Myers MJ et al. Treatment Dev Vet Pract 2020(1): 11-18. 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice OCIMUM

Research Article Non-Steroidal Anti-Inflammatory Drugs Alter Nuclear Factor- κβ in A Class Specific Manner

Abstract The capacity of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) to affect the NF-κβ dependent pathway was examined. The NSAIDs were co-cultured with RAW 264.7 cells transfected with a plasmid encoding Secreted Embryonic Alkaline Phosphatase (SEAP) under the control of NF-κβ. SEAP production was initiated by stimulating the cells with E. coli derived Lipopolysaccharide Michael J Myers*, Anne J Lewandowski and (LPS). , indomethacin, , , , Christine M Deaver and inhibited SEAP production. , , Center for Veterinary Medicine, Office of Research , and acetaminophen did not affect SEAP production. TNFα stimulation Division of Applied Veterinary Research, USA of HEK293 cells transfected with a SEAP reporter gene under the control of NF-κβ was used to determine whether the NSAIDs were working directly on Received: 16 June 2020 the NF-κβ pathway or on TLR4 signaling. TNFα stimulated HEK293 cells Accepted: 20 July 2020 showed that diclofenac and inhibited SEAP production, but aspirin Version of Record Online: 31 July 2020 did not. and diclofenac both inhibited the amount of p65 released after LPS stimulation of the RAW 264.7 cells while flunixin did not affect Citation p65 release. Flunixin had no effect on an NF-κβ gel shift assay. These results demonstrate that NSAIDs in four of the six NSAID families can inhibit NF- Myers MJ, Lewandowski AJ, Deaver CM (2020) κβ pathway, albeit via different points in the pathway. Non-Steroidal Anti-Inflammatory Drugs Alter Nuclear Factor- Κβ in A Class Specific Manner. Keywords Treatment Dev Vet Pract 2020(1): 11-18. Flunixin Meglumine; Gel Shift; Lipopolysaccharide; Non-Steroidal Anti- Correspondence should be addressed to Inflammatory Drugs; Nuclear Factor-Κβ Michael J Myers, USA E-mail: [email protected] Abbreviations Copyright NSAID : Non-Steroidal Anti-Inflammatory Drugs Copyright © 2020 Myers MJ et al. This is an open LPS : Lipopolysaccharide access article distributed under the Creative NF-κβ : Nuclear Factor-κβ Commons Attribution License which permits unrestricted use, distribution, and reproduction Introduction in any medium, provided the original author and work is properly cited. The most widely studied mechanism of action of Nonsteroidal Anti- Inflammatory Drugs (NSAIDs) is their reduction of eicosanoid production through the inhibition of the enzymatic activity of the (COX) , COX1 & COX2. As a drug class, NSAIDS can exhibit antipyretic, anti-inflammatory, and/or analgesic effects; all NSAIDs do not exhibit all three activities. COX1 is constitutively expressed, while COX2 is inducible during periods of inflammation and is the more important

Ocimum Scientific Publishers .01. Submit Manuscript

10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice form during inflammation. While inhibition of Prostaglandin [23] or to inhibit NF-κβ [17]. decreases NF-κβ E2 (PGE2) production is perhaps the most widely studied activation while flunixin meglumine has no effect on -κβNF aspect of NSAIDS, other studies have shown some NSAIDS, activation [24]. including aspirin, indomethacin, diclofenac, meloxicam, tolfenamic acid, celecoxib, and flufenamic acid can inhibit The disparate reports on the impact of various NSAIDs on NF-κβ activation [1-17]. NF-κβ activation raises the question of whether the effect of NF-κβ activation is a selective effect of the different drugs, a Previous in vivo studies from this laboratory identified three concentration dependent effect, or a cell-type specific effect. swine genomic biomarkers of inflammation that held promise of Therefore, to address this issue, we used several different clinical utility [18]. More intriguing was the finding that there cell lines, each of which had different cell surface signaling in vivo expression patterns were altered by administration of pathways with the read-out system for all being the expression the Nonsteroidal Anti-Inflammatory Drug (NSAID) flunixin of Secreted Embryonic Alkaline Phosphatase (SEAP). The meglumine [19], further suggesting these biomarkers held capacity of NSAIDs in the 5 major families to affect NF-κβ the potential to be used as surrogate endpoints to assess dependent signaling, and the mechanisms of action responsible the effectiveness of novel NSAIDs with claims to control for affecting this pathway were examined, with the expectation inflammation. Critical evaluation of these results however that the pharmacological results obtained from these cell lines raised the question of whether we were developing a model would guide future in vivo studies in swine. applicable to all NSAIDs, or if we were simply developing a flunixin meglumine specific model. The central question Materials and Methods then became how to determine if these results were more broadly applicable to other NSAIDs, which would then imply Chemicals and NSAIDs a common mechanism of action underlying their capacity to Flunixin meglumine, diclofenac, meloxicam, phenylbutazone, affect gene expression. Alterations in NF-κβ activation would ketoprofen, celecoxib, indomethacin, flufenamic acid, provide both an explanation for why flunixin meglumine piroxicam, tolfenamic acid, acetomenaphen, and aspirin were suppressed in vivo gene expression Peters et al., [19] and a all obtained from Sigma-Aldrich Chemical Co (St. Louis, MO). common mechanism of action by with other class members All drugs were >98% pure. Recombinant human TNFα was would be expected to exert similar actions in vivo. obtained from Invitrogen, Inc. (Carlsbad, CA). E. coli derived NF-κβ is an essential intracellular transcription factor with a lipopolysaccharide (LPS, E. coli 055: B5) was obtained from central role in the regulation of innate and adaptive immune Sigma-Aldrich. responses, including regulation of multiple genes induced during inflammatory responses [20]. As such, NF-KB has been Cell culture postulated as a potential target for therapeutic intervention. The impact of various NSAIDs was determined using RAW However, the ability of NSAIDs to inhibit NF-κβ activation 264.7 cells transfected with a plasmid that produces SEAP has not been a constant observation with all drugs in this class. under the transcriptional control of five NF-κβ response Neither have there been consistent observations demonstrating elements (RAW 264.7-SEAP; Novus Biologicals, Littleton, CO; the same effect on NF-κβ activation by the same NSAID. Cat # NBP2-26261). The cells were grown using Ultraculture Indomethacin has been reported to either inhibit Shen et media supplemented with 4 mM L-glutamine, 1mM Na al., [6] or not inhibit NF-κβ activation. Acetaminophen, an pyruvate, 100 units/ml penicillin & 100 µg/ml streptomycin analgesic and antipyretic NSAID, was shown in one report (all from Lonza, Walkersville MD). Media for transfected cells to directly inhibit NF-κβ activation, while this same report was supplemented with 500 µg/ml G418 (Sigma-Aldrich) found aspirin only indirectly inhibited NF-κβ activation [21]. to maintain the plasmid. The cells were passaged when they Aspirin, indomethacin, diclofenac, meloxicam, tolfenamic reached 70-80% confluency using trypsin/EDTA. The impact acid, and flufenamic acid are NSAIDS with antipyretic, anti- of NSAIDs on NF-κβ was also assessed using HEK293 cells inflammatory, and analgesic effects. Tolfenamic acid has been transfected with a SEAP reporter gene under the control of an reported to induce (rather than suppress) NF-κβ activation in IFN-β minimal promoter fused to five NF-κβ (and five AP-1) colorectal cancer cells through enhanced phosphorylation and binding sites (HEK293-TNF; Invivogen, San Diego, CA; Cat degradation of Iκβ-α Jeong et al., [22], while another study # hkb-tnfil1). These cells were grown using Ultraculture media found it suppressed inflammatory induced NF-κβ activation supplemented with 4 mM L-glutamine, 1mM Na pyruvate, by decreasing the degradation of Iκβ-α [12]. Flufenamic acid 100 units/ml penicillin & 100 µg/ml streptomycin (all from has been reported to have no impact on NF-κβ activation Lonza, Walkersville MD) and Zeocin (100 μg/ml). The cells

Submit Manuscript .02. Ocimum Scientific Publishers Treatments and Developments: Veterinary Practice 10.33513/TDVP/2001-02 were passaged when they reached 70-80% confluence using Gels were run and transferred in Bio-Rad equipment (Trans- trypsin/EDTA. Blot® Cell and Criterion™ Cell). Determination of SEAP production Chemiluminescence was read on a Typhoon™ 8610 Variable Mode Imager. Densitometry was performed and images were Test cells were plated into a 96-well plate at 5x104 cells/well colorized in ImageQuant™ software. (200 μl final volume) overnight to permit cell attachment. The media was replaced and the test NSAID (or control) was Data transformation and statistics added to the cells 1 hr prior to addition of the stimulating agent. NSAIDs were added to achieve final concentrations of SEAP assay results from each experiment were expressed 10-3 M to 10-7 M. RAW 264.7-SEAP cells were stimulated as percent of LPS stimulated values for each experiment, with LPS (1 μg/ml, final concentration). The HEK293-TNF with the LPS stimulated SEAP concentrations being 100%. cells were stimulated with recombinant human TNFα (final Percentage of LPS-induced p65 was used to perform statistical concentration, 10 ng/ml). The concentration of SEAP in tissue analysis. Data were converted into Excel and analyzed using culture fluids was determined using the SEAPorter™Assay GraphPad Prism by a One-way ANOVA and Dunnet’s post- Kit (Novus Biologicals, Cat NBP2-25285), following the test compared to LPS-induced percentages of p65 to NSAID manufacturers’ instructions. The results were read using a treated or control cultures. SPECTRAmax 250 (Molecular Devices; Sunnyvale, CA). The data was exported into Excel for further analysis and data Results transformation. Impact of NSAIDS on LPS induced SEAP Assessment for activation of NF-κβ production Alterations in NF-κβ activation were determined using the The potential for NSAIDs in the derivatives family NF-κβ p65 ELISA Kit (ActiveMotif®, cat #40096), following (diclofenac, indomethacin), the coxib family (celecoxib), the the manufacturer’s instructions. Briefly, 4 x 106 RAW 264.7- fenamate family (tolfenamic acid, flufenamic acid) and the SEAP cells/well were allowed to adhere overnight in a 6-well oxicam family (piroxicam) to inhibit production of SEAP was plate followed by pre-treatment with meloxicam, flunixin, or evaluated using RAW 264.7-SEAP cells. SEAP production diclofenac (10-3 M; diluted in sterile water) for 1 hour and was initiated following stimulation by lipopolysaccharide. subsequent stimulation with LPS (1 μg/ml) for 1 hour. The cells The results demonstrated that diclofenac, indomethacin, were processed for cytoplasmic and nuclear fractions per the celecoxib, tolfenamic acid, flufenamic acid, and piroxicam fractionation kit instructions (ActiveMotif®, cat #40010). Total inhibited SEAP production at a drug concentration of 10-3 protein assessments of the nuclear fractions were performed M (Figure 1). Celecoxib also inhibited SEAP production at a using the Pierce BCA assay (Invitrogen). Equal amounts (1.2- drug concentration of 10-4 M. Meloxicam was not evaluated 11.5 µg, determined by the smallest amount garnered from due to its low aqueous solubility [25]. These results suggest fractionation process) of total protein were loaded into the p65 that these NSAIDs either inhibit production of NF-κβ or the ELISA and the ELISA was performed as per kit instructions. activation of the NF-κβ transcription elements. In contrast, ELISAs were read on a SPECTRAmax M2e microtiter plate ketoprofen, phenylbutazone, aspirin, and acetaminophen did reader with SoftMax Pro®software. not affect SEAP production (data not shown). Electrophoretic mobility gel shift assays Impact of NSAIDS on TNFα-Induced SEAP Production Electrophoretic mobility gel shift assays (Active Motif, cat#37341) were performed per manufacturer protocol using While these results suggested these NSAIDs were impacting Flunixin meglumine at a concentration of 2 x10-3, and a NF-κβ one or more aspects of the NF-κβ pathway, another possibility oligonucleotide (consensus sequence: AGTTGA-G-G-G-G-A- was a direct effect on the TLR4 molecules responsible for the C-T-T-T-C-C-CAGGC) was biotinylated at the 5’ end (custom, initiation of the NF-κβ signaling. Assessing whether NSAIDs ThermoFisher). Biotinylated NF-κβ oligonucleotide was added impacted TLR4 or the intracellular NF-κβ pathway was at 40ng/well. Active NF-κβ (6uL) was used in the form of a determined using HEK293-TNF. The HEK293-TNF cells Raji nuclear extract (Active Motif, cat#36023). We used a have just the single TNF receptor signal transduction molecule 5% TBE gel (Criterion, cat#3450048), 10X TBE (Bio-Rad, whereas RAW 264.7-SEAP have multiple cell surface signal cat#161-0770), and a nylon membrane (Biodyne, cat#77016). transduction molecules. TNFα stimulated HEK293-TNF cells

Ocimum Scientific Publishers .03. Submit Manuscript 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice

co-cultured with either diclofenac, flunixin, or aspirin showed production in HEK293-TNF cells stimulated with TNFα, just that diclofenac and flunixin inhibited SEAP production in the as it failed to inhibit SEAP production in RAW 264.7-SEAP HEK293-TNF cells (Figure 2). Aspirin did not inhibit SEAP cells stimulated with LPS.

Figure 1: Impact of NSAIDs on LPS Stimulated RAW 264.7 Cells Expressing SEAP Under NF-κβ Control. Figure 1: Capacity of NSAIDs to reduce the production of secreted embryonic alkaline phosphatase (SEAP) following stimulation of RAW 264.9 cells. The RAW cells 264.7-SEAP cells were place in 96 well plates at a concentration of 5x104 cells/well (200 μl final volume) overnight to permit attachment. The cells were incubated with the indicated NSAIDs (10-3 to 10-5 M, final conc.) for 1 hr prior to initiation of SEAP production. SEAP production was initiated by stimulating the RAW cells with E. coli derived LPS at a final concentration of 1 μg/ml. The supernatants were collected 24 hr after initiation of SEAP production. Results expressed are the mean of at least three different experiments, with all conditions conducted in triplicate. Results with NSAIDs at 10-6 & 10-7 M are not shown as there was no effect on SEAP production.

Figure 2: Effect of NSAIDs on TNFα Stimulated SEAP Transfected HEK293 Cells. Figure 2: Effect of NSAIDs on TNFα stimulation of HEK293-TNF cells. The cells were place in 96 well plates at a concentration of 5x104 cells/well (200 μl final vol) overnight to permit attachment. The cells were incubated with the ASA, flunixin, or diclofenac (10-3 to 10-5 M, final conc.) for 1 hr prior to initiation of SEAP production. SEAP production was initiated by stimulating the HEK cells with recombinant human TNFα (final concentration, 10 ng/ml). The supernatants were collected 24 hr after initiation of SEAP production. Results expressed are representative of two different experiments, with all conditions conducted in triplicate. Results with NSAIDs at 10-6 & 10-7M are not shown as there was no effect on SEAP production.

Submit Manuscript .04. Ocimum Scientific Publishers Treatments and Developments: Veterinary Practice 10.33513/TDVP/2001-02

Effect of meloxicam, diclofenac, and flunixin κβ activation (p65 release) suggested this NSAID might be meglumine on NF-κβ p65 activation acting downstream from NF-κβ activation. Two possible points of impact were direct binding to the NF-κβ DNA binding Confirmation of NSAID inhibition of the NF-κβ pathway domains or interfering with NF-κβ binding to the NF-κβ DNA and identification of one possible point of inhibition was binding domains. Accordingly, the potential for flunixin to determined by measuring p65 release in the nuclear fraction inhibit NF-κβ DNA binding interaction was examined using of LPS stimulated cell cultures. Meloxicam (Figure 3A) and a gel shift assay. The capacity of flunixin to directly bind to the diclofenac (Figure 3B) both inhibited the amount of p65 NF-κβ binding domain was assessed by substituting in flunixin released after LPS stimulation compared to the amount of for activated p65 and determining if there was a gel shift in the p65 released by LPS stimulation, yielding p65 release values NF-κβ binding domain oligonucleotide. Flunixin was unable comparable to control levels. In contrast, flunixin (Figure 3A) to affect the binding of activated NF-κβ to the binding of an did not have an effect on p65 release, even though it inhibited NF-κβ domain oligonucleotide, nor was it able directly alter NF-κβ-driven SEAP production (Figure 1). Extension of the migration of the NF-κβ domain oligonucleotide (data flunixin exposure to 5 hr also did not result in an inhibition not shown). of p65 release (data not shown). Based on these results, we hypothesize that meloxicam and diclofenac inhibit gene Discussion expression by inhibiting the activation of NF-κβ, but flunixin acts in another portion of the NF-κβ pathway. The results of this study show that within the confines of a

Figure 3: NSAID Control of NF-κβ Activation. Figure 3: Activation of NF-κβ was assessed by measuring the release NF-κβ p65. 4 x 106 RAW 264.7-SEAP cells/well were allowed to adhere overnight in a 6-well plate followed by pre-treatment with meloxicam, flunixin, or diclofenac (10-3 M final) for 1 hour prior to cell activation to assess their capacity to inhibition of the release of p65. The cells were stimulated with LPS (1 μg/ml) for 1 hour. The cells were processed for cytoplasmic and nuclear fractions according to the fractionation kit instructions. The results are a representative experiment (mean ± S.D. of replicate determinations) from two independent experiments with each NSAID. ***p<0.001 ****p<0.0001 compared to control values.

Impact of flunixin meglumine on NF-κβ -DNA single cell type, the impact of NSAIDs on NF-κβ activation is binding confined to just those class members with anti-inflammatory activity, with all these NSAIDs consistently reducing NF-κβ The capacity of flunixin to inhibit NF-κβ driven production activation. What differs is the precise mechanism of action by of SEAP in the absence of its capacity to interfere with NF- which they inhibit NF-κβ activation. As stated at the outset

Ocimum Scientific Publishers .05. Submit Manuscript 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice of this work, the core questions on any effect of NSAIDs on also explain why the present study could not demonstrate an NF-κβ activation was whether 1) it was a selective effect of the effect of aspirin to inhibit NF-κβ activation while other studies different NSAIDs, 2) a concentration dependent effect, or 2), have reported on its capacity to inhibit NF-κβ activation. a cell specific effect. Towards addressing those questions, the Aspirin has been reported to inhibit NF-κβ activation by results of this study demonstrate that NSAID inhibition of decreasing the degradation of Iκβ-α [3,8,22]. NF-κβ activation is a common feature of all members of this drug class, and within a single cell type, all NSAIDs inhibit Previous studies from this laboratory identified a series of activation. The impact of drug concentration towards inhibiting genomic biomarkers whose expression patterns were not only NF-κβ activation cannot be fully addressed by the results of altered during LPS-induced inflammation but were inversely this study but has been postulated by others to be related to regulated by the NSAID flunixin [18,19]. Flunixin reversed the potency of the NSAID. the LPS-induced changes in these genomic biomarkers in a manner consistent with an anti-inflammatory capacity. It also This report is the first to look at a broad swath of NSAIDs, completely suppressed in vivo PGE2 production [19]. While the using the same cell read-out system and identical concentration expression of these biomarkers occurred after PGE2 production, ranges. In contrast to previous reports, this study could not some of those biomarkers are not associated with regulation by demonstrate any impact on NF-κβ activation by ketoprofen, PGE2, which suggested that the flunixin-moderated changes to phenylbutazone, aspirin, or acetaminophen. Acetaminophen these LPS-induced genes might be occurring through another and phenylbutazone have no anti-inflammatory activity, only pathway, with NF-κβ being a prime candidate. anti-pyretic and analgesic properties. This would suggest that the capacity of NSAIDs to inhibit the NF-κβ mediated Demonstrating that flunixin suppresses NF-κβ activation in pathway of gene activation is related to their anti-inflammatory this present study provides evidence for a mechanism of action actions. While aspirin has all three activities, it is much weaker on how this NSAID can suppress gene expression independent anti-inflammatory drug than the other NSAIDs evaluated of an effect on PGE2 production. Befitting this uniqueness in this study. However, ketoprofen also showed no impact among NSAIDS, it has a unique mechanism of action on how it on the activation of NF-κβ. The lack of impact on NF-κβ inhibits the NF-κβ pathway. The inhibition of activated NF-κβ activation could suggest that the relative strength of the anti- binding to its DNA binding domain represents a novel point inflammatory activity may be related to the suppression of of control for this pathway by this class of drugs. Together with the NF-κβ pathway. Alternatively, the capacity of NSAIDs other reports that some NSAIDs such as aspirin and diclofenac to inhibit NF-κβ activation may be related to the tissue/cell inhibit NF-κβ activation by decreasing the degradation of type under study. In the current study, acetaminophen had no Iκβ-α Paik et al., [3], Fredriksson et al., [8], Jeong et al., [22], impact on altering NF-κβ activation in the kidney (HEK293) further suggest that no single mechanism of action exists for and macrophage cell lines used in this study, while diclofenac how the different members of this drug family impact the did suppress activation in these two cell lines. However, in NF-κβ pathway of gene activation. However, as inhibition of a study using the liver cell line HepG2, acetaminophen was NF-κβ activation is the common theme for all NSAID classes slightlymore potent in suppressing NF-κβ activation than regardless of the precise mechanism of action. As such, this is diclofenac [26]. There may also be dose-related differences in information that can be used to develop future in vivo studies the impact of NSAIDs on NF-κβ activation. While therapeutic seeking to further develop and qualify inflammatory biomarkers doses of diclofenac inhibit NF-κβ activation, toxic doses of as surrogate endpoints that can substantiate NSAID claims to diclofenac given in vivo to mice resulted in enhanced NF-κβ control inflammation in food animals. activation in renal tissues [27]. The ability of flunixin to interfere with the binding of NF-κβ One of the unstated goals of this study was to determine if to its DNA binding domain raises the question of whether this cell reporter system could be used as a high-throughput interference with the interaction of this protein-DNA pair screening method to assess NSAID potency with respect to is a unique capacity of this NSAID, and whether flunixin inhibition of NF-κβ activation. The yes/no results of the might be capable of inhibiting a similar interaction by other current study suggest this is not possible with the current cell transcription factors. Central to answering this question is a reporter system. The inability to establish a relative potency deeper understanding of the precise nature by which flunixin approach with this reporter system may be due to the relatively interferes with NF-κβ-DNA binding. The results of this study large number of NF-κβ binding domains in the SEAP gene notwithstanding, we cannot definitively state flunixin does not containing plasmid. It is plausible that the number of binding actively bind to the NF-κβ DNA binding domain. While the domains represents a non-physiological situation that precludes results of our modified gel shift assay provided no evidence development of a dose/potency assay for NSAIDs. This could to this effect, it is equally likely that any subtle increase in

Submit Manuscript .06. O c i m u m S c i e n t i fi c P u b l i s h e r s Treatments and Developments: Veterinary Practice 10.33513/TDVP/2001-02 molecular weight caused by flunixin bind to the oligonucleotide hepatocyte apoptosis. Hepatology 53: 2027-2041. was insufficiently small to be detected by this assay system. 9. El-Shitany N, El-Bastawissy EA, El-Desoky K (2014) Ellagic acid protects against carrageenan-induced acute Meloxicam is poorly soluble in water, with an extremely low inflammation through inhibition of nuclear factor kappa B, partition co-efficient at physiological pH [25]. As such, it could inducible cyclooxygenase and proinflammatory cytokines and not be evaluated using the SEAP assays. However, it could enhancement of interleukin-10 via an antioxidant mechanism. inhibit the activation of p65 (a short-term assay), suggesting Intl Immunopharm 19: 290-299. that it would inhibit NF-κβ mediated gene expression just 10. Hassan MH, Ghobar MM (2016) Antifibrotic effect of meloxicam like the other NSAIDS did in the SEAP assay. in rat liver: role of nuclear factor kappa B, proinflammatory cytokines, and oxidative stress. Naunyn-Schmiedeberg’s Arch In conclusion, the results of this study demonstrate that Pharmacol 389: 971-983. NSAIDs with anti-inflammatory activities exhibit the same 11. Mattei RA, Dalmarco EM, Tânia Silvia Fröde TS (2015) trend towards NF-κβ activation, namely an inhibition in the Etanercept administration prevents the inflammatory response activation of this pathway. The identification of a common induced by carrageenan in the murine air pouch model. pathway on how NSAIDs affect gene expression during periods Naunyn-Schmiedeberg’s Arch Pharmacol. of inflammation, albeit via slightly different mechanisms of 12. Shao HJ, Lou Z, Jeong JB, Kim KJ, Lee J, et al. (2015) action, will greatly facility future in vivo efforts in swine to Tolfenamic acid suppresses inflammatory stimuli-mediated qualify surrogate endpoints that can substantiate NSAID activation of NF-κB signaling. Biomol Ther 23: 39-44. claims to control inflammation. 13. Barcelos RP, Bresciani G, Rodriguez-Miguelez P, Cuevas MJ, Soares FAA, et al. (2016) Diclofenac pretreatment effects Conflict of Interest on the toll-like receptor 4/nuclear factor kappa B-mediated inflammatory response to eccentric exercise in rat liver. Life The authors have no conflicts of interest. Sci 148: 247-253.

14. Pongkorpsakola P, Satitsri S, Wongkrasant P, Chittavanich References P, Kittayaruksakul S, et al. (2017) Flufenamic acid protects 1. Kopp E, Ghosh S (1994) Inhibition of NF-kappa B by sodium against intestinal fluid secretion and barrier leakage in a mouse salicylate and aspirin. Science 265: 956-959. model of Vibrio cholerae infection through NF-κB inhibition and AMPK activation Eur J Pharmacol 798: 94-104. 2. Weber C, Erl W, Pietsch A, Weber PC (1995) Aspirin inhibits nuclear factor-κβ mobilization and monocyte adhesion in 15. Gugliandolo E, Fusco R, D’Amico R, Militi, A, Oteri G, et al. stimulated human endothelial cells. Circulation 91: 1914-1917. (2018) Anti-inflammatory effect of ATB-352, a H2S-releasing ketoprofen derivative, on lipopolysaccharide-induced 3. Paik JH, Ju JH, Lee JY, Boudreau MD, Hwang DH (2000) periodontitis in rats. Pharmacol Res 132: 220-231. Two opposing effects of non-steroidal anti-inflammatory drugs on the expression of inducible cyclooxygenase. J Biol Chem 16. Zuo C, Hong Y, Qiu X, Yang D, Liu N, et al. (2018) Celecoxib 275: 28173-28179. suppresses proliferation and metastasis of pancreatic cancer cells by down-regulating STAT3 / NF-kB and L1CAM activities. 4. Tegeder I, Pfeilschifter J, Geisslinger G (2001) Cyclooxygenase- Pancreatol 18: 328-333. independent actions of cyclooxygenase inhibitors. FASEB J 15: 2057-2072. 17. Liu X, Li Z, Liu H, Zhu Y, Xia D, et al. (2019) Low concentration flufenamic acid enhances osteogenic differentiation of 5. Takada Y, Bhardwaj A, Potdar P, Aggarwal BB (2004) mesenchymal stem cells and suppresses bone loss by Nonsteroidal anti-inflammatory agents differ in their ability inhibition of the NF-κB signalling pathway. Stem Cell Res to suppress NF-κβ activation, inhibition of expression of Ther 10: 213. cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 23: 9247-9258. 18. Peters SM, Yancy H, Bremer E, Monroe J, Paul D, et al. (2011) In vitro identification and verification of inflammatory 6. Shen Y, Yang T, Wang J, Xu Q, Li R, et al. (2007) Indomethacin biomarkers in swine. Vet Immunol Immunopath 139: 67-72. enhances the cytotoxicity of recombinant human lymphotoxin α on tumor cells by suppressing NF-κβ signalling. Cancer Biol 19. Peters SM, Yancy HF, Deaver CM, Jones YL, Kenyon E, et Ther 6: 1428-1433. al. (2012) In vivo characterization of inflammatory biomarkers in swine and the impact of flunixin meglumine administration. 7. Karakawa A, Fukawa Y, Okazaki M, Takahashi K, Sano T, et Vet Immunol Immunopath 148: 236-242. al. (2009) Diclofenac sodium inhibits NF-κβ transcription in osteoclasts. J Dent Res 88: 1042-1047. 20. Mitchell JP, Carmody RJ (2018) Chapter two - NF-κB and the transcriptional control of inflammation. International Rev Cell 8. Fredriksson L, Herpers B, Benedetti G, Matadin Q, Puigvert Mol Biol 335: 41-84. JC, et al. (2011) Diclofenac inhibits tumor necrosis factor- α-induced nuclear factor-κβ activation causing synergistic 21. Ryu YS, Lee JH, Seok JH, Hong JH, Lee YS, et al. (2000)

Ocimum Scientific Publishers .07. Submit Manuscript 10.33513/TDVP/2001-02 Treatments and Developments: Veterinary Practice

Acetaminophen inhibits iNOS gene expression in RAW 3: 2864-2872. 264.7 macrophages: differential regulation of NF-κβ by acetaminophen and salicylates. Biochem Biophys Res Comm 25. Luger P, Daneck K, Engel W, Trummlitz G, Wagner K (1996) 272: 758-764. Structure and physiochemical properties of meloxicam, a new NSAID. Eur J Pharm Sci 4: 175-187. 22. Jeong JB, Yang X, Clark R, Choi J, Back SJ, et al. (2013) A mechanistic study of the proapoptotic effect of tolfenamic 26. Herpers B, Wink S, Fredriksson L, Di Z, Hendriks G, et al. acid: involvement of NF-κβ activation. Carcinogenesis 34: (2016) Activation of the Nrf2 response by intrinsic hepatotoxic 2350-2360. drugs correlates with suppression of NF‑κB activation and sensitizes toward TNFα‑induced cytotoxicity. Arch Toxicol 23. Bryant CE, Farnfield BA, Janicke HJ (2003) Evaluation of the 90: 1163. ability of carprofen and flunixin meglumine to inhibit activation of nuclear factor kappa B. Am J Vet Res 64: 211-216. 27. Borghi SM, Fattori V, Ruiz-Miyazawa KW, Bertozzi MM, Lourenco-Gonzalez Y, et al. (2018) Pyrrolidine dithiocarbamate 24. Modhave DT, Handa T, Shah RP, Singh S (2011) Successful inhibits mouse acute kidney injury induced by diclofenac by characterization of degradation products of drugs using LC- targeting oxidative damage, cytokines and NF-κB activity. MS tools; application to piroxicam and meloxicam. Anal Meth Life Sci 208: 221-231.

Submit Manuscript .08. Ocimum Scientific Publishers