Galangin ameliorated pulmonary fibrosis in vivo and in vitro by regulating epithelial-mesenchymal transition

Pulmonary fibrosis (PF) is a disease that is characterized by abnormal epithelial-mesenchymal transition (EMT) and persistent inflammatory injury, with high mortality and poor prognosis, but the current therapies are accompanied by certain adverse side effects. In this study, we investigated the role of galangin (GA), an anti-inflammatory and anti-tumoral phytochemical extracted from galangal, in preventing and curing bleomycin (BLM)-induced pulmonary fibrosis and the underlying mechanism. Histopathological staining confirmed that GA dramatically moderated bleomycin-induced pulmonary fibrosis in mice. Compared with the vehicle treatment, GA treatment inhibited the expression of vimentin and increased the expression of E-cadherin. The expression of α-Smooth muscle actin (α-SMA), which is a myofibroblast marker, was also suppressed. In addition, GA diminished the increase in the numbers of CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells and dendritic cells induced by bleomycin, and reduced the residence of inflammatory cells in the lung tissues. Notably, GA inhibited the TGF-β1-induced EMT and fibroblast differentiation in vitro, which further confirmed the potential protective effect of GA on pulmonary fibrosis. Taken together, our results suggest that GA exerts a beneficial effect on bleomycin-induced pulmonary fibrosis by attenuating EMT and inflammatory damage and may have prevent potential of pulmonary fibrosis.     

Thunbergia laurifolia leaf extract partially recovers lead-induced renotoxicity through modulating the cell signaling pathways

This research investigated the reno-protective effect of Thunbergia laurifolia Linn. (TL) in a lead-induced toxicity test through the modulation of cell signaling pathways. The study carried out to evaluate the effect of TL leaf extracts in Swiss Albino mice exposed to lead acetate (PbAc). Prior to in vivo study, a probable kidney-protective effect of the plant leaf extract was presumed through an activity-specific (PASS) molecular docking analysis. In animal model study, albino mice were divided in seven groups and co-treated with PbAc and TL (100, 200 mg/kgBW) or vitamin E (100 mg/kgBW) for 38 days, whereas the untreated control, TL control, and vehicle control groups received sodium acetate, PbAc, sodium acetate plus mineral oil, respectively. At the end of treatment, blood and kidney tissue were collected for investigating Pb concentration, estimating biochemical profile, evaluating oxidative stress and inflammatory parameters. The histopathological change of kidney along with apoptosis was assessed from kidney sections using H & E staining and TUNEL assay. Pb-exposed mice were found to be increased concentration of Pb in the blood and kidney sample, which further led to increased MDA levels in the plasma, blood, and tissue. Followed by kidney damage, increased expression of TNF-α, iNOS, and COX-2 in kidney tissues were noticed, which were related to elevated TNF-α in the systemic circulation of Pb-treated mice. Co-treatment with TL or vitamin E significantly reduced altered structure and apoptosis of kidney tissues. Downregulation of inflammatory markers especially TNF-α, iNOS, and COX-2 with simultaneous improvement of renal function through reduced plasma BUN and creatinine levels demonstrate that TL act as a potential dietary supplement to detoxify Pb in kidney showing an antioxidant and anti-inflammatory effect.     

C-Phycocyanin-a novel protein from Spirulina platensis- In vivo toxicity,antioxidant and immunomodulatory studies

A pigment-protein highly dominant in Spirulina is known as C-Phycocyanin. Earlier, in vitro studies has shown that C-phycocyanin is having many biological activities like antioxidant and anti-inflammatory activities, antiplatelet, hepatoprotective, and cholesterol-lowering properties. Interestingly, there are scanty in vivo experimental findings on the immunomodulatory and antioxidant effects of C-phycocyanin. This work is aimed at in vivo evaluation of the effects of C-phycocyanin on immunomodulation and antioxidant potential in Balb/c mice. Our results of in vivo toxicity, immunomodulatory and antioxidant effects of C-Phycocyanin suggests that C-phycocyanin is very safe for consumption and having substantial antioxidant potential and also possess immunomodulatory activities in Balb/c mice in a dosage dependent manner. C-phycocyanin doesn’t cause acute and subacute toxicity in the animal model (male, Balb/c mice) studied. We have reported that C-phycocyanin exhibited in vivo immunomodulation performance in this animal model.     

Paeonol alleviates primary dysmenorrhea in mice via activating CB2R in the uterus

Background and purposePrimary dysmenorrhea is the most common gynaecologic problem in menstruating women and is characterized by spasmodic uterine contraction and pain symptoms associated with inflammatory disturbances. Paeonol is an active phytochemical component that has shown anti-inflammatory and analgesic effects in several animal models. The aim of this study was to explore whether paeonol is effective against dysmenorrhea and to investigate the potential mechanism of cannabinoid receptor signalling.Experimental approachDysmenorrhea was established by injecting oestradiol benzoate into female mice. The effects of paeonol on writhing time and latency, uterine pathology and inflammatory mediators were explored. Isolated uterine smooth muscle was used to evaluate the direct effect of paeonol on uterine contraction.Key resultsThe oral administration of paeonol reduced dysmenorrhea pain and PGE2 and TNF-α expression in the uterine tissues of mice, and paeonol was found to be distributed in lesions of the uterus. Paeonol almost completely inhibited oxytocin-, high potassium- and Ca²⁺-induced contractions in isolated uteri. Antagonists of CB2R (AM630) and the MAPK pathway (U0126), but not of CB1R (AM251), reversed the inhibitory effect of paeonol on uterine contraction. Paeonol significantly blocked L-type Ca²⁺ channels and calcium influx in uterine smooth muscle cells via CB2R. Molecular docking results showed that paeonol fits well with the binding site of CB2R.Conclusions and implicationsPaeonol partially acts through CB2R to restrain calcium influx and uterine contraction to alleviate dysmenorrhea in mice. These results suggest that paeonol has therapeutic potential for the treatment of dysmenorrhea.     

Histological changes in placental rat apoptosis via FasL and cytochrome c by the nano-herbal Zanthoxylum acanthopodium

Zanthoxylum acanthopodium has several biological activities, such as antimicrobial, anti-inflammatory, and antioxidant properties. This strong antioxidant herb can be used as a drug for hypertension. FasL and cytochrome c expression play roles in the apoptotic pathway in the placenta. This study focused on the histological change in apoptosis via cytochrome c and Fas ligand expression by investigating whether Zanthoxylum acanthopodium (ZA) fruits affect apoptosis. The present study consisted of five treatments: Normal pregnant rats (C), Hypertension rats (C + ), hypertension rats + extra virgin olive oil (EVOO) (T1), Hypertension rats + ZA (T2), and hypertension rats + EVOO + ZA (T3). Hypertension was induced in rats by injecting 3 mlml of 6% NaCl. Nanoherbal of ZA (100 mg/kg BW) and EVOO (1 ml) were given on the 13th–19th days of pregnancy. Pregnant rats were dissected on the 20th day of pregnancy by cervical dislocation. ELISA assays were performed for the analysis of HSP-70 expression. Immunohistochemistry and TUNEL assays were used to analyse the histological changes in placental tissue. The results showed that cytochrome c and FasL protein exposure levels in the labyrinth, basal, and yolk sac zones were increased during hypertensive pregnancy (P < 0.0001) in rats. The administration of nanoherbal of ZA decreased the expression of cytochrome c and FasL. A significant difference was found in the combination of nanoherbal of ZA and EVOO.     

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels,systemic inflammation,and atherosclerosis

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr^−/− mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr^−/−/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr^−/− mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

Regenerating islet-derived protein (Reg)3β plays a crucial role in attenuation of ileitis and colitis in mice

Regenerating islet-derived protein (Reg)3β belongs to a member of the Reg family of proteins and has pleiotropic functions, including antimicrobial activity and tissue repair. However, whether Reg3β plays a protective role in the development of colitis and ileitis has not been fully investigated. We generated transgenic mice expressing a short form of cellular FLICE-inhibitory protein (cFLIPs) that promotes necroptosis, a regulated form of cell death. cFLIPs transgenic (CFLARs Tg) mice develop severe ileitis in utero. Although Reg3β is undetectable in the small intestine of wild-type embryos, its expression is aberrantly elevated in the small intestine of CFLARs Tg embryos. To test whether elevated Reg3β attenuates or exacerbates ileitis in CFLARs Tg mice, we generated a Reg3b^?/? strain. Reg3b^?/? mice grew to adulthood without apparent abnormalities. Deletion of Reg3b in CFLARs Tg mice exacerbated the embryonic lethality of CFLARs Tg mice. Dextran sulfate sodium-induced colitis, characterized by body weight loss and infiltration of neutrophils, was exacerbated in Reg3b^?/? compared to wild-type mice. Moreover, the expression of Interleukin 6, an inflammatory cytokine and Chitinase-like 3, a marker for tissue repair macrophages was elevated in the colon of Reg3b^?/? mice compared to wild-type mice after DSS treatment. Together, these results suggest that attenuation of colitis and ileitis is a result of Reg3β′s real function.     

Application of Magnetic Resonance Imaging in the Evaluation of Disease Activity in Graves’ Ophthalmopathy

ObjectiveTo evaluate magnetic resonance imaging parameters, T2 signal intensity ratios (SIRs), and normalized apparent diffusion coefficients (n-ADC) of the extraocular muscles (EOMs) in the identification of different stages of Graves’ ophthalmopathy (GO) and to find out the correlation of T2-SIRs and n-ADC values with disease changes after anti-inflammatory treatment.MethodsAltogether, 43 patients (86 orbits) were enrolled and classified into “active” or “inactive” stages by clinical activity score (CAS). Twenty-three (53.5%) patients received anti-inflammatory treatment and underwent a follow-up evaluation. Fifteen age- and gender-matched control participants (30 orbits) were included. T2-SIRs and n-ADC values of EOMs were calculated among GO and healthy controls and were correlated with CAS. Changes in these parameters were also evaluated before and after anti-inflammatory treatment.ResultsMean T2-SIRs and n-ADC values were both significantly higher in GO patients than in controls and higher in active GO than in inactive GO. In the inactive stage, n-ADC values of inferior rectus muscles were still higher than those in healthy controls. Both T2-SIRs and n-ADC values decreased after intravenous steroid pulse therapy. The cutoff value of pretreatment n-ADC was 1.780 to detect stages with specificity of 93.7% and sensitivity of 48.3% (P = .035).ConclusionT2-SIRs and n-ADC values are valuable magnetic resonance imaging indicators of the inflammatory activity in GO by detecting involvement of EOMs. They are also ideal tools to monitor the efficacy of anti-inflammatory treatment in patients with active stage GO. n-ADC values, when combined with CAS, can be promising predictive factors in the detection of stages of diseases.     

O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano–liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut–microbiota interface.     

B cells from Patients with Rheumatoid Arthritis Show Conserved CD39-Mediated Regulatory Function and increased CD39 Expression After Positive Response to Therapy

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction associated with increased pro-inflammatory mediators. In inflammatory microenvironments, exogenous ATP (eATP) is hydrolyzed to adenosine, which exerts immunosuppressive effects, by the consecutive action of the ectonucleotidases CD39 and CD73. Mature B cells constitutively express both ectonucleotidases, converting these cells to potential suppressors. Here, we assessed CD39 and CD73 expression on B cells from treated or untreated patients with RA. Neither the frequency of CD73⁺CD39⁺ and CD73^-CD39⁺ B cell subsets nor the levels of CD73 and CD39 expression on B cells from untreated or treated RA patients showed significant changes in comparison to healthy controls (HC). CpG+IL-2-stimulated B cells from HC or untreated RA patients increased their CD39 expression, and suppressed CD4⁺ and CD8⁺ T cell proliferation and intracellular TNF-production. A CD39 inhibitor significantly restored proliferation and TNF-producing capacity in CD4⁺ T cells, but not in CD8⁺ T cells, from HC and untreated RA patients, indicating that B cells from untreated RA patients conserved CD39-mediated regulatory function. Good responder patients to therapy (R-RA) exhibited an increased CD39 but not CD73 expression on B cells after treatment, while most of the non-responder (NR) patients showed a reduction in ectoenzyme expression. The positive changes of CD39 expression on B cells exhibited a negative correlation with disease activity and rheumatoid factor levels. Our results suggest modulating the ectoenzymes/ADO pathway as a potential therapy target for improving the course of RA.     

Molecular detection and phylogenetic analyses of Arsenophonus endosymbiont in wild specimens of phlebotomine sand flies from Colombia

Endosymbionts have gained prominence as a potential tool for biological control strategies in reducing vector-borne diseases. This study aimed to evaluate the presence of Arsenophonus, Spiroplasma, and Rickettsia endosymbionts in wild specimens of phlebotomine sand flies, as well as in culicids collected in different regions of Colombia. Analyses were conducted through conventional PCR, Sanger sequencing of the 16S rRNA gene, and phylogenetic analyses. Individuals from among 946 phlebotomine sand flies and 143 mosquitoes were selected for taxonomic identification confirmed through the analysis of the cytochrome oxidase subunit I gene sequences. Results showed the presence of Arsenophonus bacteria in samples of Lutzomyia longipalpis, Psychodopygus panamensis, and Pintomyia evansi. Arsenophonus sequences associated with Lu. longipalpis and Ps. panamensis are phylogenetically located near to sequences of louse flies, with K2P genetic distances of 0.006. In contrast, sequences obtained from Pi. evansi are phylogenetically located near Arsenophonus nasoniae (K2P 0.001–0.014). Other sequences of endosymbionts similar to Arsenophonus with high K2P genetic distances (0.056–0.097), when compared to different reference strains of this endosymbiont, were also found in other samples of Lu. longipalpis and Ae. aegypti. To the best of our knowledge, this is the first successful attempt to detect and elucidate the phylogenetic relationship of Arsenophonus in phlebotomine sand flies, yet its role within these insect vectors remains to be fully determined; therefore, the importance of entomological surveys that help better understand its behavior and potential use as a control agent is required to enable the proactive reduction of sand fly populations.     

Psidium guajava Linn leaf ethanolic extract: In vivo giardicidal potential with ultrastructural damage,anti-inflammatory and antioxidant effects

Introduction and aimConsidering the magnitude of giardiasis problem, the side-effects of the used anti-giardia drugs and the resistance posed against them, the current study aimed to evaluate the in-vivo giardicidal effect of Psidium guajava leaf extract (PGLE).MethodsFor fulfilling this aim, five Swiss-albino mice groups were included; GI: non-infected, GII: Giardia-infected and non-treated, GIII: Giardia-infected and metronidazole-treated, GIV: Giardia-infected and PGLE-treated, and GV: Giardia-infected and treated with both metronidazole and PGLE. Treatment efficacy was assessed via; Giardia cyst viability and trophozoite count, trophozoite electron microscopic ultrastructure, duodenal histopathological scoring, immunohistochemistry for TNF-α and duodenal scanning electron microscopy. Moreover, mice serum liver enzymes, total bilirubin, albumin, lipid profile including; total cholesterol, HDL, LDL and triglycerides were assessed. Additionally, hepatic oxidative stress markers including; malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH) and superoxide dismutase (SOD) were measured.ResultsResults showed that PGLE whether alone or combined with metronidazole has induced significant trophozoite count reduction and major architectural changes. Duodenal histological improvement, and local protective anti-inflammatory effect were confirmed. PGLE has also helped in healing of Giardia-induced gut atrophy. Thus, offered a comprehensive therapy for both the pathogen and the resultant pathological sequalae. Serum markers showed favorable hepatoprotective effect. Total cholesterol, LDL and triglycerides levels were less in PGLE-treated group than in metronidazole-treated group. Hepatic oxidative stress markers revealed the promising extract antioxidant effect. This study highlights, the promising in-vivo giardicidal PGLE activity, that was comparable to metronidazole, thus, the extract would be an ideal strongly recommended treatment for giardiasis. When combined with metronidazole, the extract potentiated its therapeutic effect. Besides, having hepatoprotective, anti-inflammatory, and antioxidant properties, the extract can combat the major side effects of metronidazole therapy.     

β-Carotene enhances the expression of inflammation-related genes and histone H3 K9 acetylation,K4 dimethylation,and K36 trimethylation around these genes in juvenile macrophage-like THP-1 cells

β-Carotene is converted into vitamin A in the body and can remove reactive oxygen species. However, it is still unclear whether β-carotene alters the expression levels of inflammation-related genes in macrophages and how this is regulated. In the present study, we investigated whether the administration of β-carotene under hyperglycemic conditions altered the expression level of inflammation-related genes and whether any observed differences were associated with changes in histone modifications in juvenile macrophage-like THP-1 cells. THP-1 cells (from a human monocytic leukemia cell line) were cultured in low glucose (5 mM), high glucose (25 mM), or high glucose (25 mM) + β-carotene (5 μM) media for 1 day, and mRNA expression levels of genes related to oxidative stress and inflammation, and histone modifications were determined by mRNA microarray and qRT-PCR analyses, and chromatin immunoprecipitation assays, respectively. The expression of inflammation-related genes, such as IL31RA, CD38, and NCF1B, and inflammation-associated signaling pathway genes, such as ITGAL, PRAM1, and CSF3R, were upregulated by β-carotene under high-glucose conditions. Under these conditions, histone H3 lysine 4 (K4) demethylation, H3K36 trimethylation, and H3K9 acetylation around the CD38, NCF1B, and ITGAL genes were higher in β-carotene-treated cells than in untreated cells. Treatment of juvenile macrophage-like THP-1 cells with β-carotene under these high glucose conditions induced the expression of inflammation-related genes, K9 acetylation, and K4 di- and K36 trimethylation of histone H3 around these genes.