Anti-inflammatory effects of quercetin, rutin, and troxerutin result from the inhibition of NO production and the reduction of COX-2 levels in RAW 264.7 cells treated with LPS

doi:10.21203/rs.3.rs-3914566/v1

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Research Article

Anti-inflammatory effects of quercetin, rutin, and troxerutin result from the inhibition of NO production and the reduction of COX-2 levels in RAW 264.7 cells treated with LPS

https://doi.org/10.21203/rs.3.rs-3914566/v1

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published 03 Aug, 2024

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Flavonols effectively scavenge the reactive nitrogen species (RNS) and reactive oxygen species (ROS) and act as immune-enhancing, anti-inflammatory, anti-diabetic, and anti-carcinogenic agents. Here, we explored the comparative antioxidant and anti-inflammatory properties of plant-originating flavonols, like quercetin, rutin, and troxerutin against acetylsalicylic acid. Quercetin and rutin showed a high ability to remove active ROS, but troxerutin and acetylsalicylic acid exhibited little such function. In RAW 264.7 cells, quercetin, rutin, and troxerutin did not exhibit cellular toxicity at low concentrations. In addition, quercetin, rutin, and troxerutin considerably (p < 0.05) lowered the protein expression of cyclooxygenase 2 (COX-2) as compared to acetylsalicylic acid in cells inflamed with lipopolysaccharides (LPS). Additionally, in inflamed cells, quercetin and rutin significantly down-regulated the nitrogen oxide (NO) level (p < 0.05) at higher concentrations, whereas Troxerutin did not reduce the NO level. In addition, Troxerutin down-regulated the pro-inflammatory protein markers, such as TNF-α, COX-2, NF-κB, and IL-1β better than quercetin, rutin, and acetylsalicylic acid. We observed that troxerutin exhibited a significantly greater anti-inflammatory effect than acetylsalicylic acid did. Acetylsalicylic acid did not significantly down-regulated the expression of COX-2 and TNF-α (p < 0.05) compared to troxerutin. Hence, it can be concluded that the down-regulation of NO levels and the expression of COX-2 and TNF-α proteins could be mechanisms of action for the natural compounds quercetin, rutin, and troxerutin in preventing inflammation.

Antiinflammation

Acetylsalicylic acid

Down-regulation

Antioxidant

Bioactive compounds

Concentration-dependent

Heat, swelling, redness, and pain are all symptoms of inflammation, which are the body's natural defense against infection or tissue damage (55, 33). In the inflammation phase, macrophages control the inflammatory response through the synthesis of several inflammatory mediators, such as prostaglandin, tumor necrosis factor, cyclooxygenase, and interleukin (50, 46, 37). COX-1 and COX-2 are two isoforms of cyclooxygenase. COX-1 is generally inducible, and its function is to maintain blood flow in the gastrointestinal tract and kidney, and also involved in physiological activities such as vascular homeostasis and antithrombosis (42). On the other hand, lipopolysaccharides (LPS) induced the production of COX-2, Interleukin-1 (IL-1), and Tumor Necrosis Factor-α (TNF-α) which causes inflammation, high fever, and pain (32). Activated COX-2 induces prostaglandins, prostacyclin, and thromboxane production, which has a significant role in generating and maintaining inflammatory reactions (22, 23, 45).

When a normal inflammatory response lasts for an abnormally long time, inflammation-related diseases occur, which is called chronic inflammation. Acute inflammation is caused by neutrophils, while chronic inflammation is a response dominated by macrophages. Activated macrophages secrete inflammatory mediators like Eicosanoid and Collagenase, which leads to tissue remodeling and tissue destruction (5, 17, 28, 27).

Some representative chronic inflammatory illnesses are inflammatory bowel disease, chronic nephritis, rheumatoid arthritis, asthma, cancer, cardiovascular disease, and many more (25, 43). To treat these, steroids and Nonsteroidal anti- inflammatory drugs (NSAIDs) are employed, which inhibit the production of prostaglandin, prostacyclin, and thromboxane by suppressing protein and gene expression of COX-2 or blocking the active site of COX-2 (18, 4, 35, 31). A very popular NSAID is acetylsalicylic acid (ASA) also known as aspirin, which is a salicylic acid derivative. Its broad spectrum of effects is an anti-inflammatory, antipyretic, and analgesic medicine. The key mechanism of action is the prevention of the manufacture of prostanoids, which are chemicals engaged in inflammatory processes and have a variety of actions in the body. Aspirin has also been shown to block several signaling pathways in cancer cells, including the Ras/c-Raf, NF- κB, extracellular signal-regulated kinase (ERK)/MAPK, and mTOR pathways (15). Aspirin's chemopreventive effect may also result in a rise in tumor-infiltrating lymphocytes in tumor microenvironment (16).

In addition, ROS and RNS are produced through metabolism using oxygen within the cells (24). They have high activity on cells or mitochondrial membranes and consequently damage cells. Furthermore, a large concentration of ROS or RNS oxidizes biomolecules or modifies proteins or genes, resulting in inflammatory disorders. Therefore, rapid removal of ROS and RNS through antioxidant action plays an important role in preventing inflammation. Flavonoids are secondary metabolites widely spread in the plant kingdom and have been used as folk remedies or anti-inflammatory agents. So, flavonoid or polyphenol has excellent antioxidant activity which suppresses inflammation, and inflammatory diseases (2, 19, 38).

Quercetin is a polyphenol of the flavonol class and is contained in grains, berries, vegetables and fruits, buckwheat seeds, capers, and lettuce (7, 20). It has also been implicated as one of the nutraceuticals in preventing various chronic diseases. Quercetin has immune-enhancing, anti-inflammatory, antioxidant, anti-diabetic, anti-carcinogenic, and antibacterial properties (20). Chemically, quercetin (Fig. 1) is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one.

On the other hand, rutin (Fig. 1), also called Quercetin-3-O-rutinoside or rutoside, is a substance obtained by combining the disaccharide rutinose with Quercetin and chemically called as 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one. Naturally, rutin is abundantly found in buckwheat, red grapes, asparagus, capers, and olives (48). Numerous studies have demonstrated rutin has excellent health benefits of preventing neurodegenerative diseases, cardiovascular diseases, skin cancer, and more (14).

Troxerutin is a flavanol among flavonoids derived from rutin and is a semi-synthetic rutin derivative (9, 53). Chemically, troxerutin (Fig. 1) is 2-[3,4-bis(2-hydroxyethoxy)phenyl]-5-hydroxy-7-(2-hydroxyethoxy)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-2-yl]oxychromen-4-one.

Troxerutin (Fig. 1), also called vitamin P4, is a derivative of rutin, and can be obtained in coffee, tea, cereal grains, and different fruits and vegetables (1). Due to its high-aqueous solubility, it has been found to be readily absorbed by the gastrointestinal system and to produce protective effects without being cytotoxic (40). The naturally occurring troxerutin has variety of biological roles, for example fighting against cancer, reduces inflammation, free radicals scavenging, and to treat diabetes (44).

Here, we investigated a study of the antioxidant activities of quercetin, rutin, and troxerutin as compared to ascorbic acid and their anti-inflammatory influences in RAW 264.7 cells against acetylsalicylic acid. The novelty of the work is that this type of study was not previously performed that checked the tentative mechanism of action selected against acetylsalicylic acid to prevent inflammation.

Chemicals and reagents

Quercetin, Rutin, and Troxerutin were purchased from Pingyu Yimeikang Plant Technology Co., LTD (Pinggyu, Henan, China). RAW 264.7 (KTCC No.40071) cells were purchased from Korea Cell Line Bank, Seoul, Korea. Dulbecco's Modified Eagle Medium (DMEM)) (Welgene, Gyeongsan, Republic of Korea), and 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) were purchased. Methanol (Thermo Fisher Scientific, Waltham, USA), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) from Sigma Aldrich, USA were purchased. All other reagents were of analytical grades.

Determination of Antioxidant Capacity

The activity of free radical scavenging of test samples was measured by a previously reported DPPH assay protocol [1]. Briefly, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging activity was measured by mixing 100 µL of 0.2 mM DPPH (Sigma Aldrich, USA) solution in ethanol with 200 µL of each diluted test sample of acetylsalicylic acid, quercetin, rutin, and troxerutin in different concentration (10, 50, 100, 250, 500, and 1000 µg/mL). The prepared solutions were kept at room temperature for 30 min. After the reaction, absorbance was measured at 517 nm by utilizing an ELISA reader (Infinite™ F200, TECAN, Switzerland). Furthermore, ascorbic acid was employed as a positive control. Ethanol was utilized as a control instead of DPPH. Eq. (1) was used to calculate the DPPH˙ free radical scavenging capability of test agents.

DPPH % scavenging activity = [1 − Absorbance test sample/Absorbance control] × 100 (1)

ABTS⁺ scavenging radical activity was also performed as formerly reported (20). In short, 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity was calculated by dissolving 7 mM ABTS (Sigma Aldrich, USA) in distilled water and then 2.45 mM potassium persulfate added to obtain a ratio of 1:1. After mixing, the solution was left at an ambient temperature of the room in a dark for 12 to 18 h for the formation of ABTS radicals. Then ABTS radicals’ solution was further diluted by using ethanol, and the absorbance was taken at 734 nm, which was accustomed to 0.706 ± 0.002. The scavenging ability was determined by mixing 150 µL of ABTS solution and 150 µL mixing of test samples with each concentration (10, 50, 100, 250, 500, and 1000 µg/mL). Afterward, the solutions were left to complete the reaction at room temperature for 6 min, and then absorbance was measured at 734 nm using an ELISA reader. While ascorbic acid was used as a positive control. Eq. (2) was used to determine the DPPH˙ free-radical-scavenging activity.

ABTS % scavenging activity = [1 − Absorbance test sample/Absorbance control] ×100 (2)

Cell culture

RAW 264.7 (KTCC No.40071) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)) with 10% fetal bovine serum (FBS) and penicillin (100 units/mL)/streptomycin (100 µg/mL). The cells were cultured in an incubator (NU-4750G, NuAire, Plymouth, MN, USA) under a 5% CO₂ atmosphere and subcultures were performed at intervals of 2 to 3 days.

Measurement of cell viability of quercetin, rutin, and troxerutin

The cytotoxicity or cell proliferation rate of quercetin, rutin, and troxerutin were measured. Cell Titer 96^®AQ_ueous One Solution Cell Proliferation Assay Kit (Promega, USA) was used to determine the effect on cell proliferation. The cell proliferation assay reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine ethosulfate; PES). To assess the cytotoxicity and proliferation, the RAW 264.7 cells were uniformly distributed in a 96-well plate at 1×10⁴ cell/well and cultured on DMEM medium with 10% FBS at 37℃ for 24 h in a 5% CO₂ environment. The test sample medium was then replaced with fresh DMEM medium without FBS and incubated at 37℃ for 96 h in a 5% CO₂ atmosphere. Each test sample medium contained each substance at concentrations of 10, 50, and 100 µg/mL. After 96 h, the sample medium was taken out from each well, and then 120 µL of DMEM medium with MTS reagent was added and incubated for 3h at 37℃ in 5% CO₂ conditions. The 96-well plate was read for absorbance at 490 nm by applying an ELISA reader.

Cell Titer 96®AQueous One Solution 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS). Measurement of cell viability of quercetin, rutin, and troxerutin with LPS was performed as the same cell proliferation assay method described above. Meanwhile, LPS was applied 48 h after sample treatment.

Quantification of nitric oxide production by quercetin, rutin, troxerutin, and acetylsalicylic acid

Nitric oxide (NO) assay was implemented to compute the effect of quercetin, rutin, and troxerutin on nitric oxide synthesis as compared to control and acetylsalicylic acid. Briefly, RAW 264.7 cells were grown in a 24-well plate at 2 × 10⁵ cells/well in DMEM medium supplemented with 10% FBS at 37℃ for 24 h under 5% CO₂ conditions. The test sample medium was then reinstated with fresh DMEM media without FBS and at 37 ℃ incubated for 48 h in a 5% CO₂ atmosphere. Each test sample was tested at concentrations of 10 and 100 µg/ml. Then LPS (1 µg/ml) was added to the wells treated with test samples and incubated at 37℃ for 48 h more under a 5% CO₂ atmosphere. To measure NO synthesis, the concentration of NO₂⁻ was calculated as follows: 50 µL of the cell supernatant and 50 µL of 1% Sulfanilamide in 5% phosphoric acid were mixed and reacted at 25℃ for 10 minutes. Thereafter, 50 µL of 0.1% N-1-naphthylenediamine was mixed and again reacted at 25℃ for 10 minutes, and then absorbance was taken at 548 nm by using an ELISA apparatus. NO₂⁻ concentration was calculated by employing a sodium nitrite standard curve. Eq. (3) was used to determine the inhibition of NO₂⁻ production.

Inhibition of NO₂⁻ (%) = [1 – NO₂⁻ production with sample/NO₂⁻ production without sample] ×100 (3)

Anti-inflammatory activity of acetylsalicylic acid, quercetin, rutin, and troxerutin with and without LPS-induced RAW 264.7 cells

Inflammation biomarkers were determined by applying a formerly stated method (20). Shortly, RAW 264.7 cells were uniformly distributed in 4.5 × 10⁷ cells per plate in a 100 mm dish. Cells were cultured in DMEM media containing 10% FBS at 37℃ for 24 h in a 5% CO₂ environment. The test sample media was then reinstated with fresh DMEM media without FBS and at 37℃ incubated for 96 h in 5% CO₂ conditions. Each test sample was added at concentrations of 10 and 100 µg/ml.

Similarly, for induction of inflammation by lipopolysaccharides (LPS), the cells were cultured in DMEM media with 10% FBS at 37℃ for 24 h in a 5% CO₂ environment. Then the test sample media was changed with fresh DMEM media without FBS and incubated at 37℃ for 48 h. Then 1µg/mL of LPS was mixed in each plate and incubated at 37℃ for 48 h in 5% CO₂ conditions. Cells were scraped off with a scraper and harvested by centrifugation. After harvesting, proteins were extracted by adding an M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific Waltham, USA) with Halt Protease Inhibitor Cocktail, EDTA-Free (Thermo Scientific, Waltham, USA). Thereafter, the protein concentration was measured and stored at -80°C.

Analysis of Western blot

In short, 30 µg of proteins were taken apart by electrophoresis by employing 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transported to a PVDF (polyvinylidene fluoride) membrane. The PVDF membrane was kept at ambient room temperature for 1 h with Western Enhanced Buffer (NEOSCIENCE, Seoul, Korea). Then, a western blot was performed with primary antibodies at 25℃ for 2 h with continuous rocking. Primary antibodies in each PVDF membrane were used at a ratio of COX-2 (1:1000), IL-1β (1:500), NFκB p65 (1:500), TNF-α (1:1000), and β-Actin (1:1000). The PVDF membrane of each sample was washed with TBS-T for 10 min and repeated three times. The washed PVDF membranes were blotted with secondary antibody at 25℃ for 1 h with continuous rocking. The secondary antibodies were incubated with a ratio of 1:10000 of Goat Anti-Rabbit IgG antibody (HRP) (GeneTex, Irvine, USA) for polyclonal antibodies or m-IgGκ BP-HRP (Santa Cruz Biotechnology, Dallas, USA) for monoclonal antibodies. After completion of the secondary antibody reaction, SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific Waltham, USA) solution was applied on the PVDF membrane (Sigma Aldrich, St. Louis, USA), and the film was sensitized in the dark and then developed. The relative intensities of specific protein bands were quantified using ImageJ®.

Primary antibodies were used as follows: COX-2 (GeneTex, Irvine, USA), IL-1β (Santa Cruz Biotechnology, Dallas, USA), NFκB p65 (Santa Cruz Biotechnology, Dallas, USA), TNF-α (GeneTex, Irvine, USA) and β-Actin (Santa Cruz Biotechnology, Dallas, USA). Secondary antibodies were used Goat Anti-Rabbit IgG antibody (HRP) (GeneTex, Irvine, USA) and m-IgGκ BP-HRP (Santa Cruz Biotechnology, Dallas, USA).

Statistical analysis

The experiment was performed three times and statistical analysis expressed as mean ± standard deviation, by utilizing the Student's t-test, and a p-value of < 0.05 was considered statistically significant.

A new generation of natural anti-inflammatory agents has drawn researchers' interest due to their potential anti-inflammatory benefits and safety. Lipopolysaccharides (LPS) is located in gram-negative bacteria on the outer membrane (54). When microorganisms are infected, cells are continuously exposed to LPS, causing inflammation. Numerous distinct cell types, including macrophages and neutrophils, can react to LPS by generating strong inflammatory mediators through activating macrophages, such as TNF-α, IL-1, IL-6, and other inflammatory mediators like NO, and prostaglandins (34). Smooth muscle cells, epithelial and endothelial cells, and other cells have also been demonstrated as the target for LPS stimulation. The activation of cells by LPS leads to the pathophysiology of several disorders, for instance, atherosclerosis, rheumatoid arthritis, asthma, and pulmonary fibrosis. Furthermore, the RAW 264.7 cells are macrophage murine cell lines and are used as a good model for the screening of anti-inflammatory drug(s) and consequently can be assessed the routes of inhibition that identify the production of pro-inflammatory cytokines. In this investigation, specifically LPS-stimulated and without LPS-stimulated RAW 264.7 cells were used as an in vitro model to examine the effects of quercetin, rutin, troxerutin, and acetylsalicylic acid on the production of pro-inflammatory chemicals such as NO, IL-1β, COX-2, NF-κB (p65) and TNF-α.

Antioxidant activity

Radical scavenging activity of acetylsalicylic acid, quercetin, rutin, and troxerutin

The DPPH and ABTS scavenging activity of quercetin, rutin, and troxerutin against control and acetylsalicylic acid were measured and results are shown in Figures 2a, and b. Quercetin showed a high antioxidant effect like that of the positive control i.e., ascorbic acid at all concentrations in DPPH assay (Fig. 2a).

In the case of rutin, 40.4% of the scavenging activity was found at 10 μg/mL, while 50, 100, 250, 500, and 1000 μg/mL concentrations showed excellent antioxidant effects similar to those of the positive control group. Troxerutin exhibited very low DPPH scavenging action and showed concentration-dependent DPPH radical scavenging properties with percentages of 3.7%, 12.8%, 13.1%, 17.9%, 19.9%, and 23.3% at concentrations of 10, 100, 250, 500, and 1000 μg/mL, respectively (Fig. 2a). Hence, it was confirmed that troxerutin has a low antioxidant effect. Also, acetylsalicylic acid, which was utilized as an anti-inflammatory drug, showed a scavenging activity of 13.1% at a concentration of 1000 μg/mL, indicating a low antioxidant effect. Therefore, it can be assumed that quercetin and rutin displayed significantly (p<0.05) better antioxidant effects than troxerutin and acetylsalicylic acid (Fig. 2a). In our interpretation, the high antioxidant activity of quercetin and rutin may be due to their ability to release electrons to hydrogen radicals or electron discharge to DPPH.

The ABTS assay was employed to determine the anti-radical ability. After bonding with ABTS in dark conditions (12–14 h), potassium persulphate makes the blue chromophore recognized as ABTS. The results of measuring the ABTS scavenging activity by employing quercetin, rutin, and troxerutin against control and acetylsalicylic acid are shown in Figure 2b. Quercetin and rutin showed excellent ABTS radical scavenging activity effects almost that of the positive control (ascorbic acid). Troxerutin exhibited concentration dependent ABTS radical scavenging activity with percentages of 26.5%, 27.7%, 30.2%, 37.2%, 38.7%, and 42.9% at concentrations of 10, 100, 250, 500, and 1000 μg/mL, respectively (Fig. 2b). Also, acetylsalicylic acid exhibited a very low antioxidant effect with 17.7% of the ABTS+ radical scavenging activity at a concentration of 1000 μg/mL as compared to quercetin, rutin, and ascorbic acid. Hence, as compared to ascorbic acid, quercetin, and rutin, troxerutin, and acetylsalicylic acid showed lower antioxidant effects.

The antioxidant property of rutin was found lower than that of quercetin which may be due to glycosylation, and also the antioxidant activity of troxerutin was observed very low which may be due to the ethylation of the hydroxyl group (8). Also, quercetin and rutin have phenolic hydroxyl groups, hence showing better antioxidant activity than troxerutin and acetylsalicylic acid. Thus, quercetin and rutin are known to acquire powerful antioxidant activities and radical scavenging properties. Furthermore, cell membrane lipids, DNA, proteins, and lipoproteins are all damaged due to stress-induced oxidation or excessive production of ROS in the cell's metabolism. The overproduction of ROS in these cells triggers an inflammatory response. Many studies have shown that plant phenolic compounds contain phenolic hydroxyl groups that can offer hydrogen to lower the free radicals and stop the oxidation of lipids, proteins, and DNA (41, 10). Polyphenols and flavonols have high antioxidant activity due to the elimination of LPS-induced ROS, thereby suppressing the inflammatory response markers (6). Therefore, ROS scavenging by using natural products would be useful as a fundamental target for various inflammatory diseases (10).

Cell viability studies

The MTT test is frequently employed in determining the level of toxicity of any natural or synthetic compound(s) by assessing cell viability. To investigate the impact of quercetin, rutin, and troxerutin on cell viability, RAW 264.7 cells were used. Cell viability experiment was performed with and without LPS. Various concentrations of quercetin, rutin, and troxerutin were added to the cell culture medium and cultured, followed by cell viability experiments. The cell viability of quercetin, rutin, and troxerutin was found in a dose-dependent manner (Fig. 3a and b).

Cell viability without LPS of quercetin, rutin, and troxerutin on RAW 264.7 cells

The results of cell viability without LPS of RAW 264.7 cells by treating with quercetin, rutin, and troxerutin are shown in Figure 3a. Quercetin displayed 99.2% and 94.9% at 5 and 10 μg/ml concentrations, respectively, and results were found like the untreated control group. However, the cell viability reduced to 64.2% and 56.9% at concentrations of 50 and 100 μg/ml, respectively. The cell viability of rutin was found 109.5% and 83.3% at 5 and 10 μg/ml concentrations, respectively. However, cell viability down to 61.3% and 51.7% at concentrations of 50 and 100 μg/ml, respectively. Troxerutin showed a survival rate of 95% or more at concentrations of 5, 10, and 50 μg/ml against the untreated control, while an 82.9% survival rate was observed at 100 μg/ml. Results indicated that as the concentrations of test compounds increased that cell viability decreased.

Cell viability in LPS-induced RAW 264.7 cells of quercetin, rutin, and troxerutin

The results of measuring the cell viability of RAW 264.7 cells after treatment with quercetin, rutin, and troxerutin followed by LPS treatment (1 μg/ml) are shown in Figure 3b. In all experimental groups, the survival rate was found to be more than 95% at all concentrations against the untreated control, while 100 μg/ml concentration of rutin showed a survival rate of 94.5%.

In the presence of LPS, our findings suggested that quercetin, rutin, and troxerutin do not cause significant toxicity to cells. Importantly, in this outcome, it was found that troxerutin was showed significantly less toxic than quercetin and rutin at high concentrations. However, quercetin, rutin, and troxerutin treatments maintained the viability of macrophages to LPS stimulation. According to earlier investigations, mild effects of LPS stimulation on macrophage viability were seen (13, 49, 3). This may be because of LPS induction that potentiates macrophages to induce proteins associated with autophagy and death.

Production of nitric oxide by quercetin, rutin, troxerutin, and acetylsalicylic acid

The highly reactive free radical nitric oxide (NO) has a role in a variety of pathological and physiological inflammatory routes. Hence, NO might have a significant impact on the pathophysiology of several diseases. Several mammalian tissues and cells use distinct NO synthases (NOS) to catalyze the oxidation of terminal guanidino nitrogen of l-arginine (Arg) to make NO and citrulline. Also, NO has immunological cytotoxic effects on tumor cells or invasive pathogens. Likewise, NO either alone or in combination with other free radical ions, causes cell injury during inflammation.

Also, LPS potentiated macrophages for NO production and triggered an inflammatory response. Hence, we studied the inhibition or reduction of NO production by acetylsalicylic acid, quercetin, rutin, and troxerutin in RAW 264.7 cells in the absence and presence of LPS (Table 1, Fig. 4). Cultured RAW 264.7 cells were treated with acetylsalicylic acid, quercetin, rutin, and troxerutin at concentrations of 10 and 100 μg/ml, respectively. Acetylsalicylic acid showed comparable NO inhibition at concentrations of 10 and 100 μg/ml against the LPS-treated control group. However, quercetin exhibited less NO inhibition in comparison to a control group treated with only LPS and acetylsalicylic acid at 10 μg/mL (Table 1). While, at 100 μg/mL, quercetin showed substantial (p<0.05) inhibition of NO production against acetylsalicylic acid and control group (Table 1). In the case of rutin, at 10 μg/mL concentration, it showed very low NO inhibition contrary to the control group, but at 100 μg/mL, it displayed better inhibition of NO production than control treated with LPS. However, Troxerutin showed no significant NO inhibition at any of concentrations.

Table 1. Nitric oxide (NO) assay results in the absence and presence of LPS of selected agents

Group	Concentration (㎍/mL)	No LPS	+ LPS (1㎍/mL)
Group	Concentration (㎍/mL)	NO₂^- production (μM)* Mean±SD	NO₂^- production (μM)* Mean±SD

Control	0	0.0	27.58±1.3
Acetylsalicylic acid	10	0.19±0.3	24.08±0.1
	100	0.15±0.3	24.29±0.3
Quercetin	10	0.27±0.4	25.57±0.6
	100	0.23±0.4	22.71±0.9
Rutin	10	0.20±0.1	27.67±0.2
	100	0.03±0.3	22.20±1.1
Troxerutin	10	0.23±0.9	27.03±0.7
	100	0.06±0.6	27.15±0.5

*NO₂^- production (μM) experiments were repeated three times.

In our findings, in the absence of LPS, quercetin, rutin, and troxerutin did not produce NO, so they did not induce inflammation (Table 1). Furthermore, in the presence of LPS, quercetin, and rutin notably (p<0.05) diminished the NO production at 100 μg/mL (Fig. 4). Hence, it can be confirmed that quercetin and rutin reduced the NO production, but Troxerutin did not significantly reduce the NO production. The difference in NO inhibition may be due to the presence or absence of polyphenol groups in the compounds. Hence, quercetin and rutin can be used to treat diseases that are induced by NO production.

Western blot analysis

Changes in anti-inflammatory protein levels by quercetin, rutin, troxerutin, and acetylsalicylic acid without LPS

An intricate physiological process is called inflammation. Unidentified inflammation triggered by macrophages and other immune cells can lead to organ failure and damage to surrounding tissues (30). To date, safe drugs to treat basic long-term inflammation are lacking and new therapies are needed. Hence, we performed the study to confirm the impacts of quercetin, rutin, and troxerutin on the transformation of RAW 264.7 cells in the absence and presence of LPS (Fig. 5), and the protein expression levels were analyzed using Western blot in the absence of LPS (Fig. 6a and b, Fig. 7a and b). The cultured RAW 264.7 cells were treated with acetylsalicylic acid (Aspirin, ASA), quercetin, rutin, and troxerutin at 10 and 100 μg/ml concentration, followed by culturing for 96 h to confirm changes in the transformation of cells and protein expression.

The expression of COX-2 and TNF-α was not detected in the control group as well as with acetylsalicylic acid, quercetin, rutin, and troxerutin (Fig. 6a and b). This indicated that inflammation was not induced by the treated compounds in the experimental group against the control group. Conversely, we observed the changes in IL-1β and NF-κB levels with selected compounds (Fig. 6a and b).

The level of IL-1β by acetylsalicylic acid was enhanced to 1.7 and 1.6-fold, while quercetin showed the 2.0 and 2.4-fold more level, and rutin exhibited 2.0 and 2.2-fold high level against control group at 10 and 100 μg/ml concentration, respectively (Fig. 6a and b). Furthermore, troxerutin showed only 2.0 times the increased level at a concentration of 10 μg/mL, for 100 μg/mL it exhibited protein expression like the untreated control group (Fig. 6a and b).

Cytokines including TNF-α and IL-1β secreted by immune cells such as macrophages are important pro-inflammatory cytokines for inflammation and are linked to several inflammatory diseases. In terms of physiology, IL-1 is a key player in the inflammation response and is necessary for several cell functions, including proliferation, cell division, and death. Amongst the proinflammatory cytokines, TNFα is a chief agent that facilitates the stimulation of proinflammatory gene transcription and the signaling cascades that are central to inflammation (11). Furthermore, TNF-α is a mediator for cell signaling, and when macrophages recognize antigens, then TNF-α is released to other immune cells as part of an inflammatory response.

Also, the high level of biomarkers, for instance, IL-6 and TNFα increase the synthesis of ROS and cause inflammation (47). Also, the amount of TNF-α was found constant in the treated cells, hence the expression of COX-2 was not produced (Fig. 6a and b). Consequently, the expression of NF-κB level was notable (p<0.05) and found less than the control in all experiment groups. Consecutively, NF-κB is a pro-inflammatory protein that controls the genes for inducible enzymes, for example, iNOS and COX-2. The levels of NF-κB were decreased in all treatment groups, but troxerutin most considerably lowered the expression level of NF-κB by 3-fold. Whereas the IL-1β expression was increased in all groups of treatment except troxerutin at 100 µg/ml. Therefore, results confirmed that quercetin, rutin, troxerutin, and acetylsalicylic acid did not induce inflammation in cells.

Changes in anti-inflammatory protein levels in LPS treated RAW 264.7 cells by quercetin, rutin, troxerutin, and acetylsalicylic acid

The effect of quercetin, rutin, troxerutin, and acetylsalicylic acid in RAW 264.7 cells on the transformation and protein expression levels in the presence of LPS was analyzed (Fig. 7a and b). For this, RAW 264.7 cells were treated with test samples at concentrations of 10 and 100 μg/ml (48 h), and then added LPS at a concentration of 1 μg/ml for an additional 48 h and afterward changes in cells and various protein expressions were observed (Fig. 7a and b). In comparison to the LPS-treated control group, the acetylsalicylic acid increased COX-2 protein expression to 1.1 and 2.6 fold at 10 and 100 μg/mL concentration, respectively. Quercetin increased to 1.7 fold and decreased to 0.3 fold COX-2 protein expression at 10 μg/ml and 100 μg/ml concentration, respectively as compared to control. Further, rutin also augmented to 2.6 times and reduced to 0.3 times COX-2 protein expression at 10 μg/ml, and 100 μg/ml concentration, respectively. Troxerutin also increased to 2.9 times and decreased to 0.6 times COX-2 protein expression at 10 μg/ml and 100 μg/ml concentration, respectively.

Furthermore, IL-1β expression compared to the LPS-treated control group at concentrations of 10 and 100 μg/ml by acetylsalicylic acid was augmented to 1.4 and 1.7 folds, respectively, and by quercetin to 1.7 and 2.1 folds, respectively. While rutin enhanced to 2.2 and 1.9 folds IL-1β expression, and IL-1β expression increased to 1.5 fold and lowered to 0.7 fold by troxerutin at concentrations of 10 μg/ml, and 100 μg/ml, respectively against the control group (Fig. 7b).

Also, it was confirmed that the expression level of NFκB was increased in all experimental groups in comparison with the control group. As compared to the LPS-treated control group, acetylsalicylic acid showed 1.9 and 2.2 times increment in NFκB expression at 10 μg/mL and 100 μg/mL, respectively. While quercetin exhibited 2.1- and 2.4-times enhancement in NFκB expression. Furthermore, rutin augmented NFκB expression by 2.6 and 2.6 times, and 2.0 and 1.7 times increased by troxerutin at 10 μg/mL and 100 μg/mL concentration, respectively against control group. Furthermore, as compared to the control group, acetylsalicylic acid lowered the TNF-α protein expression by 0.7 and 0.3 times at 10 and 100 μg/ml concentration, respectively. Whereas quercetin showed a 0.7 times decrement in TNF-α protein expression at 10, and 100 μg/ml, respectively against the control group. However, rutin reduced the TNF-α protein expression by 0.7 and 0.6 times at concentrations of 10 and 100 μg/ml, respectively. Troxerutin reduced the TNF-α protein expression by 0.1 and 0.4 times at 10 and 100 μg/ml concentration, respectively.

Additionally, the expression of pro-inflammatory genes such as COX-2, IL-1β, NF-κB, and TNF-α by LPS treatment in RAW 264.7 cells are shown in Figure 6A and B. Since, the NF-κB controls many genes involved in the immunological, acute phase, and inflammatory responses, as well as cell survival (21). Subsequently, NF-κB can induce the production of TNF-α, COX-2, iNOS, and IL-6. In the presence of LPS, the expression level of NF-κB enhanced in all experimental groups against the untreated control group (7a and b). When NF-κB expressed more, it is indicated the release of inflammatory biomarkers such as TNF-α and COX-2.

COX-2 is observed in polymorphonuclear leukocytes and is a pro-inflammatory enzyme whose expression is increased in inflammation. Specifically, COX-2 is not expressed in most cells under ordinary conditions, but it is expressed at high levels by physiological stimuli, chemical stress, or wounds and infections (51). The COX-2 enzyme converts arachidonic acid to prostaglandin endoperoxide H2 (PGH2) (52, 36). Also, excessive production of COX-2 increases prostaglandin E2 (PGE2), which causes inflammation, pain, fever, bronchoconstriction, and gastric mucosal acid secretion. It has been testified that acetylsalicylic acid reduces the inflammatory reaction by acetylating the Ser residue in the active site of the COX-2 protein (12, 26). In our experiment, COX-2 expression level by quercetin, rutin, and troxerutin in the presence of LPS was substantially (p<0.05) down-regulated than those of control and acetylsalicylic acid (Fig. 7b). Furthermore, we found that quercetin, rutin, and troxerutin reduced the COX-2 protein levels by suppressing the expression of COX-2 and showed anti-inflammatory activity that may be due to by suppressing the expression of inflammation-related COX-2 protein. Whether quercetin, rutin and troxerutin are direct inhibitors of the COX-2 like aspirin remains to be tested.

It was also noted that TNF-α level decreased by acetylsalicylic acid, quercetin, rutin, and troxerutin as compared to the control group in LPS-treated cells (Fig. 7b). However, Troxerutin showed a significant (p<0.05) reduction in TNF-α expression against the without LPS treated cells. However, increasing the concentration of Troxerutin increased the amount of TNF- α expression but found less than that of LPS alone. Troxerutin displayed good anti-inflammatory effects that may be via managing the levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α, and together with proapoptotic markers such as Bax, p53, and caspase-3. Also, it was found that flavanols may inhibit IKK and MAPK in the LPS-induced inflammatory response, thus showing an anti-inflammatory response (56, 39). Besides, flavanols showed that anti-inflammatory mechanisms may be also by regulating PI3K/AKT/JNK and MAPK/NF-κB pathways to reduce inflammation (29). Further research on the influences and molecular mechanisms of troxerutin is required to identify the structure-activity relationship in numerous molecular regulatory mechanisms. Although Troxerutin's antioxidant capacity was found to be lower than that of quercetin and rutin, however troxerutin showed good anti-inflammatory effect in LPS-induced RAW 264.7 cells. Therefore, out of tested flavanols, troxerutin can be useful in the future to cure inflammatory-centered disorders, for instance, arthritis, colitis, myocarditis, nephritis, and many more.

Quercetin, rutin, and troxerutin suppressed the expression of inflammatory cytokines in LPS-stimulated RAW 264.7 cells. The anti-inflammatory mechanisms of quercetin, rutin, and troxerutin may be via their regulatory consequences on the inflammatory signal pathways like NO, COX-2, and TNF-α inhibition. Moreover, the selected compounds did not show any cytotoxic impacts in the presence of LPS, even at a higher concentration. Briefly, our results concluded that quercetin, rutin, and troxerutin showed anti-inflammatory properties by down-regulating the expression of COX-2 and TNF-α markers significantly than acetylsalicylic acid, by suppressing the synthesis of inflammatory mediators, for example, pro-inflammatory prostaglandins. Among the selected compounds, troxerutin showed less cytotoxicity and better downregulation of inflammatory markers like COX-2 and TNF-α than quercetin and rutin. Therefore, it is presumed that troxerutin has the potential to be used as a highly effective and safe compound to treat inflammation-related disorders. Furthermore, there is a need for further investigations into the specific molecular mechanisms of the anti-inflammatory actions of troxerutin and how it can be applied in the treatment of inflammatory disorders.

Author Contributions

S.G.K., and K.E.L.; designed the concept and study; M.S., G.B.L., and Y.K.; acquisition, analysis, and interpretation of data; M.S. and S.G.K.; drafted the article; V.R., and M.S.; data curation and writing—review and editing, S.G.K.; funding acquisition M.S., S.G.K., K.E.L., V.R., and M.S.; final approval of the version to be submitted. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The authors are thankful to the Core Research Support Center for Natural Products and Medical Materials (CRCNM) at Yeungnam University, Gyeongsan, the Republic of Korea. CRCNM center supported Lyophilizer (FDA5518) and RT-PCR technical analysis. This work has been supported by Stemforce Inc., Gyeongsan, Republic of Korea. Also acknowledge the Researchers Supporting Project number (RSPD2023R674), King Saud University, Riyadh, Saudi Arabia for funding this research work.

Funding

No funding

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Ethics Approval and Consent to Participate

No ethical approval or informed consent was required for this study.

Consent for Publication

Not applicable.

Conflict of Interest

The authors declare no conflicts of interest.

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Anti-inflammatory effects of quercetin, rutin, and troxerutin result from the inhibition of NO production and the reduction of COX-2 levels in RAW 264.7 cells treated with LPS

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Abstract

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Introduction

Materials and Methods

Chemicals and reagents

Determination of Antioxidant Capacity

Cell culture

Measurement of cell viability of quercetin, rutin, and troxerutin

Quantification of nitric oxide production by quercetin, rutin, troxerutin, and acetylsalicylic acid

Analysis of Western blot

Statistical analysis

Results and discussion

Conclusions

Declarations

References

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