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-1α is 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-1α while not affecting Mut HIF-1α, suggesting that ARG-311, GLY-312, GLN-347, and GLN-387 may be involved in the interaction between HIF-1α and 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.

Safflower yellow for injection enhances anti-coagulation of warfarin in rats: implications in pharmacodynamics and pharmacokinetics.

This study investigated whether Safflower Yellow for injection (SYI) would affect the anticoagulation of warfarin in rats.Wistar male rats were divided into six groups randomly and administered with SYI (9 mg/kg, intraperitoneal injection) in single-dose and steady-dose warfarin (0.2 mg/kg, oral gavage), respectively. The pharmacodynamic parameters of PT and APTT were measured by a coagulation analyser. R/S-warfarin concentration was measured by UHPLC-MS/MS, and pharmacokinetic parameters calculated using DAS 2.0 software.The single-dose study demonstrated that SYI, alone or co-administered with warfarin, could significantly increase PT, INR, and APTT values (p < 0.01). R-warfarin Cmax, AUC, and t1/2 values increased by 9.25% (p > 0.05), 25.96% (p < 0.01), and 26.17% (p < 0.01), respectively, whereas the CL/F value reduced by 22.22% (p < 0.01) in the presence of SYI. Meanwhile, S-warfarin Cmax, AUC, and t1/2 values increased by 37.41%, 32.11%, and 31.73% (all p < 0.01), respectively, whereas the CL/F value reduced by 33.33% (p < 0.01). The steady-dose study showed that PT, INR, APTT, and the concentrations of R/S-warfarin increased significantly when SYI was co-administered with warfarin (p < 0.01).SYI can enhance warfarin's anticoagulation intensity and decelerate its metabolism in rats.

Insights into Q-markers of honey-fried licorice in treating spleen deficiency based on substance and energy metabolism regulation.

BACKGROUND: Honey-fried Licorice (HFL) is a dosage form of Glycyrrhizae Radix et Rhizome processed with honey, which has been recorded to exhibit better efficacy in tonifying the spleen compared to the raw product. In contrast, different processing methods of Glycyrrhizae Radix et Rhizome exhibit different efficacies and applications, but their current quality control index components remain consistent. PURPOSE: Based on the discovery and research strategy of traditional Chinese medicine decoction piece quality marker (Q-marker), this study aimed to conduct a multidimensional integration of constituents absorbed into the body and metabolomics based on the tonifying spleen and stomach effects of HFL to effectively identify the Q-marker of HFL. METHODS: In this study, a spleen deficiency rat model was established using the "exhausted swimming + poor diet" method to investigate the pharmacodynamics of tonifying the spleen and stomach by HFL. The constituents absorbed into blood was conducted using UPLC-Q-TOF/MS, correlation analysis between metabolomics and constituents absorbed into blood recognized the Q-Marker of HFL. RESULTS: The pharmacodynamic data demonstrated that HFL exhibited a significant regulatory effect on the disordered levels of PP, trypsin, chymase, PL, α-Glu, MTL, GAS, VIP, IL-2, IFN-γ, and IgA in the spleen deficiency model. Furthermore, HFL was found to improve the pathological changes in the spleen and intestine in the spleen deficiency model, highlighting its significant "tonifying spleen and stomach" effect. In the serum containing HFL, a total of 17 constituents were identified as being absorbed into the blood. Among these, 11 were prototypical components, while 6 were metabolites. Metabolomics data revealed that 9 differentially expressed metabolic markers were observed. Furthermore, the analysis of endogenous metabolic markers indicated that 10 components exhibited significant correlations with these biomarkers. CONCLUSION: The effect of "tonifying spleen and stomach" of HFL is closely related to the regulation of the material and energy metabolism pathway. The Q-Marker of HFL is glycyrrhizic acid and 18ß-glycyrrhetinic acid as the main control standards and liquiritin, isoliquiritin, liquiritin, isoliquiritin, isolicorice flavonol, licorice chalcone C and Formononetin were used as auxiliary standards.

Hydroxy-Safflower Yellow A Mitigates Vascular Remodeling in Rat Pulmonary Arterial Hypertension.

Purpose: The underlying causes of pulmonary arterial hypertension (PAH) often remain obscure. Addressing PAH with effective treatments presents a formidable challenge. Studies have shown that Hydroxysafflor yellow A (HSYA) has a potential role in PAH, While the mechanism underlies its protective role is still unclear. The study was conducted to investigate the potential mechanisms of the protective effects of HSYA. Methods: Using databases such as PharmMapper and GeneCards, we identified active components of HSYA and associated PAH targets, pinpointed intersecting genes, and constructed a protein-protein interaction (PPI) network. Core targets were singled out using Cytoscape for the development of a model illustrating drug-component-target-disease interactions. Intersection targets underwent analysis for Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Selected components were then modeled for target interaction using Autodock and Pymol. In vivo validation in a monocrotaline-induced PAH (MCT-PAH) animal model was utilized to substantiate the predictions made by network pharmacology. Results: We associated HSYA with 113 targets, and PAH with 1737 targets, identifying 34 mutual targets for treatment by HSYA. HSYA predominantly affects 9 core targets. Molecular docking unveiled hydrogen bond interactions between HSYA and several PAH-related proteins such as ANXA5, EGFR, SRC, PPARG, PGR, and ESR1. Conclusion: Utilizing network pharmacology and molecular docking approaches, we investigated potential targets and relevant human disease pathways implicating HSYA in PAH therapy, such as the chemical carcinogenesis receptor activation pathway and the cancer pathway. Our findings were corroborated by the efficacious use of HSYA in an MCT-induced rat PAH model, confirming its therapeutic potential.

Isoliquiritin can cause mitochondrial dysfunction and regulate Nrf2 to affect the development of mouse oocytes.

IsoliQuirtigenin (ILG) has been widely studied in somatic cells and tissues, but less in reproductive development. It is a kind of widely used food additive. In this study, it was found that ILG could significantly increase the levels of ROS,GSH and MMP in mouse oocytes (P < 0.01). In order to explore the cause of this phenomenon, it was found that the abnormal distribution of mitochondria and ATP synthesis levels were significantly increased (P < 0.05). At this time, we made a reasonable hypothesis that ILG affected mitochondrial function. In subsequent studies, it was found that the endogenous ROS accumulation level in mitochondria was significantly increased. After continuous RT-PCR screening, it was found that the expression of Nrf2 was significantly inhibited (P < 0.01). Its upstream and downstream FOXO3 GPX1, CAT, SOD2, SIRT1 gene also appear different degree of significant change (P < 0.05), in which the lower expression of NADP + (P < 0.05) illustrates the mitochondrial ATP synthesis electronic chain were suppressed, it also has the reason, By inhibiting electron chain and ATP synthesis, ILG leads to oocyte apoptosis and initiation of autophagy, reducing oocyte and its subsequent developmental potential.

Isoliquiritin counteracts cadmium-induced intestinal damage in mice through enhancing intestinal barrier function and inhibiting apoptosis.

Cadmium (Cd), a crucial toxic environmental pollutant, can induce damage to many organs, especially the gastrointestinal tract. Isoliquiritin (ISO), a critical flavonoid glycoside compound isolated from Glycyrrhiza uralensis, has anti-inflammatory, anticancer, antioxidant and other pharmaceutical value. However, the potential roles of ISO in Cd-induced intestinal damage have not been reported yet. This study aimed to research the beneficial effects of ISO on Cd-induced intestinal damage and identify its underlying mechanisms. Our results showed that ISO reduced inflammation by suppressing the production of pro-inflammatory cytokines and the activity of serum Lipopolysaccharide (LPS) in mice with Cd exposure. In terms of mechanism, ISO administration protected the intestinal barrier function through increasing the expression of tight junction proteins and Muc2. Furthermore, ISO could significantly suppress Cd-induced intestinal apoptosis and activation of NLRP3 inflammasome. Interestingly, inhibiting the activation of NLRP3 by nigericin completely blocking the effect of ISO on apoptosis. Most importantly, ISO markedly abrogated Cd-induced cell damage and NLRP3 inflammasome activation in vitro. Taken together, these findings suggest that ISO reduces Cd-induced intestinal damage by increasing the goblet cells, improving intestinal barrier, suppressing NLRP3 inflammasome activation and inhibiting apoptosis, which may offer a novel strategy against the toxic effects of heavy metals.

Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

SCOPE: Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. METHODS AND RESULTS: In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. CONCLUSION: In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

Safflower Yellow Inhibits Progression of Hepatocellular Carcinoma by Modulating Immunological Tolerance via FAK Pathway.

OBJECTIVE: To explore the anti-tumor effect of safflower yellow (SY) against hepatocellular carcinoma (HCC) and the underlying potential mechanism. METHODS: An in vitro model was established by mixing Luc-Hepa1-6 cells and CD3+CD8+ T cells, followed by adding programmed cell death protein 1 (PD-1) antibody (Anti-mPD-1) with or without SY. The apoptosis was detected by flow cytometry and the level of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. The protein levels of programmed cell death 1 ligand 1 (PD-L1), chemokine ligand (CCL5), C-X-C motif chemokine ligand 10 (CXCL10) were measured by Western blot. An in situ animal model was established in mice followed by treatment with anti-mPD-1 with or without SY. Bioluminescence imaging was monitored with an AniView 100 imaging system. To establish the FAK-overexpressed Luc-Hepa1-6 cells, cells were transfected with adenovirus containing pcDNA3.1-FAK for 48 h. RESULTS: The fluorescence intensity, apoptotic rate, release of inflammatory cytokines, and CCL5/CXCL10 secretion were dramatically facilitated by anti-mPD-1 (P<0.01), accompanied by an inactivation of PD-1/PD-L1 axis, which were extremely further enhanced by SY (P<0.05 or P<0.01). Increased fluorescence intensity, elevated percentage of CD3+CD8+ T cells, facilitated release of inflammatory cytokines, inactivated PD-1/PD-L1 axis, and increased CCL5/CXCL10 secretion were observed in Anti-mPD-1 treated mice (P<0.01), which were markedly enhanced by SY (P<0.05 or P<0.01). Furthermore, the enhanced effects of SY on inhibiting tumor cell growth, facilitating apoptosis and inflammatory cytokine releasing, suppressing the PD-1/PD-L1 axis, and inducing the CCL5/CXCL10 secretion in Anti-mPD-1 treated mixture of Luc-Hepa1-6 cells and CD3+CD8+ T cells were abolished by FAK overexpression (P<0.01). CONCLUSION: SY inhibited the progression of HCC by mediating immunological tolerance through inhibiting FAK.

Isoliquiritin Ameliorates Ulcerative Colitis in Rats through Caspase 3/HMGB1/TLR4 Dependent Signaling Pathway.

BACKGROUND: Isoliquiritin belongs to flavanol glycosides and has a strong antiinflammatory activity. This study sought to investigate the anti-inflammatory effect of isoliquiritin and its underlying mechanism. METHODS: The inflammatory (trinitro-benzene-sulfonic acid-TNBS-induced ulcerative colitis (UC)) model was established to ascertain the effect of isoliquiritin on the caspase-3/HMGB1/TLR4 pathway in rats. We also explored its protective effect on intestinal inflammation and its underlying mechanism using the LPS-induced inflammation model of Caco-2 cells. Besides, Deseq2 was used to analyze UCassociated protein levels. RESULTS: Isoliquiritin treatment significantly attenuated shortened colon length (induced by TNBS), disease activity index (DAI) score, and body weight loss in rats. A decrease in the levels of inflammatory mediators (IL-1ß, I IL-4, L-6, IL-10, PGE2, and TNF-α), coupled with malondialdehyde (MDA) and superoxide dismutase (SOD), was observed in colon tissue and serum of rats after they have received isoliquiritin. Results of techniques (like western blotting, real-time PCR, immunohistochemistry, and immunofluorescence-IF) demonstrated the potential of isoliquiritin to decrease expressions of key genes in the TLR4 downstream pathways, viz., MyD88, IRAK1, TRAF6, NF-κB, p38, and JNK at mRNA and protein levels as well as inhibit HMGB1 expression, which is the upstream ligand of TLR4. Bioinformational analysis showed enteritis to be associated with a high expression of HMGB1, TLR4, and caspase-3. CONCLUSION: Isoliquiritin could reduce intestinal inflammation and mucosal damage of TNBS-induced colitis in rats with a certain anti-UC effect. Meanwhile, isoliquiritin treatment also inhibited the expression of HMGB1, TLR4, and MyD88 in LPS-induced Caco-2 cells. These results indicated that isoliquiritin could ameliorate UC through the caspase-3/HMGB1/TLR4-dependent signaling pathway.

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<0.05 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.

Isoliquiritin treatment of osteoporosis by promoting osteogenic differentiation and autophagy of bone marrow mesenchymal stem cells.

Osteoporosis is a chronic progressive bone disease characterized by the decreased osteogenic ability of osteoblasts coupled with increased osteoclast activity. Natural products showing promising therapeutic potential for postmenopausal osteoporosis remain underexplored. In this study, we aimed to analyze the therapeutic effects of isoliquiritin (ISL) on osteoporosis in mice and its possible mechanism of action. An ovariectomy-induced osteoporosis mouse model and bone marrow mesenchymal stem cells (BMSCs) were used to analyze the effects of ISL on bone regeneration in vivo and in vitro, respectively. Mitogen-activated protein kinase (MAPK) and autophagy inhibitors were used, to investigate whether the MAPK signaling pathway and autophagy affect the osteogenic differentiation of BMSCs. ISL significantly improved bone formation and reduced bone resorption in mouse femurs without inducing any detectable toxicity in critical organs such as the liver, kidney, brain, heart, and spleen. In vitro experiments showed that ISL enhanced the proliferation and osteogenic differentiation of BMSCs and that its osteogenic effect was attenuated by p38/extracellular regulated protein kinase (ERK) and autophagy inhibitors. Further studies showed that the inhibition of phosphorylated p38/ERK blocked ISL autophagy in BMSCs. ISL promoted the osteogenic differentiation of BMSCs through the p38/ERK-autophagy pathway and was therapeutically effective in treating osteoporosis in ovariectomized mice without any observed toxicity to vital organs. These results strongly suggest the promising potential of ISL as a safe and efficacious candidate drug for the treatment of osteoporosis.

Hydroxysafflor Yellow A-Induced Osteoblast Differentiation and Proliferation of BM-MSCs by Up-Regulating Nuclear Vitamin D Receptor.

BACKGROUND: Vitamin D receptor (VDR) is critical for mineral and bone homeostasis since it plays an essential role in the osteoblast differentiation of bone marrow mesenchymal stem cells (BM-MSCs). Hydroxysafflor yellow A (HSYA) has the potential to promote bone mineralization and inhibit bone resorption, while its detailed mechanism needs to be elaborated. OBJECTIVE: This study intends to explore the action of HSYA on the proliferation and differentiation of BM-MSC and the underlying mechanism. METHODS: Different concentrations of HSYA to BM-MSC and CCK-8, and EdU were used to detect cell viability and proliferation. The alkaline phosphatase (ALP) was used to observe the differentiation ability of BM-MSC osteoblasts. The calcium uptake and mineralization of osteoblast-like cells were observed by alizarin red staining. The level of calcium ion uptake in cells was detected by flow cytometry. AutoDock was performed for molecular docking of HSYA to VDR protein. Immunofluorescence and western blotting were performed to detect the expression of VDR expression levels. Finally, the effect of VDR was verified by a VDR inhibitor. RESULTS: After treatment with HSYA, the proliferation and calcium uptake of BM-MSC were increased. The level of ALP increased significantly and reached its peak on the 12th day. HSYA promoted calcium uptake and calcium deposition, and mineralization of osteoblasts. The western blotting and immunofluorescence showed that HSYA increased the expression of VDR in the osteoblast-like cell's nucleus and upregulated Osteocalcin, S100 calcium-binding protein G, and CYP24A1. In addition, HYSA treatment increased the expression of osteopontin and the synthesis of osteogenic proteins, such as Type 1 collagen. After the addition of the VDR inhibitor, the effect of HSYA was weakened. CONCLUSION: HSYA could significantly promote the activity and proliferation of osteoblasts and increase the expression level of VDR in osteoblasts. HSYA may also improve calcium absorption by osteoblasts by regulating the synthesis of calciumbinding protein and vitamin D metabolic pathway-related proteins.

Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer.

BACKGROUND: Protein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear. METHODS: We first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process. RESULTS: Among PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5. CONCLUSIONS: In summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.

LC-MS/MS combined with blood-brain dual channel microdialysis for simultaneous determination of active components of astragali radix-safflower combination and neurotransmitters in rats with cerebral ischemia reperfusion injury: Application in pharmacokinetic and pharmacodynamic study.

BACKGROUND: Astragali Radix-Safflower combination (ARSC) is widely utilized in clinic to treat cerebral ischemia/reperfusion injury (CI/RI). Whereas, there is no in-depth research of the pharmacokinetics (PK) and pharmacodynamics (PD) analysis of ARSC after intragastric administration in rats with CI/RI. PURPOSE: The purpose of this research is to investigate the PK characteristics of eight active ingredients (astragaloside IV, calycosin, calycosin-7-O-ß-glucoside, formononetin, ononin, hydroxysafflor yellow A, syringin and vernine) of ARSC, and the regulation of neurotransmitters disorders, revealing the pharmacodynamic substance basis and the mechanism of ARSC in treating CI/RI from the molecular level. METHODS: We established a new method which based on blood-brain dual channel microdialysis (MD) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to continuously gather, and determine the components of ARSC and neurotransmitters related to CI/RI in vivo. The collected data were analyzed by sigmoid-Emax function. The neurotransmitters primarily regulated in CI/RI rat were discussed by principal component analysis and the compound most associated with total pharmacodynamics was chosen by partial least squares regression. RESULTS: The validated LC-MS/MS method had specificity and selectivity to simultaneously analyze the concentration of eight active components of ARSC extract and five neurotransmitters of CI/RI rats. The recovery rates of brain MD probe and blood MD probe were stable within six hours. The MD probes recovery rates decreased with the increase of flow rates, but the solution concentration had little effect on the probes recovery rates. It was feasible to correct the recovery rates of probes in vivo by using reverse dialysis method. All eight active ingredients of ARSC could pass across the blood brain barrier after CI/RI. ARSC regulated the release of glutamate (Glu), γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and aspartic acid (Asp). Notably, astragaloside IV and hydroxysafflor yellow A might have better regulatory effect on neurotransmitters in comparison with other six measured components of ARSC, and Glu was the neurotransmitter mainly regulated in CI/RI rats. CONCLUSION: The ARSC was able to treat CI/RI through ameliorating neurotransmitters disorders. There was a hysteresis between the peaked drug concentration and maximum therapeutic effect of ARSC. The drug effective concentrations range of ASIV, calycosin, calycosin-7-O-ß-glucoside, syringin and vernine in blood microdialysate and calycosin, syringin, vernine in brain microdialysate were narrow, which need be paid attention in clinical use.

[Research progress on mechanism of Carthamus tinctorius in ischemic stroke therapy].

Carthamus tinctorius is proved potent in treating ischemic stroke. Flavonoids, such as safflower yellow, hydroxysafflor yellow A(HSYA), nicotiflorin, safflower yellow B, and kaempferol-3-O-rutinoside, are the main substance basis of C. tinctorius in the treatment of ischemic stroke, and HSYA is the research hotspot. Current studies have shown that C. tinctorius can prevent and treat ischemic stroke by reducing inflammation, oxidative stress, and endoplasmic reticulum stress, inhibiting neuronal apoptosis and platelet aggregation, as well as increasing blood flow. C. tinctorius can regulate the pathways including nuclear factor(NF)-κB, mitogen-activated protein kinase(MAPK), signal transducer and activator of transcription protein 3(STAT3), and NF-κB/NLR family pyrin domain containing 3(NLRP3), and inhibit the activation of cyclooxygenase-2(COX-2)/prostaglandin D2/D prostanoid receptor pathway to alleviate the inflammatory development during ischemic stroke. Additionally, C. tinctorius can relieve oxidative stress injury by inhibiting oxidation and nitrification, regulating free radicals, and mediating nitric oxide(NO)/inducible nitric oxide synthase(iNOS) signals. Furthermore, mediating the activation of Janus kinase 2(JAK2)/STAT3/suppressor of cytokine signaling 3(SOCS3) signaling pathway and phosphoinositide 3-kinase(PI3 K)/protein kinase B(Akt)/glycogen synthase kinase-3ß(GSK3ß) signaling pathway and regulating the release of matrix metalloproteinase(MMP) inhibitor/MMP are main ways that C. tinctorius inhibits neuronal apoptosis. In addition, C. tinctorius exerts the therapeutic effect on ischemic stroke by regulating autophagy and endoplasmic reticulum stress. The present study reviewed the molecular mechanisms of C. tinctorius in the treatment of ischemic stroke to provide references for the clinical application of C. tinctorius.

Antitumor Proliferation and Related Mechanism of Ultrasound Irradiation Combined with Safflower Yellow.

Ultrasound irradiation is now the best method for evaluating benign and malignant tumor nodules. Chemotherapy has always played an important role in the treatment of malignant tumors. With the large-scale application of chemotherapy drugs, the problem of multidrug resistance of tumors has become more and more prominent, which has become one of the difficulties in tumor chemotherapy. This study mainly explores the antitumor proliferation and related mechanisms of ultrasound irradiation combined with safflower yellow. The breast cancer cell line 4T1 derived from BALB/c mice was selected. BALB/c is an albino laboratory mouse, which, like many commonly used sublines, originated from Mus musculus. BALB/c mice have been bred for more than 200 generations in research institutions around the world and are widely used in animal experiments in immunology and physiology. When the cell proliferation reached 80%-90% of the bottom area of the culture flask, it was resuspended, passaged, frozen, and resuscitated according to experimental needs. The 4T1 breast cancer cell line was cultured by conventional methods. 4T1 breast cancer cells in the logarithmic proliferation phase were collected. After 0.25% was digested with pancreatin, it was washed twice with PBS to adjust the concentration to 1 × 107/mL. A 0.1 mL tumor cell suspension was subcutaneously inoculated on the edge of the mouse chest, thereby establishing a breast cancer model of BALB/c mice. After 6-15 days, the tumor volume grew rapidly and became larger. When the length of the tumor is 2.5 × 2.5, the modeling is successful. Ultrasound-targeted microbubble destruction technology, as a novel drug delivery method with high efficiency and low toxicity, can form transient pores (sonoporation effect) on the cell surface, widen the intercellular space, and increase the membrane permeability, and thus effectively. The transport of drugs, genes, proteins, etc., is promoted to target organs and tissues. Tumor-forming mice were randomly divided into the following four groups: control group, safflower yellow group, ultrasound irradiation group, and ultrasound irradiation combined with safflower yellow group. From the second day of inoculation to the end of the experiment, the body weight of the mice successfully inoculated with 4T1 cells was measured every day; from the 5th day, tumors in each group were calculated body volume and tumor inhibition rate (TIR) of each group. The combined treatment group has a higher tumor inhibition rate than the ultrasound irradiation group, and the difference is statistically significant (P < 0.05). Ultrasound irradiation combined with safflower yellow pigment can effectively inhibit tumor proliferation, maintain, or even improve the efficacy of chemotherapy, thereby improving the patient's tolerance to chemotherapy.

Protective effect of hydroxysafflor yellow A on cyclosporin A-induced renal oxidative stress in vitro and in vivo.

PURPOSE: To explore the mechanism and investigate the protective effect of hydroxysafflor yellow A (HSYA) on renal oxidative stress, which cyclosporine A (CsA) induces. METHODS: HK-2 cells were treated with CsA to get CsA-induced oxidative stress. The effects on oxidative stress and apoptosis of HK-2 cells were detected. The contents of SOD, MDA, GSH-Px, ROS, and CAT in serum were measured, and the expression of apoptosis-related proteins was detected by western blot. Then, established the renal oxidative stress injury rats to verify the efficacy of HSYA. RESULTS: HSYA could reduce the ROS and MDA contents induced by CsA. Compared with the CsA group, the activities of SOD, CAT, and GSH-Px increased significantly when treated with HSYA. HSYA could inhibit CsA-induced apoptosis in HK-2 cells, and promote the protein of Bcl-2 and inhibit the expression of Bax. Animal experiments showed that HSYA could reduce CsA-induced renal cell injury by reducing glomerular cell vacuoles and inflammatory factors in tissues. It also decreased serum creatinine (Crea) and blood urea nitrogen, increased Crea clearance significantly. CONCLUSIONS: HSYA could significantly improve the antioxidant capacity of the kidney cells and inhibit cell apoptosis, thereby effectively ameliorating CsA-induced oxidative stress in vitro and in vivo.

Hydroxy-safflower yellow A composites: An effective strategy to enhance anti-myocardial ischemia by improving intestinal permeability.

Hydroxy-safflower yellow A (HSYA) is the chief component of safflower against myocardial ischemia (MI), and belongs to biopharmaceutics classification system (BCS) III drugs. Its structure contains multiple hydroxyl groups, contributing to its high polarity and poor oral bioavailability. The main objective of this study was to probe the potential of oral penetration enhancer n-[8-(2-hydroxybenzoyl) amino] sodium octanoate (SNAC) and cationic copolymer Eudragit®EPO (EPO) to promote absorption of HSYA. HSYA composites (SNAC-HSYA-EPO) were formed by hydrogen bonding and van der Waals force. SNAC-HSYA-EPO has biocompatibility, and can improve the membrane fluidity, uptake, transport, and penetration of Caco-2 cells. The mechanism of promoting of SNAC-HSYA-EPO may be related to energy and P-glycoprotein (P-gp) when compared with the inhibitor NaN3 and verapamil group. In the pharmacokinetic (PK) results, SNAC-HSYA-EPO significantly improved oral bioavailability. Pharmacodynamics (PD) results determined that SNAC-HSYA-EPO could improve the symptoms of MI. The mechanism of the SNAC-HSYA-EPO anti-MI is related to alleviating inflammation and anti-apoptosis to protect the heart. In summary, SNAC-HSYA-EPO prepared in this study possessed a complete appearance, high recombination rate and excellent oral permeability promoting ability. SNAC-HSYA-EPO has the potential to improve oral bioavailability and further enhance the anti-MI effect of HSYA.

Pharmacological mechanism of xanthoangelol underlying Nrf-2/TRPV1 and anti-apoptotic pathway against scopolamine-induced amnesia in mice.

Alzheimer's disease (AD) is a well-known type of age-related dementia. The present study was conducted to investigate the effect of xanthoangelol against memory deficit and neurodegeneration associated with AD. Preliminarily, xanthoangelol produced neuroprotective effect against H2O2-induced HT-22 cells. Furthermore, effect of xanthoangelol against scopolamine-induced amnesia in mice was determined by intraperitoneally (i.p.) administering xanthoangelol (1, 10 and 20 mg/kg), 30 min prior to induction. Mice were administered scopolamine at a concentration of 1 mg/kg; i.p. for the induction of amnesia associated with AD. Xanthoangelol dose dependently reduced the symptoms of Alzheimer's disease as observed by the results obtained from the behavioral analysis performed using Morris water maze and Y-maze test. The immunohistochemical analysis suggested that xanthoangelol significantly improved Keap-1/Nrf-2 signaling pathway. It greatly reduced the effects of oxidative stress and showed improvement in the anti-oxidant enzyme such as GSH, GST, SOD and catalase. Additionally, xanthoangelol decreased the expression of transient receptor potential vanilloid 1 (TRPV-1), a nonselective cation channel, involved in synaptic plasticity and memory. It activated the anti-oxidants and attenuated the apoptotic (Bax/Bcl-2) pathway. Xanthoangelol also significantly attenuated the scopolamine-induced neuroinflammation by the inhibition of interleukin-1 beta (IL-1ß), and tumor necrosis factor-α (TNF-α) levels. The histological analysis, showed a significant reduction in amyloid plaques by xanthoangelol. Therefore, the present study indicated that xanthoangelol has the ability to ameliorate the AD symptoms by attenuating neuroinflammation and neurodegeneration induced by scopolamine.

Isoliquiritin apioside relieves intestinal ischemia/reperfusion-induced acute lung injury by blocking Hif-1α-mediated ferroptosis.

Isoliquiritin apioside (IA), a critical ingredient of Glycyrrhizae radix et rhizoma, has been unveiled to possess remarkable pharmacological activity against oxidative stress and inflammation. However, the potential roles of IA in intestinal ischemia/reperfusion (I/R)-induced acute lung injury (ALI) have not been reported yet. In the present study, we explored the effects of IA on I/R-induced ALI, and also clarified the possible mechanisms. To mimic intestinal I/R-induced ALI, the mice were subjected to 60 min of intestinal ischemia via clamping of the superior mesenteric artery followed by 60 min of reperfusion. IA was administered orally (20 mg/kg/day and 50 mg/kg/day) for 7 consecutive days before intestinal I/R. Lung epithelial MLE-2 cells were subjected to hypoxia for 2 h and regeneration for 3 h to mimic in vitro ALI. The results showed that IA administration prevented intestinal I/R-induced lung injury, inflammation and edema. Also, IA administration decreased the level of ferroptosis in murine lung tissues challenged with intestinal I/R. In terms of mechanism, IA administration inhibited the protein upregulation of Hif-1α and HO-1 in mice with ALI. In vitro experiments further demonstrated that IA treatment could inhibit the mRNA and protein levels of Hif-1α in hypoxia/regeneration (H/R)-induced MLE-2 cells in a concentration-dependent manner. Hif-1α stabilizer molidustat itself also significantly promoted ferroptosis of MLE-2 cells. And Hif-1α activation increased the mRNA levels of Ptgs2 and Acsl4 but decreased the mRNA level of Gpx4 in H/R-induced MLE-2 cells treated with IA. Taken together, our study unveiled IA could protect against intestinal I/R-induced ALI by decreasing lung epithelial ferroptosis in a Hif-1α-dependent manner.