OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; ErbB Receptors/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; RNA, Messenger/genetics* ; Cell Movement ; Cell Line, Tumor ; Chalcone/analogs & derivatives* ; Quinones

OBJECTIVE: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25-160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 β, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. RESULTS: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 β, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). CONCLUSIONS The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Chalcone/analogs & derivatives* ; Quinones/pharmacology* ; Pyroptosis/drug effects* ; Caspase 1/metabolism* ; Glucose ; Phosphate-Binding Proteins/metabolism* ; Signal Transduction/drug effects* ; Intracellular Signaling Peptides and Proteins/metabolism* ; Oxygen/metabolism* ; Cytokines/metabolism* ; Gasdermins

This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Chalcone ; analogs & derivatives ; pharmacology ; Creatine Kinase ; blood ; Interleukin-6 ; metabolism ; L-Lactate Dehydrogenase ; blood ; Male ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Wistar ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

This paper was aimed to establish screening methods of anaphylactoid reaction caused by safflower yellow for injection based on RBL-2 H3 cell degranulation model and mice model for acute anaphylactoid reaction,and evaluate the hypersensitivity caused by safflower yellow for injection from different batches. An in vitro cell model was used to keep the cells stimulated for an hour with different batches of safflower yellow for injection as the drug group,serum-free MEM medium as negative control group and 30 mg·L-1 C48/80 as positive control group respectively. The supernatant was then absorbed,and neutral red staining technique was used to detect the effect of safflower yellow injection on the degranulation of RBL-2 H3 cells with the positive cell rate of degranulation as the indicator.An in vivo model was established to validate the experimental results,and mice model for acute anaphylactoid reaction and ELISA method were adopted to detect the plasma histamine content,and screen the hypersensitivity caused by safflower yellow for injection at the animal level by using plasma histamine content as a test index. The results of the neutral red staining experiments showed that the positive control C48/80 could cause cell degranulation,and most of the cells were deeply stained. There was significant difference in positive cell rate between different batches of safflower yellow and positive control group. In the mice model for acute anaphylactoid reaction,it was found that the positive control C48/80 significantly increased the histamine content in the plasma of mice,while the safflower yellow in each batch did not cause a significant increase in plasma histamine( P<0. 000 1). The mechanism of anaphylactoid reaction is relatively complicated. This study was mainly based on the release of histamine and other active substances by degranulation of mast cells. No significant degranulation reaction of RBL-2 H3 cells induced by safflower yellow for injection was detected,nor was the plasma histamine level significantly increased in mice from the in vitro and in vivo aspects.
Anaphylaxis ; chemically induced ; Animals ; Cell Degranulation ; drug effects ; Cells, Cultured ; Chalcone ; adverse effects ; analogs & derivatives ; Histamine ; blood ; Mast Cells ; drug effects ; Mice

OBJECTIVEThis study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.

METHODSEahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.

RESULTSHSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).

CONCLUSIONHSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; therapeutic use ; E-Selectin ; genetics ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inflammation ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Leukocytes ; cytology ; drug effects ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Protein Binding ; drug effects ; Quinones ; chemistry ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

The effect of amygdalin joint hydroxysafflor yellow A (HSYA) on the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and the possible mechanism were studied and explored. Chondrocytes were obtained from endplate of one-month SD rat intervertebral discs and cultured primary endplate chondrocytes. After identification, they were divided into normal group, induced group, amygdalin group, HSYA group and combined group. CCK-8 kit was adopted to detect the proliferation of the endplate chondrocytes. FCM was measured to detect the apoptosis. Real-time PCR method was adopted to observe the mRNA expression of Aggrecan, Col 2 alpha1, Col 10 alpha1, MMP-13 and the inflammatory cytokines IL-1beta. The protein expression of Col II, Col X was tested through immunofluorescence. Compared with the normal group, the proliferation of the endplate chondrocytes decreased while the apoptosis increased (P < 0.05). With down regulation of the mRNA expressions of Aggrecan, Col 2 alpha1 and up regulation of the mRNA expressions of Col 10 alpha1, MMP-13, IL-1beta (P < 0.05), the protein expression of Col II decreased while the protein expression of Col X increased. Compared with the induced group, amygdalin group, HSYA group, the combined group could inhibit the apoptosis and promote the proliferation (P < 0.05). They could increase the mRNA expressions of Aggrecan and Col 2 alpha1 while decrease the mRNA expressions of Col 10 alpha1, MMP-13 and IL-1beta (P < 0.05). They could also enhance the protein expression of Col II while reduce the protein expression of Col X. The effect of the combined group was significantly better than that of amygdalin and HSYA. Amygdalin joint HSYA could inhibit the degeneration of the endplate chondrocytes derived from intervertebral discs of rats induced by IL-1beta and better than the single use of amygdalin or HSYA.
Amygdalin ; pharmacology ; Animals ; Apoptosis ; Cells, Cultured ; Chalcone ; analogs & derivatives ; pharmacology ; Chondrocytes ; drug effects ; Collagen ; metabolism ; Drug Synergism ; Interleukin-1beta ; Intervertebral Disc ; cytology ; Quinones ; pharmacology ; Rats

To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.
Achyranthes ; chemistry ; Animals ; Chalcone ; administration & dosage ; analogs & derivatives ; blood ; pharmacokinetics ; Chlorogenic Acid ; administration & dosage ; blood ; pharmacokinetics ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Glucosides ; administration & dosage ; blood ; pharmacokinetics ; Glycosides ; administration & dosage ; blood ; pharmacokinetics ; Glycyrrhizic Acid ; administration & dosage ; pharmacokinetics ; Herb-Drug Interactions ; Male ; Pyrans ; administration & dosage ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

OBJECTIVETo observe the effect of Hydroxy Safflower Yellow A (HSYA) on the expression of osteogenic markers, such as alkaline phosphatase, Cbf(alpha)l and type I collagen, and explore the mechanism of HSYA in the prevention and treatment of glucocorticoid-induced ischemic necrosis of femoral head.

METHODSFifteen healthy and adult New Zealand white rabbits were collected and weighted 0.9 to 1.3 kg. The rabbits were injected abdominally with anesthetic drugs, then received marrow cavity puncture of tibia and anterior superior iliac spine to get bone marrow blood. Rabbits bone marrow mesenchymal stem cells (BMSCs) were separated from the bone marrow blood, cultured in vitro and passaged. The 3rd generation of BMSCs which had good growth condition were randomly divided into blank group, model group and HSYA groups with different doses. The BMSCs in model group were treated with high dose of dexamethasone to induce adipogenic differentiation of cells cultured in vitro, and inhibit osteogenic differentiation. The BMSCs in HSYA groups received high dose of dexamethasone and different concentrations of HSYA simultaneously. The blank group received not any special handling. After a week,the expressions of alkaline phosphatase, Cbf(alpha)l and type I collagen mRNA were detected.

RESULTSThe alkaline phosphatase activity was significantly decreased in BMSCs of the model group as compared with the blank group (P < 0.01), and the expression of Cbf(alpha)l and type I collagen mRNA were also decreased significantly (P<0.01). The alkaline phosphatase activity was significantly increased in BMSCs of each HSYA group as compared with the model group (P < 0.05 or P < 0.01), and the expression of Cbf(alpha)l and type I collagen mRNA were also increased significantly (P < 0.05 or P < 0.01).

CONCLUSIONThe mechanism of HSYA may be related to the effect of antagonism to the reduced osteogenic differentiation induced by glucocorticoid.

Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; Collagen Type I ; genetics ; metabolism ; Core Binding Factor alpha Subunits ; genetics ; metabolism ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Glucocorticoids ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteogenesis ; drug effects ; Rabbits

Bio-active components from Carthamus tinctorius were separated on the basis of antioxidant capacities in vitro. The antioxidant capacity was investigated on the basis of the ability to scavenge DPPH radical, ABTS radical and reduce Fe3+ of different polar fractions. Furthermore, the chemical compounds were isolated from bio-active fraction, and were evaluated for the antioxidative effects. Five major components were isolated and identified from water extract as 6-hydroxykaempferol 3,6,7-tri-O-β-D-glucoside(1), 6-hydroxykaempferol 3-O-β-rutinoside-6-O-β-D-glucoside (2), 6-hydroxykaempferol 3-O-β-D-glucoside (3), hydroxysafflor yellow A (4) and anhydrosafflor yellow B (5). By evaluating and comparing the antioxidative effects of different fractions and obtained compounds, the results showed that water extract displayed significantly high antioxidative activities and 6-hydroxykaempferol glycosides and quinochalcone C-glycosides were found as main contribution for antioxidant property.
Antioxidants ; isolation & purification ; metabolism ; pharmacology ; Benzothiazoles ; metabolism ; Biphenyl Compounds ; metabolism ; Carthamus tinctorius ; chemistry ; Chalcone ; analogs & derivatives ; isolation & purification ; metabolism ; pharmacology ; Ferric Compounds ; metabolism ; Free Radicals ; metabolism ; Kaempferols ; isolation & purification ; metabolism ; pharmacology ; Oxidation-Reduction ; drug effects ; Picrates ; metabolism ; Plant Extracts ; isolation & purification ; metabolism ; pharmacology ; Plants, Medicinal ; chemistry ; Quinones ; isolation & purification ; metabolism ; pharmacology ; Sulfonic Acids ; metabolism ; Water ; chemistry

OBJECTIVETo develop an HPLC method to determine the contents of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in the water extract of mixed Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos simultaneously.

METHODThe separation were carried out at 30 degrees C on a ZORBAX Eclipse Plus C18 column (4.6 mm x 100 mm, 1.8 microm) with formic acid-500 mmol x L(-1) ammonium formate-water solution (0.5:10:90) as mobile phase A and acetonitrile-formic acid solution (100: 0.5) as mobile phase B in gradient mode at a flow rate of 0.5 mL x min(-1). Detection wavelengths were 280 nm for danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, and 380 nm for hydroxysafflor yellow A.

RESULTThe 5 components were separated well with a good linearity (R2 > 0.999 3) in the range of the test concentration. The average recoveries of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, and salvianolic acid B were 99.1%, 102%, 102%, 98.5% and 101%, respectively.

CONCLUSIONThis method is simple, accurate, and repeatable.

Benzofurans ; analysis ; Chalcone ; analogs & derivatives ; analysis ; Chromatography, High Pressure Liquid ; methods ; Cinnamates ; analysis ; Depsides ; analysis ; Lactates ; analysis ; Quinones ; analysis ; Rhizome ; chemistry ; Salvia miltiorrhiza ; chemistry