Anti-Inflammatory Effects of Modified Buyang Huanwu Decoction

Methods A cytotoxicity assay for BHD was performed using the MTT assay. Following treatment with BHD, mBHD-1, and mBHD-2 in the presence of lipopolysaccharide (LPS), nitric oxide (NO) secretion was detected in cell supernatants using a NO detection kit. The expression of proinflammatory mediators was detected using RT-PCR and western blotting. To verify the mechanism of mBHD, specific inhibitors of JNK (SP600125) or p38 (SB203580) were used for co-treatment with mBHD, and then the changes in NO and nitric oxide synthase (iNOS) were measured. Results Both mBHD-1 and mBHD-2 showed greater anti-inflammatory effects than BHD. Both mBHD-1 and mBHD-2 inhibited NO secretion and decreased the expression of IL-1β, IL-6, TNF-α, and iNOS. Treatment with a p38 inhibitor and a JNK inhibitor in mBHD-1- and mBHD-2-treated cells resulted in inhibition of NO and iNOS. Conclusion We provided the first experimental evidence that mBHD may be a more useful anti-inflammatory than BHD. High concentrations or long-term use of BHD may be harmful to inflammatory status. Therefore, the length of treatment and concentration should be considered depending on the targeted disease.


Introduction
e inflammatory process is considered a major driving force in the ischemic process, brain repair, neuropathic pain, neuronal injury, and gynecological disease [1]. During the inflammatory response, diverse chemical mediators released by activated immune cells have effects on excitability and synaptic function, and these changes can contribute to the initiation and maintenance of persistent neuropathic pain [2,3]. Cerebral ischemia is accompanied by a marked inflammatory reaction that is initiated by ischemia-induced expression of cytokines, adhesion molecules, and other inflammatory mediators, including prostanoids and nitric oxide [4,5]. us, the control of the inflammatory process is a logical therapeutic target.
Nitric oxide (NO) has bifunctional roles as a free radical biological messenger in inflammatory responses. It improves the blood supply to injured tissue and is secreted as free radicals that are toxic to bacteria but can cause tissue damage by initiating apoptosis and killing otherwise healthy cells [6]. In rodents, NO can both promote [2,7] and inhibit inflammatory pain [3,7]. NO is synthesized by nitric oxide synthase (iNOS), which is mainly upregulated in activated immune cells such as macrophages and glial cells. erefore, NO and iNOS are major molecules that are generated by macrophages and other phagocytes in inflammation [8].
Traditional Chinese and Korean medicine has been prescribed for health protection and disease control for thousands of years in Eastern Asia. e Buyang Huanwu decoction (BHD) is a traditional medicine mainly used to treat circulatory disorders such as cerebral ischemia, angina, and myocardial infarction, as well as multiple neuritis and thrombophlebitis. According to experimental reports, BHD improves blood circulation, controls pain, regenerates neuronal cells [9][10][11], and protects against the neuronal damage caused by ischemic and oxidative stress [12,13].
ese ingredients have effects mainly upon improving blood circulation and protecting thrombogenesis. Recently, various versions of modified BHD (mBHD), a combination of BHD and other effective herbal components, have been used for a synergistic therapeutic effect. Previous studies have reported the effects of mBHD on sciatic nerve injury [11], focal cerebral ischemia [14], and vascular dementia [15].
Considering those reports, we developed two types of mBHD and verified their efficacy according to comparative studies of the anti-inflammatory effect. One was manufactured by eliminating Lumbricus (renamed to mBHD-1). Lumbricus is a genus of earthworm, and among the seven ingredients of BHD is the only animal material. us, we omitted this animal-derived ingredient to avoid the possibility of adverse effects.
Another formulation reduced the concentration of Astragalus (renamed mBHD-2). Traditional BHD contains 120 g of Astragalus. Potentially adverse effects of Astragalus have been reported when used in high doses, despite the enhanced immune system, anti-inflammatory, and antioxidant effects. Astragalus can interact with prescription medications, including anti-hypertensives and immune suppressants and can decrease medication efficacies. It can cause bleeding when used with other anticoagulant, antiplatelet, or antithrombotic agents [16]. Some exporters have been concerned that Astragalus is a legume and thus can cause allergic reactions in people with legume allergies (such as beans and peanuts). For these reasons, we investigated the effects of BHD, mBHD-1, and mBHD-2 on NO secretion and inflammation-associated factors and the signaling pathways involved in the inflammatory responses mediated by lipopolysaccharide-(LPS-) exposed macrophages.

Chemicals and Reagents.
e composition of BHD and modified-BHD (named as mBHD-1 or mBHD-2) is described in Table 1. e mBHD-1 formulation omitted Lumbricus from the other seven herbs comprising BHD, and mBHD-2 reduced the amount of Astragali Radix used, compared with BHD. e mixture of dried ingredients was boiled in purified saline for 2 h and concentrated under vacuum pressure at 700 mm Hg for 15 h, after which it was freeze-dried. e lyophilized powder of BHD and mBHD were then stored at − 20°C until use. e LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against total extracellular signal-related kinase (ERK), phospho-ERK (p-ERK), total N-terminal kinase (JNK), phospho-JNK (p-JNK), p38 mitogen-activated protein kinase (p38-MAPK), phospho-p38 (p-p38), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). e antibodies against iNOS and Cox-2 were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell Line and Treatments.
e murine macrophage cell line RAW264.7 was acquired from the Korean Cell Line Bank (Seoul, Republic of Korea) and was cultured in highglucose DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and maintained at 37°C in a humidified incubator with 5% CO 2 . BHD, mBHD-1, and mBHD-2 were dissolved in phosphate-buffered saline and filtrated. RAW264.7 cells were pretreated with various concentrations of BHD, mBHD-1, and mBHD-2 or co-pretreated with the inhibitors p38 or JNK for 1 h and then were stimulated with LPS (100 ng/mL) for a period of 30 min to 24 h, depending on the experiment.

Cell Viability Assays.
e cytotoxic effect of BHD, mBHD-1, mBHD-2, and several inhibitors on RAW264.7 cells was examined using an EZ-cytox assay kit (Daeil Lab Serve Co, Seoul, Republic of Korea) according to the manufacturer's instructions. Briefly, RAW264.7 cells (1 × 10 5 cells per well) were seeded in 96-well plates and were treated with various concentrations of materials. After 24 h, 10 μL of EZ-cytox was added into each well and then incubated for another 2 h under standard culture conditions. Cell viability was quantified with a spectrophotometer at 450 nm.

Measurement of NO
Production. NO secreted from cells was detected using a NO detection kit (Intron Biotechnology, Seongnam, Republic of Korea). Briefly, Table 1: Raw materials of BHD, mBHD-1, and mBHD-2.
2 Evidence-Based Complementary and Alternative Medicine RAW264.7 cells (1 × 10 5 per well) were pretreated with various concentrations of BHD, mBHD-1, and mBHD-2, or co-pretreated with SB203580 (a p38 inhibitor) or SP300125 (a JNK inhibitor) for 1 h and then stimulated with 100 ng/mL of LPS for 24 h. en, 100 μL of conditioned media was mixed with an equal volume of Griess reagent and incubated at room temperature for 10 min. Absorbance values were measured at 560 nm using a plate reader. e nitrate concentration was calculated based on a nitrite standard curve.

Statistical
Analysis. Data were expressed as mean-± standard error of the mean (SEM). e statistical differences between the groups were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test using GraphPad Prism 7.0 (GraphPad Software Inc., CA, USA). A p value <0.05 was considered statistically significant.

Effect of BHD, mBHD-1, and mBHD-2 on the Production of NO in LPS-Stimulated RAW264.7 Cells.
It is well known that a large amount of NO is produced by macrophages in an inflammatory environment. We investigated whether BHD, mBHD-1, or mBHD-2 could control the NO released by LPS. Both mBHD-1 and mBHD-2 gradually suppressed the NO induced by LPS at 1,000, 2,500, and 5,000 μg/mL (p < 0.001). Conversely, BHDpretreated cells showed the opposite results. Treatment of cells with BHD gradually increased NO at 100, 1,000, 2,500, and 5,000 μg/mL, reaching an approximately 40% increase at 5,000 μg/mL, compared with LPS (p < 0.001) ( Figure 2).

Effect of BHD, mBHD-1, or mBHD-2 on Morphological
Changes in LPS-Exposed RAW264.7 Cells. e morphology of RAW264.7 cells reportedly changes following stimulation with LPS. us, we examined whether BHD affected morphologic changes in LPS-stimulated cells. e morphology of LPS-exposed RAW264.7 cells was spindle-like, and the cells pretreated with BHD, mBHD-1, or mBHD-2 were observed to possess similar shapes to LPS-stimulated cells (Figure 3).

Effect of BHD, mBHD-1, or mBHD-2 on the Expression of Proinflammatory Cytokines in LPS-Exposed RAW264.7 Cells.
To determine whether BHD could regulate proinflammatory mediators, we examined the transcriptional level of   Figure 2: Effects of BHD, mBHD-1, and mBHD-2 on NO production in LPS-exposed RAW264.7 cells. Cells were pretreated with different concentrations of BHD (a), mBHD-1 (b), and mBHD-2 (c) for 1 h and then were stimulated with 100 ng/mL LPS. After 24 h, secreted NO was measured in conditioned media using a NO detection kit. * * * p < 0.001 vs. LPS. Statistical differences were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Error bars indicate SEM.
proinflammatory cytokines in LPS-exposed RAW264.7 cells. As shown in Figure 4(a), cells pretreated with BHD showed decreased expression of interleukin-(IL-) 1β at 2.5 mg/mL (p < 0.05), and IL-6 at 1, 2.5, and 5 mg/mL (p < 0.001) compared with that of cells stimulated only with LPS. e cells pretreated with mBHD-1 showed decreased expression of IL-1β at 2.5 (p < 0.05) and 5 mg/mL (p < 0.01) and IL-6 at 1, 2.5, and 5 mg/mL (p < 0.001) compared to the LPS-treated cells. On the other hand, the cells pretreated with mBHD-2 showed decreased expression of IL-1β at 2.5 and 5 mg/mL (p < 0.01) and IL-6 at 1 (p < 0.05), 2.5, and 5 mg/mL (p < 0.001) compared to the LPS-treated cells. mBHD-2 tended to decrease expression of tissue necrosis factor-(TNF-) α at 5 mg/mL in a dose-dependent manner, but there was no statistical significance. Cyclooxygenase-(Cox-) 2 and inducible iNOS are induced by proinflammatory cytokines in an inflammatory environment [17]. We examined the expression of Cox-2 and iNOS using western blotting. Compared to the LPStreated cells, the expression of iNOS was significantly decreased in the cells pretreated with mBHD-1 and mBHD-2 at every dose (p < 0.001), while it was highly induced in the BHD-pretreated cells (p < 0.001). e expression of Cox-2 was increased in the BHD-pretreated cells at 1 (p < 0.05) and 5 mg/mL (p < 0.001) and decreased in the mBHD-2-pretreated cells at 2.5 mg/mL (p < 0.01) compared to LPS-treated cells (Figure 4(b)).

Effect of mBHD-1 and mBHD-2 on Activation of p38, JNK, and ERK1/2 Pathways in LPS-Exposed RAW264.7 Cells.
p38, JNK, and ERK play an important role in inflammatory responses such as iNOS expression. To elucidate the molecular mechanism of the anti-inflammatory effects of mBHD-1 and mBHD-2, we examined the activities of ERK, p38, and JNK using western blotting. e activation of p38 and JNK was decreased in mBHD-1 or mBHD-2 pretreated cells, compared with LPS-stimulated cells. However, mBHD-1 and mBHD-2 did not affect the activation of the ERK pathway in LPS-stimulated cells ( Figure 5).
To confirm whether mBHD-1 or mBHD-2 regulate NO and iNOS through stimulate the JNK and p38 signaling, we co-treated cells with a JNK inhibitor (SP600125) or a p38 inhibitor (SB203580) with mBHDs. After treatment of LPSstimulated cells with SP600125 alone, NO was reduced by approximately 20%. When cells were co-treated with LPS and mBHD-1 (or mBHD-2), NO increased by approximately 15% compared with that in cells treated with  Figure 3: Effects of BHD, mBHD-1, and mBHD-2 on the morphology of LPS-exposed RAW264.7 cells. Cells were pretreated with 1, 2.5, or 5 mg/mL of each treatment, and their morphologic changes were observed using light microscopy.   Figure 4: Effect of BHD, mBHD-1, and mBHD-2 on the expression of proinflammatory cytokines in LPS-exposed RAW264.7 cells. Cells were pretreated with 1, 2.5, or 5 mg/mL of each drug for 1 h and then were stimulated with LPS for 4 h. Transcriptional levels of inflammatory cytokines of IL-1β, IL-6, and TNF-α were detected using RT-PCR (a) and the protein expression of iNOS and Cox-2 was detected using western blotting (b). IL: interleukin; TNF: tumor necrosis factor; iNOS: inducible nitric oxide; Cox-2: cyclooxygenase-2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LPS: lipopolysaccharide. C, control; * p < 005; * * p < 0.01; * * * p < 0.001 vs. control; # p < 005; ## p < 0.01; ### p < 0.001 vs. LPS. Statistical differences were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Error bars indicate SEM. 6 Evidence-Based Complementary and Alternative Medicine SP600125 alone (p < 0.001). In cells treated with SB203580 alone, NO was reduced by about 40%, and the NO reduction between the SB203580 treatment alone and the SB203580 + mBHD-1 (or mBHD-2) treatment showed similar results, compared with that in cells treated with LPS alone (Figure 6(a)). e expression of iNOS was significantly decreased in both SP600125-and SB203580-treated cells, compared with that of the control group ( Figure 6(b)). ese results indicated that mBHD-1 and mBHD-2 regulate NO and iNOS expression through the p38 MAPK and JNK pathway (Figure 7).

Discussion
Traditional BHD consists of seven natural herbal components that function to improve the circulatory system and immunity in humans [18][19][20] and protect neural activity and regeneration [21,22]. us, this traditional medicine has been prescribed mainly to patients with autoimmune encephalomyelitis [23] or ischemic brain disorders [12,24] for more than 300 years. Recent studies on the use of various mBHD, which have reduced BHD components or are combined with other effective herbs, have provided reports regarding their therapeutic advantage. In a rat model of Alzheimer's disease, mBHD with a reduced quantity of all components improved memory deficiency and decreased the cerebral infarction in the hippocampus [25,26]. Another version of mBHD with added Polygalae radix and Acori Graminei Rhizoma showed anti-inflammatory and antioxidant effects on microglial cells [20]. Two studies using modified BHD with Achyranthis Radix, Acori Graminei Rhizoma, Polygalae Radix, Cinnamomi Ramulus, and Salviae Miltiorrhizae Radix reported reduction of the cerebral infarction size and neurological deficit in ischemia/reperfusion models [14] and hippocampal inflammatory responses in vascular dementia models [15]. A BHD preparation including Lycopi Herba, Typae pollen, Trogopterori feces, and Corydalis tubers showed antithrombotic activities and can be applied to diverse female diseases caused by thrombosis and inflammation such as endometriosis, myoma, and chronic pelvic inflammatory disease [27]. According to a clinical report in 2011, BHD without Lumbricus improved symptoms in patients with hyperhidrosis resulting from sequela of spinal cord injuries. Tritici Fructus Levis is utilized mainly for inhibiting spontaneous perspiration and night sweats by securing the exterior.
Astragalus not only has various therapeutic effects, including enhanced immune system function as well as antiinflammatory and antioxidant effects, but also poses potential risk when used at a high dose [28][29][30][31]. Lumbricus is an earthworm genus considered to possess therapeutic effects in epilepsy and stroke [32]. Until now, the side effects of Lumbricus have not been reported, but out of the seven ingredients composing BHD, this represents the only animal-derived component, whereas the other components of BHD are plant-derived.
Based on these reports, we developed two types of mBHD, the mBHD-1 preparation (without Lumbricus) and the mBHD-2 preparation (reduced amount of Astragalus) and verified the improvement of anti-inflammatory effects. We compared the anti-inflammatory effects of BHD and mBHDs in LPS-stimulated murine macrophage RAW264.7 cells. e activity of the iNOS and NO system is mediated by macrophages and other phagocytes during inflammatory episodes [8]. In immune-defense status, iNOS is activated by proinflammatory cytokines such as IL-1β,  Figure 6: Both mBHD-1 and mBHD-2 inhibit NO secretion by regulating the p38 signaling pathway in LPS-exposed RAW264.7 cells. Cells were pretreated with SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor) with or without BHD, mBHD-1, or mBHD-2 for 1 h and then were stimulated with LPS (100 ng/mL). After 24 h, NO was detected in conditioned media using a NO detection kit (a), and expression of iNOS and Cox-2 was measured in total protein using western blotting (b). LPS + SP: LPS and SP600125 treatment; LPS + SB: LPS and SB203580 treatment. * * p < 0.01; * * * p < 0.001 vs. LPS; ### p < 0.001 vs. LPS + SP. Statistical differences were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. Error bars indicate SEM.  On the basis of those reports, we examined the effect of BHD, mBHD-1, and mBHD-2 on changes in NO secretion and expression of iNOS and proinflammatory cytokines in LPS-stimulated RAW264.7 cells. According to our results, the cells treated with mBHD-1 and mBHD-2 showed significantly and dose-dependently decreased NO secretion and expression of IL1-β, TNF-α, Cox-2, and iNOS. e results of mBHD-1 and mBHD-2 treatments showed similar results. However, BHD-treated cells showed opposite results. BHD increased NO secretion and the expression of these factors. As mentioned above, BHD is composed of ingredients that are useful in blood circulation.
NO has bifunctional roles as a free radical biological messenger and a modulator of inflammatory responses; these roles can be separated as defensive mechanisms and tissue damage mechanisms. Furthermore, NO improves the blood supply to injured tissue and is secreted as a free radical that is toxic to bacteria and modulates the actions of macrophage-derived cytokines on target cells. However, NO can cause tissue damage by inducing apoptosis [33]. In an oxidative environment, high levels of NO released by iNOS have the opportunity to react with superoxide, leading to peroxynitrite formation and cell toxicity. ese properties may define the roles of iNOS in host immunity, enabling its participation in antimicrobial and antitumor activities as part of the oxidative burst produced by macrophages [34]. Proinflammatory cytokines (TNF-α, IL-6, and IL-1β) are important mediators in the inflammatory process and are mainly produced by activated macrophages [35]. Excessive production of these proinflammatory cytokines will lead to clinical problems associated with autoimmune disorders (rheumatoid arthritis) and inflammatory response syndrome (septic shock) [36].
To further investigate the mechanism of the anti-inflammatory effects of mBHD, we examined major signaling molecules associated with the inflammatory process. LPSstimulated macrophages have been widely reported to activate three different groups of MAP kinases: ERK, JNK, and p38 [37]. Our results showed that BHD, mBHD-1, and mBHD-2 significantly reduced p38 and JNK activated by LPS but did not affect ERK1/2 phosphorylation. Activation of p38 and JNK inhibited by mBHD-1 and mBHD-2 was stably maintained 24 h later but that inhibited by BHD was recovered in 24 h.
Finally, we confirmed whether mBHD-1 and mBHD-2 control NO secretion and iNOS expression through the regulation of the p38 and JNK pathway using the inhibitors SB203580 for p38 and SP600125 for JNK in LPS-induced RAW264.7 cells. SB203580 is a selective inhibitor of p38 [38], and SP600125 is a potent, selective, and reversible inhibitor of JNK [39]. As our results demonstrate, the cells co-treated with mBHD and SB203580 showed significantly reduced NO and iNOS expression in the presence of LPS, compared with those exposed to LPS alone. e inhibitory effects on NO secretion produced by SB203580 were higher than those produced by SP600125.

Conclusion
In conclusion, mBHD-1 and mBHD-2 were more effective in suppressing the NO/iNOS system following activation by LPS through the regulation of the p38 and JNK pathway in LPS-exposed RAW264.7 cells. Both mBHD-1 and mBHD-2 showed greater anti-inflammatory activity than BHD. ese results may indicate that the prescribed concentration of Astragalus in BHD should be determined depending on the target disease.
is study provided the first experimental evidence of an anti-inflammatory effect of mBHD. However, further studies are still needed to elucidate the fundamental mechanism of mBHD in various types of in vitro and in vivo disease models. Moreover, further research should continue to develop various combinations of BHD and develop specific prescriptions for each targeted disease.

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

Conflicts of Interest
e authors declare no conflicts of interest.