Effects of total flavone of oldenlandia diffusa on the proliferation and apoptosis of hepatocellular carcinoma stem cell / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of total flavone of oldenlandia diffusa (FOD) on the proliferation and apoptosis of hepatocellular carcinoma (HCC) stem cells sorted from Huh7. 【Methods】 Human HCC cell lines Huh7 was cultured in vitro; CD133 positive (CD133+) stem cells in Huh7 cell line were sorted by flow cytometry, and stem cell markers such as Nanog, Oct4 and Sox2 were tested by Western blotting. CD133+-Huh7 was stimulated by different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL and 400 μg/mL) of FOD for different time (24 h, 48 h, 72 h and 96 h). CCK8 and plate cell cloning assay were used to detect the effect of FOD on CD133+-Huh7 proliferation while Annexin V-PE/7-AAD was used to detect the effect of FOD on CD133+-Huh7 apoptosis. Western blotting was used to detect the protein expressions of protein 53 (P53), factor associated suicide-Fas-associating protein with a novel death domain (Fas-FADD), B-cell lymphoma-2 (Bcl-2), Cleaved-Caspase3, and Bcl-2 associated X protein (Bax). 【Results】 More than 95% of stem cells were purified for further experiments. Cell proliferation of CD133+-Huh7 was significantly inhibited by FOD, with the significant suppression at the concentration of 100 μg/mL for 72 h compared with negative control group (P<0.05). The apoptosis rate was significantly upregulated than that in the negative control group (P<0.05). The protein expression of Bcl2 decreased while Bax and Cleaved-Caspae3 increased via FAS/FADDD and P53 axis. 【Conclusion】 FOD can significantly inhibit the proliferation and promote the apoptosis of CD133+-Huh7.

Effect of total flavone of oldenlandia diffusa on the proliferation, apoptosis and stemness of breast cancer MDA-MB-231 cell lines / 西安交通大学学报(医学版)

【Objective】 To investigate the effect of total flavone of oldenlandia diffusa(FOD) on the stemness, proliferation and apoptosis of breast cancer(BC) stem cells sorted from MDA-MB-231. 【Methods】 Human BC cell lines MDA-MB-231 was cultured in vitro; MDA-MB-231 was stimulated by different concentrations(0 μg/mL, 100 μg/mL, 200 μg/mL and 400 μg/mL) of FOD for different time (24 h, 48 h and 72 h). CCK8 and plate cell cloning assay were used to detect the effect of FOD on MDA-MB-231 proliferation; CD44+/CD24-MDA-MB-231 cell line were tested by flow cytometry and stem cell markers such as Nanog, Oct4 and Sox2 were tested by Western blotting; Annexin V-PE/7-AAD was used to detect the effect of FOD on MDA-MB-231 apoptosis and Bcl2, cleaved-caspase3 and Bax were tested by Western blotting. 【Results】 Cell proliferation of MDA-MB-231 was significantly inhibited by FOD, with the significant suppression at concentrations of 400 μg/mL for 72 h compared with negative control group(P<0.05). The apoptosis rate was significantly upregulated than the negative control group (P<0.05). The protein expression of Bcl2 decreased while Bax and cleaved-caspae3 increased, and stemness markers such as Nanog, Sox2 and Oct4 decreased in FOD-treated cells. Moverover, Akt-GSK3β-β-catenin axis was inhibited in FOD-treated cells. 【Conclusion】 FOD could significantly inhibit the stemness and proliferation and promote the apoptosis of MDA-MB-231.

Mechanism of active components of "Notoginseng Radix et Rhizoma-Drynariae Rhizoma" in treatment of osteoporosis based on network pharmacology and in vitro cell experiment / 中国中药杂志

The present study aimed to explore the main active components and potential mechanisms of Panax notoginseng saponins(PNS) and osteopractic total flavone(OTF) in the treatment of osteoporosis(OP) through network pharmacology, molecular docking and in vitro cell experiments, which was expected to provide a theoretical basis for clinical applications. The blood-entering components of PNS and OTF were obtained from literature search and online database, and their potential targets were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The OP targets were obtained by means of searching Online Mendelian Inheritance in Man(OMIM) and GeneCards. The common targets of the drug and disease were screened by Venn. Cytoscape was used to construct a "drug-component-target-disease" network, and the core components were screened according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened according to the node degree. GO and KEGG enrichment analysis of potential therapeutic targets were carried out by R language. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock Vina. Finally, HIF-1 signaling pathway was selected for in vitro experimental verification according to the results of KEGG pathway analysis. Network pharmacology showed that there were 45 active components such as leachianone A, kurarinone, 20(R)-protopanaxatriol, 20(S)-protopanaxatriol, and kaempferol, and 103 therapeutic targets such as IL6, AKT1, TNF, VEGFA and MAPK3 involved. PI3K-AKT, HIF-1, TNF and other signaling pathways were enriched. Molecular docking revealed that the core components had good binding ability to the core targets. In vitro experiments found that PNS-OTF could up-regulate the mRNA expression levels of HIF-1α, VEGFA and Runx2, indicating that the mechanism of PNS-OTF in treating OP may be related to the activation of HIF-1 signaling pathway, and thus PNS-OTF played a role in promoting angiogenesis and osteogenic differentiation. In conclusion, this study predicted the core targets and pathways of PNS-OTF in treating OP based on network pharmacology and carried out in vitro experimental verification, which reflected the characteristics of multi-component, multi-target and multi-pathway synergy of PNS-OTF, and provided new ideas for the future clinical treatment of OP.

Efficacy of Getong Tongluo Capsule () for Convalescent-Phase of Ischemic Stroke and Primary Hypertension: A Multicenter, Randomized, Double-Blind, Controlled Trial / 中国结合医学杂志

OBJECTIVE@#To evaluate whether the efficacy of Getong Tongluo Capsule (, GTC, consisted of total flavone of Radix Puerariae) on improving patients' quality of life and lowering blood pressure are superior to the extract of Ginkgo biloba (EGB) for patients with convalescent-phase ischemic stroke and primary hypertension.@*METHODS@#This randomized, positive-drug- and placebo-controlled, double-blind trial was conducted from September 2015 to October 2017. Totally 477 eligible patients from 18 hospitals in China were randomly assigned in a 2:1:1 ratio to the following interventions, twice a day for 12 weeks: (1) GTC 250 mg plus EGB-matching placebo 40 mg (237 cases, GTC group), (2) EGB 40 mg plus GTC-matching placebo 250 mg (120 cases, EGB group) or (3) GTC-matching placebo 250 mg plus EGB-matching placebo 40 mg (120 cases, placebo group). Moreover, all patients were orally administered aspirin enteric-coated tablets 100 mg, once a day for 12 weeks. The primary outcome was the Barthel Index (BI). The secondary outcomes included the control rate of blood pressure and National Institutes of Health Stroke Scale (NIHSS) scores. The incidence and severity of adverse events (AEs) were calculated and assessed.@*RESULTS@#The BI relative independence rates, the clinical recovery rates of NIHSS, and the total effective rates of NIHSS in the GTC and EGB groups were significantly higher than the placebo group at 12 weeks after treatment (P0.05). The control rate of blood pressure in the GTC group was significantly higher than the EGB and placebo groups at 12, 18 and 24 weeks after treatment (P0.05).@*CONCLUSION@#GTC exhibited significant efficacy in improving patients' quality of life as well as neurological function and controlling hypertension. (Registration No. ChiCTR1800016667).

Promoting effect of osteopractic total flavone combined with nano-bone materials on proliferationand differentiation of MC3T3-E1 cells / 中国组织工程研究

BACKGROUND: Preliminary study has found that osteopractic total flavone can promote osteogenic differentiation of MC3T3-E1 cells on the surface of nano-bone material, but the underlying mechanism needs to be studied in depth. OBJECTIVE: To investigate the actin mechanism of osteopractic total flavone combined with nano-bone material on MC3T3-E1 cells. METHODS: MC3T3-E1 cells were co-cultured with nano-bone material, and 100 mg/L and 250 mg/L osteopractic total flavone were treated as drug intervention, including 10 μg/L transforming growth factor-β as positive control. The samples were divided into eight groups: (1) Normal group; (2) DKK1 group: Wnt pathway inhibitor DKK1 (0.1 mg/L) blocks Wnt/β-catenin signaling pathway; (3) DKK1+transforming growth factor-β group; (4) DKK1+100 mg/L osteopractic total flavone group; (5) DKK1+250 mg/L osteopractic total flavone group; (6) DKK1+ nano-hydroxyapatite/collagen+transforming growth factor-β group; (7) DKK1+nano-hydroxyapatite/collagen+100 mg/L osteopractic total flavone group; (8) DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group. Cells in each group were harvested after 24 and 48 hours of intervention. Immunofluorescence labeling was used to observe the binding of Wnt and LRP in osteoblasts in the Wnt/β-catenin pathway. The expression of β-catenin, LRP 5, GSK-3β, Cyclin D1, and RUNX2 was detected by real-time polymerase chain reaction and western blot assay. RESULTS AND CONCLUSION: (1) Confocal laser scanning microscope showed that obvious brown and yellow staining was shown in the DKK1+transforming growth factor-β group, DKK1+250 mg/L osteopractic total flavone group, DKK1+nano-hydroxyapatite/ collagen+transforming growth factor-β group, and DKK1+nano-hydroxyapatite/collagen+250 mg/L osteopractic total flavone group, indicating that Wnt and LRP combined better than other groups. (2) Real-time polymerase chain reaction and western blot assay results showed that osteopractic total flavone could promote the expression of β-catenin, LRP5 and RUNX2, and downregulated GSK3β expression. These findings confirm that osteopractic total flavone can promote the differentiation and proliferation of osteoblasts by activating the Wnt/β-catenin signaling pathway. Gene activation induced by osteopractic total flavone was dose-dependent.

Effects of pueraria total flavone on inflammatory cytokine and stat3 expression in brain tissue of exhaustive exercise rats / 中国组织工程研究

BACKGROUND: The physiological and pathological mechanisms underlying the inflammatory response of rat brain tissue caused by exhaustive exercise are very complicated. Studies have shown that Pueraria total flavonoids have anti-oxidative, neuroprotective, and cardio-cerebrovascular protective effects against brain injury. OBJECTIVE: To investigate the effect of Pueraria total flavone on inflammatory cytokines and signal transducer and activator of transcription-3 (STAT3) expression in rat brain tissue after exhaustive exercise. METHODS: Fifty healthy male Sprague-Dawley rats were randomly divided into a quiet control group, an exercise control group, low, middle, high dose Pueraria total flavone groups. Each training group conducted a 6-week sports training. At the end of 6-week exercise, the rats were confirmed to be exhausted. The rats were intragastrically administered 50, 100 and 200 mg/kg Pueraria total flavone in low, middle and high dose groups, respectively. Administration in each Pueraria total flavone group began at 30 minutes before exercise, once a day, and ended until the completion of the experiment. The activities of tumor necrosis factor-α, interleukin-8, interleukin-1β and interleukin-10 in serum and brain tissue of rats were determined by ELISA. The expression of STAT3 in rat brain tissue was detected by RT-PCR and western blot. The study protocol was approved by the Animal Ethics Committee of Guangxi Normal University (approval No. GXMU201703049). RESULTS AND CONCLUSION: The levels of tumor necrosis factor-α, interleukin-8, interleukin-1β and interleukin-10 in serum and brain tissue of exercise control rats were higher than those in the quiet control group (P < 0.01). The levels of tumor necrosis factor-α and interleukin-10 in serum and brain tissue of rats with middle and high dose of Pueraria total flavone were significantly lower than those in the exercise control group (P < 0.01). The levels of interleukin-8 and interleukin-1β in serum and brain tissue of rats with low, middle and high dose of Pueraria total flavone were significantly lower than those in the exercise control group (P < 0.01 or P < 0.05). The expression levels of STAT3 mRNA and protein in brain tissue of exercise control rats were significantly higher than those in the quiet control group (P < 0.01). The expression levels of STAT3 mRNA and protein in the brain tissue of rats with low, middle and high doses of Pueraria total flavone were significantly higher than those in the quiet control group (P < 0.01). To conclude, exhaustive exercise can cause inflammatory reaction and up-regulate STAT3 expression in rat brain tissue. The total flavonoids of Puerariae can regulate the expression of STAT3 in brain tissues and inhibit the inflammatory response of brain tissue, thus protecting damaged brain tissue.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway / 中国结合医学杂志

OBJECTIVE@#To test the hypothesis that the inhibition of endoplasmic reticulum (ER) stress-induced apoptosis in oxidized low-density lipoproteins (ox-LDL)-induced human aortic-vascular smooth muscle cells (HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway by Pollen Typhae total flavone (PTF).@*METHODS@#Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an HPTF group (70 μg/mL high ox-LDL+500 μg/mL PTF), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), and a LPTF group (70 μg/mL high ox-LDL+100 μg/mL PTF) in the first part; and a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), a shRNA group (transducted with PERK shRNA lentiviral particles), a scramble shRNA group (transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group (250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group (70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability, and the level of apoptosis was monitored by flow cytometry.@*RESULTS@#The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and shRNA groups. Moreover, the ox-LDL group had increased protein and mRNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, eIF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and shRNA groups.@*CONCLUSIONS@#The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

Effect of Dodder Total Flavone on Polycystic Ovary Syndrome Rat Models / 中国实验方剂学杂志

Objective: To investigate the effect of dodder total flavone on polycystic ovary syndrome (PCOS) rat models induced by dehydroepiandrosterone (DHEA) combined with human chorionic gonadotropin (HCG). Method: Except the blank group, the remaining rats were injected with DHEA 0.06 mg·g-1 in the morning on the nucha and 1.5 U HCG in the afternoon for 21 consecutive days. On the 16th day after the modeling, the vaginal smear was performed to monitor the estrus cycle. Sixty rats with successful modeling were selected and divided into model group, dacin-35 group, and high, middle and low-dose dodder total flavonoids groups, with 10 rats in each group. On the day of grouping, drugs were given respectively to the drug treatment groups, and the blank group and the model group were given equal volume solvent. The drugs were given continuously for 21 days. Blood was collected from abdominal aorta 2 h later after the final administration, serum levels of testosterone (T), estrogen (E2), luteinizing hormone (LH) and follicle stimulating hormone(FSH) were measured; rats were put to death, and the ovaries at the same part of each rat were fixed in 10% formalin solution. htoxylin eosin (HE) staining was performed, and the morphological changes in the ovaries were observed by light microscopy; the same part of the ovary was taken, and androgen receptor (AR), B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax) were measured in the ovary by immunohistochemistry to observe the expressions of relevant proteins in the ovary; the hypothalamus and pituitary were taken, and the expressions of gonadotropin-releasing hormone (GnRH) in hypothalamus and gonadorelin releasing hormone receptor (GnRHR) in pituitary were detected by immunohistochemistry. Result: PCOS rat model was successfully replicated. Serum levels of T, E2 and LH were significantly reduced, and FSH level of PCOS was significantly increased in each drug treatment group (PPPPPPConclusion: The PCOS rat model was successfully replicated. Total flavonoids of dodder may play a protective role in PCOS model rats by regulating the secretion of androgen, inhibiting the expression of ovarian apoptotic protein and impacting the hypothalamic-pituitary-ovary axis pathway.

Effect of Dodder Total Flavone on Polycystic Ovary Syndrome Rat Models Induced by Letrozole / 中国实验方剂学杂志

Objective: To investigate the effect of dodder total flavone on polycystic ovary syndrome (PCOS) rat models induced by letrozole. Method: Except the blank group, the other rats were given letrozole 1 mg·kg-1 for 21 consecutive days to replicate PCOS animal model. On the 16th day of the modeling, the estrous cycle was detected by vaginal smear, and rat with persistent keratinization of vaginal epithelial cells were selected as the PCOS model rat. The model rats were randomly divided into model group, Dacin-35 group, and high, middle, low-dose dodder total flavonoids groups. The corresponding drugs were given for 21 consecutive days. At the end of the administration, materials were collected to calculate ovary index, and enzyme-linked immunosorbent assay(ELISA) was used to measure estrogen (E2), testosterone (T), gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels of serum. The right ovary of rats was stained with haematoxylin-eosin (HE), and the pathological changes were observed by optical microscope. Androgen receptor(AR) expressions in hypothalamus, pituitary and ovary were detected by mmunohistochemistry. Result: Compared with the blank group, serum T, GnRH and LH levels, ovarian index and LH/FSH ratio were significantly increased, while FSH and E2 levels were significantly decreased (PPP2, T, GnRH and LH levels, ovarian index and LH/FSH ratio were significantly decreased in high, middle and low-dose dodder total flavonoids groups (P2 level was significantly increased (PP2 level in PCOS model rats was obviously increased in low-dose dodder total flavonoids group (PPConclusion: Dodder total flavonoids may play a protective role in PCOS model rats by regulating the secretion of estrogen and androgen and affecting the hypothalamic-pituitary-ovary axis pathway.

Effect of Total Flavone of Haw Leaves on Nuclear Factor Erythroid-2 Related Factor and Other Related Factors in Nonalcoholic Steatohepatitis Rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH.</p><p><b>METHODS</b>High-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot.</p><p><b>RESULTS</b>Lipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01).</p><p><b>CONCLUSIONS</b>Nrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.</p>

Determination of flavonoids constituents in epimedium total flavone capsule and research on its anti-osteoporosis activity / 中国中药杂志

To develop a rapid resolution liquid chromatography (RRLC) method for the simultaneous determination of epimedoside A, epimedin A1, epimedin A, epimedin B, epimedin C, icariin, baohuosideⅡ, icarisideⅠ, sagittatoside B, 2"--rhamnosyl icarisideⅡ, and baohuosideⅠin epimedium total flavone capsule. At the same time, the effects of the above 11 compounds on osteogenic differentiation of MC3T3-E1 cells were investigated by detecting the content of alkaline phosphatase (AKP). The results showed that baohuoside Ⅱ had the highest activities, and both the activities of baohuoside Ⅱ and icariside Ⅰ were stronger than those of icariin.In this study, the content determination method of flavonoid glycosides was established, and the anti-osteoporosis effect of monomers was compared, providing technical support for the study of the pharmacodynamic and mechanism of Epimedium total flavone capsule.

Study on the effects of total flavonoids from litchi nucleus on nuclear translocation of nuclear factor-kappa B and related protein expression in rat hepatic stellate cell / 中华肝脏病杂志

Objective@#The effect of total flavonoids of litchi (TFL) on nuclear translocation of nuclear factor-kappa B (NF- kappa B) in rat hepatic stellate cell line (HSC-T6) induced by transforming growth factor - beta 1 (TGF- beta 1) in vitro was studied to explore the mechanism of action of anti-hepatic fibrosis drugs.@*Methods@#HSC-T6 was cultured in vitro, induced by TGFβ1 for 24 h, and then treated with TFL at 125, 250 and 500 μg/ml for 48 h. The effect of TFL on NF-κB nuclear translocation in HSC-T6 was observed by confocal laser microscopy. The effects of TFL on the expression of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and Collagen I protein were detected by western blot. The expressions of TLR4 and p-NF-κB p65 were detected by immunofluorescence. Data were presented as mean±SEM. Homogeneity test of variance was performed and then followed by one-way analysis of variance (ANOVA). The multiple comparisons between groups were performed by LSD test. P < 0.05 was considered statistically significant.@*Results@#Confocal laser scanning microscopy showed TFL inhibited the nuclear translocation of NF-κB in activated HSC-T6 in a concentration-dependent manner and TFL down regulated the protein expression levels of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and collagen I protein in HSC-T6 in a concentration-dependent manner.@*Conclusion@#The mechanism of TFL against hepatic fibrosis may be related to the inhibition of nuclear translocation of NF-κb in the activated HSC-T6 and the expression of TLR4, P-iκbɑ, P-nf-κb p65, NF-κb and collagen I protein in HSC-T6.

Effect and mechanism of total flavone of epimedium on primary callus formation in ovariectomized rats with fractures / 中国骨伤

<p><b>OBJECTIVE</b>To explore the effects and related mechanisms of total flavone of epimedium treatment(TFE)on primary callus for mation in ovariectomized rats.</p><p><b>METHODS</b>Forty male SD rats weighted from 209 to 246 g and aged 6 to 8 weeks were selected. Six weeks after ovariectomy a femur fracture model with middiaphyseal segment fracture was established, estimated and randomly divided into TFE group (150 mg·kg⁻¹·d⁻¹) and control group(received saline). HE staining was used to evaluate the morphologic difference of primary callus during the bone callus healing between these two groups. The relative expression of Runt-related transcription factor 2(Runx2) mRNA in the callus was identified by real-time polymerase chain reaction. Immunohistochemical technique was used to observe the Casein kinase 2-interacting protein 1(CKIP-1) protein level in the callus of the two groups. Maximum fracture load was tested by three point bend test.</p><p><b>RESULTS</b>The BMD, primary callus volume, trabecular member(Tb.N) and trabecular thickness(Tb.Th) were higher in TFE group than that in control group(<0.001). The Tb.N and Tb.Th of primary callus were higher in TFE group than control group (=0.001). The volume and bone volume/tissue volume of primary callus were in TFE group than control group(<0.01). The trabecular separation(Tb.Sp) of primary callus were in control group higher than TFE group(<0.01). The HE staining of the 6 week slices showed that the degree of cartilage ossification in callus of the TFE group was significantly higher than that in control group under high magnification. Real-time PCR revealed that the comparative expression of Runx2 mRNA in control group was higher than that in TFE group(<0.001); the positive number of CKIP-1 was less in TFE group than that in control group (<0.001). TFE could increase the maximum load of the primary callus (<0.001).</p><p><b>CONCLUSIONS</b>TFE can promote the cartiage ossification of callus in ovariectomized rats, enhancing the bone strength and bone quality in the process of fracture healing via the CKIP-1/Runx2 pathway.</p>

Study on the Protective Effect Mechanism of Total Flavones from Prunus cerasifera Fruits for Alcoholic Liver Disease in Rats / 中国药房

OBJECTIVE:To study the protective effects of total flavone from Prunus cerasifera fruits(PCE)on alcoholic liver disease(ALD)in rats,and provide experimental basis for developing new medicines for anti-ALD. METHODS:40 rats were ran-domly divided into normal group(distilled water),model group(distilled water),silibinin group [positive control,30 mg/(kg·d)] and PCE high-dose,low-dose groups [80,40 mg/(kg·d)],8 in each group. All rats were intragastrically administrated(10 mL/kg) every morning,once,for 6 weeks;meanwhile,except for normal group,rats in other groups received 50% alcohol(10 mL/kg) once intragastrically every afternoon to induce ALD model. After administration,heart,liver,spleen and other organ indexes,sub-cutaneous fat,brown fat,abdominal fat indexes were determined,as well as serum biochemical indexes [glutamate transaminase (ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),direct bilirubin(DBIL),indirect bilirubin(IBIL),gamma-glu-tamyl transpeptidase(GGT),and total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL),low-density lipopro-tein (LDL)] level and liver biochemical indexes [superoxide dismutase (SOD),malondialdehyde (MDA),glutathione peroxidase (GSH-Px),TC,TG] and TC,TG levels in feces;pathological changes of liver and kidney tissues were observed. RESULTS:Compared with normal group,heart,liver,spleen indexes,subcutaneous fat,abdominal fat indexes in model group were in-creased(P<0.05 or P<0.01),brain index and brown fat index were decreased(P<0.05);HDL level and HDL/TC ratio in serum were decreased,other serum indexes were increased(P<0.05 or P<0.01);SOD,GSH-Px levels in liver tissue were decreased, other above-mentioned liver biochemical indexes were increased(P<0.01);TC,TG levels in feces were increased(P<0.01);liv-er and kidney showed obvious lesions. Compared with model group,the above-mentioned indexes in silibinin group and PCE high-dose group were significantly improved (P<0.05 or P<0.01);ALT,AST in serum and MDA level in liver tissue in PCE low-dose group were significantly decreased(P<0.01),and SOD,GSH-Px levels in liver tissue were significantly increased(P<0.05);lesion degree of liver and kidney and lipid accumulation in liver were reduced in administration groups. CONCLUSIONS:PCE may play a role in anti-ALD by anti-oxidation,promoting liver cell regeneration and regulating lipid metabolism.

Effects of total flavone of hawthorn leaf on expression of p38MAPK signaling pathway and inflam-mation factors in rats brain with chronic cerebral ischemia / 中华行为医学与脑科学杂志

Objective To investigate the therapeutic effect and mechanism of total flavone of haw-thorn leaf ( TFHL) on p38MAPK signaling pathway and inflammation factors in rats brain with chronic cere-bral ischemia.Methods SPF class healthy male SD rats were randomly divided into sham group, model group,TFHL group and Ginkgo leaf group( 12 rats in each group) .Permanent bilateral carotid artery ligation was used to prepare chronic cerebral ischemia model.Morris water maze method was used to evaluate learn-ing and memory abilities of rats.Immunohistochemistry and Western blot methods were used to measure the expression of caspase-3 and p38MAPK proteins.ELISA method was used to measure the amounts of TNF-αand IL-1βin hippocampal tissue.Results Compared with the model group,TFHL treatment (36 d) can im-prove learning and memory capabilities of vascular dementia rats,shorten the escape latency ( TFHL group(10.01±2.85) s vs Model group (19.54±6.12) s, P<0.05) and the course of searching platform(TFHL group(2.6044±0.3219)m vs model group(3.3502±0.6231)m, P<0.05),increase the numbers of crossing the platform (TFHL group(5.17±2.12) times vs Model group (3.96±1.34) time,s P<0.05) and the platform quadrant swimming distance percentage (TFHL group(48.22±7.39)%vs model group (33.42±5.32) %, P<0.01).The number of caspase-3 positive cells in the hippocampus significantly reduced (TFHL group(1.677 ±0.164) vs Model group (2.387±0.171), P<0.05),the expression level of P38MAPK protein (TFHL group (0.0161±0.0003) vs Model group (0.0254±0.0018), P<0.05),TNF-α(TFHL group(19.61±3.61) ng/10 mg vs Model group (27.82±6.57) ng/10 mg, P<0.01)and IL-1β(TFHL group(24.41±2.56) ng/10 mg vs Model group (29.43±5.26) ng/10 mg, P<0.05) were significantly decreased.Conclusion TFHL plays a protective role in nerve function of the chronic cerebral ischemia rats.The mechanism of its antia-poptosis might be associated with the activation of P 38MAPK signaling pathway,inflammation and the apoptosis of neurons in the brain.

Improving Effect of Total Flavone from Litchi chinensis on the Hepatocyte Injury in Liver Fibrosis Model Rats / 中国药房

OBJECTIVE:To study the improving effect of total flavone from Litchi chinensis (TFL) on the hepatocyte injury in rats with liver fibrosis. METHODS:The rats were given dimethylnitrosamine(DMN),ip,once a day in the first 3 d of every week,which lasted for 30 consecutive days to establish hepatocyte injury model. 60 rats were equally randomized into a normal control(isometric normal saline)group,a model(isometric normal saline)group and the groups of high and low-dose TFL(200 and 100 mg/kg). When the model was being established,drugs were administered,ig,once a day for 45 consecutive days except for normal control group. HE staining was performed,and then the rats’hepatocytes were observed under the microscope and path-ological stage (S1-S4) of liver tissue was analyzed. Masson staining and immunohistochemical staining were conducted,and then the rats’hepatocytes were observed under the microscope and calculation was made for the degree of liver fibrosis and the expres-sion of Bcl-2 and Bax. The activities of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in rats’serum were determined. RESULTS:The pathological stages of liver cell of rats in the model group were mainly stages S3 and S4 and the groups of high and low doses TFL were dominated by stages S1 and S2. Compared to the rats in the normal control group,those in the mod-el group had higher degree of liver fibrosis,expression of Bcl-2 and Bax and activities of AST and ALT in serum. Compared to the rats in the model group,those in the groups of high and low doses TFL had lower degree of liver fibrosis,higher expression of Bcl-2,lower expression of Bax,and lower activities of AST and ALT in serum. There were statistically significance (P<0.05). CONCLUSIONS:TFL can alleviate the hepatocyte injury in rats with liver fibrosis to some degree by a mechanism which may be related to the up-regulation the expression of Bcl-2 and the down-regulation of the expression of Bax.

Intervention Effects of Pollen Typhae Total Flavone on INS-1 PancreaticβCells Damage Induced by Palmitic Acid / 广州中医药大学学报

Objective To investigate the effects of Pollen Typhae total flavone (PTF) on INS-1 pancreaticβcell damage induced by palmitic acid ( PA) . Methods INS-1 pancreatic β cells were given long-term induction with PA to establish the impaired cell model, and then were intervened with PTF. Cell viability was determined by tetrazolium salt ( XTT) colorimetry. Results PA impaired the viabilities of INS-1 pancreatic β cells in concentration- and time-dependent manner, and PTF improved the impairment of INS-1 pancreatic β cells induced by PA in concentration -dependent manner. Moreover, PTF showed better improvement on the impairment when the INS-1 pancreatic β cells were impaired more seriously by PA. Conclusion PTF has effects on ameliorating the impairment of INS-1 pancreaticβcells induced by PA for long time.

Effects of the total flavone of litchi chinensis sonn on expressions of NF-κB andα-SMA in TGF-β1 activated rat hepatic stellate cells / 天津医药

Objective To investigate the effects of total flavonoids of litchi chinensis sonn (TFL) on cell proliferation and the molecular mechanism in rat hepatic stellate cells (HSC-T6) activated by growth factor-β1 (TGF-β1). Methods HSC-T6 cells were treated by 0.25%Trypsin-EDTA and then were digested into single cell suspension by DMEM (10%FBS included), which were mixed with TGF-β1 (5μg/L). (1) MTT method was used to detect the proliferation of HSC-T6 cells. Cells were cultured in 96-well plate and were treated by different concentrations of TFL including TGF-β1 group, the control group (5‰DMSO included), and different concentrations of TFL groups (80, 160, 320, 640 and 800 mg/L TFL). Each group has three wells. The absorbance (A) value was measured by enzyme standard meter at the 490 nm wavelength after 24 h, 48 h and 72 h treatment. The cell inhibitory rate was calculated. The subsequent experimental drug concentration and drug treatment time were determined according to half inhibitory concentration (IC50). (2) The expression levels of NF-κB andα-SMA mRNA were detected by PCR (for mRNA) and Western blot assay (for protein). Cells were cultured in the 10 cm culture dish and were divided into different TGF-β1 groups, including TGF-β1 group, the control group (5‰DMSO included), and different concentrations of TFL groups (125, 250 and 500 mg/L TFL). After 48 h, related indicators were measured. Results At the same treatment time point, with the increased concentrations of TFL, A values were gradually decreased, and the cell inhibitory rates were gradually increased. There were no significant differences in the expressions of NF-κB andα-SMA mRNA between TGF-β1 group and control group. And there were no significant differences in the expressions of NF-κB andα-SMA mRNA between TFL125 group, TGF-β1 group and control group. There was a gradually decrease in the expressions of NF-κB andα-SMA mRNA and protein with the increased concentrations of TFL. Conclusion TFL can inhibit TGF-β1-induced HSC-T6 cell proliferation, which is involved in the inhibited expressions of NF-κB andα-SMA to anti-fibrotic effects in liver fibrosis.

An Experimental Study of Total Flavone from Litchi Chinensis Sonn Improving Symptoms of Cholestatasis in BDL Rats / 天津医药

Objective To observe the effects of total flavone from litchi chinensis sonn (TFL) on the liver function in-cluding p16 protein, pro collagen type 3 (PC3) and pro collagen typeⅠ(PCⅠ) in model rats with liver fibrosis induced by bile duct ligation. Methods Forty rats were randomly divided into four groups:sham operation (SO) group, bile duct liga-tion (BDL) group, TFL group and silibinin (SIL) group. Rats were gavaged with saline (5 mL·kg-1·d-1) in SO and BDL group, rats were gavaged with TFL (200 mL·kg-1·d-1) in TFL group and rats were gavaged with SIL (5 mL·kg-1·d-1) in SIL group for four weeks. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin direct (BILD) and bilirubin total (BILT) were detected in four groups. The liver tissues were stained by HE and Masson methods. The ex-pression levels of p16, PC3 and PCⅠin liver tissues were determined by Western blot assay. Results The serum levels of ALT (44.6 IU/L±8.0 IU/L), AST (103.8 IU/L±18.1 IU/L), BILD (0.76 μmol/L±0.28μmol/L) and BILT (1.48μmol/L±0.35μmol/L) were lower in SO group. There was a higher level of ALT in BDL group (147.4 IU/L±86.3 IU/L) than that of TFL group (92.9 IU/L±47.3 IU/L). The serum level of ALT was higher in AST group (362.7 IU/L±106.6 IU/L) than that of TFL group (290.1 IU/L ± 171.7 IU/L) and SIL group (250.2 IU/L ± 54.9 IU/L). The serum level of BILD was lower in BDL group (99.71μmol/L±40.87μmol/L) than that of SIL group (137.01μmol/L±38.86μmol/L). The serum levels of BILD and BILT were significantly lower in TFL group (81.48μmol/L±47.50μmol/L, 106.64μmol/L±61.04μmol/L) than those of SIL group (P<0.05). There were small amount of new bile duct and no obvious cells degeneration, small amount of infiltration of in-flammatory cells and collagen deposition in TFL group. The liver fibrosis improved significantly in TFL group than that of BDL group. There were more new bile duct in hepatic portal area in SIL group than those of TFL group. The expression levels of p16, PC3 and PCⅠwere significantly higher in BDL group than those of TFL group. The expression level of PC3 was significantly lower in BDL group than that of SIL group. The expression level of PCⅠwas significantly higher in BDL group than that of SIL group (P<0.05). There was no significant difference in the expression level of p16 between BDL group and SIL group. The expression levels of PC16 and PC3 were significantly lower in TFL group than those of SIL group (P<0.05). There was no significant difference in the ex-pression level of PCⅠbetween TFL group and SIL group. Conclusion TFL can improve the liver function in model rats with choles-tatic liver fibrosis and reduce liver fibrosis, which may be related with inhibitory effects on the expressions of p 16, PC3 and PCⅠ.

Effects of Pollen Typhae Total Flavone on Inflammatory Factors and Insulin Sensitivity in Type 2 Diabetic Rats / 广州中医药大学学报

Objective To investigate the effects of Pollen Typhae total flavone ( PTF) , an extract from Pollen Typhae which has the actions of activating blood and removing stasis, on inflammatory factors and insulin sensitivity in type 2 diabetic rats. Methods SD rats were used as the experimental animal. Type 2 diabetic rats induced by high fat diet plus low dose of streptozotocin were randomly divided into model group, PTF group (in the dosage of 200 mg·kg-1·d-1) , and rosiglitazone group (in the dosage of 4 mg·kg-1·d-1) . Additionally, normal control group was set up. After treatment for 4 weeks, plasma interleukin 6 ( IL-6) and tumor necrosis factor alpha ( TNF-α) levels were detected, the insulin tolerance test ( ITT) was performed, and the protein expression of suppressor of cytokine signaling-3 ( SOCS-3) in skeletal muscle was determined. Results After treatment for 4 weeks, the plasma levels of IL-6 and TNF-α, the homeostasis model of insulin resistance ( HOMA-IR) , and expression level of SOCS-3 in skeletal muscle in the model groups were significantly increased ( P﹤0.05) as compared with those in the normal control group, and insulin tolerance was also impaired in the model group ( P﹤0.05) . Compared with the model group, IL-6 level and HOMA-IR were markedly decreased in the PTF group ( P﹤0.05) , and the impaired insulin tolerance was obviously improved (P﹤0.05) . The level of SOCS-3 in the skeletal muscle of PTF group was also much lower than that of the model group and rosiglitazone group (P﹤0.05) . Conclusion PTF has effects on decreasing the levels of plasma IL-6 and SOCS-3 in the skeletal muscle and on improving insulin sensitivity in type 2 diabetic rats.