Flavonoids are identified from the extract of Scutellariae Radix to suppress inflammatory-induced angiogenic responses in cultured RAW 264.7 macrophages

Scutellariae Radix (SR), also named Huangqin in China, is the dried root of Scutellaria baicalensis Georgi. Historically, the usage of SR was targeted to against inflammation. In fact, chronic inflammation has a close relationship with hypoxia and abnormal angiogenesis in tumor cells. Hence, we would like to probe the water extract of SR in suppressing the inflammation-induced angiogenesis. Prior to determine the pharmaceutical values of SR, the first step is to analysis the chemical compositions of SR according to China Pharmacopeia (2015). From the results, the amount of baicalin was 12.6% by weight. Furthermore, the anti-angiogenic properties of SR water extract were evaluated in lipopolysaccharide (LPS) pre-treated cultured macrophage RAW 264.7 cells by detecting the inflammatory markers, i.e. Cox-2, cytokine and iNOS, as well as the translocation activity of NFκB and angiogenic biomarker, i.e. VEGF. This herbal extract was capable of declining both inflammatory and angiogenic hallmarks in a concentration-dependent manner. Moreover, the SR-derived flavonoids, i.e. baicalin, baicalein, wogonin and wogonoside, were shown to be active chemicals in the anti-inflammatory-induced angiogenesis. Therefore, the inflammation-induced angiogenesis is believed to be suppressed by SR water extract, or its major ingredients. These results shed light in the benefiting role of SR in the inflammation-induced angiogenesis in vitro.

Shanghan Lun. According to TCMs theory, the role of this decoction is to clear heat dampness and purge fire 8 . Therefore, SR is considered as an indispensable herb in Chinese literature to eliminate heat/fire, i.e. detoxification and anti-inflammation. Angiogenesis is a critical constituent of inflammation, and, classically, tumor angiogenesis is also interpreted as an inflammation-induced angiogenesis 9 . The tumor tissue, exhibiting an excessively active process of angiogenesis, is composed of predominant inflammatory infiltrate, neoplastic and stromal cells 9 . Indeed, vascular endothelial growth factor (VEGF) is a critical player in modulating angiogenesis development, and which is believed to be secreted by immune cells 10 . The innate immune cell, particularly macrophage, is reported to express several VEGF receptors (VEGFRs) 11,12 . Macrophages are being recruited in responding to the receptor stimulation, and which significantly contribute to the process of angiogenesis 13 . Furthermore, it is estimated that approximately 15-20% of malignancies are triggered by chronic inflammation 9 . The initiation and progression of cancer are also closely linked to angiogenesis. Here, we would like to probe the possible anti-angiogenic functions of SR in cultured macrophage RAW 264.7 cells. In addition, the chemicals deriving from SR exctract responsible for this function was identified. The angiogenic biomarkers, e.g. Cox-2, cytokines, iNOS, VEGF, were determined in vitro by the challenging of SR herbal extract and/or its active ingredients.

SR suppresses inflammation.
Chemical standardization of SR water extract was required to ensure the repeatability of the below biochemical assays. According to Chinese Pharmacopeia (2015) requirement, the content of baicalin in SR extract should be higher than 9.0%. From the HPLC results, the baicalin content of the prepared SR water extract was 12.6%, which was much higher than the minimum requirement. In addition, the amount of baicalein was higher than 10% of the dry material, as determined by chemical analysis. Indeed, these two chemicals are the major flavonoids in SR. Besides, the HPLC fingerprint of SR water was achieved at an absorbance of 280 nm (Fig. 1).
Application of lipopolysaccharide (LPS) on cultured RAW 264.7 cells was a well-studied model to mimic inflammatory condition 14 . In the LPS-treated cells, the transcription factor NFκB was induced to translocate from cytosol into nucleus robustly, as illustrated here by both western blotting data of nucleus-isolated fraction and immuno-histochemical staining ( Fig. 2A). In addition, the expressions of angiogenic biomarkers, including Cox-2, iNOS, HIF-1α and VEGF, were markedly induced by LPS stimulation (Fig. 2B). These proteins are closely related to the inflammation-induced angiogenesis in cultured macrophage. In LPS-applied RAW 264.7 cells, the amount of NFκB in nucleus fraction was reduced strikingly and the deduction was linear (Fig. 2C). The maximum inhibition was at ~50%, as compared to the blank (Fig. 2C). Cox-2 is a mediator for angiogenesis and tumor growth 15 and NFκB is able to regulate Cox-2 expression in various types of cancer cells 16,17 . Once NFκB being activated, i.e. during the inflammatory situation, it could be translocated into nucleus as to regulate transcription of Cox-2 gene. The expression level of Cox-2 was shown in similar pattern with that of NFκB, and the inhibitory effect was in a dose-dependent manner (Fig. 2C). Dexamethasone served as a positive control, a well-studied synthesized drug to mitigate inflammation clinically, which could suppress NFκB translocation and Cox-2 expression, significantly (Fig. 2C).
The pro-inflammatory cytokines, i.e. tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, activate inflammation, as proposed for the deterioration of angiogenesis in tumor cells 18 . The mRNAs encoding IL-1β, IL-6 and TNF-α were restrained upon the SR treatment (Fig. 3). The SR herbal extract (1 mg/mL) showed the strongest inhibition, i.e.~50% for IL-1β, ~60% for IL-6 and ~60% for TNF-α (Fig. 3). Dexamethasone was a positive control suppressing cytokine expression at least 70% (Fig. 3).  One of the main inflammatory mediators reported to be committed in inflammation and carcinogenesis is nitric oxide (NO). There are three regimens for NO synthesis and production, i.e. neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) synthases 19 . Only iNOS is a characteristic for the pathophysiology of inflammation 19 . Here, we revealed the protein level of iNOS in LPS-stimulated RAW 264.7 cells. The expression of LPS-induced iNOS was decreased under application of various concentrations of SR herbal extract, as compared to the control (Fig. 4A). The minimal inhibition of iNOS expression was revealed at 0.03 mg/mL SR extract by ~20%. The maximal suppression was at 70% in the present of 1 mg/mL SR extract. Another method was also employed here in detecting NO production: DAF-FM DA was used for the quantification of NO. In cultured RAW 264.7 cells, LPS provoked a progressive rise in intracellular NO production, as reflected by fluorescence intensity (Fig. 4 B). The cancellation effects were also observed under the challenging of various contents of SR extract (Fig. 4B). Dexamethasone restrained the NO production robustly after 48 hours of treatment (Fig. 4B). In both cases, the amounts of iNOS and NO were significantly suppressed in LPS-stimulated RAW 264.7 cells.

SR restrains expression of tumor angiogenesis marker. The abnormal expressions of hypoxia-inducible
factor-1 (HIF-1) and VEGF are the hallmark of hypoxia, inflammation, carcinogen invasion 12,20 . HIF-1 is a highly conserved transcriptional complex, and which is a heterodimer composed of α and β subunit. The expression of HIF-1α showed a closely relationship with hypoxia condition, instead of HIF-1β 20 . Here, the transcript levels of HIF-1α and HIF-1β were determined after application of SR herbal extract. The mRNA level of HIF-1α was declined by over 30% maximum, but not for HIF-1β (Fig. 5A). The protein level of HIF-1α was also significantly decreased: the maximal decline level was at ~60% (Fig. 5B). Moreover, the expression of VEGF was determined by western blot. The amount of VEGF, revealed by western blotting in LPS-stimulated RAW 264.7 cells, was robustly decreased after application of SR extract in a concentration-dependent manner (Fig. 6A). The laser confocal method was also employed here to provide the detailed VEGF expression pattern. After high dose of SR (1.0 mg/mL, SR-H) treatment, the VEGF expression was markedly reduced, as compared to the LPS-treated control (Fig. 6B). Dexamethasone served as a positive control, and which could mitigate VEGF expression level, significantly (Fig. 6A,B). All of these results indicated that SR showed the possibilities of inhibiting inflammatory-induced angiogenesis.
The SR-derived flavonoids suppress inflammation. The anti-cancer functions of baicalin and baicalein, two major active components of SR, have been reported 21,22 . Inflammation plays an indispensable role for tumorigenesis 9,23 . Accordingly, the anti-inflammation properties of baicalin and baicalein were explored here. The results indicated that these two chemicals have potential anti-inflammatory functions by reducing the translational activities of Cox-2 and iNOS, at different levels (Fig. 7). Higher concentrations of baicalin and baicalein showed stronger suppressive activities on the LPS-induced inflammation (Fig. 7). Suppression in inflammatory-specific genes can be attributed to inhibition of angiogenesis activity in LPS-induced RAW 264.7 cells (Fig. 7). In addition, other flavonoids in SR, e.g. wogonin, wogonoside, were also reported to mediate the inflammatory processes both in vitro and in vivo [24][25][26][27] . Therefore, we analyzed the anti-inflammation functions of these two flavonoidic compounds in cultured RAW 264.7 cells (Fig. 8). A promoter construct having NFκB activating DNA elements upstream of a luciferase gene was used in transfecting RAW 264.7 cells. In the DNA transfected cells, wogonin or wogonoside could suppress the LPS-induced transcriptional activity of pNFκB-Luc in a dose-dependent manner. The maximal suppression levels were at ~17% for wogonin and ~25% for wogonoside (Fig. 8). These data suggested that SR and its major flavonoidic compounds, e.g. baicalein, baicalin, wogonin and wogonoside, could modulate inflammatory-induced angiogenesis in cultured macrophage.

Discussion
According to TCM theory, health preservation is to preserve individual body to maintain health, prevent from diseases and prolong life expectancy. About 1,892 types of TCMs and over 11,000 Chinese herbal medicine prescriptions were recorded in Bencao Gangmu by Li Shizhen (AD ~1596). Today's practices of TCMs are the culmination of theoretical development and clinical investigation over thousands of years in China. Indeed, TCM practices are believed to be based on the cosmologic principles of Chinese philosophy viewing disease as an imbalance of the living system, and therefore TCM treatments aim to maintain a balance. It is argued that this regimen is more suitable for chronic disease prevention and treatment 28 . In contrast, western medicine is relying on a detailed classification of disease based on empirical investigations and treatments. Synthesized drugs, usually in the single-chemical form, have been successful in treatment of acute conditions: this strategy is trying to influence the entire system by perturbing the single action. Many diseases are multi-factorial. In the case of chronic diseases, i.e. inflammation, the patients are suggested to intake medicines for a long and indefinite period of time, which often results in triggering serious side effects. Therefore, there is an increasing interest in the pharmaceutical industry by utilizing the herbal medicine as the novel candidates for the regimen of chronic diseases, i.e. inflammation.
The immune system has several defense mechanisms with increasing specificity to against the entry of pathogens and to avoid diseases. Innate immune cells, e.g. macrophages, usually orchestrate the rapid immune response by secreting different kinds of cytokines 29 . These cytokines play vital roles in monitoring immune response under pathogen infections and inflammations 29 . Chronic inflammation is a long-lasting disease and finally could result in development of cancer, cardiovascular diseases, neurodegenerative diseases and respiratory diseases 30 . Studies have shown that the transcription factors NFκB and STAT3, regulating the immune-related gene expressions, are essentially active during angiogenesis. Furthermore, the activations of NFκB and STAT3 could result in cancer cell proliferation, survival, invasion and metastasis by reducing the sensitivity to chemotherapy 30,31 . In fact, inflammation and angiogenesis are two closely related processes, and both of them could be triggered by hypoxia 32 . The anti-inflammatory agents are able to mitigate hypoxia condition and thereafter to alleviate angiogenesis. Moreover, the anti-inflammatory medicines are now widely accepted for angiogenic treatment in cancer therapy 32 . Together with our current results, the anti-angiogenic effect of SR in inflammatory macrophages could be mediated by multiple mechanisms: (i) suppressing pro-inflammatory cytokine expression; (ii) alleviating hypoxia condition by decreasing HIF-1α; and (iii) reducing angiogenesis inducer VEGF. Thus, SR, or its active floavonoids, baicalein, baicalin, wogonin and wogonoside, could be a promising target in developing drugs for relieving the syndromes of chronic inflammatory-triggered angiogenesis. SR is a well-known herb found within many multi-herb formulations, and the major aims of these formulae are to reduce inflammation and anti-cancer 33 . SR is a key herb found within Xiaochai Hutang, a herbal formula written by Zhang Zhongjing in AD ~200. The major functions of this herbal decoction were to enhance immune system and to clear the "fire". After intake of Xiaochai Hutang for 5 years in hepatitis patients, the liver function was greatly improved by 78%, and in parallel the serum levels of liver enzymes were reduced 34 . The in vitro study reported that this herbal formula not only suppressed wild-type virus number but also lamivudine-resistant HBV mutant 35 . The mechanistic study has revealed that the treatment with Xiaochai Hutang decreases the DNA-binding activity of nuclear extract of HepA2 cells to a specific cis-element of the HBV core promoter 35 . In cultured macrophages, the combination of SR and Liriopis Tuber could inhibit the expressions of inflammatory protein and granulocyte colony-stimulating factor in a dose-dependent manner. In addition, this herbal combination suppressed colony-stimulating factor and tumor necrosis factor at a dose of 25 μg/mL 36 . In parallel, the water extract of SR was able to induce apoptosis and to change the ratio of Bax/Bcl in a series of cancer cells 37 . Similarly, SR is selectively toxic to lung cancer cell lines by enhancing the expressions of p53 and Bax 38 . Oral administration of SR water extract for 10 days significantly inhibited tumor size in mouse xenograft model 39 . After oral administration of SR for 5 days in rat model, the level of PGE2 in LPS-stimulated macrophages was reduced robustly, and the pharmacodynamic interaction was proposed to be via the Cox-2 pathway 40 . The major metabolites were reported to be baicalin and baicalein by utilizing HPLC coupled with electrochemical detector 41 . From the results, AUC 0-24 hour values were 1.66 ± 0.34 µM and 19.8 ± 3.9 µM for baicalin, and 0.853 ± 0.065 µM and 10.0 ± 3.1 µM for baicalein, respectively 41 . Furthermore, the pharmacokinetic parameters of baicalin and baicalein, after oral administration of SR, were calculated and analyzed by the pharmacokinetic program 42 .
The anti-cancer effects of SR have been suggested to be triggered by baicalin and baicalein. Baicalin suppressed the growth of lymphoma and myeloma cells by regulating transcriptional and translational levels of phospholipid scramblase 1, a regulator of cell cycle and differentiation-related genes 43 . The anti-cancer functions of baicalein are contributed for ROS scavenging ability, abolishing NFκB activity and affecting cell cycle genes 43,44 . More importantly, baicalin and baicalein are believed to be promising candidates for chemotherapy adjuvant by not inducing possible mutations, a major problem of conventional anti-cancer drugs 21,22 . In addition, wogonin and wogonoside inhibited cancer cell proliferation in a concentration-dependent manner in various cell models 37 . Moreover, wogonin was capable of inducing HL-60 cell death both by stimulating DNA fragmentation and up-regulating apoptosis marker expressions 45 . Besides the anti-inflammatory functions of wogonoside and wogonin, the anti-hepatitis B virus (HBV) pharmaceutical values of these compounds were also demonstrated 27,46 .

Materials and Methods
Raw material and HPLC condition. The raw material of SR (the dried root of S. baicalensis) was obtained from Hebei province, which was authenticated by Dr. Tina Dong, one of the authors. The voucher specimen of SR (# 02-09-06) was kept in Centre for Chinese Medicine of HKUST. The raw material of SR was weighed and boiled in water for 2 hours, twice: the volume was 8 times and 6 times, respectively. Baicalin, baicalein, wogonin and wogonoside were purchased from TLCM (HKUST, Hong Kong China). The purities of these chemicals were, confirmed by HPLC, higher than 98.0%. HPLC analysis was conducted on an Agilent 1200 series system (Agilent, Waldbronn, Germany), equipped with a degasser, a binary pump, an auto-sampler, and a thermo-stated column compartment. Chromatographic separations were carried out on a Phenomenex C18 column (particle size 5 μm, 4.60 mm × 250 mm) with 1% acetate acid in water (as solvent A): acetonitrile (as solvent B) in the mobile phase at Cultures were seeded onto 6-well plate for 48 hours. High salt lysis buffer (1 M NaCl, 10 mM HEPES, pH 7.5, 1 mM EDTA, 0.5% Triton X-100) was utilized for collecting cells. Total protein of each sample was adjusted by 2X lysis buffer (0.125 M HCl, pH 6.8, 4% SDS, 20% glycerol, 2% 2-meracptoethanol and 0.02% bromophenol blue), and which was subjected to SDS-PAGE analysis. The membranes were incubated with different antibodies, i.e. anti-Cox-2 (~72 kDa), iNOS (~135 kDa), HIF-1α (~120 kDa) and VEGF (~22 kDa) (CST, Danvers, MA). The above mentioned antibodies were at 1: 2,000 dilutions in the 2.5% fat-free milk. GAPDH was employed for the internal control at 1: 5,000,000 dilutions dissolved in the 2.5% fat-free milk. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies (Zymed, South San Francisco, CA) were employed here as secondary antibody at 1: 5,000 dilutions for 3 hours at room temperature.
Transfection analysis. The DNA construct containing 5X NFκB response element, named as pNFκB-Luc, was utilized here to detect the transcriptional activities upon the drug treatment. Lipofectamine 3000 (Invitrogen) was as a transfection kit, the transfection efficiency was over 30%. Cells were lysed by luciferase buffer containing 0.2% Triton X-100, 1 mM dithiothreitol (DTT) and 100 mM potassium phosphate buffer (pH 7.8) for luciferase assay after treated with LPS for 24 hours, then challenging with herbal extracts or chemicals for another 48 hours.
Statistical analysis and other assays. Protein levels were evaluated by Bradford's method (Herculues, CA). Statistical tests have been done by using one-way analysis of variance. Data were expressed as Mean ± SEM, where n = 3-5. Statistically significant changes were classified as significant (*) where p < 0.05, more significant (**) where p < 0.01 and highly significant (***) where p < 0.001 as compared with control group.