Artesunate Attenuates Pro-Inflammatory Cytokine Release from Macrophages by Inhibiting TLR4-Mediated Autophagic Activation via the TRAF6-Beclin1-PI3KC3 Pathway

Background/Aims: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production during sepsis. Previously, artesunate (AS) was reported to protect septic mice by reducing LPS-induced pro-inflammatory cytokine release. In the present study, the possible mechanism of the anti-inflammatory effect of AS was further investigated. Methods: An enzyme-linked immunosorbent assay was used to detect TNF-α and IL-6 release from macrophages. Specific small interfering RNAs (siRNAs) were used to knockdown the mRNA expression of target genes. Transmission electron microscopy and laser confocal microscopy were used to observe changes in autophagy. Western blotting was performed to detect the protein levels of tumor necrosis factor receptor-associated factor6 (TRAF6), Beclin1, phosphatidylinositol 3-kinase class III (PI3KC3), autophagy-related protein 5 (ATG5), and sequestosome 1. Immunoprecipitation (IP) and fluorescent co-localization were used to detect the interactions between TRAF6–Beclin1 and Beclin1–PI3KC3, and the ubiquitination of Beclin1. Results: AS inhibited TNF-α and IL-6 release from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs) induced by LPS. However, the inhibition by AS of LPS-induced cytokine release decreased when autophagy was inhibited using 3-MA, bafilomycin A1, or a siRNA targeting the Atg5 gene. Notably, AS showed an inhibition of LPS-induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages with down-regulated TRAF6, indicating that AS inhibited LPS-induced cytokine release and autophagic activation via TLR4-TRAF6 signaling. Western blotting results showed AS could reduce the levels of TRAF6, Beclin1, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclin1 by acting on TRAF6. This interrupted the TRAF6–Beclin1 interaction and subsequent the formation of Beclin1– PI3KC3 core complex of the PI3K-III complex. Conclusion: AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclin1-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclin1 and then interrupting the formation of Beclin1-PI3KC3 core complex of the PI3K-III complex. Our findings reveal the mechanism of AS’s anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment.

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Anti-Inflammatory Effect of Cherry Extract Loaded in Polymeric Nanoparticles: Relevance of Particle Internalization in Endothelial Cells

Pharmaceutics ◽

10.3390/pharmaceutics11100500 ◽

2019 ◽

Vol 11 (10) ◽

pp. 500 ◽

Cited By ~ 6

Author(s):

Denise Beconcini ◽

Francesca Felice ◽

Ylenia Zambito ◽

Angela Fabiano ◽

Anna Maria Piras ◽

...

Keyword(s):

Endothelial Cells ◽

Prunus Avium ◽

Umbilical Vein ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Tetrazolium Salt ◽

Tnf Α ◽

Anti Inflammatory ◽

Inflammatory Effect

This study aimed at evaluating the anti-inflammatory effect of natural cherry extract (CE), either free or encapsulated in nanoparticles (NPs) based on chitosan derivatives (Ch-der) or poly(lactic-co-glycolic acid) (PLGA), on human umbilical vein endothelial cells (HUVEC). CE from Prunus avium L. was characterized for total polyphenols, flavonoids, and anthocyanins content. CE and CE-loaded NP cytotoxicity and protective effect on lipopolysaccharide (LPS)-stressed HUVEC were tested by water-soluble tetrazolium salt (WST-1) assay. Pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-10, and PGE2) released by HUVEC were quantified by enzyme-linked immunosorbent assay (ELISA). All NP types were internalized into HUVEC after 2 h incubation and promoted the anti-inflammatory effect of free CE at the concentration of 2 µg gallic acid equivalents (GAE)/mL. CE-loaded Ch-der NPs showed the highest in vitro uptake and anti-inflammatory activity, blunting the secretion of IL-6, TNF-α, and PGE2 cytokines. Moreover, all NPs reduced the production of nitric oxide and NLRP3 inflammasome, and had a stronger anti-inflammatory effect than the major corticosteroid dexamethasone. In particular, the results demonstrate that natural CE protects endothelial cells from inflammatory stress when encapsulated in NPs based on quaternary ammonium chitosan. The CE beneficial effects were directly related with in vitro internalization of CE-loaded NPs.

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The Effects of New Zealand Grown Ginseng Fractions on Cytokine Production from Human Monocytic THP-1 Cells

Molecules ◽

10.3390/molecules26041158 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1158

Author(s):

Wei Chen ◽

Prabhu Balan ◽

David G. Popovich

Keyword(s):

New Zealand ◽

Inflammatory Cytokines ◽

Transforming Growth Factor Beta ◽

Inflammatory Cytokine ◽

Transforming Growth Factor ◽

Enzyme Linked Immunosorbent Assay ◽

High Dose ◽

Tnf Α ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Pro-inflammatory cytokines and anti-inflammatory cytokines are important mediators that regulate the inflammatory response in inflammation-related diseases. The aim of this study is to evaluate different New Zealand (NZ)-grown ginseng fractions on the productions of pro-inflammatory and anti-inflammatory cytokines in human monocytic THP-1 cells. Four NZ-grown ginseng fractions, including total ginseng extract (TGE), non-ginsenoside fraction extract (NGE), high-polar ginsenoside fraction extract (HPG), and less-polar ginsenoside fraction extract (LPG), were prepared and the ginsenoside compositions of extracts were analyzed by HPLC using 19 ginsenoside reference standards. The THP-1 cells were pre-treated with different concentrations of TGE, NGE, HPG, and LPG, and were then stimulated with lipopolysaccharide (LPS). The levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and anti-inflammatory cytokines, such as interleukin-10 (IL-10), and transforming growth factor beta-1 (TGF-β1), were determined by enzyme-linked immunosorbent assay (ELISA). TGE at 400 µg/mL significantly inhibited LPS-induced TNF-α and IL-6 productions. NGE did not show any effects on inflammatory secretion except inhibited IL-6 production at a high dose. Furthermore, LPG displayed a stronger effect than HPG on inhibiting pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) productions. Particularly, 100 µg/mL LPG not only significantly inhibited the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, but also remarkably enhanced the production of anti-inflammatory cytokine IL-10. NZ-grown ginseng exhibited anti-inflammatory effects in vitro, which is mainly attributed to ginsenoside fractions (particularly less-polar ginsenosides) rather than non-saponin fractions.

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Cytoprotective Chaperone Proteins Are Novel Anti-Inflammatory Targets in Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.1292.1292 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1292-1292

Author(s):

Sanjiv Kumar ◽

Ciprian Anea ◽

Itia Lee ◽

Aluya Oseghale ◽

Julia Brittain

Keyword(s):

Inflammatory Response ◽

Inflammatory Cytokine ◽

Chaperone Proteins ◽

Cytokine Release ◽

Cell Disease ◽

Coagulation Activation ◽

Tnf Α ◽

Anti Inflammatory ◽

Proof Of Principle

Abstract Sickle cell disease (SCD) is a pro-inflammatory condition. Levels of TNF-α, IL-6, IL-8, and IL-10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non-crisis "steady state" condition. Additional insult, either via infection or vaso-occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro-inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro-coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti-inflammatory, anti-coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY-922 could block the bacterial toxin lipopolysaccharide (LPS) - induced TF expression and pro-inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY-922 in a pre-clinical study. Townes mice with SCD or without were administered AUY-922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS-mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro-inflammatory response in the AA mice (n=24). However, LPS-induced an exaggerated response in the SS mice. Levels of TNF-α, IL-6, IL-8, and IL-10 were elevated up to 40,000 fold over control treated SS mice. Pre-treatment with AUY-922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY-922 doubled the amount of the anti-inflammatory chaperone molecule HSP70 in the livers of the SS mice. This particular result suggested that the function of HSP90 could be spared, and the induction of HSP70 was potentially sufficient to protect against the LPS-induced insult. Of note, the main function of HSP70 is cytoprotection in response to oxidative and febrile stress. Therefore, we next sought to determine, in a proof of principle in vitro study, whether induction of HSP70 alone was sufficient to block LPS-induced cytokine release and coagulation activation. We treated human monocytes with the HSP70 inducer, celastrol for 24h, followed by treatment with LPS (1µg/ml). We observed a significant release of the cytokines IL-6 and TNF-α with LPS treatment. However, induction of HSP70 via celastrol was sufficient to block this inflammatory response. Furthermore, we observed that celastrol blocked the LPS-induced, TF-specific clotting of plasma in vitro. Interestingly, we also observed that conditioned media from celastrol treated monocytes could block LPS-induced IL-6 release in an HSP70 dependent manner. Thus, secreted HSP70 was an active participant in cellular protection from LPS-induced insult. Initial studies suggest that secreted HSP70 levels may be lower in patients with SCD than in unaffected individuals. Therefore, replacement of this chaperone may be of significant benefit as therapeutic. Thus, taken together, our data demonstrate in both a pre-clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD. Disclosures No relevant conflicts of interest to declare.

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Abstract 088: Chronic Efferent Vagal Stimulation Enhances Norepinephrine-mediated Inhibition of Splenic Pro-inflammatory Cytokine Release in Lupus Hypertension

Hypertension ◽

10.1161/hyp.68.suppl_1.088 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Grace S Pham ◽

Amber S Fairley ◽

Keisa W Mathis

Keyword(s):

Inflammatory Cytokines ◽

Lupus Erythematosus ◽

Inflammatory Cytokine ◽

Vagal Stimulation ◽

Reproductive Age ◽

Cytokine Release ◽

Tnf Α ◽

Anti Inflammatory ◽

Stimulation Of ◽

Pro Inflammatory Cytokines

Hypertension is prevalent in the autoimmune disease systemic lupus erythematosus (SLE), occurring with alarming frequency in reproductive-age women. Recent studies implicate the adaptive immune system in the development and maintenance of hypertension, and neuroimmune pathways may regulate this source of inflammation. One example is the cholinergic anti-inflammatory pathway (CAP), an endogenous nerve-to-spleen mechanism that regulates splenic pro-inflammatory cytokine release. We hypothesized that this pathway is impaired in SLE and that chronic stimulation of the CAP at the level of the efferent vagus nerve would attenuate hypertension in SLE. Starting at 30 and 32 weeks of age, female NZBWF1 SLE mice and NZW control mice were treated with the pharmacologic efferent vagal stimulators CNI-1493 (CNI; 8mg/kg; twice weekly; i.p.) or galantamine (GAL; 4mg/kg; daily; i.p.), or saline. At 34 weeks of age, we measured mean arterial pressure (MAP), finding that MAP (mmHg) in SLE mice was elevated compared to controls (139.83 ± 4.56 vs. 120.70 ± 2.96; n=4-6/group, p = 0.002), while the rise in MAP was prevented by CNI (134.45 ± 3.07)and GAL (129.25 ± 3.97) in SLE mice. We further hypothesized that splenocytes isolated from SLE mice conditioned by efferent vagal stimulation would release fewer pro-inflammatory cytokines in the presence of norepinephrine, which stimulates splenic β2 adrenergic receptors. We incubated isolated splenocytes for 24 hours at 37°C with and without norepinephrine (100 μM), then measured pro-inflammatory cytokines in the supernatant via ELISA. Compared to control mice, splenocytes from SLE mice secreted 70.7% and 146.5% higher concentrations of IL-6 and TNF-α (8.24 vs. 4.83 and 2.79 vs. 1.13 pg/mL, respectively; n=2/group) in the presence of norepinephrine. Compared to saline-treated SLE mice, splenocytes from CNI and GAL-treated SLE mice released fewer cytokines when incubated with norepinephrine (8.24 vs. 5.31 and 5.79 pg/mL IL-6; 2.79 vs. 2.18 and 0.81 pg/mL TNF-α; n=2/group). These in vivo and in vitro data suggest that stimulation of the CAP at the level of the efferent vagus may promote anti-inflammatory splenocyte activity, which may be protective against hypertension in the setting of chronic inflammation.

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Anti-Inflammatory Effect of Polyherbal Formulation (PHF) on Carrageenan and Lipopolysaccharide-Induced Acute Inflammation in Rats

Biomedical & Pharmacology Journal ◽

10.13005/bpj/1811 ◽

2019 ◽

Vol 12 (04) ◽

pp. 1801-1809

Author(s):

Bat-Erdene Jargalsaikhan ◽

Narangerel Ganbaatar ◽

Myadagbadam Urtnasan ◽

Nyamdolgor Uranbileg ◽

Dagvatseren Begzsuren

Keyword(s):

High Mobility ◽

Ethanol Extract ◽

Total Phenolic ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Inflammatory Disorders ◽

Polyherbal Formulation ◽

Tnf Α ◽

Anti Inflammatory ◽

Inflammatory Effect

Polyherbal formulation (PHF) is composed of Artemisia santolinifolia Turcz, Saussurea salicifolia L. and Hippophae rhamnoides L., which mainly used for inflammatory disorders in traditional Mongolian medicine. The aim of the study was to evaluate the anti-inflammatory effect of PHF in carrageenan and lipopolysaccharide (LPS) induced models of inflammation. The total active constituents of 20% ethanol extract of PHF was determined, using Folin-Ciocalteu reagent and aluminum chloride reagent, respectively. Inflammation models were induced by 1% carrageenan and LPS 7.5 mg/kg in the experimental groups. The levels of serum tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and high mobility group box 1 protein (HMGB-1) were measured in PHF pretreatment groups by enzyme-linked immunosorbent assay (ELISA). The lungs were harvested and evaluated for histopathological assessment on 12 hours after LPS administration. The content of total phenolic was 28.5±0.12 mg/g and flavonoids 12.4±0.42 mg/g. After 60, 120, 180, 240 and 300 min, the data indicate that PHF 75, 150 and 300 mg/kg was significantly effective reducing paw edema volumes induced by carrageenan compared to control (p<0.01). PHF pretreatment significantly reduced levels of serum TNF-α, IL-1β and IL-6 at 300 minutes after carrageenan injection. Moreover, pretreated with PHF 150 mg/kg groups serum levels of TNF-α, IL-1β and HMGB-1 were significantly (p<0.01) reduced compared with the control group after LPS injection. It showed less inﬂammation and change of pulmonary structure compared with the LPS group at 12 hours after LPS injection. From the results of the study, it was demonstrated that PHF had sufficient potential to treat inflammatory disorders by reducing pro-inflammatory cytokines.

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Anti-inflammatory Effect of Triterpenoic Acids of Eriobotrya japonica (Thunb.) Lindl. Leaf on Rat Model of Chronic Bronchitis

The American Journal of Chinese Medicine ◽

10.1142/s0192415x09006862 ◽

2009 ◽

Vol 37 (02) ◽

pp. 309-321 ◽

Cited By ~ 18

Author(s):

Jin-Fang Ge ◽

Ting-Yu Wang ◽

Bin Zhao ◽

Xiong-Wen Lv ◽

Yong Jin ◽

...

Keyword(s):

Chronic Bronchitis ◽

Bronchial Epithelium ◽

Lung Homogenate ◽

Eriobotrya Japonica ◽

Intercellular Adhesion Molecule ◽

Enzyme Linked Immunosorbent Assay ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Anti Inflammatory ◽

Inflammatory Effect

This study was designed to investigate the anti-inflammatory effect of Triterpenoic Acids from Eriobotrya japonica (Thunb.) Lindl. (TAL) on chronic bronchitis (CB) in rats. CB model was established by combination of Bacillus Calmette-Guerin (BCG, 5 mg/kg, injected through the caudal vein) and lipopolysaccharide (LPS, 1 g/L, injected through endotracheal intubation). Rats with CB model were treated with TAL (50, 150 and 450 mg/kg) for 3 weeks. The leukocytes in bronchoalveolar lavage fluid (BALF) were counted after Wright staining, the levels of cytokine tumor necrosis factor alpha (TNF-α), interleukin (IL)-8, and IL-10 in the supernatants of lung homogenate were assessed by enzyme-linked immunosorbent assay (ELISA), and the protein expression of nuclear factor kappaB (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelium were tested by immunohistochemical staining. As compared to the normal and sham groups, the total number of leukocyte, the differential counts of neutrophils and alveolar macrophage (AM) in BALF, the levels of TNF-α and IL-8 in the supernatants of lung homogenate, and the expression of NF-κB and ICAM-1 on bronchial epithelium in CB rats were significantly increased, while the level of IL-10 was decreased. TAL (50, 150 and 450 mg/kg) attenuated these alterations in model CB rats, which indicates that TAL has anti-inflammatory effect in the rats with CB.

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In vivo anti-inflammatory activity of Liquidambar formosana Hance infructescence extract

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v16i10.13 ◽

2017 ◽

Vol 16 (10) ◽

pp. 2403-2410

Author(s):

Haoran Ma ◽

Fuqian Wang ◽

Jie Jiang ◽

Lu Cheng ◽

Hong Zhang ◽

...

Keyword(s):

Inflammatory Activity ◽

Enzyme Linked Immunosorbent Assay ◽

Paw Edema ◽

Tnf Α ◽

Cox 2 ◽

Anti Inflammatory ◽

Underlying Mechanisms ◽

Anti Inflammatory Cytokine ◽

Inflammatory Effect

Purpose: To evaluate the anti-inflammatory activity of Liquidambar formosana Hance infructescence (Liquidambaris fructus, ELF) in vivo, and clarify its underlying mechanisms. Methods: The in vivo anti-inflammatory activity of ELF was examined by xylene-induced ear swelling test in mice as well as carrageenan-induced paw edema method in rats. The levels of inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-10) in serum were measured by enzyme-linked immunosorbent assay (ELISA), while the expressions of COX-2, iNOS and NF-κB p65 in paw tissue of rats were evaluated by western blot.Results: After ELF treatment, the levels of TNF-α (p < 0.001), IL-1β (p < 0.001) and IL-6 (p < 0.001) in serum decreased and the levels of anti-inflammatory cytokine IL-10 increased (p < 0.01). In addition, ELF treatment resulted in decrease of COX-2 (p < 0.01), iNOS (p < 0.01) and NF-κB p65 (p < 0.01) expressions in Wistar rats.Conclusion: The results reveal that ELF possesses significant anti-inflammatory effect in vivo. The anti-inflammatory activity is associated with the levels of TNF-α, IL-1β, IL-6 and IL-10 in serum. Furthermore, the suppression of NF-κB p65, iNOS and COX-2 is linked to its anti-inflammatory effect. These results provide a rationale for the use of Liquidambaris fructus in inflammatory disease in traditional medicine.Keywords: Anti-inflammatory activity, Liquidambaris fructus, Cytokines, Ear swelling test, Paw edema

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Pentoxifylline and Oxypurinol: Potential Drugs to Prevent the “Cytokine Release (Storm) Syndrome” Caused by SARS-CoV-2?

Current Pharmaceutical Design ◽

10.2174/1381612826666200811180232 ◽

2020 ◽

Vol 26 (35) ◽

pp. 4515-4521

Author(s):

Francisco J. López-Iranzo ◽

Ana M. López-Rodas ◽

Luis Franco ◽

Gerardo López-Rodas

Keyword(s):

Lung Parenchyma ◽

Interstitial Tissue ◽

Cytokine Release ◽

Phase I Clinical Trials ◽

Infected People ◽

Tnf Α ◽

Transient Loss ◽

Anti Inflammatory ◽

And Control ◽

Inflammatory Action

Background: COVID-19, caused by SARS-CoV-2, is a potentially lethal, rapidly-expanding pandemic and many efforts are being carried out worldwide to understand and control the disease. COVID-19 patients may display a cytokine release syndrome, which causes severe lung inflammation, leading, in many instances, to death. Objective: This paper is intended to explore the possibilities of controlling the COVID-19-associated hyperinflammation by using licensed drugs with anti-inflammatory effects. Hypothesis: We have previously described that pentoxifylline alone, or in combination with oxypurinol, reduces the systemic inflammation caused by experimentally-induced pancreatitis in rats. Pentoxifylline is an inhibitor of TNF-α production and oxypurinol inhibits xanthine oxidase. TNF-α, in turn, activates other inflammatory genes such as Nos2, Icam or IL-6, which regulate migration and infiltration of neutrophils into the pulmonary interstitial tissue, causing injury to the lung parenchyma. In acute pancreatitis, the anti-inflammatory action of pentoxifylline seems to be mediated by the prevention of the rapid and presumably transient loss of PP2A activity. This may also occur in the hyperinflammatory -cytokine releasing phase- of SARS-CoV-2 infection. Therefore, it may be hypothesized that early treatment of COVID-19 patients with pentoxifylline, alone or in combination with oxypurinol, would prevent the potentially lethal acute respiratory distress syndrome. Conclusion: Pentoxifylline and oxypurinol are licensed drugs used for diseases other than COVID-19 and, therefore, phase I clinical trials would not be necessary for the administration to SARS-CoV-2- infected people. It would be worth investigating their potential effects against the hyperinflammatory response to SARS-CoV-2 infection.

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Anti-Inflammatory Effect of Simonsinol on Lipopolysaccharide Stimulated RAW264.7 Cells through Inactivation of NF-κB Signaling Pathway

Molecules ◽

10.3390/molecules25163573 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3573

Author(s):

Lian-Chun Li ◽

Zheng-Hong Pan ◽

De-Sheng Ning ◽

Yu-Xia Fu

Keyword(s):

Nitric Oxide ◽

Signaling Pathway ◽

Morphological Changes ◽

Raw264.7 Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Tnf Α ◽

Anti Inflammatory ◽

Factor Α ◽

Oxide Synthase ◽

Necrosis Factor

Simonsinol is a natural sesqui-neolignan firstly isolated from the bark of Illicium simonsii. In this study, the anti-inflammatory activity of simonsinol was investigated with a lipopolysaccharide (LPS)-stimulated murine macrophages RAW264.7 cells model. The results demonstrated that simonsinol could antagonize the effect of LPS on morphological changes of RAW264.7 cells, and decrease the production of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW264.7 cells, as determined by Griess assay and enzyme-linked immunosorbent assay (ELISA). Furthermore, simonsinol could downregulate transcription of inducible nitric oxide synthase (iNOS), TNF-α, and IL-6 as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibit phosphorylation of the alpha inhibitor of NF-κB (IκBα) as assayed by Western blot. In conclusion, these data demonstrate that simonsinol could inhibit inflammation response in LPS-stimulated RAW264.7 cells through the inactivation of the nuclear transcription factor kappa-B (NF-κB) signaling pathway.

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Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

Journal of Translational Medicine ◽

10.1186/s12967-021-02823-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Qilu Wei ◽

Ning Kong ◽

Xiaohui Liu ◽

Run Tian ◽

Ming Jiao ◽

...

Keyword(s):

Protein Expression ◽

Cell Counting ◽

Enzyme Linked Immunosorbent Assay ◽

Fast Green ◽

Expression Levels ◽

Tnf Α ◽

Anti Inflammatory ◽

Safranin O ◽

Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

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