Effect of Benzoylsalicylic Acid on IKK-Beta Kinase and NF-κB Pathway in Murine Macrophage raw 264.7 Cells

The transcription factor NF-κB regulates a large array of genes of immune and inammatory responses. Deregulated NF-κB signalling is implicated in the pathogenesis and broad spectrum of human inammatory disorders and malignancies. The mechanism for NF-κB activation is the inducible degradation of IκB, triggered through its site-specic phosphorylation by a multi-subunit IκB kinase (IKK) complex. Aspirin (acetylsalicylic acid) a well-known anti-inammatory agent that binds to ATP binding pocket of IKKβ and inhibits its kinase activity. However, several side effects of aspirin due to the inactivation of COX-1 limits the therapeutic usage of ASA. Here we have demonstrate the effect of a plant phenolic compound benzoylsalicylic acid (BzSA) isolated rst time in plants a potent anti-viral compound inhibits Tobacco mosaic virus (TMV) and enhance the plant defense response (Samuel et all 2016&2017) inhibit the IKKβ mediated NF-κB pathway higher than aspirin. Our In-vitro COX enzymatic assays with BzSA have shown less COX-1 and high COX-2 inhibition as compared to ASA. Western blotting analysis of Raw 264.7 cells that were pre-treated with BzSA down-regulated LPS stimulated pIKK-β, pIκB, NF-κBp65, TNF-α, COX-1, COX-2, 5-LOX, IL-1β, and IL-6 higher than ASA. Therefore, our observations suggested that the potencial therapeutic value of BzSA an upcoming new inhibitor of NF-KB pathway and the dual inhibitor of COX2/5-LOX without effecting the usefull COX-1. Hence useful as an antiinammatory agent like ASA.

and thus acts as a transcription factor for the induction of TNFα, COX-2, iNOS as well as cytokines like IL-1, IL-2 and IL-6 (Aggarwal, 2006;Esposito and Cuzzocrea, 2009;Liu et al., 2013;Pahl, 1999). Thus, identi cation of new NF-κB inhibitors are the promising therapeutics to prevent in ammation and cancer diseases (Andres et al., 2013;Xu et al., 2011). Previously, it was reported that ASA and sodium salicylate inhibit the IKK-β and therefore blocks the activation of NF-κB pathway (Yin et al., 1998). NF-κB is a hallmark of in ammatory responses and plays a fundamental role in in ammation and associated cancers (Pikarsky et al., 2004). Literature survey suggested that a number of chemical activators targets the genes of NF-κB pathway (Pahl, 1999). Chebulagic acid (CA) a natural plant compound shown the anti-in ammatory effects on LPS-stimulated RAW 264.7 macrophages through NF-κB inhibition and also MAP kinase phosphorylation (Reddy and Reddanna, 2009). NF-κB controls many physiological processes including in ammation, immunity, apoptosis and angiogenesis (Aggarwal, 2006;Hayden and Ghosh, 2008; Vallabhapurapu and Karin, 2009). Baicalein a natural compound suppressed TNF-α-induced NF-κB activation and its target gene products (Li et al., 2016).
Cyclooxygenases (COX-1 and COX-2) and lipoxygenases  are the key enzymes of arachidonic acid (AA) metabolism (Dannhardt and Kiefer, 2001; Greene et al., 2011). COX enzymes catalyses the conversion of AA to prostaglandins (PGs) and thromboxanes . COX-1 constitutively expressed in mast cells and also different organs whereas, COX-2 localized primarily in in ammatory cells and tissues and the selective COX-2 inhibitors are exceedingly bene cial anti-in ammatory drugs (Seibert and Masferrer, 1994;Seibert et al., 1994). Literature suggested that offensive up-regulation of COX-2 and iNOS has been associated with pathophysiology of certain types of human cancers as well as in ammatory disorders (Surh et al., 2001). ASA is a widely used non-steroidal anti-in ammatory (NSAID) drug world wide (Alfonso et al., 2014;Wentz et al., 1976). The anti-thrombic and anti-ulcerogenic side effects of ASA largely due to the acetylation of Ser 530 in COX-1 and Ser 516 in COX-2 (Kalgutkar et al., 1998). Inhibition of COX-1 by ASA and other NSAIDs causes side effects (Brune and Patrignani, 2015).
Several reports suggested that the phytochemicals like curcumin, epigallocatechin gallate (EGCG), resveratrol inhibit COX-2 and are proven to be effective anti-in ammatory and anti-cancer agents (Surh et al., 2001).
To the best of my knowledge we isolated rst time BzSA from the seed coats of Givotiarottleriformis and shown its role in plant systemic defense against TMV virus (S. . The plant G.rottleriformisis tree species belongs to Euphorbiaceae family and are known have anti-rheumatism, anti-psoriasis and anti-dandruff medicinal properties. Phytochemical analysis of G.rottleriformis seed coats provides the evidences to the medicinal value of this plant as we identi ed important pharmaceutical molecules such as salicylic acid (SA), benzoic acid (BA), gallic acid (GA) and methylgallate (MG) Samuel Kamatham, 2015). The puri ed GA and MG from seed coats of this plant exhibit anti-cancer potential against the proliferation of A431skin cancer cellline without effecting the normal HaCaTcellline (Samuel Kamatham, 2015).
The present study have shown the anti-in ammatory and anti-cancer potential of BzSA in RAW 264.7 cells. Structurally BzSA is a natural and ASA is a synthetic analogue of SA (Fig. 1). The effect of BzSA on COX-1 and COX-2 inhibition was studied and compared with ASA. This study shown the evedances of BzSA down-regulated IKK-β and the down-stream NF-κB pathway higher as compare to ASA in a dosedependent manner.

Cell culture
RAW 264.7 murine macrophase cells were cultured as a monolayer in petridish and supplemented with DMEM medium containing 10% heat inactivated FBS, 100IU/ml penicillin,100µg/ml streptomycin, 2mM L-glutamine and maintained in a humidi ed atmosphere with 5% CO2 at 37 C. The cells were subcultured alternative days and the exponentially growing cells were used for the treatments.

MTT assay
RAW 264.7 cells (5X10 3 cells per well) were seeded in 96 well plates and incubated in the presence or absence of BzSA or ASA (0.001, 0.01, 0.1, 1.0, and 10 mM) for 24h and 48h. In a nal volume of 100µl, 20µl of MTT (5mg/ml in PBS) was added to each well and incubated for an additional 3h at 37 ºC. Then the culture medium was removed from the wells and added 200µl of DMSO followed by dissolved the purple blue formazan crystals. The optical color density was quanti ed at 570nm on ELISA multi-mode plate reader (SYNERGYMX, Biotech). All the experiments are repeated three times under the same conditions. 2.4 Preparation of whole cell extract and Western bloting analysis RAW 264.7 cells were treated either with BzSA or ASA (200 and 400µM; dissolved DMEM medium containing 0.01% DMSO) for 24h followed by stimulated with 25ng/mL LPS for 2h. DMSO and LPS treated cells were maintained as positive and negative controls. After the pre-treatment, the cells were washed with 1X PBS and re-suspended in a RIPA lysis and extraction buffer containing 1X protease inhibitor cocktail followed by incubation for 30min at 4°C with a frequent vortexing. Then the lysate was centrifuged at 12,000 rpm for 20min and collected the supernatant and the total protein concentration was estimated by Bradford protein assay (Bradford, 1976) and stored at -20 o C for further use. The protein samples (20µg) was resolved on 12% SDS-PAGE and then transferred onto nitrocellulose (NC) membrane. Then the NC membrane were incubatedin 5% (w/v) non-fat dry milk powder at RT for 1h to block nonspeci c sites and incubated with primary antibody of interest ( both phospho and total IKK-α, IKK-β, IκBα, IκBα, NF-κBp65 and TNF-α, COX-1, COX-2, iNOS and IL1β) overnight at 4°C under shaking followed by washing with TBST for 3 times 10 min each. The NC membrane were then incubated with respective secondary antibody conjugated with HRP for 1h at RT followed by washing with TBST for 3 times10 min each. Finally, the blot were developed by adding HRP substrate followed by recorded using gel documentation system (Bio-rad).

Isolation of COX-1 enzyme
COX-1 enzyme was isolated from Ram seminal vesicles according to Hemleret al; 1976 (Hemler and Lands, 1976), with a slight modi cations. In brief, Ram seminal vesicles were homogenized with a grinder in Tris-HCl (pH 8.0) buffer for 1min and then the homogenate was ltered through cheese cloth and centrifuged at 13,000g at 4°C for 30 min. Finally, 0.01% sodium azide was added and stored in small aliquots at -80°C and used as a COX-1 enzyme.

Isolation of COX-2 enzyme
The enzyme COX-2 was isolated according to Reddy et al; 2000 (Reddy et al., 2000) with slight modi cations. In brief, the human recombinant COX-2 enzyme was expressed in Sf-9 cells, harvested the cells and sonicated for 3min in 50mM Tris-HCl buffer (pH 7.2) followed by centrifuged at 100,000 g at 4°C for 80 min using ultracentrifuge (Hitachi, Himac CP-100α). Then the cell pellet was resuspended in 2.5 mM Tris-HCl buffer (pH 7.2), 0.8% Tween-20, 1mM phenol, and 0.5% glycerol, and stored in small aliquots at -80°C and used as a COX-2 enzyme.

COX-1 and COX-2 enzyme activity
The enzymatic activities of both COX-1 and COX-2 were measured according to Copeland et al;(Copeland et al., 1994 with slight modi cations based on the a chromogenic assay and oxidation of N,N,N",N"-tetramethyl-p-phenylene diamine (TMPD) during the reduction of PGG 2 to PGH 2 . In brief, the assay mixture contained Tris-HCl buffer (100mM, pH 8.0), hematin (15µM), EDTA (3µM), enzyme (COX-1 or COX-2) and the test compounds (BzSA or ASA). Then the assay mixture was pre-incubated for 15min at 25°C and the reaction was initiated by addition of arachidonic acid (AA) and TMPD in a nal volume of 1ml. The enzyme activity was measured after 1min by estimated initial TMPD oxidation by monitoring absorbance at 603nm. A low rate of non-enzymatic TMPD oxidation was observed in the absence of COX-1 or COX-2 enzymes and are treated as control reaction and were normalised from the test experimental values while calculating the percentage of inhibition. Each experiment were repeated three times under the same conditions.

5-LOX assay
Similarly, we puri ed the 5-LOX enzyme from potato tubers and assayed according to (Reddanna et al., 1990. Enzyme activity was measured using polarographic method with a Clark's oxygen electrode on Strathkelvin Instruments, model 782, RC-300. The typical reaction mixture contained 50-100µl of enzyme and 10µl of the substrate (133µΜ of AA) in a total volume of 3ml with 100 mM phosphate buffer pH 6.3. The rate of decrease in oxygen concentration was taken as a measure of enzyme activity. Stock solutions of BzSA and ASA were prepared freshly in DMSO before use. Various concentrations of BzSA and ASA were prepared and the LOX reaction was initiated by the addition of substrate. The reaction was allowed to proceed at 25ºC and the maximum slope generated was taken for calculating the enzyme activity. The percentageof inhibition was calculated by comparison of LOX activity in the presence or absence of inhibitor. The concentration of the test compound causing 50% inhibition (IC 50 ) was calculated from the concentration-inhibition response curve. The experiment were repeated for three times under the same conditions.

Cytotoxicity assay
The cytotoxic effect of BzSA was determined and compared with ASA in Raw 264.7 cells (Fig. 2a-f). Raw 264.7 cell that were pre-treated with BzSA have shown its cytotoxic effect with an IC 50 value of 3.0 mM at 24h and 48h (Fig. 2a-c). In contrast, ASA shown its cytotoxic affect with an IC 50 value of 5.0 mM at 24h and 48h respectively (Fig. 2d-f).

Effect of BzSA on COX-1/COX-2 and 5-LOX enzyme activity
In order to determine the effect of BzSA on COX-1, COX-2 and 5-LOX enzyme activity we perform in vitro enzymatic assays. Interestingly, 4.2 mM BzSA showed 10% COX-1 and 35% COX-2 inhibition (Table 1). Whereas, 4.2 mM ASA shown 95% COX-1 and 11% COX-2 enzyme (Table 1). However, BzSA and ASA have no inhibition effect on 5-LOX enzyme activity even at increasing concentrations (Table 1). To examine the effect of BzSA on COX-1 expression we performed western bloting in RAW 264.7 cells that were stimulated with LPS. Previous reports has been suggested that COX-1 required to maintaine the body physiology and constitutively expressed in the gastrointestinal tract and many other tissues in the body including lung, kidney, stomach, platelets and monocytes etc. Interestingly, in our results pretreatment of BzSA and ASA does not effect the COX-1 expression upon LPS stimulation (Fig. 3).

COX-2
COX-2/5-LOX dual inhibitors are the promising pharmaceutical value for the development of potent drugs to cancer and various in ammatory diseases. RAW 264.7 cells that were pretreated with BzSA downredulated LPS stimulated COX-2 completely at 200µM (Fig. 3). Whereas cells that were pretreated with ASA down-regulated LPS stimulated COX-2 less than BSA in a dose dedendent manner (Fig. 3). These results highlite BzSA is a potant COX-2 inhibitor than ASA.
3.1.5 5-LOX 5-lipoxygenase (5-LOX) pathway is the major source of potent proin ammatory leukotrienes (LTs) issued from the metabolism of arachidonic acid (AA), and the best known for their roles in the pathogenesis of asthma. Dual COX-2/5-LOX inhibitors are promising drugs to treat in ammatory diseases. In our results, RAW 264.7 cells that were pretreated with 200µM BzSA down-regulated the expression of 5-LOX enzyme (Fig. 3). Whereas, RAW 264.7 cells that were pretreated with ASA reduced 5-LOX expression low as compared to BzSA (Fig. 3). These results suggest that BzSA is a potant 5-LOX inhibitor.

Inhibition of IKK complex by BzSA
The activation of catalytic kinase subunits of IKK kinase complex (IKKα and IKKβ) is a regulatory step in two signalling pathways known as classical (canonical) pathway and the alternative (non-canonical) pathway, leading to the activation of NF-κB. The IKK mediated phosphorylation of IκB and proteosomal degradation are leading to the activation of NF-κB dimers, nuclear translocation and induction of target gene expression. Here we have shown the effect of BzSA on catalytic kinase subunits of IKK kinase complex (IKKα and IKKβ) in RAW 264.7 cells in a dose responsive manner.

IKK-α/β inhibition by BzSA
In order to determine the effect of BzSA on NF-κB pathway and its regulatory IKK-α/β complex, we examine the effect of BzSA and ASA on LPS stimulated IKK-α/β in RAW 264.7 cells. Interestingly, BzSA down-regulated LPS stimulated phospho IKK-α/β higher than ASA in a dose responsive manner (Fig. 4). However, The total IKKα and IKK-β levels were remains same in BzSA, ASA and the controls (Fig. 4).

NF-κB
In order to determine the effect of BzSA on NF-κB activation, RAW 264.7 cells were pre-treated with BzSA and assess the inhibition of LPS stimulated p-NF-κBp65. Interestingly, down-regulation of p-NF-κB-p65 levels in BzSA pre-treated cells were reduced in a dose dependent manner upon LPS stimulation (Fig. 4). Whereas, in ASA pre-treated cells there was no much reduction of LPS stimulated p-NF-κB-p65 levels were noticed as compared to BzSA (Fig. 4).

BzSA inhibits NF-κB-and its responsive gene expression in RAW 264.7 cells
Nuclear translocation of p-NF-κBp65 triggers the expression of in ammatory mediators such as COX-1, COX-2, 5-LOX, TNF-α, iNOS and cytokines such as IL-1β and IL-6, whose expression play an important roles in immune, stress, apoptosis, cell proliferation, cell differentiation and development. Here pretreatment of BzSA down-regulated the expression of LPS stimulated in ammation mediators and cytokines in RAW 264.7 cells.

TNF-α
TNF-α is an in ammation responsive marker protein and TNF-α antagonists may be effective in treating various in ammatory disorders. Inhibition of TNF-α proved to be an effective therapy for patients with rheumatoid arthritis and other forms of in ammatory disease including psoriasis, psoriatic arthritis, ankylospondylitis and in ammatory bowel disease. A moderate reduction of reduction of TNF-α in BzSA and ASA pretreated RAW 264.7 cells were noticed (Fig. 5).

iNOS
Inducible nitric oxide synthase (iNOS) is one of the three key enzymes that generate nitric oxide (NO) from the amino acid arginine. iNOS-derived NO is a free radical, whose predominant function is that of a messenger through cGMP. iNOS-derived NO plays an important role in numerous physiological (e.g. blood pressure regulation, wound repair and host defence mechanisms) and pathophysiological (in ammation, infection, neoplastic diseases, liver cirrhosis, diabetes) conditions and associated with malignant disease. In particular, prevital effects such as malignant transformation, angiogenesis, and metastasis are modulated by iNOS. Interestingly, BzSA pre-treatment dow-regulated LPS stimulated iNOS in RAW 264.7 cells in a dose wise (Fig. 5). Similarly, ASA pre-treatment were also down-regulated iNOS completely at 4mM (Fig. 5).

BzSA inhibits IL-1β and IL-6
Interleukin-1β (IL-1β) and interleukin-6 (IL-6) are major inducers of hepatic in ammation and the acute phase response. Over expression of IL-6 has been implicated in the pathologyof a number of diseases including multiple myeloma, rheumatoid arthritis, castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence selective antagonists of IL-6 may offer therapeutic bene ts. Interestingly, BzSA pretreatment completely down-regulated LPS stimulated IL-6 in RAW 264.7 cells even at 200µM concentration (Fig. 5 ). Whereas ASA inhibit the expression of LPS stimulated IL-6 in a dose dependent manner, however less effective compared to BzSA (Fig. 5). Similarly, our results shown that BzSA pretreated cells down-regulated IL-1β and IL-6in a dose dependent manner and found to be better response compared to ASA (Fig. 5).

Discussion
The The cytotoxicity MTT assay results of RAW264.7 cells were shown that the IC 50 value of BzSA slight higher than aspirin (Fig. 2). Aspirin indued a decrease in cell viability in a time and deose dependent manner (Bellosillo et al., 1998). Interestingly, BzSA have shown more COX-2 enzyme inhibition activity over ASA (Table 1). NSAIDs including aspirin, indomethacin and ibuprofen are non-selective inhibitors of COX-1 and COX-2, whereas, celecoxib and rofecoxib selectively inhibit COX-2 enzyme activity (Rao and Knaus, 2008). NSAIDs are potent anti-in ammatory and anti-cancer agents that acts through the inactivation of the COX enzymes, mostly COX-2 and thus directed the prostaglandins (PGs) synthesis at the site of in ammation (Rao and Knaus, 2008;Willoughby et al., 2000). BzSA have no effect on 5-LOX enzyme inhibition (Table 1). In view of the importance of dual COX-2/5-LOX, identi cation of speci c inhibitors of COX-2/5-LOX proteins have therapeutic advantage (Fiorucci et al., 2001;Ranjbar et al., 2016). Interestingly, BzSA inhibits COX-2 and 5-LOX expression higher than aspirin (Fig. 3). COX-2 expression is highly restricted and selectively induced by pro-in ammatory cytokines at the site of in ammation and thus involved in the production of pro-in ammatory PGs (Crofford, 1997;Rao and Knaus, 2008;Seibert and Masferrer, 1994).
NF-κB induce 5-LOX enzyme expression during the in ammation process (Pahl, 1999). 5-LOX enzyme catalyzes AA metabolism in the leucocytes (Steinhilber, 1999). Leukotrienes (LTs) are the metabolic products of AA metabolism, which possess a potent pro-in ammatory activity and thus might be involved in cardiovascular diseases (de Gaetano et al., 2003). Decreased expression of 5-LOX gene enhanced cell death in breast cancer cells and therefore plays an essential role in reducing the tumor proliferation (Kumar et al., 2016). The 5-LOX enzyme inhibitor zileuton reduces in ammatory reaction in the ischemic brain damage through the activation of P13K/Akt signaling pathway (Tu et al., 2016). A report suggest that 7-subsituted coumarin derivatives are potential 5-LOX inhibitors (Srivastava et al., 2016).
Interestingly, the inhibition effect of BzSA on COX-1 enzymatic activity very low as compared to ASA (Table 1). However, BzSA and ASA have no effect at COX-1 expression even increasing concentrations (Fig. 3). One of the major disadvantage of ASA is inhibition of COX-1 and associated anti-thrombic and ulcerogenic side effects (Bianchi Porro and Pace, 1988; Undas et al., 2007). COX-1 expressed constitutively in almost all cell types in the human body and thus produced anti-in ammatory PGs which are important to maintain the homeostatic functions such as integrity of the gastric mucosa, platelet function, and renal blood ow (Allison et al., 1992). These results encouraging that the denti cation of selective inhibitors of COX-2 with out effecting COX-1 are useful therapeutics.
In order to validate the effect of BzSA on NF-κB pathway we tested a series of NF-κB targeted in ammatory mediaters. BzSA suppressed COX-2 (Fig. 3 ). COX-2 expression depends on NF-κB activation (Lim et al., 2001). Previosely it was reported that pro and anti-in ammatory nature of COX-2 (Poligone and Baldwin, 2001). The expression of in ammatory mediators such as TNF-α, COX-2, 5-LOX, iNOS and cytokines such as IL-1β and IL-6 require translocation of NF-κB from cytosol to nucleus (Rao and Knaus, 2008). TNF-α is a pleiotropic cytokine, which is a therapeutic target for in ammatory diseases therefore TNF-α inhibitors are therapeutic advantages in in ammatory disorders and malignant diseases (Esposito and Cuzzocrea, 2009;Parameswaran and Patial, 2010). Previously, it was reported that ASA inhibits TNF-α gene expression in RAW 264.7 cells through the suppression of active NF-κB binding to the TNF-α promotor (Shackelford et al., 1997). No signi cant inhibition of TNF-α by BzSA and ASA noticed (Fig. 5). Literature suggested that induced expression of TNF-α activated by NF-κB in different cell lines (Ke et al., 2007;Parameswaran and Patial, 2010;Wajant et al., 2003). Similarly, the expression of iNOS depends on the nuclear localization of activated NF-κB. BzSA suppressed iNOS higher than ASA (Fig. 5).
Previously, it was suggested that inducible nitric oxide synthase (iNOS) is activated by several immunological stimuli and the resultant accumulated nitric oxide cytotoxic to the cells. Furthermore, iNOS mutants exhibited reduced immune response to carrageenin and showed resistance to LPS induced mortality (Vig et al., 2004;Wei et al., 1995). Increased iNOS associated with malignant diseases, particularly malignant transformation, angiogenesis and metastasis (Lechner et al., 2005).
Interestingly, in our study we observed more suppression of IL-1β in BzSA treated cells as compared to ASA (Fig. 5). Interleukins are the cytokines important in the regulation of immune responses, in ammatory reactions and hematopoiesis. Over activation of inmmune system causes several in ammatory disorders. Anti-in ammatory agents including ASA, anti-cytokine therapies, small molecules and many drugs under clenical trails. IL-1β and IL-6 are the the pro-in ammatory cytokines responds to in ammation. IL-1β induces IL-6 in peripheral blood monocytes (Tosato and Jones, 1990). Proin ammatory cytokines such as TNF-α, IL-1β and vascular growth factor (VEGF) plays a central role inin ammation (Dinarello, 2010). In our results we also noticed remarkable reduction in IL-6 by BzSA in a dose dependent manner over ASA (Fig. 5). Previously reports demonstrated that IL-1β and TNF-α induce IL-6 and most of the biological activities associated with IL-1 and IL-6 (Tosato and Jones, 1990).

Conclusions
Our ndings highlighte the anti-in ammatory and anti-cancer therapeutic proterties of BzSA in RAW 264.7 cells. Interestingly, BzSA exhibited more COX-2 inhibition activity and low COX-1 inhibition activity over ASA. BzSA suppressed NF-κB pathway and the mechanism involves the inhibition of up-stream IKKcomplex is similar to ASA (Fig. 6). Hence we suggested that the BzSA is a potencial anti-in ammatory and anti-caner agent like aspirin.

Declarations
Con ict of interest Statement: We wish to con rm that there is no con icts of interest associated with this publication and no signi cant nancial support for this work that could have in uenced its outcome.     Effect of BzSA on TNF-α, iNOS, IL-1β and IL-6 expression in RAW 264.7 cells. Raw 264.7 cells pre-treated with BzSA and ASA for 24h followed by stimulated with LPS. The whole cell protein lysate was prepared and performed western blotting analysis. Down-regulation of TNF-α, iNOS, IL-1β and IL-6 in BzSA pretreated cells upon stimulation with LPS as compared to ASA. DMSO 0.01% /ml and 0.25µg/ml LPS treated RAW264.7 cells was used as controls. Beta-actin was served as a loading control.

Figure 6
Proposed mechanism of NF-κB pathway inhibition in BzSA pre-treated RAW 264.7 cells upon LPS stimulation.