Hydroxysafflor yellow A induced ferroptosis of Osteosarcoma cancer cells by HIF-1α/HK2 and SLC7A11 pathway

Osteosarcoma is a very serious primary bone cancer with a high death rate and a dismal prognosis.Since there is no permanent therapy for this condition,it is necessary to develop a cure.Therefore,this investigation was carried out to assess the impacts and biological functions of hydroxysafflor yellow A(HYSA)in osteosarcoma cell lines(MG63).In this investigational study,MG63 cells were utilized.Microarray experiments,quantitative polymerase chain reaction(qPCR),immunofluorescent staining,extracellular acidification rate(ECAR),oxygen consumption rate(OCR),glucose consumption,lactate production,and ATP levels,proliferation assay,5-Ethynyl-2′-deoxyuridine(EDU)staining,and Western blot were performed.In MG63 cells,HYSA lowered cell proliferation and metastasis rates,suppressed EDU cell number,and enhanced caspase-3/9 activity levels.HYSA reduced the Warburg effect and induced ferroptosis(FPT)in MG63 cells.Inhibiting ferroptosis diminished HYSA’s anti-cancer activities in MG63 cells.The stimulation of the HIF-1α/SLC7A11 pathway decreased HYSA’s anti-cancer activities in MG63 cells.HIF-1αis one target spot for HYSA in a model of osteosarcoma cancer(OC).HYSA altered HIF-1α’s thermophoretic activity;following binding with HYSA,HIF-1α’s melting point increased from~55°C to~60°C.HYSA significantly enhanced the thermal stability of exogenous WT HIF-1αwhile not affecting Mut HIF-1α,suggesting that ARG-311,GLY-312,GLN-347,and GLN-387 may be involved in the interaction between HIF-1αand HYSA.Conclusively,our study revealed that HYSA induced FPT and reduced the Warburg effect of OC through mitochondrial damage by HIF-1α/HK2/SLC7A11 pathway.HYSA is a possible therapeutic option for OC or other cancers.

Hydroxysafflor yellow A protects against thioacetamide-induced liver fibrosis in rats via suppressing proinflammatory/fibrogenic mediators and promoting hepatic stellate cell senescence and apoptosis

Objective:To evaluate the effect of hydroxysafflor yellow A(HSYA)on thioacetamide-induced liver fibrosis.Methods:Thioacetamide was administered to rats intraperitoneally in doses of 200 mg/kg twice a week for 12 weeks.Thioacetamide-intoxicated rats were given silymarin(50 mg/kg)or HSYA(5 mg/kg)orally every day for 8 weeks.Liver enzymes,fibrosis markers,histological changes as well as immunohistochemistry of TNF-α,IL-6,p21,α-SMA,and caspase-3 were examined.The effect of HSYA on HSC-T6 activation/proliferation and apoptosis was also determined in vitro.Results:HSYA decreased liver enzymes,TNF-α,IL-6,and p21 expressions,hepatic PDGF-B,TIMP-1,TGF-β1,and hydroxyproline levels,as well as fibrosis score(S2 vs.S4)compared to the thioacetamide group.HSYA also downregulatedα-SMA while increasing caspase-3 expression.Surprisingly,at 500μg/mL,HSYA had only a slightly suppressive effect on HSC proliferation,with a 9.5%reduction.However,it significantly reduced TGF-β1,inhibitedα-SMA expression,induced caspase-3 expression,and promoted cell senescence.Conclusions:HSYA may be a potential therapeutic agent for delaying and reversing the progression of liver fibrosis.More research on HSYA at higher doses and for a longer period is warranted.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443

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<0.05or 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/m TOR 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_0084443on HBEGF,EGFR,PI3K,AKT,m TOR 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.

Hydroxysafflor Yellow A Inhibits Pyroptosis and Protecting HUVECs from OGD/R via NLRP3/Caspase-1/GSDMD Pathway

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(1.25-160μmol/L)of HSYA on HUVECs after OGD/R.HSYA 80μmol/L was used for follow-up experiments.The contents of inflammatory cytokines interleukin(IL)-18,IL-1β,monocyte chemotactic protein 1(MCP-1),tumor necrosis factorα(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-1β,MCP-1,TNF-αandIL-6(P<0.01or 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.

Protective effect of hydroxysafflor yellow A on MSCs against senescence induced by D-galactose

Objective: To examine the protective effects of hydroxysafflor yellow A(HSYA) against the senescence of mesenchymal stem cells(MSCs) induced by D-galactose(D-gal) in vitro, and investigate the potential mechanism involved.Methods: Grouping experiment, Normal control(NC) group: conventional culture with complete medium;Senescence group: MSCs were cultured for 48 h with complete medium containing 10 g/L D-gal;HSYA group: on the basis of senescence induction, HSYA with the suitable concentration was used to protect MSCs. The key experimental indices associated with oxidative stress, inflammatory response, cell senescence, proliferation and apoptosis were measured through chemical colorimetry, β-galactosidase staining, Ed U incorporation and flow cytometry, respectively. The relative quantity(RQ) of proteins related closely to cell proliferation, apoptosis, and NF-κB signaling were measured by Western blotting.Results: As compared with Senescence group, treatment with HSYA(120 mg/L) effectively ameliorated the adverse situation of MSCs. Oxidation stress and inflammation along with D-Gal induction was dramatically alleviated in MSCs;The β-Gal-positive staining indicated that MSC senescence was significantly mitigated;The proliferative capability of MSCs was significantly increased by up-regulating PCNA and inhibiting p16 expression;The anti-apoptotic effect on MSCs was exerted by down-regulating the RQ of cleaved Caspase-3 and Bax;The activity of NF-κB signaling in MSCs was notably suppressed through inhibiting phosphorylation of IKKβ and p65.Conclusion: HSYA(120 mg/L) significantly delayed the D-Gal-induced senescence process in MSCs through attenuating inflammatory reaction and oxidative stress, and suppressing the activity of NF-κB signaling.

Hydroxysafflor yellow A improves learning and memory in a rat model of vascular dementia by increasing VEGF and NR1 in the hippocampus

Hydroxysafflor yellow A （HSYA） has angiogenesis- regulating and neuro-protective effects, but its effects on vascular dementia （VaD） are unknown. In this study, 30 adult Sprague-Dawley rats were. randomly allocated to five groups： normal, sham-operation, VaD alone （bilateral carotid artery occlusion）, VaD plus saline （control）, and VaD plus HSYA. One week after operation, the HSYA group received one daily tail-vein injection of 0.6 mg/100 g HSYA for two weeks. Five weeks after operation, the spatial memory of all five groups was evaluated by the water maze task, and synaptic plasticity in the hippocampus was assessed by the long-term potentiation （LTP） method. Vascular endothelial growth factor （VEGF） and N-methyi-D- aspartic acid receptor 1 （NR1） expression in the hippocampus was detected via Western blot. We found that, compared with the group with VaD alone, the group with HSYA had a reduced escape latency in the water maze （P 〈0.05）, and the LTP at CA3- CA1 synapses in the hippocampus was enhanced （P 〈0.05）. Western blot in the late-phase VaD group showed slight up-regulation of VEGF and down- regulation of NR1 in the hippocampus, while HSYA significantly up-regulated both VEGF and NRI. These results suggested that HSYA promotes angiogenesis and increases synaptic plasticity, thus improving spatial learning and memory in the rat model of VaD.

Protective Effect of Hydroxysafflor Yellow A on Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Rats

Objective： To observe the effect of hydroxysafflor yellow A （HSYA）, an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflammation and pulmonary fibrosis induced by bleomycin （BLM） in rats. Methods： Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone （DXM） group. Three doses of HSYA （35.6, 53.3, and 80.0 mg？kg–1？day–1） were intraperitoneally （i.p.） injected in rats for 3 weeks after BLM administration and DXM was used as the positive control （n=8 or 12）. Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor （TNF）-α, interleukin （IL）-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin （α-SMA） protein distribution in rat lung tissue was observed by immunohistochemistry. Results： On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure （PaO2） increased （HSYA 80.0 mg？kg–1, P〈0.01） and CO2 partial pressure （PaCO2） decreased （HSYA 53.3, 80.0 mg？kg–1, P〈0.05）. Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg？kg–1 groups than those in the model group （all P〈0.05）. Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 （HSYA 53.3, 80.0 mg？kg–1, P〈0.01）, and decreased PaCO2 （53.3 and 80.0 mg？kg–1, P〈0.05）. Further, the mRNA expression of TGF-β1, α-SMA, and collagen Ⅰ as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes （53.3, 80.0 mg？kg–1, P〈0.05）. Hematoxylin and eosin and Masson＇s trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups （53.3, 80.0 mg？kg–1, P〈0.05）. Conclusion： HSYA could alleviate acute lung inflammation and chronic pulmonary fibrosis induced by BLM in rats.

Effect and Safety of Hydroxysafflor Yellow A for Injection in Patients with Acute Ischemic Stroke of Blood Stasis Syndrome: A Phase Ⅱ , Multicenter, Randomized, Double-Blind, Multiple-Dose, Active-Controlled Clinical Trial

Objective:To assess the effect and safety of Hydroxysafflor Yellow A for Injection(HSYAI)in treating patients with acute ischemic stroke(AIS)and blood stasis syndrome(BSS).Methods:A multicenter,randomized,double-blind,multiple-dose,active-controlled phaseⅡtrial was conducted at 9 centers in China from July 2013 to September 2015.Patients with moderate or severe AIS and BSS were randomly assigned to low-,medium-,high-dose HSYAI groups(25,50 and 70 mg/d HSYAI by intravenous infusion,respectively),and a control group(Dengzhan Xixin Injection(灯盏细辛注射液,DZXXI)30 mL/d by intravenous infusion),for 14 consecutive days.The primary outcome was the Modified Rankin Scale(mRS)score 1 at days 90 after treatment.The secondary outcomes included the National Institute of Health Stroke Scale(NIHSS)score 1,Barthel Index(BI)score 95,and BSS score reduced 30%from baseline at days 14,30,60,and 90 after treatment.The safety outcomes included any adverse events during 90 days after treatment.Results:Of the 266 patients included in the effectiveness analysis,66,67,65 and 68 cases were in the low-,medium-,and high-dose HSYAI and control groups,respectively.The proportions of patients in the medium-and high-dose HSYAI groups with mRS score 1 at days 90 after treatment were significantly larger than the control group(P<0.05).The incidences of favorable outcomes of NIHSS and BI at days 90 after treatment as well as satisfactory improvement of BSS at days 30 and 60 after treatment in the medium-and high-dose HSYAI groups were all significantly higher than the control group(P<0.05).No significant difference was reported among the 4 groups in any specific adverse events(P>0.05).Conclusions:HSYAI was safe and well-tolerated at all doses for treating AIS patients with BSS.The medium(50 mg/d)or high dose(75 mg/d)might be the optimal dose for a phaseⅢtrial.

Hydroxysafflor Yellow A Attenuate LipopolysaccharideInduced Endothelium Inflammatory Injury

Objective： This 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. Methods： Eahy926 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- κ B） p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase （MAPKs） and NF- κ B 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. Results： HSYA protected EC viability against LPS-induced injury （P〈0.05）. LPS-induced NF- κ B p65 subunit DNA binding （P〈0.01） and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α （I κ B α） 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）. Conclusion： HSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Protective effect of hydroxysafflor yellow A on Parkinson cell model and its network pharmacology analysis

As an effective component of safflower,hydroxysafflor yellow A(HSYA)has the effect of promoting blood circulation,removing blood stasis,and relieving pain,and it has a certain effect on blood stasis and wind-induced Parkinson‘s disease(PD).However,the current research mostly involves a single intervention mechanism,which is not conducive to the clinical transformation of this type of drugs.In the present study,rotenone was used to construct a PD cell model,and the protective effect of HSYA on the PD cell model was evaluated by cell viability and mitochondrial membrane potential.TTD database was used to query PD-related therapeutic targets,Swiss Target Prediction database to query HSYA-related targets,STRING database was used to search the gene interaction relationship of common targets,and ClueGO pathway was adopted to enrich and analyze common targets and their interaction targets,as well as to explore the comprehensive intervention mechanism.The results showed that rotenone could successfully establish the PD cell model,and HSYA had significant protective effect on PD cell model.Through the network pharmacological analysis,36 PD-related therapeutic targets and 88 HSYA-related targets were queried.The common targets of PD and HSYA were FKBP1A,HTR1A,SLC6A4 and SLC6A3.To enrich four common targets and their interaction targets through REACTOME pathway,eight cell signal pathways were obtained,and six cell biological processes were obtained through biological process pathway enrichment.

Protective Effect of Hydroxy safflor Yellow A against Chronic Mild Stress-induced Memory Impairments by Suppressing Tau Phosphorylation in Mice

Chronic stress plays a critical role in the etiology of sporadic Alzheimer's disease(AD).However,there are currently no effective drugs that can target chronic stress to prevent AD.In this study,we explored the neuroprotective effect of hydroxysafflor yellow A(HSYA)against chronic mild stress(CMS)-induced memory impairments in mice and the underlying mechanism.The Morris water maze test showed that HSYA significantly reduced CMS-induced learning and memory impairments in mice.HSYA increased the expression of brain-derived neurotrophic factor(BDNF)and activated downstream tropomyosin-related kinase B(TrkB)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling.HSYA decreased the expression of regulator of calcineurin 1-1L(RCAN1-1L)that could promote the activity of glycogen synthase kinase-3β(GSK-3β).HSYA also attenuated tau phosphorylation by inhibiting the activity of GSK-3βand cyclin-dependent kinase-5(Cdk5).Our data indicated that HSYA has protective effects against CMS-induced BDNF downregulation,tau phosphorylation and memory impairments.HSYA may be a promising therapeutic candidate for AD by targeting chronic stress.

The safflower MBW complex regulates HYSA accumulation through degradation by the E3 ligase CtBB1

The regulatory mechanism of the MBW(MYB-bHLH-WD40) complex in safflower(Carthamus tinctorius) remains unclear. In the present study,we show that the separate overexpression of the genes CtbHLH41, CtMYB63, and CtWD40-6 in Arabidopsis thaliana increased anthocyanin and procyanidin contents in the transgenic plants and partially rescued the trichome reduction phenotype of the corresponding bhlh41, myb63,and wd40-6 single mutants. Overexpression of CtbHLH41, CtMYB63, or CtWD40-6 in safflower significantly increased the content of the natural pigment hydroxysafflor yellow A(HYSA)and negatively regulated safflower petal size.Yeast-two-hybrid, functional, and genetic assays demonstrated that the safflower E3 ligase CtBB1(BIG BROTHER 1) can ubiquitinate CtbHLH41,marking it for degradation through the 26S proteasome and negatively regulating flavonoid accumulation. CtMYB63/CtWD40-6 enhanced the transcriptional activity of CtbHLH41 on the CtDFR(dihydroflavonol 4-reductase) promoter.We propose that the MBW-CtBB1 regulatory module may play an important role in coordinating HYSA accumulation with other response mechanisms.

Effects of Danhong Injection(丹红注射液) and Its Main Components on Anticoagulation and Fibrinolysis in Cultured Vein Endothelial Cells

Objective： To observe the effects of Danhong Injection （丹红注射液） and its main components, including daiclzein and hydroxysafflor yellow A （HSYA）, on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells （VECs）. Methods： VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N2 and 5% CO2 mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α （6-keto-PGF1 α） level was measured by the method of radioimmunoassay （RIA）. Superoxide dismutase （SOD） activity was tested by water soluble tetrazolium salt. The content of malondialdehyde （MDA） was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator （t-PA） and plasminogen activator inhibitor （PAl） were measured by the method of chromogenic substrate. The contents of endothelin （ET） and nitric oxide （NO） were detected by non-equilibrium RIA and enzyme- linked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. Results： Compared with the normal control group, the floating cells number, PAl activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1 α and NO contents, and t-PA and SOD activities were decreased significantly （P〈0.01）. Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1 α content and SOD activity, regulated PAl and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate （P〈0.05 or P〈0.01）. Conclusions： Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.