Rat Macrophages Research Articles

Abstract B011: Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis

Abstract Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion, modulation of the tumor microenvironment (TME), plasticity, and ability to colonize secondary tissues. Therefore, a viable anti-metastasis strategy needs to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, the purpose of this study was to test the hypothesis that Rac and Cdc42 inhibitors target both migratory cancer cells and immunosuppressive cells in the TME. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects. Since the targets of these inhibitors may also affect immune cell behavior, we first tested their potential to target Rac/Cdc42 function in human and rat macrophage cell lines and show that EHop-016 and MBQ-167 inhibit Rac activation (at 5X higher concentration than the IC50 in breast cancer cells), actin cytoskeletal extensions, and migration, without affecting cell viability. EHop-016 and MBQ-167 significantly decrease the proinflammatory cytokine Interleukin-6 (Il-6) from plasma and the TME. Next, we tested the in vivo effect of MBQ-167 on immune cells from breast cancer mouse models. Tumors from immunodeficient mice bearing mammary tumors that were treated with vehicle or 50mg/kg MBQ-167 5x a week for 5 weeks, were analyzed by flow cytometry to quantify different immune cell types. MBQ-167 treated mice demonstrated elevated CD11+F4/80+CD86+ M1 macrophages and reduced MDSCs in mammary tumors. In the immunocompetent 4T-1 breast cancer model, spleens contained a lower frequency of macrophages and neutrophils, accompanied by an increase in activated cytotoxic CD69+, CD8+T cells. Moreover, when metastasis to the lungs was reduced by ~90% following MBQ-167 treatment, granzyme B+ CD8+ T cells were elevated in the lung extracts, without affecting the immunosuppressive CD4+ regulatory T cell counts. In vitro, we also demonstrated that increasing doses of MBQ-167 induced T cell activation and interferon-gamma secretion, a cytokine known to have antitumor properties. Taken together, Rac/Cdc42 inhibitors induce anti-metastatic cancer effects via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells, while promoting the activation of cytotoxic T cells, in the tumor and the metastasis microenvironment. Therefore, MBQ-167 promises to be an effective antimetastatic cancer therapeutic that is poised to enter Phase I clinical trials. Citation Format: Stephanie Dorta-Estremera, Anamaris Torres, Michael Rivera-Robles, Ailed Cruz-Collazo, Luis Borrero-Garcia, Suranganie Dharmawardhane. Rac and Cdc42 inhibition in the tumor microenvironment as a strategy to prevent metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B011.

In vitro screening of the potential of six Cameroonian medicinal plants on male reproductive biomarkers

Background: Male infertility and erectile dysfunction are the main conditions associated with male reproductive function disorders. Testosterone is the main hormone that regulates male reproductive function, while nitric oxide is a potent vasodilator involved in the erectile process. Methods: This study was conducted to evaluate in vitro the potential of the aqueous extract of six Cameroonian medicinal plants traditionally used for male reproductive function: Palisota ambigua, Terminalia superba, Rauvolfia macrophylla, Pycnanthus angolensis, Pausynistalia yohimbe, and Shumanniophyton magnificum. Results: Their effect on testosterone and nitric oxide production was evaluated using rat interstitial and macrophage cells, respectively. Then, the antioxidant properties, flavonoids, and total phenolic content were assessed. The phytochemical screening was also performed. Conclusion: Each plant extract significantly increased (p<0.05) testosterone production. At 100 µg/mL, the extract of Schumanniophyton magnificum showed the highest level (3.730 ng/mL). Each plant extract significantly increased (p<0.05) the production of nitric oxide. The aqueous extract of Rauvolfia macrophylla showed the highest level of nitric oxide with an EC50 = 8.554 µg/mL. For antioxidant activities, Terminalia superba showed the highest antiradical activity (SC50 = 4.796 µg/mL), the highest reducing power (OD = 2.235 at 700 nm), and the highest total antioxidant capacity (276.035 mgEAA/g of plant extract) and while Pausynistalia yohimbe, showed the highest inhibition on lipid peroxidation (15.170 µg/mL). Terminalia superba also showed the highest flavonoid (13.419 mgEQ/g) and total phenolic (300.813 mgEGA/g) content. The phytochemical screening of the plant extracts showed many active secondary metabolites such as alkaloids, phenols, flavonoids, saponins, and glycosides. All the extracts showed androgenic effects and antioxidant activities without significant cytotoxicity, thus supporting the use of these medicinal plants by traditional healers for managing male infertility.

Effects of Acalypha indica L. Extract on Inflammatory Response in The Pathogenesis of Nonalcoholic Fatty Liver Disease: An Overview of TLR9, NFκB and TNFα Expression in Hepatocytes and Macrophages of Sprague-Dawley Rats

Effects of Acalypha indica L. Extract on Inflammatory Response in The Pathogenesis of Nonalcoholic Fatty Liver Disease: An Overview of TLR9, NFκB and TNFα Expression in Hepatocytes and Macrophages of Sprague-Dawley Rats

The effect of hyperglycemia on the activation of peritoneal macrophages of albino rats.

Hyperglycemia is one of the main damaging factors of diabetes mellitus (DM). The severity of this disease is most clearly manifested under conditions of the inflammatory process. In this work, we have studied the activation features of rat peritoneal macrophages (MPs) under conditions of high glucose concentration in vitro. Comparison of the independent and combined effects of streptozotocin-induced DM and hyperglycemia on proliferation and accumulation of nitrites in the MPs culture medium revealed similarity of their effects. Elevated glucose levels and, to a lesser extent, DM decreased basal proliferation and NO production by MPs in vitro. The use of the protein kinase C (PKC) activator, phorbol ester (PMA), abolished the proinflammatory effect of thrombin on PMs. This suggests the involvement of PKC in the effects of the protease. At the same time, the effect of thrombin on the level of nitrites in the culture medium demonstrates a pronounced dose-dependence, which was not recognized during evaluation of proliferation. Proinflammatory activation of MPs is potentiated by hyperglycemia, one of the main pathological factors of diabetes. Despite the fact that high concentrations of glucose have a significant effect on proliferation and NO production, no statistically significant differences were found between the responses of MPs obtained from healthy animals and from animals with streptozotocin-induced DM. This ratio was observed for all parameters studied in the work, during analysis of cell proliferation and measurement of nitrites in the culture medium. Thus, the results obtained indicate the leading role of elevated glucose levels in the regulation of MPs activation, which is comparable to the effect of DM and even "masks" it.

Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

Abstract 10753: Phosphodiesterase Type 5 Inhibition and C-c Chemokine Receptor-2 Disruption Synergistically Protect Against Pulmonary Arterial Hypertension in Monocrotaline-Treated Rats: Potential Synergism of Anti-inflammatory Agents and Pulmonary Vasodilator Therapy

BACKGROUND: Whether conventional pulmonary vasodilator therapy and anti-inflammatory agents synergistically protect against pulmonary arterial hypertension (PAH) is unknown. Hypothesis: We tested the hypothesis that a phosphodiesterase type 5 (PDE5) inhibitor and disruption of C-C chemokine receptor (CCR)-2, a receptor for MCP-1, synergistically protect against PAH in monocrotaline (MCT)-treated rats. Methods: Study1:Seven-week-old male Ccr2 (-/-) rats, generated by using the CRISPAR/ Cas9 genome editing technology, or wild-type littermates, treated with MCT (60mg/kg) or vehicle, were subjected to hemodynamic, morphometric and immunohistochemical analyses and mRNA and protein expression studies on day21 (n=40, 4 groups). Study 2: Tadarafil (10mg/kg/d), a PDE5 inhibitor, or vehicle was administered in Ccr2 (-/-) or wild-type MCT rats on days 21-42 (n=71, 4 groups) for evaluating survival and were evaluated hemodynamically on day28. Results: Study1: MCT treatment increased right ventricular systolic pressure (RVSP), an index of right ventricular hypertrophy (Fulton index), indices of pulmonary vascular diseases (%medial wall thickness, %muscularization) and the number of perivascular CD68-positive macrophages in WT rats, all of which were decreased by Ccr2 (-/-) (p<.05). Similarly, an increase in mRNA expression of inflammatory cytokines (IL6 ,TNFa, MCP1, IL1b, TGFb) and a decrease in BMPR2 pathways (BMPR2, pSmad1/5/9 and ID1) as well as an increase in endothelial apoptosis (% cleaved Caspase 3 positive CD31 cells) and a decrease in NO pathways (eNOS and phospho-eNOS/eNOS) in lungs in wild-type MCT rats were reversed by Ccr2 (-/-) on day21 (p<.05) with comparable PDE5 expression. Study2: Tadarafil administration or CCR2(-/-)(p=.03 and p=.02) alone improved the survival in wild type MCT rats. A double intervention synergistically improved the survival in separately treated groups (p=.02 vs CCR2(-/-), p=.04 vs Tadarafil) in MCT rats. Tadarafil treatment on days21-28 decreased RVSP and Fulton index in CCR2(-/-) MCT rats (p<.05). Conclusions PDE5 inhibition and CCR-2 disruption synergistically protect against PAH in MCT-treated rats, suggesting an adjunctive role of a CCR2 inhibitor on top of administering a PDE5 inhibitor for PAH.

Bioactive diterpenoids and sesquiterpenoids with different skeletons from Salvia digitaloides Diels

Salvia has been regarded as a beneficial healing herb in ancient Egypt, Rome and Greece, and is listed as an official medicine in the pharmacopoeias of many countries worldwide. Currently, Salvia is widely used to flavor and preserve food. Here, two undescribed norabietane-type diterpenoids, sadigitaloides A and B, two undescribed germacrane-type sesquiterpenoids, sadigitaloides C and D, five undescribed guaiane-type sesquiterpenoid lactones, sadigitaloides E-I, two undescribed noreudesmane-type sesquiterpenoids, sadigitaloides J and K, one known diterpenoid, three known sesquiterpenoids, and three other types of known compounds were isolated from the 95% ethanol extract of the whole plants of Salvia digitaloides. Their structures and absolute configurations were characterized using 1D and 2D NMR spectroscopic techniques, electronic circular dichroism (ECD) calculations, HRESIMS experiments, and single-crystal X-ray diffraction analysis. Some compounds were evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced TNF-α production in rat macrophage NR8383 cells. Sadigitaloide A showed noticeable anti-inflammatory activity at a concentration of 100.0 μM. At a concentration of 60 μM, sadigitaloide B exhibited better protection of dopaminergic neurons than the positive control n-butylidenephthalide in the Caenorhabditis elegans model injured by 6-OHDA. The phytotoxic activities of some compounds were attributed to considerable inhibitory effects on the growth of the roots and hypocotyls of Raphanus sativus L seedlings, especially cis, trans-abscisic acid, whose inhibition rates were much higher than those of glyphosate at concentrations ranging from 50 to 400 ppm. These results indicated that abietane-type diterpenoids possessed excellent anti-inflammatory and neuroprotective activities and further suggested that the low-molecular-weight compounds exhibited outstanding phytotoxic activities.

Alcohol impairs recognition and uptake of Mycobacterium tuberculosis by suppressing toll-like receptor 2 expression.

Alcohol impairs pulmonary innate immune function and is associated with an increased risk of tuberculosis (TB). Toll-like receptor 2 (TLR2) is a pattern recognition receptor on alveolar macrophages that recognizes Mycobacterium tuberculosis (Mtb). The expression of TLR2 depends, in part, on granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. Given our prior work demonstrating the suppression of GM-CSF signaling following chronic alcohol ingestion, we hypothesized that alcohol impairs TLR2 expression via the suppression of GM-CSF and thereby reduces the ability of the macrophage to recognize and phagocytose Mtb. Primary alveolar macrophages were isolated from control-fed and alcohol-fed rats. Prior to cell isolation, some alcohol-fed rats were treated with intranasal GM-CSF and then endotracheally inoculated with an attenuated strain of Mtb. Primary macrophages were then isolated and immunofluorescence was used to determine phagocytic efficiency and TLR2 expression in the presence and absence of GM-CSF treatment and phagocytic efficiency in the presence and absence of TLR2 neutralization. TLR2 expression and phagocytosis of Mtb were significantly lower in the alveolar macrophages of alcohol-fed rats than control-fed rats. In parallel, blocking TLR2 signaling recapitulated this decreased phagocytosis of Mtb. In contrast, intranasal GM-CSF treatment restored TLR2 expression and Mtb phagocytosis in the alveolar macrophages of alcohol-fed rats to levels comparable to those of control-fed rats. Chronic alcohol ingestion reduces TLR2 protein expression and phagocytosis of Mtb, likely due to impaired GM-CSF signaling. GM-CSF restores membrane-bound TLR2 expression and phagocytic function.

Assessment of the influence of the composition of atmospheric microparticles on redox homeostasis of alveolar macrophages

Introduction. The components and dimensions of suspended particulate matter (SPM) depend on territory, the sources of generation. The aim is to assess the relationship between the quality and dispersed composition of atmospheric SPMs with oxidative and antioxidant processes in alveolar macrophages. Materials and methods. The impact of actual multi component pollution of the surface layer of atmospheric air on alveolar macrophages (AMs) of Wistar rats was modelled. Correlations between the characteristics of AMs and pollution were assessed using the Spearman correlation coefficient. Results. The predominant contribution of the quality characteristics of SPM has been founded. The effect of the dispersed composition increases with an increase of the finely dispersed particles fraction. Metal particles have caused oxidative damage to lipids, with the formation of stable lipid products. The antioxidant system (AOS) has been characterized by the activation of compensatory response. Mineral components have caused irreversible oxidative damage to lipid and protein compounds, activating the thioredoxin formation. Soot particles have showed the most pathogenic effect, leading to oxidative damage to lipids, proteins, and nucleic acids. The AOS stress was accompanied by an increase in the formation of oxidized glutathione and thioredoxin, which performs restoration of proteins and DNA. Limitations. Our study characterizes the response of the AMs of a particular territory. Conclusion. The established tendencies characterize the impact of composition of SPMs of the urban atmospheric on AMs. The study made it possible to identify the most sensitive criteria for the response of AMs upon contact with atmospheric SPMs (thioredoxin, protein carbonyl, 8-hydroxy-2’-deoxyguanosine).

The Mitigatory Effect of Shen-Qi Compound on the Diabetic Thoracic Aortic Complications through Inhibiting the Inflammatory Microenvironment by miR-223-3p/RBP-J/IRF8 Axis.

Background Disruption of the vascular immunological inflammatory microenvironment is linked to metabolic memory impairment. Even though it has been proven that the Shen-Qi compound (SQC) can efficiently halt metabolic memory and preserve vascular endothelial cells, extensive studies need to be done to investigate if it can also change the vascular immune-inflammatory microenvironment by regulating the immune system. This will help figure out the role of stopping metabolic memory. Methods After 4 weeks on a high-fat diet (HFD), GK rats were used to create a model for diabetic thoracic aortic problems. The effect and mechanisms of SQC on diabetic thoracic aortic complications were assessed by hematoxylin-eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), biochemical analysis, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), reverse transcription, real-time polymerase chain reaction (RT-qPCR), immunofluorescence (IF), western blot, and luciferase reporter assays. Results SQC treatment ameliorates the HFD-induced pathological symptoms as well as the HFD-induced increased concentrations of fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) and decreased concentrations of high-density lipoprotein cholesterol (HDL-C). Besides, SQC counteracted the HFD-induced average fluorescence intensity of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), as well as the concentrations of endothelin-1 (ET-1) and monocyte chemoattractant protein-1 (MCP-1), while rescuing the HFD-induced concentrations of nitric oxide (NO) and nitric oxide synthetase (NOS). Also, SQC decreases apoptosis and oxidative stress in rats with diabetic thoracic aortic complications. In addition, SQC facilitated the polarization of macrophages, stimulated the activation of dendritic cells, and regulated the inflammatory milieu in rats with diabetic thoracic aortic complications. Furthermore, SQC also modulated the miR-223-3p/RBP-J/IRF8 axis in the macrophages of rats with diabetic thoracic aortic complications. Conclusion SQC ameliorated diabetic thoracic aortic complications through the regulation of apoptosis, oxidative stress, and inflammatory microenvironment mediating by the miR-223-3p/RBP-J/IRF8 axis.

Mechanism of Response of Alveolar Macrophages in Wistar Rats to the Composition of Atmospheric Suspensions

Atmospheric air quality is a crucial factor in the health of human populations. Suspended particulate matter (SPM) is one of the most dangerous components polluting urban air. The aim of the present article is to study the effect of model suspensions (MS) of SPM that are characteristic of the composition of atmospheric air at locations with various anthropogenic loads on redox processes in alveolar macrophages (AM). Atmospheric air sampling was carried out in the breathing zone according to the method developed by one of the authors. AM were isolated from bronchoalveolar lavage fluid of experimental animals. The MS of SPM were prepared in accordance with the actual air pollution: MS No. 1 corresponded to an area with a low man-made load, and MS No. 2 corresponded to an area with a high man-made load. Load tests with model suspensions were carried out for 2 days. Parameters of oxidant processes and antioxidant system (AOS) were determined in cells and culture media. The proportion of the influence of the qualitative and dispersed composition of MS and the indicator of intra-system tension were calculated based on correlation dependencies. The atmospheric air with a high man-made load was dominated by particles up to 10 µm, whereas air with an insignificant man-made load contained SPM of more than 10 µm in size. Unidirectional changes were observed due to an exposure to both model suspensions, but the most pronounced oxidative modifications of lipids, proteins and genetic structures were caused by the exposure to MS No. 2. When exposed to MS No. 1, the AOS maintained the redox balance at the physiological level, localizing the resulting destruction inside the cells. MS No. 2 caused the redox balance to shift towards oxidants, potentiating the generalization of the destruction process. An increase in the content or a longer stress-inducing effect of PM2.5 causes a depletion in the reserve capacity of the AOS and the transition of destruction processes to the systemic level, which contributes to the development of the preconditions for environmentally dependent pathology.

Exosomes from dysfunctional chondrocytes affect osteoarthritis in Sprague-Dawley rats through FTO-dependent regulation of PIK3R5 mRNA stability.

AimsExosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages.MethodsRat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot.ResultsDC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001).ConclusionExosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA.Cite this article: Bone Joint Res 2022;11(9):652–668.

Glycyrrhizic Acid Protects Experimental Sepsis Rats against Acute Lung Injury and Inflammation.

Background The incidence of acute lung injury/acute respiratory distress (ALI/ARDS) is high in sepsis aggravating morbidity and mortality. Glycyrrhizic acid (GA) has pharmacological activities in the treatment of inflammation and antiviral. Materials and Methods Sepsis rats were constructed by the cecal ligation and puncture (CLP) surgery. After GA (25 and 50 mg/kg) injection, the survival rate, blood oxygen, biochemical indexes, myeloperoxidase (MPO) activity, and wet/dry weight ratio of the lung were observed. The bronchoalveolar lavage fluid was collected to count the cells and measure the level of TNF-α, IL-1β, IL-10, and high mobility group box-1 protein (HMGB1). Lung tissue sections were taken to observe the levels of histopathological injury and apoptosis by HE and TUNEL staining. The levels of HMGB1, TLR4, p-38 MAPK, NF-κB, and ERK1/2 proteins were observed by immunohistochemistry and Western blot. Results GA treatment improved the survival rate, blood oxygen, ALT, AST, BUN, and Scr of CLP rats. It could advance the MPO activity, the wet/dry weight ratio, histopathological injury, apoptosis, and the IL-10 level in the lung. After GA injection, the number of total cells, neutrophils, and macrophages in the CLP rats was reduced and the levels of TNF-α, IL-1β, HMGB1, TLR4, p-38 MAPK, and ERK1/2 in the CLP rat were also repressed. Conclusions GA treatment may improve the sepsis-induced ALI/ARDS and inflammation by inhibiting HMBG1. This study provided an experimental basis for the prevention and treatment of ALI/ARDS caused by sepsis.

Silica exposure activates non-canonical inflammasome complex in intratracheal instilled rat model.

Inhalation of silica crystals in occupational settings is a main cause of silicosis, a chronic irreversible pulmonary disorder. Our prior studies demonstrated the activation of inflammasome sensors AIM2 and NLRP3, effector protein caspase-1, and significant increase in IL-1β in silica exposed rats, suggesting that the canonical inflammasome activation may be associated with silica-induced tissue damage and inflammation. In our current study using the same animal model system, we further evaluated the components of non-canonical inflammasome, including NEK7, caspase-11, and GSDMD following silica exposure. We demonstrated sustained NEK7 elevation in the rat lung epithelial cells and macrophages following 1- and 3-day exposure. Enhanced NEK7 expression was also detected in lung homogenate by western blot. Similarly, caspase-11 expression was induced by silica exposure in lung sections and homogenate. Elevated GSDMD was observed both in lung sections by immunohistochemical staining and in lung tissue homogenate by western blot. In summary, our current study demonstrated increase in NEK7, caspase-11, and GSDMD in silica exposed rats, indicating activation of non-canonical inflammasome complex, thereby providing a broad inflammasome activation pathway caused by silica exposure.

Role of bone marrow mesenchymal stem cell-conditioned media combined with polystyrene nanoparticles in rat osteomyelitis and macrophage polarization

This study aimed at investigating the potential role of bone marrow mesenchymal stem cell-conditioned media (BMSC-CM) incorporated with polystyrene nanoparticles (PS-NPs) in inflammatory response in osteomyelitis and clarifying its underlying mechanism. BMSCs were collected from 25 osteomyelitis and healthy rats, and their conditioned media (CM) were collected. Enzyme-linked Immunosorbent Assay (ELISA) was used to determine the expression levels of inflammatory factor IL-10, while the BMSCs were identified by surface antigens. After treatment of BMSCs, BMSC-CM, and BMSC-CM with PS-NPs, flow cytometry was conducted to assess apoptosis of inflammatory cells and macrophage polarization-specific protein, and mRNA expressions were detected. The expression level of IL-10 was up-regulated in the osteomyelitis rats. With primary BMSCs in a long spindle shape, the MSCs were positive for CD44 (99.87%) and negative for antigen CD34 (4.32%). The BMSC-CM combined with PS-NPs caused increased macrophage polarization-related genes (BMP-2, MCP-1, iNOS, CD206, Arg-1, TGF-β) with decreased IL-10 expression. The BMSC-CM combined with PSNPs might inhibit the polarization of BMSCs to macrophages and alleviate inflammation in osteomyelitis.

Effect of Er Miao San on peritoneal macrophage polarisation through the miRNA-33/NLRP3 signalling pathway in a rat model of adjuvant arthritis

Context Er Miao San (EMS) is a formulation that contains Atractylodis Rhizoma and Phellodendri Cortex in 1:1 ratio, and is commonly used to treat rheumatoid arthritis (RA) and other inflammatory diseases. Objective We investigated the mechanism of action and effects of EMS on peritoneal macrophage differentiation in a rat model of adjuvant arthritis (AA). Materials and methods EMS (3, 1.5 and 0.75 g/kg; once daily) and methotrexate (0.5 mg/kg; once every 3 days) were administered orally from days 21 to 35 after immunisation. Paw swelling and arthritis index were measured; pathological changes in the ankle joint were observed using x-ray and haematoxylin eosin staining. The ratio of CD86/CD206 in macrophages was detected by flow cytometry. Examination of the miRNA-33/NLRP3 signalling pathway was examined by RT-qPCR and western blotting. The levels of cytokines in the serum and cell supernatants were tested by ELISA. Results EMS significantly reduced the AA index in rats (from 11.0 to 9.3) and pathological changes in the ankle joint (from 3.8 to 1.4). The ratio of CD86/CD206 was reduced, and polarisation to M1 improved (from 0.9 to 0.6) in macrophages of EMS-treated rats. EMS downregulated the miRNA-33/NLRP3 pathway. Furthermore, EMS treatment increased IL-10 and TGF-β levels in the serum and supernatant of macrophages of AA rats and simultaneously decreased the levels of IL-1β and TNF-α. Discussion and conclusions Our results suggest that EMS may reduce macrophage polarisation to the M1 inflammatory phenotype by downregulating the miRNA-33/NLRP3 pathway in AA rats. These findings may provide new insights into the treatment of RA.

Butyric Acid Ameliorates Myocardial Fibrosis by Regulating M1/M2 Polarization of Macrophages and Promoting Recovery of Mitochondrial Function

BackgroundWe aimed to investigate the effect and mechanism of butyric acid on rat myocardial fibrosis (MF).Methods16S rRNA sequencing was used to analyze the gut microbiota characteristics of the Sham group and MF group. HPLC was applied to measure butyric acid in the feces and serum. In vitro, rat macrophages RMa-bm were stimulated with LPS and IL-4, respectively, and then butyrate was added to study the influences of butyrate on M1/M2 polarization and mitochondrial function of rat macrophages. The rat macrophages and rat myocardial fibroblasts were co-cultured to explore the effect of butyrate on rat myocardial fibroblasts. In addition, MF rats were fed with butyric acid diet.ResultsCompared with the Sham group, collagen deposition in the MF group was increased, and fibrosis was serious. The abundance of Desulfovibrionaceae and Helicobacteraceae in the MF group was increased compared with the Sham group. Gut epithelial cells were destroyed in the MF group compared with the Sham group. Compared with the Sham group, LPS content in the MF group was increased and butyric acid was decreased. Butyrate inhibited M1 and promoted M2. Furthermore, butyrate may promote mitochondrial function recovery by regulating M1/M2 polarization of macrophages. After adding butyrate, cell proliferation ability was decreased, and aging and apoptosis were increased, which indicated that butyrate inhibited rat myocardial fibroblasts activity. Moreover, butyric acid could protect mitochondria and improve the symptoms of rats with MF.ConclusionsButyric acid ameliorated MF by regulating M1/M2 polarization of macrophages and promoting recovery of mitochondrial function.

Phenotypic Characterization of Macrophage and Monocyte Populations in the NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi Rat Model

BackgroundThe NLR family pyrin domain containing 3 (NLRP3) inflammasome is a multimeric protein complex that is activated in response to pathogen and damage associated molecular patterns (PAMPs &amp; DAMPs) to initiate inflammatory responses through IL‐1ß, IL‐18, and pyroptosis. Pharmacological inhibition of NLRP3 attenuates inflammatory responses after myocardial infarction and hypertension in rodents. However, the effect of NLRP3 gene loss on basal macrophage and monocyte populations in rats is unknown. We hypothesized that NLRP3 gene loss would not alter macrophage and monocyte phenotype under basal conditions.MethodsCRISPR‐Cas9 was used to induce a 14 base pair deletion in exon 1 of the NLRP3 gene in the DAHL/SSJrHsdMcwi strain to produce NLRP3 KO DAHL/SS‐Nlrp3em2Mcwi rats. NLRP3 KO male (n=11), NLRP3 KO female (n=10), wild type (WT) male (n=5), and WT female (n=10) were studied at 4‐5 mo. Teklad 7034 low salt (0.12% NaCl) diet was provided ad libitum. Proinflammatory M1 (CD11b+CD68+) and anti‐inflammatory M2 (CD11b+CD206+) macrophages (Mɸs) were quantified in CD45+ cells from left ventricle (LV), peripheral blood monocytes (PBMCs), bone marrow (BMDMs), and splenocytes using density gradient centrifugation and flow cytometry.ResultsSex (p&lt;0.01) and genotype (p&lt;0.01) main effects were observed in several morphological characteristics. NLRP3 KO and male rats exhibited significantly increased rat, kidney and LV weights, and LV/tibial length vs WT and female, respectively. Males also displayed significantly increased spleen length and visceral adipose weight vs females (p&lt;0.01). In LV, the percent of CD11b+CD68+ cells was 28% greater in NLRP3 KO vs WT (n=1‐2/group). In PBMCs, the percent of CD11b+CD206+ cells was 13% greater in WT female vs males and NLRP3 KO (n=2‐4/group), while NLRP3 KO females exhibited ~20% less CD11b+CD68+ in splenocytes (n=1‐4/group). In BMDMs, ~60% were positive for CD11b+CD68+, while ~2% were CD11b+CD206+ across all groups (n=1‐2/group).ConclusionPreliminary data suggest that NLRP3 gene loss on a Dahl salt sensitive background is associated with increased proinflammatory M1 Mɸs in LV concomitant with increased LV mass, independent of sex. We hypothesize that NLRP3 may possess an inflammasome independent role in regulating resident LV macrophages under basal conditions.

L‐Citrulline Modulates Macrophage Polarization to an M2 Phenotype in a Model of Lipopolysaccharide‐Induced Lung Injury in Neonatal Rats

IntroductionBronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants resulting from inflammation and oxidative stress, leads to arrested pulmonary development and lifelong respiratory morbidities. Macrophages are the most abundant immune cells in the neonatal lung, orchestrating inflammation, maintaining homeostasis, and supporting tissue remodeling and repair. Macrophages can polarize into specific phenotypes in response to the micro‐environment. M1 macrophages are pro‐inflammatory and pathogen killing whereas, M2 macrophages are anti‐inflammatory and support normal lung development. Existing therapies for BPD have inherent side effects. L‐Citrulline (L‐CIT), a non‐essential amino acid, that is safe and well‐tolerated by infants, reduces oxidative stress and lung inflammation. Thus, we hypothesize that L‐CIT modulates macrophage polarization to an M2 phenotype in the presence of lipopolysaccharide (LPS) induced lung injury during early development.MethodsSprague Dawley rat pups received saline (SAL) or L‐CIT (2.5 g/kg) intraperitoneally during postnatal day (PND) 4‐9. In addition, pups were treated with LPS (5mg/kg) or SAL intrapharyngeally during PND 5‐8. Thus, we had 4 treatment groups, ie., SAL+SAL (controls), LPS+SAL, LPS+L‐CIT and L‐CIT+SAL. Pups were euthanized on PND 9. Bronchoalveolar Lavage Fluid (BALF): BALF was cytospinned, and cells were counted following hematoxylin and eosin staining. BALF supernatant was collected, and an arginase activity assay was performed for M2 marker expression. Lung homogenate: Lungs were dissected, homogenized and processed for western blotting of M2 (CD206) and M1 (Arg2) markers normalized to rat macrophage marker (F4/80).ResultsBALF analysis: The number of alveolar macrophages infiltrated in the BALF of animals treated with L‐CIT+LPS (n=4) and those treated with SAL+LPS (n=4) were similar; both were significantly higher than control (p&lt;.001; p&lt;.0001, respectively). Arginase activity in the BALF was significantly higher in animals treated with L‐CIT+LPS (p&lt;.05; n= 4) compared to LPS alone (p&lt;.05; n=4). Lung homogenate analysis: LPS+SAL treated animals had lower levels of protein expression for CD206 (M2 marker) (p&lt;.05; n=4) compared to control animals. Whereas L‐CIT+LPS treated animals had increased expression of CD206 (M2 marker) (p&lt;.05; n=4) and lower expression of Arg2 (M1 marker) (p&lt;.001; n=4) compared to control animals.ConclusionIn BPD pathogenesis, there is a major shift towards M1 polarization due to the presence of pro‐inflammatory stimuli in the microenvironment, leading to arrested lung development. Our preliminary findings support a role for L‐CIT in regulating macrophage polarization, favoring an anti‐inflammatory M2 phenotype in the presence of inflammatory injury. This further suggests that L‐CIT, a safe nutritional supplement, could reduce inflammatory lung injury during early development.

Experimental and clinical substantiation of the use of xymedon as an antioxidant in the complex treatment of patients with chronic osteomyelitis

The antioxidant activity of blood plasma is studied in patients with chronic osteomyelitis. The experimental data on the effect of antioxidants of xymedone and methyluracil on the basic free radicals, peroxide oxidation of li poids and chemoluminescence of peritoneal macrophages of rats made it possible to justify the use of drugs in the complex treatment of patients in pre- and postoperative period. As a result of the use of xymedone in the complex treatment the blood indices improved, antioxidant plasma activity increased in 25 patients of the basic group. In patients of the control group taking methyluracil the efficiency of the treatment was less significant