Chemopreventive and immunomodulatory effects of phenolic-rich extract of Commiphora leptophloeos against inflammatory bowel disease: Preclinical evidence.

ETHNOPHARMACOLOGY RELEVANCE: Commiphora leptophloeos (Mart.) J.B. Gillet (Burseraceae) is a medicinal plant native to Brazil, popularly known as "imburana". Homemade leaf decoction and maceration were used to treat general inflammatory problems in the Brazilian Northeast population. Our previous research confirmed the anti-inflammatory activity of the C. leptophloeos hydroalcoholic leaf extract. AIM OF THE STUDY: Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gut with no ideal treatment to maintain the remissive status. This work aimed to characterize the phytochemical composition and physicochemical properties of the C. leptophloeos hydroalcoholic leaf extract and its efficacy in chemopreventive and immunomodulatory responses in inflammatory bowel disease in non-clinical models. MATERIALS AND METHODS: Mass spectrometry and physicochemical tests determined the phytochemical profile and physicochemical characteristics of the Commiphora leptophloeos (CL) extract. The chemopreventive and immunomodulatory effects of CL extract (50 and 125 µg/mL) were evaluated in vitro in the RAW 264.7 lipopolysaccharide (LPS) induced cell assay and in vivo in the model of intestinal inflammation induced by 2,4-Dinitrobenzenesulfonic acid (DNBS) in mice when they were treated with CL extract by intragastric gavage (i.g.) at doses of 300, 400 and 500 mg/kg. RESULTS: Phytochemical annotation of CL extract showed a complex phenolic composition, characterized as phenolic acids and flavonoids, and satisfactory physicochemical characteristics. In addition, CL extract maintained the viability of RAW macrophages, reduced ROS and NO production, and negatively regulated COX-2, iNOS, TNF-α, IL-1ß, IL-6, and IL-17 (p < 0.05). In the intestinal inflammation model, CL extract was able to downregulate NF-κB p65/COX-2, mTOR, iNOS, IL-17, decrease levels of malondialdehyde and myeloperoxidase and cytokines TNF-α, IL-1ß and IL-6 (p < 0.05). CONCLUSION: Based on these findings, CL extract reduced inflammatory responses by down-regulating pro-inflammatory markers in macrophages induced by LPS and DNBS-induced colitis in mice through NF-κB p65/COX-2 signaling. CL leaf extract requires further investigation as a candidate for treating inflammatory bowel disease.

Anti-inflammatory, healing and antiophidic potential of Jatropha mollissima (Pohl) Baill. (Euphorbiaceae): From popular use to pharmaceutical formulation in gel.

Jatropha mollissima (Pohl) Baill. (Euphorbiaceae) is widely used in traditional medicine to treat inflammatory disorders. So, a topical gel containing the hydroethanolic extract of its leaves was developed and evaluated for its anti-inflammatory, wound healing, and antiophidic properties in mice. First, the chemical profile of different parts of the plant was characterized by liquid chromatography coupled to mass spectrometry (LC-MS) using molecular networking. In the leaf extract, 11 compounds were characterized, with a particular emphasis on the identification of flavonoids. The gel efficiently inhibited carrageenan-induced paw edema, as well as acute and chronic croton oil-induced ear edema models, thereby reducing inflammatory and oxidative parameters in inflamed tissues. Besides anti-inflammatory activity, the herbal gel showed significant wound healing activity. The edematogenic, hemorrhagic and dermonecrotic activities induced by Bothrops jararaca snake venom were effectively inhibited by the treatment with J. mollissima gel. The association with the herbal gel improved in up to 90% the efficacy of commercial snake antivenom in reduce venom-induced edema. Additionally, while antivenom was not able to inhibit venom-induced dermonecrosis, treatment with herbal gel reduced in 55% the dermonocrotic halo produced. These results demonstrate the pharmacological potential of the herbal gel containing J. mollissima extract, which could be a strong candidate for the development of herbal products that can be used to complement the current antivenom therapy against snake venom local toxicity.

Lacticaseibacillus casei decrease long-chain fatty acids and most substances in an experimental model of intestinal mucositis.

PURPOSE: To evaluate the long-chain fatty acid and major compounds levels in the feces after prophylactic oral use of Lacticaseibacillus casei in an experimental model of intestinal mucositis. METHODS: Fifteen Swiss mice were randomly divided into three groups (n=5/group): The negative or positive control groups (n = 5) received saline orally for 18 days and an the intraperitoneal (i.p.) of saline or 5 Fluorouracil (450 mg/kg) in 15th day, respectability. L. casei group received oral concentration of L. casei (1x109 CFU/mL) for 18 days, the i.p. injection of 5-fluorouracil (450 mg/kg) in 15th days. Tissue samples from colon and each small intestine segment were collected for histopathological analysis. Stool samples were collected. Fecal composition of long-chain fatty acids and sterols were analysed by gas chromatography-mass spectrometry on the 15th and the 18th day. RESULTS: The mucosa layer of all small intestine segments of animals from L. casei showed well preserved epithelium and glands, without necrosis signs, but Goblet cells number decreased. Several long-chain fatty acids and sterols have been identified before and after in the groups. L. casei administration after 5-FU treatment reduced concentrations of linoleic acid (18:2) (p < 0.001) and oleic acid (18:1) (p < 0.001) in feces. CONCLUSIONS: L. casei prevented the mucosal damage associated with 5-FU-induced intestinal mucositis reduced long-chain fatty acid levels in the feces.

Inhibition of murine colorectal cancer metastasis by targeting M2-TAM through STAT3/NF-kB/AKT signaling using macrophage 1-derived extracellular vesicles loaded with oxaliplatin, retinoic acid, and Libidibia ferrea.

Colorectal cancer is still unmanageable despite advances in target therapy. However, extracellular vesicles (EVs) have shown potential in nanomedicine as drug delivery systems, especially for modulating the immune cells in the tumor microenvironment (TME). In this study, M1 Macrophage EVs (M1EVs) were used as nanocarriers of oxaliplatin (M1EV1) associated with retinoic acid (M1EV2) and Libidibia ferrea (M1EV3), alone or in combination (M1EV4) to evaluate their antiproliferative and immunomodulatory potential on CT-26 and MC-38 colorectal cancer cell lines and prevent metastasis in mice of allograft and peritoneal colorectal cancer models. Tumors were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and epithelial-mesenchymal transition process (EMT) were analyzed in vitro in colorectal cancer cell lines. Polarization of murine macrophages (RAW264.7 cells) was also carried out. M1EV2 and M1EV3 used alone or particularly M1EV4 downregulated the tumor progression by TME immunomodulation, leading to a decrease in primary tumor size and metastasis in the peritoneum, liver, and lungs. STAT3, NF-kB, and AKT were the major genes downregulated by of M1EV systems. Tumor-associated macrophages (TAMs) shifted from an M2 phenotype (CD163) to an M1 phenotype (CD68) reducing levels of IL-10, TGF-ß and CCL22. Furthermore, malignant cells showed overexpression of FADD, APAF-1, caspase-3, and E-cadherin, and decreased expression of MDR1, survivin, vimentin, and PD-L1 after treatment with systems of M1EVs. The study shows that EVs from M1 antitumor macrophages can transport drugs and enhance their immunomodulatory and antitumor activity by modulating pathways associated with cell proliferation, migration, survival, and drug resistance.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor.

PURPOSE: To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. METHODS: Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). RESULTS: WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). CONCLUSIONS: L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

Erythrocyte-cancer hybrid membrane-coated reduction-sensitive nanoparticles for enhancing chemotherapy efficacy in breast cancer.

Cell-membrane-coated biomimetic nanoparticles (NPs) have attracted great attention due to their prolonged circulation time, immune escape mechanisms and homotypic targeting properties. Biomimetic nanosystems from different types of cell -membranes (CMs) can perform increasingly complex tasks in dynamic biological environments thanks to specific proteins and other properties inherited from the source cells. Herein, we coated doxorubicin (DOX)-loaded reduction-sensitive chitosan (CS) NPs with 4T1 cancer cell -membranes (CCMs), red blood cell -membranes (RBCMs) and hybrid erythrocyte-cancer membranes (RBC-4T1CMs) to enhance the delivery of DOX to breast cancer cells. The physicochemical properties (size, zeta potential and morphology) of the resulting RBC@DOX/CS-NPs, 4T1@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs, as well as their cytotoxic effect and cellular NP uptake in vitro were thoroughly characterized. The anti-cancer therapeutic efficacy of the NPs was evaluated using the orthotopic 4T1 breast cancer model in vivo. The experimental results showed that DOX/CS-NPs had a DOX-loading capacity of 71.76 ± 0.87 %, and that coating of DOX/CS-NPs with 4T1CM significantly increased the NP uptake and cytotoxic effect in breast cancer cells. Interestingly, by optimizing the ratio of RBCMs:4T1CMs, it was possible to increase the homotypic targeting properties towards breast cancer cells. Moreover, in vivo tumor studies showed that compared to control DOX/CS-NPs and free DOX, both 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs significantly inhibited tumor growth and metastasis. However, the effect of 4T1@DOX/CS-NPs was more prominent. Moreover, CM-coating reduced the uptake of NPs by macrophages and led to rapid clearance from the liver and lungs in vivo, compared to control NPs. Our results suggest that specific self-recognition to source cells resulting in homotypic targeting increased the uptake and the cytotoxic capacity of 4T1@DOX/CS-NPs by breast cancer cells in vitro and in vivo. In conclusion, tumor-disguised CM-coated DOX/CS-NPs exhibited tumor homotypic targeting and anti-cancer properties, and were superior over targeting with RBC-CM or RBC-4T1 hybrid membranes, suggesting that the presence of 4T1-CM is critical for treatment outcome.

Phenolic-rich extract of Nopalea cochenillifera attenuates gastric lesions induced in experimental models through inhibiting oxidative stress, modulating inflammatory markers and a cytoprotective effect.

Nopalea cochenillifera (Cactaceae), popularly known as "palma" or "palma doce", is from Mexico, but it was widely introduced in Brazil through crops. It has been used as food and in traditional medicine and is a good source of phenolic compounds. In this study the phytochemical profile and gastroprotective activity of phenolic-rich extract of N. cochenillifera in acute gastric lesion models induced by ethanol and indomethacin were evaluated. High-performance liquid chromatography coupled with mass spectrometry (HPLC/ESI/MSn) allowed the characterization of 12 compounds such as sugars, phenolics and flavonoids. Among polyphenols, the main peak was assigned to isorhamnetin-3-O-(2'',3''-O-di-rhamnose)-glucoside. The TPC and TFC in the dry extract were 67.85 mg of gallic acid equivalent per g/extract and 46.16 mg quercetin equivalent per g/extract, respectively. In the in vitro MTT assay, the extract showed no cytotoxicity and suppressed ROS levels in LPS-treated RAW 264.7 cells. Preclinical models in rats showed that a dose of 100 mg kg-1 (p < 0.0001) in the ethanol model and doses of 100 mg kg-1 (p < 0.5) and 200 mg kg-1 (p < 0.01) in the indomethacin model reduced the gastric lesions. Also, the extract reduced the MPO, MDA, TNF-α and IL-1ß levels and increased the GSH and IL-10 levels. The pre-treatment with the extract led to the upregulation of SOD and the downregulation of COX-2 by immunohistochemical analysis. It also showed a cytoprotective effect in the histopathological analysis and stimulated the restoration of the mucus content as observed in the periodic acid-Schiff analysis without modifying the pH, volume or total acidity of the gastric juice. Taken together, N. cochenillifera extract can be applied as a novel gastroprotective ingredient for food or pharmaceutical products.

Toxicity and Anti-Inflammatory Activity of Phenolic-Rich Extract from Nopalea cochenillifera (Cactaceae): A Preclinical Study on the Prevention of Inflammatory Bowel Diseases.

Phenolic compounds have been scientifically recognized as beneficial to intestinal health. The cactus Nopalea cochenillifera, used as anti-inflammatory in traditional medicine, is a rich source of these bioactive compounds. The present study aimed to investigate the phytochemical profile of N. cochenillifera extract and evaluate its acute toxicity and anti-inflammatory effect on 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis in rats. The total phenolic content per gram of dry extract was 67.85 mg. Through HPLC-IES-MSn, a total of 25 compounds such as saccharides, organic acids, phenolic acids and flavonoids were characterized. The dose of 2000 mg/kg of extract by an oral route showed no signs of toxicity, mortality or significant changes in biochemical and hematological parameters. Regarding intestinal anti-inflammatory effects, animals were treated with three different doses of extract or sulfasalazine. Macroscopic analysis of the colon indicated that the extract decreased the disease activity index. Levels of IL-1ß and TNF-α decreased, IL-10 increased and MDA and MPO enzyme levels decreased when compared with the control group. In addition, a down-regulation of MAPK1/ERK2 and NF-κB p65 pathway markers in colon tissue was observed. The epithelial integrity was improved according to histopathological and immunohistological analysis. Thus, the extract provided strong preclinical evidence of being effective in maintaining the remission of colitis.

Effective breast cancer therapy based on palmitic acid-loaded PLGA nanoparticles.

Although new strategies for breast cancer treatment have yielded promising results, most drugs can lead to serious side effects when applied systemically. Doxorubicin (DOX), currently the most effective chemotherapeutic drug to treat breast cancer, is poorly selective towards tumor cells and treatment often leads to the development of drug resistance. Recent studies have indicated that several fatty acids (FAs) have beneficial effects on inhibiting tumorigenesis. The saturated FA palmitic acid (PA) showed anti-tumor activities in several types of cancer, as well as effective repolarization of M2 macrophages towards the anti-tumorigenic M1 phenotype. However, water insolubility and cellular impermeability limit the use of PA in vivo. To overcome these limitations, here, we encapsulated PA into a poly(d,l-lactic co-glycolic acid) (PLGA) nanoparticle (NP) platform, alone and in combination with DOX, to explore PA's potential as mono or combinational breast cancer therapy. Our results showed that PLGA-PA-DOX NPs and PLGA-PA NPs significantly reduced the viability and migratory capacity of breast cancer cells in vitro. In vivo studies in mice bearing mammary tumors demonstrated that PLGA-PA-NPs were as effective in reducing primary tumor growth and metastasis as NPs loaded with DOX, PA and DOX, or free DOX. At the molecular level, PLGA-PA NPs reduced the expression of genes associated with multi-drug resistance and inhibition of apoptosis, and induced apoptosis via a caspase-3-independent pathway in breast cancer cells. In addition, immunohistochemical analysis of residual tumors showed a reduction in M2 macrophage content and infiltration of leukocytes after treatment of PLGA-PA NPs and PLGA-PA-DOX NPs, suggesting immunomodulatory properties of PA in the tumor microenvironment. In conclusion, the use of PA alone or in combination with DOX may represent a promising novel strategy for the treatment of breast cancer.

Lacticaseibacillus casei decrease long-chain fatty acids and most substances in an experimental model of intestinal mucositis

Purpose: To evaluate the long-chain fatty acid and major compounds levels in the feces after prophylactic oral use of Lacticaseibacillus casei in an experimental model of intestinal mucositis. Methods: Fifteen Swiss mice were randomly divided into three groups (n=5/group): The negative or positive control groups (n = 5) received saline orally for 18 days and an the intraperitoneal (i.p.) of saline or 5 Fluorouracil (450 mg/kg) in 15th day, respectability. L. casei group received oral concentration of L. casei (1x109 CFU/mL) for 18 days, the i.p. injection of 5-fluorouracil (450 mg/kg) in 15th days. Tissue samples from colon and each small intestine segment were collected for histopathological analysis. Stool samples were collected. Fecal composition of long-chain fatty acids and sterols were analysed by gas chromatography-mass spectrometry on the 15th and the 18th day. Results: The mucosa layer of all small intestine segments of animals from L. casei showed well preserved epithelium and glands, without necrosis signs, but Goblet cells number decreased. Several long-chain fatty acids and sterols have been identified before and after in the groups. L. casei administration after 5-FU treatment reduced concentrations of linoleic acid (18:2) (p < 0.001) and oleic acid (18:1) (p < 0.001) in feces. Conclusions: L. casei prevented the mucosal damage associated with 5-FU-induced intestinal mucositis reduced long-chain fatty acid levels in the feces.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor

ABSTRACT Purpose: To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. Methods: Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). Results: WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). Conclusions: L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

The beneficial effects of Lacticaseibacillus casei on the small intestine and colon of Swiss mice against the deleterious effects of 5-fluorouracil.

Background: Intestinal mucositis is one of the most common and important side effects of 5-fluorouracil (5-FU). Currently, there are still no specific and effective protocols for its prevention and treatment. The aim of the present study was to evaluate the effect of oral administration of Lacticaseibacillus casei (L. casei) on the progression of 5-FU-induced intestinal mucositis. Methods: L. casei (1x109 CFU/ml) or saline was orally administered to Swiss mice, beginning 15 days before intestinal mucositis induction by single intraperitoneal 5-FU administration (450 mg/kg). Body weight, number of peripheral leukocytes and fecal lactic acid bacteria were monitored. After euthanasia, on day 18, tissue samples from colon and each small intestine segment were collected for histopathology. Jejunal tissues were collected and evaluated for iNOS and TNF-alpha immunoexpression, IL-1-beta, IL-6 and TNF-alpha levels, malonaldehyde (MDA) accumulation, invertase activity and factor nuclear kappa B (NFkB-P65) gene expression, toll like receptor-4 (TLR-4), mucin-2 (MUC-2), occludin and zonula occludens-1 (ZO-1). Results: The positive impact of L. casei on 5-FU-induced leukopenia was observed, but not on 5-FU-induced weight loss in mice. L. casei reduced 5-FU-induced inflammation in the colon and small intestine (p<0.05). Decreased TNF-α, IL-1ß, IL-6 (p<0.05) and MDA (p<0.05) levels, as well as decreased iNOS and TNF-alpha protein expressions (p<0.05) were found in the jejunum from L casei group. In addition, L-casei down-regulated NFKB-P65 (p<0.05) and TLR-4 (p<0.05) gene expressions and up-regulated MUC-2 and mucosal barrier proteins occludin and ZO-1 gene expressions (p<0.05). Furthermore, greater lactic acid bacteria population (p<0.05) was found in the L. casei group when compared to control groups. Conclusion: Oral L. casei administration can protect the intestine of Swiss mice from 5-FU-induced intestinal mucositis, thus contributing to overall health.

Topical gel containing phenolic-rich extract from Ipomoea pes-capre leaf (Convolvulaceae) has anti-inflammatory, wound healing, and antiophidic properties.

The growing use of phytotherapy in clinical practice arouses interest in studies using medicinal plants as active ingredients for new medicines. Ipomoea pes-caprae has a wide medicinal use in the treatment of inflammatory disorders, skin wounds, stings, and painful rheumatic processes. Assayed in this study are the physicochemical characterization of a gel developed with this extract and the evaluation of its anti-inflammatory and healing efficacy, in addition to its antiedematogenic action on Bothrops snake envenoming in mice. The qualitative and quantitative analyses of the hydroethanolic extract by mass spectrometry showed 18 phenolic compounds, highlighting a high content of chlorogenic acid (0.92 µg/g), neochlorogenic acid (6.07 µg/g), and isochlorogenic acid (0.80 µg/g) compounds. The formulation was stable in relation to the physical-chemical characteristics during the time of analysis and was considered safe for topical treatment in animals, causing no skin irritation. Although the results have shown an absence of activity in the model of ear edema induced by croton oil (acute inflammation), the herbal gel efficiently inhibited carrageenan paw edema and chronic ear edema induced by multiple applications of croton oil, which may indicate the possible performance under the kinin pathway such as bradykinin, histamine, and serotonin. Wound healing in the group treated with the I. pes-caprae gel was accelerated compared with the placebo group, also confirmed through histological data. Edema induced by Bothrops erythromelas snake venom was efficiently reduced in the treatment with I. pes-caprae gel associated with the antibothropic-crotalic serum, whereas the antivenom alone was not effective. This approach presents a promising formulation based on I. pes-caprae with potential therapeutic use for inflammatory disorders.

Combined use of systemic quercetin, glutamine and alpha-tocopherol attenuates myocardial fibrosis in diabetic rats.

This study aimed to analyze the effects of the quercetin (100 mg/kg), 1% glutamine and 1% α-tocopherol antioxidants in the myocardium of rats with streptozotocin-induced diabetes mellitus. Twenty male rats were subdivided into four groups (n = 5): N (normoglycemic); D (diabetic); NT (normoglycemic treated with antioxidants); and DT (diabetic treated with antioxidants) treated for 60 days. Clinical parameters, oxidative stress markers, inflammatory cytokines, myocardial collagen fibers and immunoexpression of superoxide dismutase 1 (SOD-1), glutathione peroxidase-1 (GPx-1), interleukin-1ß (IL-1-ß), transforming growth factor-beta (TGF-ß), and fibroblast growth factor-2 (FGF-2) were evaluated. Results showed reduced body weight, hyperphagia, polydipsia and hyperglycemic state in groups D and DT. The levels of glutathione (GSH) were higher in NT and DT compared to N (p < 0.01) and D (p < 0.001) groups, respectively. Greater GSH levels were found in DT when compared to N animals (p < 0.001). In DT, there was an increase in IL-10 in relation to N, D and NT (p < 0.05), while GPx-1 expression was similar to N and lower compared to D (p < 0.001). TGF-ß expression in DT was greater than N (p < 0.001) group, whereas FGF-2 in DT was higher than in the other groups (p < 0.001). A significant reduction in collagen fibers (type I) was found in DT compared to D (p < 0.05). The associated administration of quercetin, glutamine and α-tocopherol increased the levels of circulating interleukin-10 (IL-10) and GSH, and reduced the number of type I collagen fibers. Combined use of systemic quercetin, glutamine and alpha-tocopherol attenuates myocardial fibrosis in diabetic rats.

Extracellular Vesicles from M1-Polarized Macrophages Combined with Hyaluronic Acid and a ß-Blocker Potentiate Doxorubicin's Antitumor Activity by Downregulating Tumor-Associated Macrophages in Breast Cancer.

One of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the ß-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-κB was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-ß. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer.

Hyperbaric oxygen therapy mitigates left ventricular remodeling, upregulates MMP-2 and VEGF, and inhibits the induction of MMP-9, TGF-ß1, and TNF-α in streptozotocin-induced diabetic rat heart.

AIMS: Hyperbaric oxygen (HBO) therapy has been widely used for the adjunctive treatment of diabetic wounds, and is currently known to influence left ventricular (LV) function. However, morphological and molecular repercussions of the HBO in the diabetic myocardium remain to be described. We aimed to investigate whether HBO therapy would mitigate adverse LV remodeling caused by streptozotocin (STZ)-induced diabetes. MAIN METHODS: Sixty-day-old Male Wistar rats were divided into four groups: Control (n = 8), HBO (n = 7), STZ (n = 10), and STZ + HBO (n = 8). Diabetes was induced by a single STZ injection (60 mg/kg, i.p.). HBO treatment (100% oxygen at 2.5 atmospheres absolute, 60 min/day, 5 days/week) lasted for 5 weeks. LV morphology was evaluated using histomorphometry. Gene expression analyzes were performed for LV collagens I (Col1a1) and III (Col3a1), matrix metalloproteinases 2 (Mmp2) and 9 (Mmp9), and transforming growth factor-ß1 (Tgfb1). The Immunoexpression of cardiac tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) were also quantified. KEY FINDINGS: HBO therapy prevented LV concentric remodeling, heterogeneous myocyte hypertrophy, and fibrosis in diabetic rats associated with attenuation of leukocyte infiltration. HBO therapy also increased Mmp2 gene expression, and inhibited the induction of Tgfb1 and Mmp9 mRNAs caused by diabetes, and normalized TNF-α and VEGF protein expression. SIGNIFICANCE: HBO therapy had protective effects for the LV structure in STZ-diabetic rats and ameliorated expression levels of genes involved in cardiac collagen turnover, as well as pro-inflammatory and pro-angiogenic signaling.

Gel formulated with Bryophyllum pinnatum leaf extract promotes skin wound healing in vivo by increasing VEGF expression: A novel potential active ingredient for pharmaceuticals.

Bryophyllum pinnatum (Crassulaceae) is used in traditional medicine for treating skin wounds. In our previous study, a topical gel containing B. pinnatum aqueous leaf extract showed a preclinical anti-inflammatory effect in in vivo acute edema models. In continuation, the present study aims to evaluate the phytochemical content and the stability of a formulation in gel containing B. pinnatum aqueous leaf extract and its healing properties and mechanism of action through an experimental model of induction of skin wounds in rats and in vitro assays. The animals were treated topically for 7 or 14 days with a formulation in gel containing extract at 5% or a placebo or Fibrinase® in cream. In addition, to establish some quality control parameters, the total phenolic content (TPC), total flavonoid content (TFC), and a study focusing on the phytochemical and biological stability of a gel for 30 days at two different conditions (room temperature and 40°C/75% RH) were performed. Gel formulation containing extract showed a TPC and TFC of 2.77 ± 0.06 mg of gallic acid/g and 1.58 ± 0.03 mg of quercetin/g, respectively. Regarding the stability study, the formulation in gel showed no significant change in the following parameters: pH, water activity, chromatographic profile, and the content of the major compound identified in the extract. The gel formulation containing extract stimulated skin wound healing while reducing the wound area, as well as decreasing the inflammatory infiltrate, reducing the levels of IL-1ß and TNF-α, and stimulating angiogenesis with increased expression of VEGF, an effect similar to Fibrinase. In conclusion, the gel formulation containing extract exhibited relevant skin wound healing properties and, therefore, has the potential to be applied as a novel active ingredient for developing wound healing pharmaceuticals.

The Receptor AT1 Appears to Be Important for the Maintenance of Bone Mass and AT2 Receptor Function in Periodontal Bone Loss Appears to Be Regulated by AT1 Receptor.

A large number of experimental studies has demonstrated that angiotensin II (Ang II) is involved in key events of the inflammatory process. This study aimed to evaluate the role of Ang II type 1 (AT1) and Ang II type 2 (AT2) receptors on periodontitis. Methods: Experimental periodontitis was induced by placing a 5.0 nylon thread ligature around the second upper left molar of AT1 mice, no-ligature or ligature (AT1-NL and AT1-L), AT2 (AT2-NL or AT2-L) and wild type (WT-NL or L). Alveolar bone loss was scanned using Micro-CT. Cytokines, peptides and enzymes were analyzed from gingival tissues by Elisa and RT-PCR. Results: The blockade of AT1 receptor resulted in bone loss, even in healthy animals. Ang II receptor blockades did not prevent linear bone loss. Ang II and Ang 1-7 levels were significantly increased in the AT2-L (p < 0.01) group compared to AT2-NL and AT1-L. The genic expression of the Mas receptor was significantly increased in WT-L and AT2-L compared to (WT-NL and AT2-NL, respectively) and in AT1-L. Conclusions: Our data suggest that the receptor AT1 appears to be important for the maintenance of bone mass. AT2 receptor molecular function in periodontitis appears to be regulated by AT1.

In vitro-in vivo availability of metformin hydrochloride-PLGA nanoparticles in diabetic rats in a periodontal disease experimental model.

CONTEXT: Metformin is an important oral anti-hyperglycemic used in diabetes. Polylactic-co-glycolic acid (PLGA) has been widely used due to its reliability in controlling the release of drugs. OBJECTIVE: This study evaluates the in vitro-in vivo availability of metformin hydrochloride-loaded polylactic-co-glycolic acid. MATERIAL AND METHODS: In vitro metformin release (Met-free or PLGA + Met-12.5 mg/mL per 360 min) was evaluated using static Franz vertical diffusion cells. The in vivo study was performed with two control groups (validation bioanalytical method) and two experimental groups of diabetic male Wistar rats treated with PLGA + Met 10 mg/kg or Met 100 mg/kg by oral gavage. Diabetes was induced by streptozotocin (40 mg/kg) through the penile vein. Blood samples were collected 0.5, 1, 4, 7, 10, 12, 18, 24, 36, 48 and 72 h and analysed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: PLGA + Met 10 mg/kg was released in the in vitro assay suggesting a parabolic diffusion kinetic model (K -0.0619-0.5h) with a 100% release profile in 10 h by controlled diffusion. The in vivo assay showed the apparent volume of distribution Vz/F (PLGA + Met 10 mg/kg, 40971.8 mL/kg vs. Met 100 mg/kg, 2174.58 mL/kg) and mean residence time MRTinf (PLGA + Met 10 mg/kg, 37.66 h vs. Met 100 mg/kg, 3.34 h). DISCUSSION AND CONCLUSIONS: The formulation modifies pharmacokinetics parameters such as apparent distribution volume and mean residence time. The PLGA + Met 10 mg/kg had a slower elimination rate compared to Met 100 mg/kg in diabetic rats in a periodontal disease experimental model.

Functionalized Nanoparticles Targeting Tumor-Associated Macrophages as Cancer Therapy.

The tumor microenvironment (TME) plays a central role in regulating antitumor immune responses. As an important part of the TME, alternatively activated type 2 (M2) macrophages drive the development of primary and secondary tumors by promoting tumor cell proliferation, tumor angiogenesis, extracellular matrix remodeling and overall immunosuppression. Immunotherapy approaches targeting tumor-associated macrophages (TAMs) in order to reduce the immunosuppressive state in the TME have received great attention. Although these methods hold great potential for the treatment of several cancers, they also face some limitations, such as the fast degradation rate of drugs and drug-induced cytotoxicity of organs and tissues. Nanomedicine formulations that prevent TAM signaling and recruitment to the TME or deplete M2 TAMs to reduce tumor growth and metastasis represent encouraging novel strategies in cancer therapy. They allow the specific delivery of antitumor drugs to the tumor area, thereby reducing side effects associated with systemic application. In this review, we give an overview of TAM biology and the current state of nanomedicines that target M2 macrophages in the course of cancer immunotherapy, with a specific focus on nanoparticles (NPs). We summarize how different types of NPs target M2 TAMs, and how the physicochemical properties of NPs (size, shape, charge and targeting ligands) influence NP uptake by TAMs in vitro and in vivo in the TME. Furthermore, we provide a comparative analysis of passive and active NP-based TAM-targeting strategies and discuss their therapeutic potential.