Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus

Parasitology ◽  
1998 ◽  
Vol 116 (6) ◽  
pp. 525-532 ◽  
Author(s):  
C. LOGULLO ◽  
I. DA SILVA VAZ ◽  
M. H. F. SORGINE ◽  
G. O. PAIVA-SILVA ◽  
F. S. FARIA ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fat Body ◽  
Sodium Dodecyl ◽  
Aspartic Proteinase ◽  
Boophilus Microplus ◽  
Proteolytic Processing ◽  
Protein Sequencing ◽  
Hard Tick

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS–PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S] methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3·5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

scholarly journals Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

2012 ◽  
Vol 49 (No. 8) ◽  
pp. 305-311 ◽  
Author(s):  
G. Ozbey ◽  
H. Ongor ◽  
D. T Balik ◽  
V. Celik ◽  
A. Kilic ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Major Band ◽  
Cell Extracts ◽  
Sds Page ◽  
Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

scholarly journals Subcellular Localization of the Intracellular Survival-Enhancing Eis Protein of Mycobacterium tuberculosis

2001 ◽  
Vol 69 (7) ◽  
pp. 4295-4302 ◽  
Author(s):  
John L. Dahl ◽  
Jun Wei ◽  
James W. Moulder ◽  
Suman Laal ◽  
Richard L. Friedman
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intracellular Survival ◽  
Terminal Amino Acid ◽  
Terminal Amino

ABSTRACT Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designatedeis, was found to enhance intracellular survival ofMycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377–384, 2000). Wheneis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eisgene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product ofeis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

scholarly journals Detection of immunogenic protein from salivary gland of Aedes albopictus

2021 ◽  
Vol 40 (3) ◽  
pp. 234-242
Author(s):  
Rike Oktarianti ◽  
Rochmatul Nuryu Khasanah ◽  
Syubbanul Wathon ◽  
Kartika Senjarini
Keyword(s):  
Salivary Gland ◽  
Western Blot ◽  
Salivary Glands ◽  
Aedes Albopictus ◽  
Blot Analysis ◽  
Gel Electrophoresis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Immunogenic Proteins ◽  
Healthy Persons

BackgroundDengue virus is transmitted by several species of Aedes mosquitoes, with Aedes albopictus as secondary vector. During blood feeding, these vectors inject saliva into the vertebrate hosts. The saliva contains anticoagulant, anti-inflammatory and immunogenic factors. The objective of this research was to detect immunogenic proteins from Ae.albopictus salivary glands reacting with sera of people living in dengue endemic areas. MethodsThe identification of immunogenic proteins of Ae. albopictus salivary gland used one-dimensional gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and western blot analysis, respectively. To determine the immunogenic nature of the candidate proteins, the antigens from the salivary gland of Ae. albopictus were reacted with sera from healthy persons, dengue hemorrhagic fever (DHF) patients, and neonates, each of the groups comprising 10 samples. ResultsThe protein profiles of Ae. albopictus salivary glands showed 13 bands with molecular weights from 16 kDa up to 97 kDa, i.e. 16, 17, 26, 28, 31, 32, 45, 55, 60, 67, 73, 76, and 97 kDa. According to western blot analysis result, the 31 kDa proteins were recognized in all endemic population sera, both in DHF patients and healthy persons. In contrast, protein bands of 47 and 67 kDa were only recognized by the sera of DHF patients. ConclusionThree immunogenic proteins of 31, 47 and 67 kDa were detected from Ae. albopictus salivary glands. These immunogenic proteins may be developed as candidate biomarkers for bite exposure to Ae. albopictus and as vector-based DHF vaccines.

scholarly journals First Report of Barley yellow streak mosaic virus-Infected Barley in Alaska

Plant Disease ◽  
2000 ◽  
Vol 84 (5) ◽  
pp. 595-595 ◽  
Author(s):  
N. L. Robertson ◽  
S. K. Brumfield
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mosaic Virus ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Virus Disease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Mechanical Transmission

Barley yellow streak mosaic virus (BaYSMV) was first described and reported in Montana and Alberta, Canada, more than 17 years ago (1). Since then, it has been detected in two other locations: Pocatello Valley, ID (3), and across the border in Utah. BaYSMV has now been found in the Alaskan interior. In July 1999, dry-land barley (Hordeum vulgare L.) growing in University of Alaska-Fairbanks experimental plots exhibited symptoms similar to those described for BaYSMV, including parallel chlorotic streaks and leaf banding. Mechanical inoculation of Nicotiana benthamiana with diseased barley sap produced systemic mosaic symptoms. As previously reported for BaYSMV sap-transmission tests (2), parallel inoculations to barley plants yielded no symptoms. Electron microscopy of leaf dips and minipurifications of infected N. benthamiana revealed long filamentous particles that matched the size and shape reported for BaYSMV (1). Ultrathin sections of diseased barley and N. benthamiana leaves displayed characteristic virus particles. BaYSMV was confirmed by immuno-sorbent electron microscopy assays (4) and western blot analysis with polyclonal antiserum. Long filamentous BaYSMV particles appeared only on grids coated with BaYSMV antiserum and exposed to diseased N. benthamiana sap. Total protein extracts from diseased barley tissue and inoculated N. benthamiana, as well as with protein extracted from partially purified preparations, were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigel and stained with Coomassie blue. Diseased samples, but not healthy controls, contained a protein of ≈33 kDa that was within the size range of a previously described protein from partially purified BaYSMV particles (2). Western blot analysis with an Immuno-Blot alkaline phosphatase assay system (Bio-Rad Laboratories, Hercules, CA) confirmed that the protein reacted with polyclonal BaYSMV. This is the first serological documentation of a BaYSMV-specific protein and that the ≈33-kDa protein is the main antigen recognized by the BaYSMV polyclonal antiserum. Based on virus particle shape and size, symptomology, mechanical transmission host range, and serology, we conclude that BaYSMV is associated with the barley disease observed. Barley yellow streak mosaic virus disease outbreaks are associated with recurring drought and are accompanied by infestations of the brown wheat mite vector, Petrobia latens Müller (1), so it is not surprising that this report coincides with abnormally dry conditions occurring throughout the 1990s in the interior of Alaska. References: (1) N. L. Robertson and T. W. Carroll. Science 240:1188, 1988. (2) N. L. Robertson and T. W. Carroll. Plant Dis. 75:839, 1991. (3) J. S. Skaf et al. Plant Dis. 76:861, 1992. (4) J. S. Skaf and T. W. Carroll. Plant Dis. 79:1003, 1995.

Cytoskeletons of X-Linked GATA-1, G208S Macrothrombocytes Are Deficient in Talin.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1829-1829
Author(s):  
James G. White ◽  
Steven M. Burris ◽  
Angela Thomas
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Single Cells ◽  
Sodium Dodecyl ◽  
Normal Subjects ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Integrin Activation ◽  
Normal Platelet

Abstract Germline mutations in the X-linked transcription factor, GATA-1, cause several hematopoietic disorders resulting in anemia and/or macrothrombocytopenia. Features of the platelet ultrastructural pathology, including hypo- and agranular megathrombocytes (Mtc) in all mutations, tubular membrane sheets often in parallel association in megakaryocytes and Mtc of patients with the V205M and G208S variations, platelets sequestered in Mtc and platelets in platelets in Mtc (as many as five in one Mtc) in the V205M, G208S and R216Q disorders, together with platelets attached to platelets attached to platelets forming large Mtc in the V205M and G208S mutations have helped to explain how Mtc are formed and why they fail to separate from proplatelets of megakaryocytes and enter the circulation as single cells. However, the findings do not explain why patients with GATA-1 mutations often have serious bleeding problems. Major receptors for hemostasis and thrombosis are present on GATA-1 Mtc, although, in some cases, slightly reduced in frequency. Yet, while the cells bind to and spread fully on glass and plastic, they adhere poorly to vWF or collagen coated glass slides under flow conditions, aggregate less well than normal platelets when stirred with collagen or ristocetin in an aggregometer and fail to retract clots. The findings suggest that internal activation mechanisms for the surface receptors are defective. The present study has solubilized GATA-1 Mtc from two males with the G208S mutation and normal platelets in sodium dodecyl sulfate/ EDTA in the presence of protease inhibitors and analyzed their cytoskeletal proteins on reduced, one dimensional polyacrylamide gels using a Mini-Slab Gel Apparatus. Gels were electrophoresed by the method of Laemmli. Proteins were stained with colloidal Coomassie G250 blue. GATA-1 Mtc cytoskeletal proteins were the same as those from the normal subjects, except for the absence of Talin. Western blot analysis to prove the missing protein is Talin was carried out. Normal platelet and GATA-1 Mtc cytoskeletal proteins were transferred to nitrocellulose sheets. Identification and localization of the missing protein was accomplished using a mouse monoclonal antibody against human Talin and a secondary anti-mouse IgG peroxidase conjugate. The Western blot analysis confirmed that the missing Mtc protein is Talin. Recent studies have shown that binding of the 235–245 kD actin-binding protein Talin to the β subunit cytoplasmic tails of integrins is the final step of integrin activation. The absence of Talin in GATA-1 Mtc may result in failure of full integrin activation accounting for functional failure in hemostasis.

scholarly journals A simple and efficient purification method of native immunoreactive antigen for diagnosis of camel hydatidosis

2020 ◽  
Vol 13 (1) ◽  
pp. 141-146
Author(s):  
Nagwa I. Toaleb ◽  
Mohamed S. Helmy ◽  
Eman E. El Shanawany ◽  
Eman H. Abdel-Rahman
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Indirect Elisa ◽  
Sodium Dodecyl ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Morbidity ◽  
Diagnostic Efficacy ◽  
Sds Page

Background: Cystic echinococcosis (CE), a zoonotic disease that affects animal and human health, is of increasing economic importance due to high morbidity rates and high economic losses in the livestock industry. Aim: The present study was conducted to purify the antigen from hydatid cyst fluid (HCF) with high diagnostic efficacy of camel hydatidosis using indirect enzyme-linked immunosorbent assay (ELISA). Materials and Methods: The HCF antigen was purified using Sephacryl S-300 column chromatography. Characterization of fractions was performed using reducing and non-reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Further, antibodies against Echinococcus granulosus cysts in camel serum were detected using indirect ELISA. Results: The purification process resulted in three fractions of antigens: FI, FII, and FIII. Indirect ELISA showed that higher diagnostic efficacy was observed in FI than in FII and FIII. Indirect ELISA, in which FI was utilized, showed 88% sensitivity and 91.7% specificity. Non-reducing SDS-PAGE showed that FI had two bands of molecular weights 120 and 60 kDa. Western blot analysis of FI demonstrated that 60, 38, and 22 kDa were antigenic bands when reacted with naturally infected camel sera with E. granulosus cysts. Using indirect ELISA, F1 recorded an infection percentage of 81.7% in randomly collected camel serum samples. Conclusion: FI is a promising antigen for accurate diagnosis of camel CE using indirect ELISA.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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