Exploring lipid biomarkers of coronary heart disease for elucidating the biological effects of gelanxinning capsule by lipidomics method based on LC-MS.

High-throughput lipidomics technology was used to explore the potential therapeutic targets and mechanism of action of gelanxinning capsule on rat model with coronary heart disease (CHD). This study attempts to provide a novel method to interpret the molecular mechanism of traditional medicine. The lipid markers of CHD were determined by full-scan analysis based on ultra-performance liquid chromatography-high-definition mass spectrometry. Then, the metabolic changes associated with gelanxinning capsule treatment via the modulation of lipid biomarkers and pathway in rats were characterized. After gelanxinning treatment, the metabolic profile tended to recover compared with the model group. A total of 26 potential biomarkers were identified to represent the disorders of lipid metabolism in CHD animal model, of which 19 were regulated by gelanxinning capsule administration, and four metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, and glycerolipid metabolism were involved. From the pathway analysis, it was found that glycerophospholipid metabolism and sphingolipid metabolism with significant differences have the potential to be regarded as new targets for the treatment of CHD. Gelanxinning capsule with its good therapeutic effect protects against CHD by regulating lipid biomarkers and pathway from lipidomics-guided biochemical analysis.

Induction of apoptosis by total flavonoids from Scutellaria barbata D. Don in human hepatocarcinoma MHCC97-H cells via the mitochondrial pathway.

Scutellaria barbata D. Don, a traditional Chinese medicine, reportedly possesses antitumor activity against a variety of tumors. In the present study, we investigated the cytotoxic effect of total flavonoids from S. barbata (TF-SB) on human hepatocarcinoma cells and the underlying molecular mechanisms regarding the effect were explored. TF-SB treatment significantly reduced the cell viability of human HCC MHCC97-H cells in a dose-dependent manner. Further flow cytometric analysis showed that the apoptosis rate of MHCC97-H cells increased and the mitochondrial membrane potential (∆ψm) of MHCC97-H cells decreased after TF-SB treatment. DNA ladder showed that TF-SB induced a significant increase in DNA fragmentation in MHCC97-H cells. Reverse transcription PCR and Western blot analysis revealed that the expression levels of Smac, Apaf-1, Cytochrome c, Caspase-9, and Caspase-3 were upregulated in a dose-dependent manner and after treatment with different concentrations of TF-SB for 48 h. These results suggest that TF-SB induces apoptosis in MHCC97-H cells through the mitochondrial pathway.

Saikosaponin-d increases the radiosensitivity of smmc-7721 hepatocellular carcinoma cells by adjusting the g0/g1 and g2/m checkpoints of the cell cycle.

BACKGROUND: Saikosaponin-d (SSd), a monomer terpenoid purified from the Chinese herbal drug Radix bupleuri, has multiple effects, including anticancer properties. However, the effect of SSd on tumors exposed to radiation is largely unknown. To investigate the radiosensitizing effect of SSd and its possible mechanism, we combined SSd with radiation therapy to treat SMMC-7721 hepatocellular carcinoma cells under oxia and hypoxia. METHODS: Cell growth, apoptosis, and cell cycle distribution were examined after treatment with SSd alone, radiation alone, and their combinations under oxia and hypoxia. The protein and mRNA levels of p53, Bcl2, and BAX were measured using western blot analysis and RT-PCR, respectively. RESULTS: Treatment with SSd alone and radiation alone inhibited cell growth and increased apoptosis rate at the concentration used. These effects were enhanced when SSd was combined with radiation. Moreover, SSd potentiated the effects of radiation to induce G0/G1 arrest in SMMC-7721 cells, and reduced the G2/M-phase population under hypoxia. However, under oxia, SSd only potentiated the effects of radiation to induce G0/G1 arrest, but not G2/M-phase arrest. These effects of SSd alone, radiation alone, and their combination, were accompanied by upregulated expression of p53 and BAX and downregulation of Bcl2 expression under oxia and hypoxia. CONCLUSION: SSd potentiates the effects of radiation on SMMC-7721 cells; thus, it is a promising radiosensitizer. The radiosensitizing effect of SSd may contribute to its effect on the G0/G1 and G2/M checkpoints of the cell cycle.

Anti-angiogenic effect of the total flavonoids in Scutellaria barbata D. Don.

BACKGROUND: Angiogenesis is closely related to the growth, invasion and metastasis of tumors, also considered as the key target of anticancer therapy. Scutellaria barbata D. Don (S. barbata), a traditional Chinese medicine, is being used to treat various diseases, including cancer. However, the antitumor molecular mechanism of S. barbata was still unclear. This study aimed to investigate the inhibitory effects of the total flavones in S. barbata (TF-SB) on angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with various concentrations of TF-SB. Cell viability was examined using the MTT assay. The scratch assay was used to detect the migration of HUVECs after treatment with TF-SB. The ability of HUVECs to form network structures in vitro was demonstrated using the tube formation assay. The chick embryo chorioallantoic membrane assay was performed to detect the in vivo anti-angiogenic effect. The expression of VEGF was measured by the enzyme-linked immunosorbent. RESULTS: Results showed that TF-SB inhibited the proliferation and migration of HUVECs in a dose- dependent manner. Simultaneously, TF-SB significantly suppressed HUVEC angiogenesis in vitro and in vivo. Furthermore, VEGF was downregulated in both HUVECs and MHCC97-H cells after TF-SB treatment. CONCLUSION: TF-SB could suppress the process of angiogenesis in vitro and in vivo. TF-SB potentially suppresses angiogenesis in HUVECs by regulating VEGF. These findings suggested that TF-SB may serve as a potent anti-angiogenic agent.

The effect of RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract on radiation-induced skin injury in a rat model.

BACKGROUND: Radiation-induced skin injury is a common complication of radiotherapy. The RHIZOMA COPTIDIS and COPTIS CHINENSIS aqueous extract (RCE) can ameliorate radiation-induced skin injury in our clinical observation. But, the protective mechanism of RHIZOMA COPTIDIS and COPTIS CHINENSIS in radiation-induced skin injury remains unclear. METHODS: In this experiment, we developed a radiation-induced skin injury rat model to study the mechanism. The animals were randomly divided into control group, treatment group, radiation group, and treatment and radiation group. 5 rats in each group were separately executed on 2 d and 49 d post-radiation. The semi-quantitative skin injury score was used to measure skin reactions by unblinded observers, and hematoxylin and eosin staining was used to evaluate the damage areas by irradiation. The MDA content, SOD activity of skin and serum were measured to detect the oxidative stress. RESULTS: Acute skin reactions were caused by a single dose of 45 Gy of ß-ray irradiation, and the skin injury could be found in all rats receiving irradiation based on the observation of HE staining of skin at different time-points, while RCE could significantly ameliorate those changes. The MDA content in serum and skin of control rats was 4.13±0.12 mmol/ml and 4.95±0.35 mmol/mgprot on 2 d post-radiation. The rats receiving radiation showed an increased content of MDA (5.54±0.21 mmol/ml and 7.10±0.32 mmol/mgprot), while it was 4.57±0.21 mmol/ml and 5.95±0.24 mmol/mgprot after treated with RCE (p<0.05). Similar changes of the MDA content could be seen on 49 d post-radiation. However, the SOD activity of rats receiving radiation decreased compared with control group on both time-points, which was inhibited by RCE (p<0.05). Meanwhile, no valuable changes could be found between control group and treatment group on 2 d and 49 d. CONCLUSIONS: Our study provides evidences for the radioprotective role of RCE against radiation-induced skin damage in rats by modulating oxidative stress in skin, which may be a useful therapy for radiation-induced skin injury.

Protective effects of Scutellaria barbata against rat liver tumorigenesis.

Scutellaria barbata D. Don (S. barbata), a traditional Chinese medicine, is used to treat cancers, inflammation, and urinary diseases. This study aimed to determine any protective effects of S. barbata crude extract (CE-SB) against rat liver tumorigenesis induced by diethylnitrosamine (DENA). Liver malfunction indices in serum were measured by biochemical examination. Hematoxylin and eosin staining was performed to examine liver pathology. Contents of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured in liver homogenates to evaluate oxidative stress. The levels of liver malfunction indices in the CE-SB groups, especially in the CE-SB high dose group, were lower than that of the model group (P<0.05). The results from histological examination indicated that the number of liver nodules in the CE-SB groups decreased compared with the model group (P<0.05). Content of MDA determined in liver was significantly decreased, and level of SOD elevated by CE-SB. CE-SB can inhibit experimental liver tumorigenesis and relieve hepatic injury in rats.

Total flavonoids of Scutellaria barbata inhibit invasion of hepatocarcinoma via MMP/TIMP in vitro.

Metastasis is the major cause of cancer-related deaths. Targeting the process of metastasis has been proposed as a strategy to fight cancer. Scutellaria barbata D. Don (S. barbata), a traditional Chinese medicine, is used for treatment of many diseases, including cancer. This study aimed to determine the anti-metastatic effect of total flavonoids of S. barbata (TF-SB) using the human hepatocarcinoma MHCC97H cell line with high metastatic potential. Our results show that TF-SB could significantly inhibit the proliferation and invasion of MHCC97H cells in a dose-dependent manner. MMP-2 and MMP-9 expression were obviously decreased after TF-SB treatment at both the mRNA and protein level. TIMP-1 and TIMP-2 expression were simultaneously increased. The present study indicates that TF-SB could reduce the metastatic capability of MHCC97H cell, probably through decrease of the MMP expression, and simultaneous increase of the TIMP expression.

In vitro and in vivo antitumor activity of Scutellaria barbate extract on murine liver cancer.

In the present study, we investigated the in vitro and in vivo antitumor effects of crude extract of Scutellaria Barbate (CE-SB) on mouse hepatoma H22 cells. The MTT assay was used to determine the growth inhibition of H22 cells in vitro. The in vivo therapeutic effects of CE-SB were determined using H22 tumor bearing mice. Besides, the body weight, tumor weight, thymus index and spleen index of H22 bearing mice were also measured. The tumor inhibitory rate (IR) was calculated according to the mean weight of tumor (MWT). The phagocytotic function of macrophages was examined by observing peritoneal macrophages phagocytize chicken RBC. The results showed that CE-SB could inhibit the growth of hepatoma H22 Cells in vitro and in vivo. Furthermore, CE-SB could improve immune function of H22 tumor bearing mice. Together these results indicate that CE-SB has antitumor activity and seems to be safe and effective for the use of anti-tumor therapy.

[Inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats].

OBJECTIVE: To investigate the inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats. METHODS: Hepatocarcinoma model rats were induced by diethylnitrosamine (DEN). Sixty SD male rats were randomly divided into 4 groups: normal control group, hepatocarcinoma model group, ESB of high dose group and ESB of low dose group. All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations; Hematoxy and eosin (HE) methods were used to examine the changes of liver pathology. RESULTS: The levels of ALT, AST, TBIL, ALP, gamma-GT, AFU in hepatocarcinoma model group and ESB groups were higher than that of control group (P < 0.05). ESB could relieve hepatic injures. The levels of liver function indexes in ESB groups were lower than that of model group. Histological examination demonstrated that the number of liver cancer nodus in ESB groups were lower than that of model group. Furthermore, ESB could attenuate the grade of cancer cell differentiation. CONCLUSION: ESB could inhibit experimental hepatocarcinoma and relieve hepatic injures in rats.

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3.

AIM OF THE STUDY: Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated. MATERIALS AND METHODS: Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunofluorescence method. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Fluorescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate. CONCLUSIONS: Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

[Antitumor and immune-modulating effects of Scutellaria barbata extract in mice bearing hepatocarcinoma H22 cells-derived tumor].

OBJECTIVE: To investigate the effects of Scutellaria barbata extract (ESB) in suppressing tumor growth and modulating the immune functions in mice bearing tumors derived from hepatocarcinoma H22 cells. METHODS: Fifty mice inoculated subcutaneously with H22 cells were equally divided into the model group, high-, moderate-, and low-dose ESB groups, and 5-Fu group, with corresponding treatments for 10 days. Another 10 mice with only saline injection served as the normal control group. The body weight, tumor mass, thymus index and spleen index of the mice were measured, and the lymphocyte proliferation activity, NK cell activity and interleukin-2 (IL-2) production by the splenocytes were detected. RESULTS: Moderate- and high-dose ESB significantly suppressed the tumor growth with tumor inhibition rate of 28.68% and 36.98%, respectively. ESB treatment at moderate and high doses significantly increased the thymus index and spleen index (P < 0.01), which were decreased significantly in 5-Fu group. The lymphocyte proliferation activity, NK cell activity and IL-2 production by the splenocytes were significantly lower in the model group than in the normal group (P < 0.05). Compared with the model group, ESB at the high dose obviously increased the three indexes above mentioned. The NK cell activity was also significantly improved in moderate-dose ESB group (P < 0.05). CONCLUSION: ESB can suppress the growth of H22 implant tumor and enhance the immune function of the tumor-bearing mice.

[Scutellaria barbata extract enhances efficacy and reduces toxicity of chemotherapy in hepatoma H22-bearing mice].

OBJECTIVE: To study the assistant effect of Scutellaria barbata extract (ESB) in 5-fluorouracil (5-FU) chemotherapy. METHODS: A mouse model of transplanted hepatoma H22 was used in this study to evaluate the synergic and attenuating effects of ESB in chemotherapy. Tumor inhibition rate, life span of mice and the toxicity of chemotherapy were observed. The body weight, tumor weight, thymus index and spleen index in H22-bearing mice were also measured. The phagocytotic function of macrophages was studied by observing phagocytization of peritoneal macrophages. RESULTS: The increase of body weight in 5-FU plus ESB groups was higher than that in 5-FU group, and the side effects such as anorexia, abdominal distention and athrepsy were relieved. Compared with untreated group, prolonged lifetime in 5-FU plus high-dose ESB group and 5-FU plus low-dose ESB group was improved. Life prolongation rates in 5-FU plus high-dose ESB group and 5-FU plus low-dose ESB group were 61.46% and 23.59% respectively. High-dose ESB, 5-FU, 5-FU plus low-dose ESB and 5-FU plus high-dose ESB could inhibit the tumor growth, and the tumor inhibition rates were 36.98%, 42.26%, 52.45% and 65.28%, respectively. Thymus index and spleen index were increased significantly in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. White blood cell (WBC) count was decreased obviously in 5-FU group, while the count of WBC was increased in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. The phagocytotic function of macrophages was also increased in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. CONCLUSION: ESB can enhance the effect of chemotherapy, relieve the side effects and improve immune function of mice in chemotherapy. These results suggest that ESB, as a biochemical modulator to enhance the therapeutic effects, is useful in cancer chemotherapy.

[Effects of Scutellaria Barbata drug-containing serum on apoptosis and mitochondrial transmembrane potential of hepatoma H22 cells].

OBJECTIVE: To investigate the effects of serum containing Scutellaria Barbata extract (ESB) on apoptosis rate and mitochondrial transmembrane potential (MTP) of liver cancer cell line H22 from mice in vitro. METHODS: H22 cells were cultured in vitro and divided into 5 groups: blank control group, low-dose ESB group, medium-dose ESB group, high-dose ESB group and fluorouracil (5-Fu) group. Methyl thiazolyl tetrazolium assay was utilized to determine the proliferation rates of H22 cells. Cellular morphology was observed under a transmission electron microscope (EM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with a laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the MTP of H22 cells. RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observed in a time-dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48h. The apoptosis rates of blank control group, 5-Fu group, low-dose ESB group, medium-dose ESB group and high-dose ESB group were (0.51+/-0.32)%, (11.26+/-2.97)%, (1.07+/-0.46)%, (3.15+/-1.12)%, (7.83+/-2.25)% respectively. ESB could reduce the MTP of H22 cells from mice as compared with the untreated group. The MTPs of the blank control group, 5-Fu group, and low-, medium- and high-dose ESB groups were (245.45+/-67.37), (127.42+/-41.35), (213.68+/-65.52), (186.34+/-56.37) and (142.65+/-39.44) respectively, which were negatively correlated with the apoptosis rates. CONCLUSION: ESB-containing serum effectively induces apoptosis, which may be related to the decrease of MTP in H22 cells.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

[Effect of matrine injections on invasion and metastasis of gastric carcinoma SGC-7901 cells in vitro].

OBJECTIVE: To investigate the effect of matrine injections on invasion and metastasis of gastric carcinoma SGC-7901 cells in vitro. METHODS: MTT assay was used to examine the effect of matrine injections on proliferation of SGC-7901 cells after 24, 48, 72, 96 hours treatment; Transwell chamber assay was performed to determine the effect of matrine injection on invasion and migratory capacity of the cells; Effect on adhesion potential of SGC-7901 cells was tested by cell matrigel adhesion assay. RESULTS: Matrine injec-tions could inhibit the proliferation of SGC-7901 cells, with obvious dose-dependent and time-dependent effects. Matrine injections sig-nificantly inhibited adhesion, invasion and migration capacity of SGC-7901 cells in vitro. The inhibitory rate of them after treatment with 1.5 mg/ml matrine injections for 24 h were (45.32 +/- 3.10)%, (32.66 +/- 2.82)%, (38.35 +/- 3.41)% respectively. After treat-ment of matrine injections (1.5 mg/ml)for 24 h, the expression level of CD44(V6) in SGC-7901 cells was decreased compared with the untreated group. CONCLUSION: Matrine injections can inhibit the migration, invasion and adhesion capacity of SGC-7901 cells in vitro. The inhibition effect may be related to down-regulating the expression of CD44(V6) protein.