Effects of the Chinese Medicinal Herb Scutellaria barbata on Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression on LPS-Induced RAW 264.7 Cells

Gabriel K. Harris, Deborah Cutler Sbarra, Stephen S. Leonard, Yong Qian, Brian Y.Y. Wong, and Xianglin Shi

Effects of the Chinese Medicinal Herb Scutellaria barbata on Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression on LPS-Induced RAW 264.7 Cells

Gabriel K. Harris, Deborah Cutler Sbarra, Stephen S. Leonard, Yong Qian, Brian Y.Y. Wong, and Xianglin Shi

Many thanks to the members of the Shi and Castranova labs at NIOSH-Morgantown for their invaluable help and advice on this project.

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

Extracts of Scutellaria barbata (SB), a plant species related to mint, have been used in traditional Chinese medicine to treat cancers of the liver, lung and breast. Elevated levels of Cyclooxygenase-2 (Cox-2) and Inducible Nitric Oxide Synthase (iNOS) have also been associated with numerous cancers. This study examined the effects of an aqueous SB extract on lipopolysaccharide (LPS) induced Cox-2 protein, prostaglandin E2 (PGE2), iNOS

protein, and NOx (stable oxidation products of nitric oxide) levels in the macrophage-like RAW 264.7 mouse peritoneal cell line. Because antiinflammatory substances often demonstrate antioxidant properties, the effects of SB on H2O2 and hydroxyl radical formation were also tested via confocal microscopy and electron spin resonance,

respectively. RAW cells were treated with SB at concentrations of 0.00, 0.25, 0.50, 0.75, or 1.00 mg/ml. LPS was given at 5 μg/ml. All treatments were applied for 12 hours. We observed a dose-dependent decrease in Cox-2 and iNOS protein expression for all groups treated with SB + LPS. SB alone had no effect on the protein expression of Cox-2 or iNOS. SB treatment decreased PGE2, but not NOx, in a dose-dependent manner. SB treatment

elevated H2O2 and hydroxyl radical production in the same cell line. These data indicate that SB modulates Cox-

2, and iNOS protein expression, as well as PGE2 and NOx in vitro and that this may be related to the formation of

reactive oxygen species.

Western Blotting Analysis for Cox-2 and iNOSRAW cells were cultured in Dulbeco’s Minimal Essential Media supplemented with 5% fetal bovine serum and penicillin/streptomycin at 37oC in a 5% CO2 atmosphere. For each experiment, cells were plated into 12-well

culture plates at a concentration of 1x 106 cells per well. After 24 hours of growth, cells were treated with 0.00, 0.25, 0.50, 0.75, or 1.00 mg/ml SB, with LPS (5 μg/ml), or co-treated with a combination of SB and LPS for 12 additional hours. After correction for total protein content, cell lysates were analyzed by Western blot. All results shown are representative of at least 2 experiments.

PGE2 Analysis by ELISA

Media in which cells were grown for Western blotting analyses was assayed for PGE2 using a monoclonal ELISA

kit (Cayman Chemical).

NOx Measurement by Latchet InstrumentMedia from Western analyses was also used to determine NOx concentrations. Nitrate reductase was used to convert nitrates contained in the media to nitrites. Nitrites were then reacted with Greiss reagent and analyzed by the Latchet Instrument (Zellweger Instruments) to determine NOx concentration.

H2O2 Measurement by Confocal Microscopy

Cells were cultured on sterilized coverslips, placed into 6 well plates, and, otherwise, grown in conditions identical to those above. After 12 hours of treatment, carboxymethyl dichlorofluoroscein was added to growth media (at 5μM final concentration) in order to visualize H2O2. A Zeiss LSM 510 confocal microscope (measuring

emission at 535nm) was used to image intracellular H2O2.

Hydroxyl Radical Measurement by ESR RAW cells were grown using media and culture conditions identical to those for Western blotting analyses. Immediately before the experiment, cells were scraped, centrifuged, and suspended in phosphate-buffered saline together with the spin trap DMPO (200mM). Cells were treated with LPS (1,000 μg/ml) and 1, 5, 10, or 25μl of an 80 mg/ml SB plant extract. This mixture was incubated at 37oC for 15 minutes. Hydroxyl radical signal was read using a Bruker EMX spectrometer equipped with a flat cell assembly.

Statistics Results appearing in figures 1-4 were analyzed using the proc MIXED function of SAS, version 8.0. 

Methods and MaterialsMethods and MaterialsMethods and MaterialsMethods and Materials

National Institute for Occupational Safety and HealthHealth Effects Laboratory Division/Pathology and Physiology Research Branch

Morgantown, West Virginia

National Institute for Occupational Safety and HealthHealth Effects Laboratory Division/Pathology and Physiology Research Branch

Morgantown, West Virginia

AbstractAbstractAbstractAbstract

ResultsResultsResultsResults

Figure 6: Effects of SB Plant Extract (80 mg/ml) on LPS-Induced HydroxylRadical Signal as Measured by ESR. Treatments were as follows: (A) DMPO ONLY;(B) Cells +DMPO + LPS; (C) Cells + DMPO + LPS + 1μl SB; (D) Cells + DMPO +LPS + 5μl SB; (E)Cells + DMPO + LPS + 10μl SB; (F) Cells + DMPO + LPS + 25μlSB. The center field for each scan was set to 3480 Gauss, with a scan width of 100Gauss.

References References References References 1. Cuzzocrea, S., et al. Br J Pharmacol 2002 135:496-510.

2. Lee J.E., et al. Biochem Biophys Res Commun 2002 298:230-234.

3. Hong J., et al. Biochem Pharmacol 2001 62:1175-1183.

4. Sakihama, Y. et al. Toxicology 2002 177:67-80.

5. Wong B.Y., et al. Cancer Biother Radiopharm 1996 11:51-56.

6. Villegas I., et al. Free Radic Res 2002 36:769-777.

Results and DiscussionResults and DiscussionResults and DiscussionResults and DiscussionFigure 1 demonstrates the dose-dependent inhibitory effects of the Scutellaria barbata (SB) plant extract on cyclooxygenase-2 (Cox-2) protein expression. The SB extract also inhibited the formation of prostaglandin E2 (PGE2), a

product of the action of the Cox-2 protein on arachidonic acid, as shown in Figure 2. Additionally, SB reduced the expression of inducible nitric oxide synthase (iNOS) back to control levels (Figure 3). Figure 4 demonstrates that SB initially enhances, but at the highest concentration inhibits the mean concentration of the stable oxidation products of nitric oxide (NOx) in culture media. The same results were observed when NOx was measured using a 96-well plate method in a separate experiment (data not shown). Given these data, it is possible that the SB extract both enhanced the activity of the iNOS protein and reduced its expression. Although a preponderance of literature indicates that, in general, Cox-2 inhibition and antioxidant activity go hand in hand, the opposite may also be true. A number of published reports indicate that both pure chemicals and crude plant extracts with proven antioxidant properties have been shown to stimulate Cox-2 expression1-3. It may, therefore, be possible that extracts possessing Cox-2 inhibitory properties may also exhibit prooxidant characteristics. The SB extract inhibited several measures of inflammation (Cox-2 and iNOS protein expression, as wells as PGE2 formation) while increasing the levels of two reactive oxygen

species, H2O2 and the hydroxyl radical. Figure 5 demonstrates that LPS, SB, and SB + LPS increased intracellular H2O2

levels. Levels of H2O2 increased with dose in SB + LPS-treated cells. Figure 6 shows that increasing concentrations of

SB result in an increase in the formation of the hydroxyl radical. The hydroxyl radical formation seen in Figure 6 (which was measured over a period of 15 minutes) may reflect an ability of SB to induce an oxidative burst in RAW cells, as has been shown in another mouse macrophage cell line, J7744. Additionally, the plant extract itself may possess radical forming capacity as evidenced by its ability to form hydroxyl radicals in a cell-free system (DMPO and SB only, data not shown). Taken together, this data indicates that the SB extract may be capable of decreasing Cox-2 and iNOS gene expression and product formation via the formation of reactive oxygen species. This unexpected finding is in agreement with published reports indicating that some antioxidants are capable of inducing Cox-2 and, conversely, that some Cox-2 inhibitors are capable of inducing oxidative stress4,6.

Figure 1: Effects of SB Plant Extract and LPS Treatment on Cox-2 Protein Expression. Significant (p < 0.05) reductions in Cox-2 were observed for all LPS-treated cells co-treated with SB, relative to cells treated with LPS alone.

Figure 2: Effects of SB Plant Extract and LPS Treatment on PGE2 Expression.Significant (p < 0.05) reductions in PGE2 were observed for all LPS-treated cells co-treated with SB, relative to cells treated with LPS alone.

Figure 5: Effects of SB plant Extracts on LPS-Induced H2O2 Production.LPS, SB, and SB + LPS induced H2O2 production.

Control LPS Only (5 mg/ml) SB (1 mg/ml)

[mg/ml SB] + LPS 0.25 0.50 0.75 1.00

TM

Figure 3: Effects of SB Plant Extract and LPS Treatment on iNOS Expression.Significant (p < 0.05) reductions in iNOS protein expression were observed for all LPS-treated cells co-treated with SB , relative to cells treated with LPS alone.

130 kDaiNOS

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Figure 4: Effects of SB plant Extract and LPS Treatment on NOx Formation.Only the group co-treated with 1.0 mg/ml SB, in addition to LPS, displayed NOx levels significantly (p < 0.05) lower than the LPS Only group.

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Scutellaria barbata