages contribute to intestinal tissue damage via production of ROS and NF-κB-dependent pro-inflammatory cytokines. The introduction of anti-TNF-α therapies in the treatment of IBD patients was a seminal advance. This therapy is often limited by a loss of efficacy due to the development of adaptive immune response, underscoring the need for novel therapies targeting similar pathways. Vinpocetine is a nootropic drug and in addition to its antioxidant effect, it is known to have anti-inflammatory and analgesic properties, partly by inhibition of NF-κB and downstream cytokines. Therefore, the present study evaluated the effect of the vinpocetine in a model of acid acetic-induced colitis in mice. Treatment with vinpocetine reduced edema, MPO activity, microscopic score and macroscopic damage, and visceral mechanical hyperalgesia. Vinpocetine prevented the reduction of colonic levels of GSH, ABTS radical scavenging ability, and normalized levels of anti-inflammatory cytokine IL-10. Moreover, vinpocetine reduced NF-κB activation and thereby NF-κB-dependent pro-inflammatory cytokines IL-1ß, TNF-α, and IL-33 in the colon. Thus, we demonstrate for the first time that vinpocetine has anti-inflammatory, antioxidant, and analgesic effects in a model of acid acetic-induced colitis in mice and deserves further screening to address its suitability as an approach for the treatment of IBD.

trans-Chalcone, a flavonoid precursor, inhibits UV-induced skin inflammation and oxidative stress in mice by targeting NADPH oxidase and cytokine production.

trans-Chalcone is a plant flavonoid precursor, which lacks broad investigation on its biological activity in inflammatory processes. In the present study, anti-inflammatory and antioxidant mechanisms of systemic administration with trans-chalcone, a flavonoid precursor, on ultraviolet (UV) irradiation-induced skin inflammation and oxidative stress in hairless mice were investigated by the following parameters: skin edema, myeloperoxidase activity (neutrophil marker), matrix metalloproteinase-9 activity, reduced glutathione levels, catalase activity, lipid peroxidation products, superoxide anion production, gp91phox (NADPH oxidase subunit) mRNA expression by quantitative PCR and cytokine production by ELISA. Systemic treatment with trans-chalcone inhibited skin inflammation by reducing skin edema and neutrophil recruitment, and also inhibited matrix metalloproteinase-9 activity. trans-Chalcone also inhibited oxidative stress, gp91phox mRNA expression, and the production of a wide range of pro-inflammatory cytokines, while it did not affect anti-inflammatory cytokines induced by UV irradiation. However, trans-chalcone did not prevent oxidative stress in vitro, suggesting that its in vivo effect is more related to anti-inflammatory properties rather than a direct antioxidant effect. In conclusion, treatment with trans-chalcone inhibited UV-induced skin inflammation resulting in oxidative stress inhibition in vivo. Therefore, systemic supplementation with this compound may represent an important therapeutic approach in inflammatory skin diseases induced by UV irradiation.

Vinpocetine reduces diclofenac-induced acute kidney injury through inhibition of oxidative stress, apoptosis, cytokine production, and NF-κB activation in mice.

Acute kidney injury (AKI) represents a complex clinical condition associated with significant morbidity and mortality. Approximately, 19-33% AKI episodes in hospitalized patients are related to drug-induced nephrotoxicity. Although, considered safe, non-steroidal anti-inflammatory drugs such as diclofenac have received special attention in the past years due to the potential risk of renal damage. Vinpocetine is a nootropic drug known to have anti-inflammatory properties. In this study, we investigated the effect and mechanisms of vinpocetine in a model of diclofenac-induced AKI. We observed that diclofenac increased proteinuria and blood urea, creatinine, and oxidative stress levels 24h after its administration. In renal tissue, diclofenac also increased oxidative stress and induced morphological changes consistent with renal damage. Moreover, diclofenac induced kidney cells apoptosis, up-regulated proinflammatory cytokines, and induced the activation of NF-κB in renal tissue. On the other hand, vinpocetine reduced diclofenac-induced blood urea and creatinine. In the kidneys, vinpocetine inhibited diclofenac-induced oxidative stress, morphological changes, apoptosis, cytokine production, and NF-κB activation. To our knowledge, this is the first study demonstrating that diclofenac-induced AKI increases NF-κB activation, and that vinpocetine reduces the nephrotoxic effects of diclofenac. Therefore, vinpocetine is a promising molecule for the treatment of diclofenac-induced AKI.

Quercetin Inhibits Peripheral and Spinal Cord Nociceptive Mechanisms to Reduce Intense Acute Swimming-Induced Muscle Pain in Mice.

The present study aimed to evaluate the effects of the flavonoid quercetin (3,3´,4´,5,7-pentahydroxyflavone) in a mice model of intense acute swimming-induced muscle pain, which resembles delayed onset muscle soreness. Quercetin intraperitoneal (i.p.) treatment dose-dependently reduced muscle mechanical hyperalgesia. Quercetin inhibited myeloperoxidase (MPO) and N-acetyl-ß-D- glucosaminidase (NAG) activities, cytokine production, oxidative stress, cyclooxygenase-2 (COX-2) and gp91phox mRNA expression and muscle injury (creatinine kinase [CK] blood levels and myoblast determination protein [MyoD] mRNA expression) as well as inhibited NFκB activation and induced Nrf2 and HO-1 mRNA expression in the soleus muscle. Beyond inhibiting those peripheral effects, quercetin also inhibited spinal cord cytokine production, oxidative stress and glial cells activation (glial fibrillary acidic protein [GFAP] and ionized calcium-binding adapter molecule 1 [Iba-1] mRNA expression). Concluding, the present data demonstrate that quercetin is a potential molecule for the treatment of muscle pain conditions related to unaccustomed exercise.