STAT3/Girdin/Akt; MAPK; NF-κB

Scutellarin administration dramatically reduced HepG2 cell viability in a dose-dependent manner and inhibited HCC cell migration and invasion in vitro. Scutellarin therapy dramatically reduced the expression of STAT3 and actin filament (Girdin) and the phosphorylation of STAT3 and Akt in HCC cells. Introduction of STAT3 overexpression restored scutellarin-downregulated Girdin expression, Akt activation, motility, and invasion in HCC cells. Furthermore, activation of Girdin overexpression totally removed the inhibitory effects of scutellarin on Akt phosphorylation, migration, and invasion of HCC cells. Scutellarin can suppress HCC cell metastasis in vivo and migration and invasion in vitro by down-regulating STAT3/Girdin/Akt signaling [1]. Scutellarin preferentially stimulates Akt phosphorylation [2]. Scutellarin is a well-established therapeutic drug because it not only suppresses microglial activation, thereby ameliorating neuroinflammation, but also improves astrocyte responses. Acutellarin enhances astrocyte responses by upregulating the production of neurotrophic factors, among others, so exhibiting its neuroprotective benefits. Notably, the actions of scutellarin on reactive astrocytes are mediated by activated microglia, supporting functional “crosstalk” between the two glial types [3]. Scutellarin can suppress RANKL-mediated osteoclastogenesis, osteoclast bone resorption function, and the expression of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, c-Fos, NFATc1) level. Further investigations have revealed that scutellarin can suppress RANKL-mediated MAPK and NF-κB signaling pathways, including JNK1/2, p38, ERK1/2 and IκBα phosphorylation [5].

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Scutellarin is an active flavone from Erigeron breviscapine (vant) Hand Mass. This study aimed to investigate the potential role of scutellarin in migration and invasion of human hepatocellular carcinoma (HCC) cells and its possible mechanism. In comparison with the vehicle-treated controls, treatment with scutellarin (50 mg/kg/day) for 35 days significantly mitigated the lung and intrahepatic metastasis of HCC tumors in vivo. Scutellarin treatment significantly reduced HepG2 cell viability in a dose-dependent manner, and inhibited migration and invasion of HCC cells in vitro. Scutellarin treatment significantly reduced STAT3 and Girders of actin filaments (Girdin) expression, STAT3 and Akt phosphorylation in HCC cells. Introduction of STAT3 overexpression restored the scutellarin-downregulated Girdin expression, Akt activation, migration and invasion of HCC cells. Furthermore, induction of Girdin overexpression completely abrogated the inhibition of scutellarin on the Akt phosphorylation, migration and invasion of HCC cells. Scutellarin can inhibit HCC cell metastasis in vivo, and migration and invasion in vitro by down-regulating the STAT3/Girdin/Akt signaling.[1]

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Baicalin and Scutellarin, two flavonoid glucuronic acids isolated from Scutellaria baicalensis, exhibit beneficial effects on glucose homeostasis. Baicalin and scutellarin are similar in structure except scutellarin has an additional hydroxyl at composition C-4'. In this work, we observed that baicalin and scutellarin promoted glucose disposal in mice and in adipocytes. Baicalin selectively increased phosphorylation of AMP-activated kinase (AMPK), while scutellarin selectively enhanced Akt phosphorylation. Both of them increased AS160 phosphorylation and glucose uptake in basal condition. AMPK inhibitor or knockdown of AMPK by siRNA blocked baicalin-induced AS160 phosphorylation and glucose uptake, but showed no effects on scutellarin. In contrast, Akt inhibitor and knockdown of Akt with siRNA decreased scutellarin-stimulated glucose uptake but had no effects on baicalin. The molecular dynamic simulations analysis showed that the binding energy of baicalin to AMPK (-34.30kcal/mol) was more favorable than scutellarin (-21.27kcal/mol), while the binding energy of scutellarin (-29.81kcal/mol) to Akt was much more favorable than baicalin (4.04kcal/mol). Interestingly, a combined treatment with baicalin and scutellarin acted synergistically to enhance glucose uptake in adipocytes (combination index: 0.94-0.046). In conclusion, baicalin and scutellarin, though structurally similar, promoted glucose disposal in adipocytes by differential regulation on AMPK and Akt activity. Our data provide insight that multicomponent herbal medicines may act synergistically on multiple targets. [2]

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Aseptic prosthetic loosening is a major complication after hip joint replacement. Wear particle-induced periprosthetic osteolysis plays a key role in aseptic prosthetic loosening. Attempting to modulate receptor activator of nuclear factor-κB (RANKL) mediated signaling pathways is a promising strategy to prevent aseptic prosthetic loosening. In the present study, we determined the effect of Scutellarin (SCU) on titanium (Ti) particle-induced osteolysis in a mouse calvarial model and RANKL-mediated osteoclastogenesis. We determined that SCU, the major effective constituent of breviscapine isolated from a Chinese herb, has potential effects on preventing Ti particle-caused osteolysis in calvarial model of mouse. In vitro, SCU could suppress RANKL-mediated osteoclastogenesis, the function of osteoclast bone resorption, and the expression levels of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, c-Fos, NFATc1). Further investigation indicated that SCU could inhibit RANKL-mediated MAPK and NF-κB signaling pathway, including JNK1/2, p38, ERK1/2, and IκBα phosphorylation. Taken together, these results indicate that SCU could inhibit osteoclastogenesis and prevent Ti particle-induced osteolysis by suppressing RANKL-mediated MAPK and NF-κB signaling pathway. These results suggest that SCU is a promising therapeutic agent for preventing wear particle-induced periprosthetic osteolysis.[5]

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Scutellarin was purified from the plant Erigeron breviscapus (Vant.) Hand.-Mazz. The activity against 3 strains of human immunodeficiency virus (HIV) was determined in vitro in this study. These were laboratory-derived virus (HIV-1IIIB), drug-resistant virus (HIV-1(74V)), and low-passage clinical isolated virus (HIV-1(KM018)). From syncytia inhibition study, the EC50 of scutellarin against HIV-1IIIB direct infection in C8166 cells was 26 microM with a therapeutic index of 36. When the mode of infection changed from acute infection to cell-to-cell infection, this compound became even more potent and the EC50 reduced to 15 microM. This suggested that cell fusion might be affected by this compound. By comparing the inhibitory effects on p24 antigen, scutellarin was also found to be active against HIV-1(74V) (EC50 253 microM) and HIV-1KM018 (EC50 136 microM) infection with significant difference in potency. The mechanism of its action was also explored in this study. At a concentration of 433 microM, scutellarin inhibited 48% of the cell free recombinant HIV-1 RT activity. It also caused 82% inhibition of HIV-1 particle attachment and 45% inhibition of fusion at the concentrations of 54 microM. In summary, scutellarin was found to inhibit several strains of HIV-1 replication with different potencies. It appeared to inhibit HIV-1 RT activity, HIV-1 particle attachment and cell fusion. These are essential activities for viral transmission and replication.[6]

Scutellarin (50 mg/kg/day) can considerably lower HCC tumor metastases to the liver and lungs in vivo. In comparison to the control group, the scutellarin treatment group had a considerably lower number of metastases in the liver and lungs [1]. Rats treated with scutellarin showed a significant reduction in neurobehavioral impairments as compared to the SAH group. Scutellarin increased the expression of eNOS in comparison to SAH animals [4].

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Angiographic vasospasm, especially in the early phases (<72h) of subarachnoid hemorrhage (SAH), is one of the major complications after an aneurysm rupture and is often the cause of delayed neurological deterioration. Scutellarin (SCU), a flavonoid extracted from the traditional Chinese herb Erigeron breviscapus, has been widely accepted as an antioxidant, but the effect of SCU on vasospasm after SAH remains elusive. Endovascular perforation was conducted to induce SAH in Sprague-Dawley rats. Then, the underlying mechanism of the anti-vasospasm effect of SCU was investigated using a modified Garcia scale, India ink angiography, cross-sectional area analysis, immunohistochemistry staining and western blot. SCU (50μM, 100mg/kg) alleviated angiographic vasospasm and improved neurological function 48h after SAH and enhanced the expression of endothelial nitric oxide synthase (eNOS) at the intima of cerebral arteries. In addition, SCU upregulated the expression of phosphorylated extracellular-regulated kinase 5 (p-Erk5) and Kruppel-like factor 2 (KLF2) at 48h after SAH. However, the effects of SCU were reversed by the Erk5 inhibitor XMD8-92. Our results indicate that SCU could attenuate vasospasm and neurological deficits via modulating the Erk5-KLF2-eNOS pathway after SAH, which may provide an experimental basis for the clinical use of SCU treatment in SAH patients.[4]

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Mortality [4]
None of the sham-operated rats died (0 of 15) in the present study. Twenty-four rats died after SAH due to the severe hemorrhagic volume. The mortality was 37.5% (9 of 24 rats) in the SAH + vehicle group, 31.8% (7 of 22 rats) in the SAH + Scutellarin group, and 34.8% (8 of 23 rats) in the SAH + SCU + XMD8-92 group. No significant differences were observed among these groups (p < 0.05) (Fig. 1A). No rats were excluded due to mild hemorrhagic severity from the following experiments.

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Scutellarin treatment improves neurological function after subarachnoid hemorrhage [4]
In comparison to the sham group (16.9 ± 1.0), SAH rats suffered from significant neurological deficits (12.8 ± 1.9, p < 0.05) (Fig. 1B). The rats treated with SCU displayed a significant alleviation in neurobehavioral deficits compared to the SAH group (14.5 ± 1.3, p < 0.05) (Fig. 1B).

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Scutellarin treatment alleviates vasospasm after subarachnoid hemorrhage [4]
Vasospasm was represented by the reduction of the vascular diameter and cross-sectional areas (Fig. 1C). The diameter of the MCA in the SAH + vehicle group (96.8 ± 3.3 μm) was significantly smaller than that in the sham group (197.4 ± 4.5 μm, p < 0.05) (Fig. 1C, D) and larger than in the SAH + vehicle group (197.4 ± 4.5 μm, p < 0.05) (Fig. 1C, D). Furthermore, a similar trend was found in the results of the cross-sectional areas of the basilar artery (5495 ± 780 μm2 in the sham group, 2780 ± 491 μm2 in the SAH + vehicle group, 4476 ± 700 μm2 in the SAH + SCU group, p < 0.06) (Fig. 1C, E).

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Scutellarin treatment enhanced eNOS expression after subarachnoid hemorrhage [4]
Histological staining showed the location and expression of eNOS in the intima of the basilar artery and MCA (Fig. 2A). We also used western blot to analyze the eNOS expression in the brains of each group. In the SAH + vehicle group, the expression of eNOS was much lower than that in the sham group. The SCU treatment displayed significantly enhanced eNOS expression compared with SAH rats (p < 0.05) (Fig. 2B, C).

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Erk5 inhibition abolished the protective effects of Scutellarin on the neurological function and vasospasm after subarachnoid hemorrhage [4]
We employed Erk5 inhibitor XMD8-92 to clarify the role of the Erk5 pathway on the effects of Scutellarin/SCU treatment. Compared to the SAH + SCU group, the cross-sectional areas of the basilar artery were significantly decreased in the SAH + SCU + XMD8-92 group (2884 ± 621, p < 0.05) (Fig. 1C, D). Additionally, the SCU plus XMD8-92 treatment significantly decreased the diameter of the MCA compared with the SCU–only treatment (197.4 ± 4.5 μm, p < 0.05) (Fig. 1C, D). Furthermore, the neurological scores of the SAH + SCU + XMD8-92 group were significantly decreased compared with the SAH + SCU group (10.2 ± 2.3, p < 0.05) (Fig. 1B).

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Erk5 inhibition abolished the effects of Scutellarin on downstream signals after subarachnoid hemorrhage [4]
SCU/Scutellarin treatment significantly enhanced the expression of p-Erk5, KLF2 and eNOS compared with the SAH group, but not in Erk5 expression (p < 0.05) (Fig. 2B, C; 3A–D). However, administration of XMD8-92 plus SCU significantly reduced the expression of p-Erk5, KLF2 and eNOS compared with SCU-treated rats (p < 0.05) (Fig. 2B, C; 3A–D).

Cell proliferation assay [1]
HepG2 cells (1 × 105/well) were cultured in 96-well plates and treated in triplicate with Scutellarin at concentrations of 5, 10, 20, 30, and 100 μM or vehicle alone for 24 h. The cellular viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and was expressed as a percentage of proliferation versus controls.

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Scratch wound healing assay [1]
Different groups of HepG2 cells were cultured to 80% of confluency, and serum-starved for 2 h, followed by scratching the monolayer cells with a pipette tip. The cells were treated in triplicate with, or without, 30 μM Scutellarin in complete medium for 6, 12, and 24 h. The scratched areas were marked and photoimaged. The average migration distance of each group of cells was measured in three random regions in each well.

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In vitro invasion assay [1]
The effect of Scutellarin on invasion of HepG2 cells was measured by transwell invasion assay using matrigel invasion chamber (8 μm pore size). Different groups of HepG2 cells (1 × 105/well) were cultured in triplicate in 0.5 ml serum-free medium in the upper chamber that had been coated with matrigel. The lower chambers were filled with complete medium in the presence or absence of 30 μM scutellarin. After cultured for 24 h, the cells on the top surface of the membranes in the upper chambers were removed, and the cells on the bottom surface were stained with crystal violet. The numbers of invaded cells per field were counted in 10 random fields per transwell filter at 200 × magnification.

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Glucose consumption [2]
Differentiated 3T3-L1 cells were cultured in plates (1 × 105 cells/well) and starved in Krebs–Ringer phosphate–HEPES buffer (KRHB, containing 118-mM NaCl, 5-mM KCl, 1.3-mM CaCl2, 1.2-mM MgSO4, 1.2-mM KH2PO4, and 30-mM HEPES, containing 0.5% BSA, pH 7.4) for 4 h, then treated with baicalin and Scutellarin in KRH buffer containing 11 mM glucose. After 4 h, glucose content in the culture supernatant was examined with a glucose kit. The amount of glucose consumption was calculated by subtracting the glucose from the blank well.

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Measurement of 2-deoxyglucose (2DG) uptake and assessment of synergism and dose–response relationship [2]
Cells were seeded in 6-well plates, and treated with baicalin (2.5, 5, 10, 20, 40, 80 μM), or Scutellarin (2.5, 5, 10, 20, 40, 80 μM), or their combination at a ratio of 1:1. Glucose uptake was determined by 2DG uptake with an enzymatic photometric assay by using 2DG uptake measurement kit. The Chou–Talalay approach was employed to evaluate synergistic effects between baicalin and scutellarin for the promotion of glucose uptake in adipocytes. To calculate combination index (CI) values, the CalcuSyn software was used. Interaction was quantified based on a CI to assess synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1).

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Measurement of cellular PI3K [2]
Adipocytes were treated with baicalin or Scutellarin at concentration of 10 μM for 0.5 h, and then lysed in ice-cold cell lysis buffer, and centrifugated at 13,000g for 15 min at 4 °C. The level of PI3K in the supernatant was detected by PI3K Assay Kit.

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siRNA transfection [2]
At 70% confluence, 3T3-L1 cells in six-well dishes were incubated with fresh DMEM without antibiotics for 2 h before transfection. Cells were transfected in duplicate with siRNA targeting mouse AMPKα1/2, Akt2 or control siRNA using transfection reagents for 6 h according to the manufacturer's protocol. The medium was removed and replaced with the fresh medium in the presence or absence of baicalin or Scutellarin (10 μM) after 24 h of transfection. Then cells were collected for western blot analyses.

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Cell viability [5]
The Cell Counting Kit assay was applied to exclude the influence of Scutellarin/SCU on viability of BMMs and Raw264.7 cells. Briefly, BMMs and RAW264.7 cells were cultured in a 96-well plate and stimulated with M-CSF (30 ng/mL) and RANKL (60 ng/mL), and subsequently treated with the indicated doses of SCU for 48 h. Next, 10 μL of CCK-8 assay solution was added to each well, and the cells were incubated for an additional 2 h. The absorbance values of each well were measured using an enzyme standard instrument, and the following formula was used to test the cell viability: [experimental group OD-zeroing OD]/[control group OD-zeroing OD].

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TRAP staining [5]
TRAP staining was preformed to examine the inhibiting effects of Scutellarin/SCU on osteoclast differentiation. BMMs were plated into a 6-well-plate at a density of 1.2 × 105 cells/well and different concentrations of SCU (0, 2.5, 5 or 10 μM), as well as M-CSF (30 ng/mL) and RANKL (60 ng/mL), were added. Every 2 days, the culture medium was replaced until mature osteoclasts were formed on day 7. Subsequently, the original medium was removed, and 4% (w/v) paraformaldehyde was used to fix cells for 20 min. TRAP staining was performed in accordance with the instructions. The number of TRAP staining positive osteoclasts with more than three nuclei were calculated.

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Resorption pit assay [5]
Because Scutellarin/SCU could inhibit RANKL-mediated osteoclastogenesis, we hypothesized that it could also inhibit osteoclastic bone resorption. Therefore, BMMs were seeded onto bone slices at a density of 8 × 103 cells/well and stimulated with M-CSF (30 ng/mL) and RANKL (60 ng/mL), and SCU (0, 2.5, 5, or 10 μM) until osteoclasts were observed on day 7. Next, ultrasound and mechanical stimulation were used to remove the cells attached to the surface of the bone slices. Scanning electron microscope (SEM) was used to test the resorption pits in each bone slice, and we randomly choose three areas to calculate the bone resorption using Image J software.

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F-actin ring staining [5]
Considering that the formation of mature F-actin ring is critical for the activity of osteoclasts bone-resorption, we used F-actin ring staining to determine the effect of SCU on F-actin ring formation. BMMs were stimulated with M-CSF (30 ng/mL) and RANKL (60 ng/mL), and various concentrations of Scutellarin/SCU (0, 2.5, 5, or 10 μM) until osteoclasts were observed on day 7. To fix the cells, the original medium was removed and 4% (w/v) paraformaldehyde was added for 20 min before 0.1% (v/v) Triton X-100 solution was added for 5 min to permeabilize the cells. Alexa-Fluor 647 phalloidin solution, diluted in 0.2% (w/v) BSA-PBS, was used to incubate cells at room temperature for 1 h. Next, 0.2% (w/v) BSA-PBS and PBS was used to wash cells to remove the excess dye, and anti-fade mounting medium was applied to avoid fluorescence quenching. The ZEN fluorescence microscope was used to detect the length of fluorescence.

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RT-PCR analysis [5]
During the process of osteoclastogenesis, some osteoclast differentiation associated genes, including TRAP, Cathepsin K, c-Fos, and NFATc1 and so on are upregulated. Therefore, to confirm the effect of Scutellarin/SCU on inhibiting osteoclast differentiation gene expression, TRAP, Cathepsin K, c-Fos, and NFATc1 were investigated. BMMs were seeded in 24-well plates at a density of 10 × 104 cells/well and cultured in complete a-MEM supplemented with 30 ng/mL M-CSF and 60 ng/mL RANKL. RANKL induced osteoclastogenesis from mouse BMMs and was administered with either different doses of SCU (0, 2.5, 5.0, or 10 μM) for 5 days. Briefly, a reverse transcriptase kit was used to extract the total RNA from BMMs; polymerase chain reaction (PCR) was used to synthesize and amplify cDNA from total RNA using a reverse transcription kit.

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Western blot analysis [5]
Next, to determine the possible mechanism that mediates the effect of Scutellarin/SCU on inhibiting osteoclastogenesis. Raw264.7 cells were treated for the indicated time with various concentrations of SCU and to determine whether SCU can regulate the expression of c-Fos and NFATc1 after inhibiting the NF-κB and MAPKs signaling pathways, Raw264.7 cells were treated with or without 10 μM SCU, as well as RANKL for 3, 5, and 7 days. Next, cells were lysed, and supernatants were collected as samples. Protein (30 μg) was separated on 10% SDS–PAGE and transferred to PVDF membranes, and 5% skim milk dissolved in TBST was used to block the membranes. Next, rabbit primary anti-p-IκBα, p-JNK, JNK, p-p38, p38, p-ERK, ERK, NFATc1, c-Fos and β-actin were probed successively overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-mouse IgG antibodies were used as secondary antibodies for 2 h at room temperature. The signals were detected by exposure in an BIO-RAD imaging system. The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and NFATc1, p-P38, p-JNK, and p-ERK, p-IκBα. (*p < 0.05,**p < 0.01).

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Cytotoxicity assay. [6]
Cytotoxicity was measured by MTT method as described previously. Briefly, cells were seeded in the absence or presence of various concentrations of Scutellarin in triplicate for 3–7 days. The percentage of viable cells was quantified at 595/630 nm (A595/630) in an ELISA reader. The cytotoxic concentration that caused the reduction of viable cells by 50% (CC50) was determined from dose–response curve.

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Syncytium reduction assay. [6]
Different concentrations of Scutellarin were added in a 96-well microtitre plate. C8166 or MT-2 (3 × 104 cells/well) were seeded and inoculated with 100 TCID50 HIV-1 and then incubated at 37 °C in a humidified incubator with 5% CO2 for a period of 72 h. Control assays were performed without the testing compounds in HIV-1-infected and uninfected cultures. AZT was used for drug control. The number of syncytium (multinucleated giant cell) in each well was counted under an inverted microscope. Percentage inhibition of syncytial cell formation was estimated from the percentage of syncytial cell number in treated culture to that in infected control culture.

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Inhibition of HIV-1 p24 antigen production in acute infection. [6]
The effect of Scutellarin on HIV-1 replication in vitro was also measured by p24 expression using capture ELISA as described previously. Briefly, MT-2 or C8166 cells were inoculated with HIV-174V or HIV-1IIIB at an MOI of 0.03, respectively, at 37 °C for 2 h to allow for viral absorption. It was then washed three times with PBS. The cells were plated at 3 × 104/well with or without the addition of scutellarin. HIV-1 p24 expression was assayed in cell-free supernatants harvested at day 4.

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Inhibition of HIV-1 p24 antigen production in chronically infected cell lines. [6]
H9 cells chronically infected with HIV-1IIIB were washed three times with PBS to remove free virus particle. 200 μl/well (3 × 105 cell/ml) of the cell suspension was cultured for 3 days in a 96-well culture plate with different concentrations of Scutellarin. Three wells without scutellarin were used as negative control. After 3 days of incubation, p24 antigen in the culture supernatants was tested by ELISA.

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Inhibition of HIV-1 p24 antigen production in PBMC. [6]
Adequate numbers of PHA-activated normal PBMC were inoculated with HIV-1KM018 (MOI = 0.03). After 2 h of virus adsorption, the cells were washed twice with PBS and incubated with or without Scutellarin in culture medium supplemented with 50 U/ml human recombinant IL-2 at 1 × 106 cells/ml for 7 days. Half of the medium was changed twice per week with corresponding scutellarin concentrations. At 7 days post-infection, HIV-1 p24 antigen in the culture supernatants was analyzed by ELISA. The inhibition of HIV-1 p24 antigen production in PBMC was calculated.

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Assay of HIV-1 particle attachment and entry. [6]
HIV-1 entry was estimated from the concentration of intracellular virus RNA by real-time RT-PCR. C8166 cells were pretreated with different concentrations of Scutellarin for 1 h. It was then inoculated with HIV-1IIIB and allowed to adsorb with virus for 2 h at 37 °C. HIV-1 bound on the cell surface was removed by trypsinization, and then washed three times with PBS. The attachment of HIV-1 to cells was monitored after 1 h of incubation with HIV-1IIIB at 4 °C, and then washed extensively with PBS to eliminate unbound HIV-1 particles. T20 and DS were used as control. The amount of RNA in cell extracts was measured by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).

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Cell fusion assay. [6]
C8166 cells (2 × 105) were pretreated for 1 h with varying concentrations of Scutellarin prior to mixing with 3 × 104 HIV-1IIIB chronically infected H9 cells in 96-well plate. After co-culture for 6 h at 37 °C in 5% CO2, syncytia formation was scored through an inverted microscope.

Experimental groups and drug administration [4]
Rats were randomly assigned into four experimental groups: sham (n = 15), not subjected to any treatment or intervention; SAH + vehicle (n = 24), intracerebroventricularly administered normal saline after SAH surgery; SAH + Scutellarin/SCU (n = 22), intracerebroventricularly injected 100 mg/kg SCU/Scutellarin at the concentration of 50 μM immediately after SAH; and SAH + SCU + XMD8-92 (n = 23), intracerebroventricularly injected same amount of SCU plus 10 μM XMD8-92 (100 mg/kg) immediately after the SAH surgery.

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In vivo orthotopic liver xenograft model and treatment [1]
Male BALB/c nude (H-2d, 5–6 weeks old) mice were housed in a specific pathogen-free facility in a 12-h light/dark cycle with free access to sterilized food and water. To establish an orthotopic liver xenograft model, individual mice were anesthetized with isoflurane and a small incision was made in their abdomen. Individual mice were injected with 2 × 106 SK-Hep1 cells in 30 μl Matrigel into their left lobe of the liver. Twenty-four hours after orthotopic liver implantation, the mice were randomized and injected intraperitoneally with Scutellarin (50 mg/kg/day) or vehicle (0.9% NaCl, normal saline) daily for 35 consecutive days (n = 10 per group). Subsequently, the mice were sacrificed, and their lungs and livers were excised, fixed in 10% buffered formalin and paraffin-embedded for hematoxylin and eosin staining.

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Oral glucose tolerance test and insulin sensitivity assay in mice [2]
Mice deprived of food overnight were orally administered with baicalin (50 mg·kg− 1), Scutellarin (50 mg·kg− 1) or metformin (200 mg·kg− 1), respectively. After 1.0 h, the mice were given glucose (2 g·kg− 1) by gavage. Blood was collected from the orbital sinus at regular intervals after glucose load, and blood glucose and insulin levels were measured with commercial kit. Area under the curve for glucose (AUC-G) and insulin sensitivity index (ISI) was calculated, as described.
Diabetic mice model was induced by macrophages-derived conditioned medium (Mac–CM) according to a previous method the authors established. Mice were administrated with drugs, and 30 min later, were intraperitoneally (i.p.) injected with Mac–CM (0.1 mL 10 g− 1, diluted with saline, 1:1, v·v− 1). After another 0.5 h, mice were given glucose, and glucose intolerance and insulin sensitivity were tested.

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Titanium particle-induced calvarial osteolysis [5]
To determine the effect of Scutellarin/SCU on titanium (Ti) particle-induced osteolysis, we built a mouse calvarial osteolysis model according to previous studies. Briefly, 20 healthy 6–8-week-old male C57BL/J6 mice were randomly assigned into four groups: sham PBS control group (sham group), SCU-low and Ti particles group, SCU-high and Ti particles group, and pure Ti particles group (vehicle group). After animals were anesthetized, the cranial periosteum was separated from the calvarium. Subsequently, approximately 30 mg Ti particles were planted under the periosteum at the middle suture of the calvaria and the wounds were sewn up. Animals in the SCU-low group and SCU-high group were intraperitoneally treated with Scutellarin/SCU at 5 or 10 mg/kg/day, respectively, for 10 days starting the first day after operation. For the sham and vehicle groups, animals were treated with equal amount of PBS daily. After 10 days, the mice were sacrificed and 4% paraformaldehyde was used to fix the calvarias and prepared for micro-CT analysis.

mouse LD50 intravenous 1314 mg/kg Zhongcaoyao. Chinese Traditional and Herbal Medicine., 14(33), 1983

[1]. Scutellarin suppresses migration and invasion of human hepatocellular carcinoma by inhibiting the STAT3/Girdin/Akt activity. Biochem Biophys Res Commun. 2017 Jan 29;483(1):509-515.

[2]. Differential regulation of baicalin and scutellarin on AMPK and Akt in promoting adipose cell glucose disposal. Biochim Biophys Acta. 2016 Nov 27;1863(2):598-606.

[3]. Scutellarin attenuates microglia-mediated neuroinflammation and promotes astrogliosis in cerebral ischemia - a therapeutic consideration. Curr Med Chem. 2017;24(7):718-727.

[4]. Scutellarin attenuates vasospasm through the Erk5-KLF2-eNOS pathway after subarachnoid hemorrhage in rats. J Clin Neurosci. 2016 Dec;34:264-270.

[5]. Scutellarin inhibits RANKL-mediated osteoclastogenesis and titanium particle-induced osteolysis via suppression of NF-κB and MAPK signaling pathway. Int Immunopharmacol. 2016 Nov;40:458-465.

[6]. The anti-HIV-1 effect of scutellarin. Biochem Biophys Res Commun. 2005 Sep 2;334(3):812-6.

Scutellarin is the glycosyloxyflavone which is the 7-O-glucuronide of scutellarein. It has a role as an antineoplastic agent and a proteasome inhibitor. It is a glycosyloxyflavone, a glucosiduronic acid, a trihydroxyflavone and a monosaccharide derivative. It is functionally related to a scutellarein. It is a conjugate acid of a scutellarin(1-).
Scutellarin has been reported in Perilla frutescens, Scutellaria indica, and other organisms with data available.

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In conclusion, the findings from this study demonstrated that scutellarin, an active ingredient of Erigeron breviscapine (vant) Hand Mass, effectively inhibited HCC metastasis in vivo, and migration and invasion of HCC cells in vitro. The inhibition of HCC metastasis by scutellarin was at least partially related to its inhibition on the STAT3/Girdin/Akt signaling, different from sorafenib targets in HCC. Thus, scutellarin may be a promising adjuvant or alternative agent for combination with sorafenib therapy for advanced HCC patients.[1]

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In conclusion, this study demonstrated the positive roles of baicalin and scutellarin in regulation of glucose uptake in adipocytes. Baicalin and scutellarin could promote glucose uptake in adipocytes by differential management of AMP-kinase or Akt activity, and a combination of the two compounds produced synergistic effects potentially through a multi-component and multi-target mode in the absence of insulin. This study may lead to a promising drug combination for patients with glucose metabolism disorders.[2]

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Neuroinflammation plays an important role in different brain diseases including acute brain injuries such as cerebral ischemic stroke and chronic neurodegenerative diseases e.g. Alzheimer's disease etc. The central player in this is the activated microglia, which produce substantial amounts of proinflammatory mediators that may exacerbate the disease. Associated with microglia activation is astrogliosis characterized by hypertrophic astrocytes with increased expression of proinflammatory cytokines, neurotrophic factors, stem cell, neuronal and proliferation markers, all these are crucial for reconstruction of damaged tissue and ultimate restoration of neurological functions. Here, we review the roles of activated microglia and reactive astrocytes in brain diseases with special reference to cerebral ischemia, and the effects of scutellarin, a Chinese herbal extract on both glial cells. We first reviewed the close spatial relation between activated microglia and reactive astrocytes as it suggests that both glial cells work in concert for tissue reconstruction and repair. Secondly, we have identified scutellarin as a putative therapeutic agent as it has been found to not only suppress microglial activation thus ameliorating neuroinflammation, but also enhance astrocytic reaction. In the latter, scutellarin amplified the astrocytic reaction by upregulating the expression of neurotrophic factors among others thus indicating its neuroprotective role. Remarkably, the effects of scutellarin on reactive astrocytes were mediated by activated microglia supporting a functional "cross-talk" between the two glial types. This review highlights some of our recent findings taking into consideration of others demonstrating the beneficial effects of scutellarin on both glial cell types in cerebral ischemia as manifested by improvement of neurological functions.[3]

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In conclusion, the present study demonstrated that SCU induced vasorelaxation and neuroprotective effects against SAH-induced vasospasm via the Erk5-KLF2-eNOS pathway. Our results may provide an experimental basis for the clinical use of SCU treatment in SAH patients.[4]

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Taken together, our study indicated that SCU has inhibitory effects on Ti particle-induced osteolysis in vivo. This effect may be mediated by suppressing RANKL-mediated MAPK (p38, JNK1/2, ERK1/2) and NF-κB signaling pathways. These findings may provide a new method for preventing wear particle-induced aseptic prosthesis loosening.
Our study indicated that SCU has inhibitory effects on osteoclastogenesis and Ti particle-induced osteolysis in vivo. This effect was mediated by suppressing RANKL-mediated MAPKs (p38, JNK1/2, ERK1/2) and NF-κB signaling pathways. These findings may provide a new method for preventing wear particle-induced aseptic prosthesis loosening.[5]

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Scutellarin is one of the flavonoides used clinically to treat cerebral vascular patients in China. There are large numbers of natural compounds belonging to flavonoides family. Some of them exhibit anti-HIV-1 effect in vitro and often with more than one mode of actions. They can interact at different steps in the life cycle of HIV-1, including viral entry, RT, integrase, and Vpr. Many anti-HIV-1 agents (e.g., suramine, michellamine, and glycyrrhizin) also showed significant PKC inhibition. Scutellarin also has an inhibitory effect on PKC and therefore PKC inhibition may be one of the mechanisms by which scutellarin inhibits HIV-1 replication. It is premature at this stage to define the mechanism of action of scutellarin. There were some evidences that scutellarin interfere with viral attachment, cell fusion, RT inhibition, and PKC inhibition. The anti-HIV-1 action of scutellarin may be related to one or more of these activities. In conclusion, scutellarin was shown to inhibit HIV-1 replication. This action may be related to cell entry and/or RT inhibition.[6]

C21H18O12

462.3604

462.079

C, 54.55; H, 3.92; O, 41.52

27740-01-8

185617

Light yellow to green yellow solid powder

1.8±0.1 g/cm3

891.6±65.0 °C at 760 mmHg

314.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.764

-0.46

7

12

4

33

777

5

C1=CC(=CC=C1C2=CC(=O)C3=C(C(=C(C=C3O2)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)C(=O)O)O)O)O)O)O)O

DJSISFGPUUYILV-ZFORQUDYSA-N

InChI=1S/C21H18O12/c22-8-3-1-7(2-4-8)10-5-9(23)13-11(31-10)6-12(14(24)15(13)25)32-21-18(28)16(26)17(27)19(33-21)20(29)30/h1-6,16-19,21-22,24-28H,(H,29,30)/t16-,17-,18+,19-,21+/m0/s1

(2S,3S,4S,5R,6S)-6-[5,6-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-7-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid

Scutellarin; Scutellarin B; Scutellarein-7-glucuronide; Breviscapine; Scutellarein-7beta-D-glucuronide; Scutellarein 7-beta-D-glucuronide; Scutellarein-7beta-D-glucuronoside; ...; 27740-01-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~216.28 mM)

Solubility in Formulation 1: 5 mg/mL (10.81 mM) in 15% Cremophor EL 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10 mg/mL (21.63 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)