Breviscapine Reduces Sepsis-Induced Acute Lung Injury by Targeting CASP8 to Regulate Neutrophil Apoptosis and Inflammation.

Background: Breviscapine has been demonstrated to have beneficial effects in ameliorating acute lung injury (ALI), yet its potential therapeutic value and molecular mechanisms in sepsis-induced ALI remain unexplored. Methods: We utilized network pharmacology approach to identify the potential targets and mechanisms of breviscapine in treating sepsis-induced ALI. To construct a murine model of sepsis, we performed cecal ligation and puncture (CLP). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) were employed to respectively determine the pathologic changes and levels of inflammatory factors. Neutrophil count and total protein level in bronchoalveolar lavage fluid (BALF) were detected by corresponding kit. Additionally, we utilized flow cytometry, immunofluorescence, Western blotting, and real-time reverse transcription PCR (qRT-PCR) to detect cell apoptosis, protein expression, and gene expression. Finally, we used ELISA kits to detect the activity of myeloperoxidase (MPO) and caspase-8 (CASP8). Results: Breviscapine was revealed to target 81 potential proteins in the treatment of sepsis-induced ALI, while CASP8 was the most important one as demonstrated by network analysis. In vivo experiments demonstrated that breviscapine effectively reduced the severity of sepsis-induced ALI and inflammation, and significantly suppressed neutrophil infiltration in the lung tissues of CLP mice and promoted neutrophil apoptosis in the peripheral blood. In vitro experiments revealed that lipopolysaccharide (LPS)-induced neutrophil apoptosis was inhibited, and the expression and activity of CASP8 were down-regulated. Breviscapine intervention markedly up-regulated the expression and activity of CASP8, consequently activating neutrophil apoptosis and inhibiting inflammatory response by activating the NF-κB signaling pathway. Conclusion: Breviscapine is remarkably effective in improving sepsis-induced ALI, and its mechanism of action may be to induce neutrophil apoptosis, inhibit inflammatory overreaction and reduce its infiltration in pulmonary tissues by up-regulating the expression and activity of CASP8.

Breviscapine ameliorates autophagy by activating the JAK2/STAT5/BCL2 pathway in a transient cerebral ischemia rat model.

Breviscapine (Bre), an extract from Erigeron breviscapus, has been widely used to treat cerebral ischemia but the mechanisms of its neuroprotective effects need to be clarified. The present study investigated whether Bre could alleviate excessive autophagy induced by cerebral ischemia in the rat middle cerebral artery occlusion (MCAO) ischemia model via activating the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5)/B-cell lymphoma 2 (BCL2) pathway. Rats were randomly divided into 5 groups, i.e. Sham group, MCAO+saline group, MCAO+Bre group, MCAO+DMSO (Dimethyl sulfoxide) group, and MCAO+Bre+AG490 (Tyrphostin AG490, the inhibitor of STAT5) group. The model was established and neuroprotection was evaluated by determining infarct volumes and conducting neurological behavioral tests. Autophagy levels in the infarct penumbra were detected using transmission electron microscopy and Western blotting. The expression of proteins in the JAK2/STAT5/BCL2 pathway was tested by Western blotting. Compared to the MCAO+saline group, the infarct volumes in the MCAO+Bre group were significantly reduced and neurological behavior improved. Breviscapine administration also significantly increased p-JAK2, p-STAT5, and BCL2 expression but decreased autolysosome numbers; it also downregulated Beclin-1 expression and the LC3II/LCI ratio. The JAK2 inhibitor AG490 reversed these effects. These findings indicate that breviscapine can improve neural recovery following ischemia through alleviating excessive autophagy and activation of the JAK2/STAT5/BCL2 axis.

Portable wireless electrochemical sensing of breviscapine using core-shell ZIFs-derived Co nanoparticles embedded in N-doped carbon nanotube polyhedra-modified electrode.

A core-shell ZIF-67@ZIF-8-derived Co nanoparticles embedded in N-doped carbon nanotube polyhedra (Co/C-NCNP) hybrid nanostructure was prepared by a pyrolysis method. The synthesized Co/C-NCNP was modified on the screen-printed carbon electrode and used for the portable wireless sensitive determination of breviscapine (BVC) by differential pulse voltammetry. The Co/C-NCNP had a large surface area and excellent catalytic activity with increasing Co sites to combine with BVC for selective determination, which led to the improvement of the sensitivity of the electrochemical sensor. Under optimized conditions, the constructed sensor had linear ranges from 0.15 to 20.0 µmol/L and 20.0 to 100.0 µmol/L with the limit of detection of 0.014 µmol/L (3S0/S). The sensor was successfully applied to BVC tablet sample analysis with satisfactory results. This work provided the potential applications of zeolitic imidazolate framework-derived nanomaterials in the fabrication of electrochemical sensors for the sensitive detection of drug samples.

Preparation, Characterization, and Evaluation of Mesoporous Silica Nanoparticles in Enhancing Oral Bioavailability of Poorly Water-Soluble Drugs.

BACKGROUND: Breviscapine (BVP) is one of the extracts of several flavonoids of Erigeron breviscapus, which has been widely used in the treatment of cerebral infarction and its sequelae, cerebral thrombus, coronary heart disease, and angina pectoris. But BVP has poor solubility. OBJECTIVE: The objective of the study is to develop mesoporous silica nanoparticles (MSNs) that can be loaded with a drug with poor water solubility. The MSNs, which were designed for oral administration, enhanced both the dissolution rate and drug loading capacity. METHODS: The use of MSNs as an oral drug delivery system was investigated by SEM, TEM, BETBJH, XRD, FT-IR, and HPLC. Additionally, we examined the oral bioavailability of BVP loaded onto MSNs and examined the cellular cytotoxicity of MSNs. RESULTS: The results indicate that the oral bioavailability of BVP after loading onto MSNs was greater than that of a marketed product. Furthermore, we studied the mechanism by which MSNs enhance the oral absorption of BVP. CONCLUSION: MSNs have the potential to enhance the oral bioavailability of poorly water-soluble drugs by accelerating the drug dissolution rate.

Breviscapine protects against pathological cardiac hypertrophy by targeting FOXO3a-mitofusin-1 mediated mitochondrial fusion.

Forkhead box O3a (FOXO3a)-mediated mitochondrial dysfunction plays a pivotal effect on cardiac hypertrophy and heart failure (HF). However, the role and underlying mechanisms of FOXO3a, regulated by breviscapine (BRE), on mitochondrial function in HF therapy remain unclear. This study reveals that BRE-induced nuclear translocation of FOXO3a facilitates mitofusin-1 (MFN-1)-dependent mitochondrial fusion in cardiac hypertrophy and HF. BRE effectively promotes cardiac function and ameliorates cardiac remodeling in pressure overload-induced mice. In addition, BRE mitigates phenylephrine (PE)-induced cardiac hypertrophy in cardiomyocytes and fibrosis remodeling in fibroblasts by inhibiting ROS production and promoting mitochondrial fusion, respectively. Transcriptomics analysis underscores the close association between the FOXO pathway and the protective effect of BRE against HF, with FOXO3a emerging as a potential target of BRE. BRE potentiates the nuclear translocation of FOXO3a by attenuating its phosphorylation, other than its acetylation in cardiac hypertrophy. Mechanistically, over-expression of FOXO3a significantly inhibits cardiac hypertrophy and mitochondrial injury by promoting MFN-1-mediated mitochondrial fusion. Furthermore, BRE demonstrates its ability to substantially curb cardiac hypertrophy, reduce mitochondrial ROS production, and enhance MFN-1-mediated mitochondrial fusion through a FOXO3a-dependent mechanism. In conclusion, nuclear FOXO3a translocation induced by BRE presents a successful therapeutic avenue for addressing cardiac hypertrophy and HF through promoting MFN-1-dependent mitochondrial fusion.

Intervention effect and mechanism of breviscapine on hepatic fibrosis in rats / 中国药房

OBJECTIVE To investigate the intervention effect and potential mechanism of breviscapine on hepatic fibrosis （HF） in rats based on the transforming growth factor-β（1 TGF-β1）/Smad2/extracellular signal-regulated protein kinase 1（ERK1） and Kelch-like epichlorohydrin-associated protein 1（Keap1）/nuclear factor-erythroid 2-related factor 2（Nrf2）/heme oxygenase-1（HO-1） pathways. METHODS Totally 60 rats were randomly divided into normal control group， model group， breviscapine low-dose， medium-dose and high-dose groups （5.4， 10.8， 21.6 mg/kg）， and colchicine group （positive control， 0.45 mg/kg）， with 10 rats in each group， half male and half female. Except for the normal control group， HF model of the other groups was induced by carbon tetrachloride. Subsequently， each drug group was given corresponding medicine by gavage once a day for 28 days. The liver appearance of rats in each group was observed and their liver coefficients were calculated. The levels of alanineaminotransferase （ALT） and aspartate aminotransferase （AST）in serum， those of ALT， AST， superoxide dismutase （SOD），malondialdehyde （MDA） and glutathione peroxidase （GSH- Px） in liver tissue were detected. The liver tissue inflammatory and fibrotic changes were observed. The protein and mRNA expressions of TGF-β1， Smad2， ERK1， Nrf2， Keap1 and HO-in liver tissue were detected. RESULTS Compared with the normal control group， the model group showed large areas of white nodular lesions in the liver， obvious inflammatory cell infiltration and collagen fiber deposition. The body weight， the levels of SOD and GSH-Px in liver tissue， the protein and mRNA expressions of Nrf2 and HO-1 were significantly lowered in the model group （P＜0.05）； the liver coefficient， the percentage of Masson staining positive area， ALT and AST levels of serum and liver tissue， MDA level of liver tissue， the protein and mRNA expressions of TGF-β1， Smad2， ERK1 and Keap1 were significantly increased （P＜0.05）. Compared with the model group， the liver lesions of rats in each drug group were improved， and the above quantitative indexes were generally reversed （P＜0.05）. CONCLUSIONS Breviscapine has a good intervention effect on HF rats， which may be related to inhibiting TGF-β1/Smad2/ERK1 pathway for anti-fibrosis and regulating Keap1/Nrf2/HO-1 pathway to inhibit oxidative stress.

Identification of m6A methylation-related genes in cerebral ischaemia‒reperfusion of Breviscapus therapy based on bioinformatics methods.

BACKGROUND: Cerebral ischaemia‒reperfusion (I/R) frequently causes late-onset neuronal damage. Breviscapine promotes autophagy in microvascular endothelial cells in I/R and can inhibit oxidative damage and apoptosis. However, the mediation mechanism of breviscapine on neuronal cell death is unclear. METHODS: First, transcriptome sequencing was performed on three groups of mice: the neuronal normal group (Control group), the oxygen-glucose deprivation/ reoxygenation group (OGD/R group) and the breviscapine administration group (Therapy group). Differentially expressed genes (DEGs) between the OGD/R and control groups and between the Therapy and OGD/R groups were obtained by the limma package. N6-methyladenosine (m6A) methylation-related DEGs were selected by Pearson correlation analysis. Then, prediction and confirmation of drug targets were performed by Swiss Target Prediction and UniProt Knowledgebase (UniProtKB) database, and key genes were obtained by Pearson correlation analysis between m6A-related DEGs and drug target genes. Next, gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA) were used to obtain the pathways of key genes. Finally, a circRNA-miRNA‒mRNA network was constructed based on the mRNAs, circRNAs and miRNAs. RESULTS: A total of 2250 DEGs between the OGD/R and control groups and 757 DEGs between the Therapy and OGD/R groups were selected by differential analysis. A total of 7 m6A-related DEGs, including Arl4d, Gm10653, Gm1113, Kcns3, Olfml2a, Stk26 and Tfcp2l1, were obtained by Pearson correlation analysis. Four key genes (Tfcp2l1, Kcns3, Olfml2a and Arl4d) were acquired, and GSEA showed that these key genes significantly participated in DNA repair, e2f targets and the g2m checkpoint. IPA revealed that Tfcp2l1 played a significant role in human embryonic stem cell pluripotency. The circRNA-miRNA‒mRNA network showed that mmu_circ_0001258 regulated Tfcp2l1 by mmu-miR-301b-3p. CONCLUSIONS: In conclusion, four key genes, Tfcp2l1, Kcns3, Olfml2a and Arl4d, significantly associated with the treatment of OGD/R by breviscapine were identified, which provides a theoretical basis for clinical trials.

Breviscapine reverses doxorubicin resistance in breast cancer and its related mechanisms.

BACKGROUND: Based on the effect of breviscapine (BRE) on reversing drug resistance of human breast cancer cell line MCF-7/doxorubicin (Dox), the mechanism was preliminarily explored. METHODS: The methyl thiazolyl tetrazolium (MTT) method was introduced to detect inhibitory effect of Dox alone or in combination with BRE on MCF-7 (M) and MCF-7/Dox (MD) cells, and the inhibitory concentration (IC50 ) was obtained. Cell apoptosis and Dox concentration was assessed by flow cytometry. The drug resistance multiple and reversal fold were calculated. Western blot was performed to evaluate the expression of Bcl-2, Bax, EGFR, p-EGFR, P-gp, caspase-3, and cleaved-caspase-3 protein in cells. The efflux of Rho-123 was measured by flow cytometry and fluorescence microscopy. RESULTS: The IC50 of Dox on MD and M cells was 16.67 and 0.71 µg/mL, respectively, with a drug resistance ratio of 23.48 times. The IC50 of Dox combined with BRE on MD cells was 5.62 µg/mL, with a reversal ratio of 2.97 times. BRE greatly enhanced Dox-induced apoptosis of MD cells. Bax and cleaved-caspase-3 (proapoptotic protein) expression were obviously increased, while Bcl-2 (antiapoptotic protein) expression was significantly decreased after BRE treatment. BRE inhibited EGFR activation and P-gp expression. BRE increased the intracellular accumulation of Dox in MD cells by P-gp. CONCLUSION: BRE could increase the MD sensitivity to Dox via increasing Bax and cleaved-caspase-3 expression and inhibiting Bcl-2 expression, thereby promoting cell apoptosis. BRE reversed Dox resistance of MD cells by increasing Dox intracellular accumulation through inhibiting P-gp expression via EGFR.

Breviscapine Ameliorates Fear Extinction and Anxiety in BALB/cJ Mice.

OBJECTIVE: In this research, the influence of breviscapine on anxiety, fear elimination, and aggression and the potential mechanism was investigated. METHODS: Anxiety and locomotion were analyzed by elevated plus maze and open field test in mice. Bussey-Saksida Mouse Touch Screen Chambers were used to perform fear conditioning. Territorial aggression was assessed by resident intruder test. Protein levels were evaluated by Western blot. Breviscapine improved fear-extinction learning in BALB/cJ mice. RESULTS: Breviscapine at 20-100 mg/kg increased center cross number, total distance traveled, and velocity in a dose-dependent manner. On the other hand, breviscapine at 20-100 mg/kg decreased the immobility time in open field test. In addition, breviscapine at 20-100 mg/kg increased the ratio of time on the open arm, time on the distal parts of the open arm, and total distance traveled in elevated plus maze. Breviscapine at 100 mg/kg increased the average attack latency and decreased the number of attacks over the last 3 days of resident intruder test. In hippocampus, protein levels of postsynaptic density protein-95 and synaptophysin were elevated by breviscapine at these three doses. CONCLUSION: The administration of breviscapine alleviates fear extinction, anxiety, and aggression, while increases locomotor in a dose-dependent manner, which might be associated with its influence on synaptic function.

Breviscapine alleviates podocyte injury by inhibiting NF-κB/NLRP3-mediated pyroptosis in diabetic nephropathy.

Podocyte injury is a critical factor in the pathogenesis of diabeticnephropathy (DN). Emerging evidence has demonstrated that breviscapine (Bre) exerts a renoprotective effect on diabetic rats. However, the effects of Bre on regulating podocyte injury under high glucose (HG) conditions remain unclear. In this study, an experimental mouse model of DN was induced by intraperitoneal injections of streptozotocin (STZ) in vivo. The effects of Bre on podocyte injury were assessed using cell counting kit-8 (CCK-8) assay, TdT-mediated dUTPnick-endlabelling (TUNEL) staining, quantitative real-time PCR (qRT‒PCR) and western blot analysis. We found that renal function was significantly decreased in diabetic mice, and this effect was blocked by Bre treatment. Bre effectively increased podocyte viability and inhibited HG-induced cell apoptosis. Furthermore, Bre ameliorated HG-induced podocyte injury, as evidenced by decreased α-smooth muscle actin (α-SMA) expression and increased podocin and synaptopodin expression. Mechanistically, Bre inhibited HG-induced nuclear factorkappaB (NF-κB) signalling activation and subsequently decreased NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, resulting in a decrease in pyroptosis. Pharmacological inhibition of NLRP3 decreased HG-induced podocyte injury, whereas the NLRP3 agonist abrogated the effects of Bre on inhibiting podocyte injury. In summary, these results demonstrate that Bre alleviates HG-induced podocyte injury and improves renal function in diabetic mice, at least in part by inhibiting NF-κB/NLRP3-mediated pyroptosis.

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OBJECTIVES: To study the protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism. METHODS: A preterm rat model of brain injury caused by intrauterine inflammation was prepared by intraperitoneal injections of lipopolysaccharide in pregnant rats. The pregnant rats and preterm rats were respectively randomly divided into 5 groups: control, model, low-dose breviscapine (45 mg/kg), high-dose breviscapine (90 mg/kg), and high-dose breviscapine (90 mg/kg)+ML385 [a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg] (n=10 each). The number and body weight of the live offspring rats were measured for each group. Hematoxylin-eosin staining was used to observe the pathological morphology of the uterus and placenta of pregnant rats and the pathological morphology of the brain tissue of offspring rats. Immunofluorescent staining was used to measure the co-expression of ionized calcium binding adaptor molecule-1 (IBA-1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex of offspring rats. ELISA was used to measure the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1ß (IL-1ß) in the brain tissue of offspring rats. Western blotting was used to measure the expression of Nrf2 pathway-related proteins in the brain tissue of offspring rats. RESULTS: Pathological injury was found in the uterus, and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, and severe microglia pyroptosis occurred in the cerebral cortex of the offspring rats in the model group. Compared with the control group, the model group had significant reductions in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain tissue of the offspring rats (P<0.05), but significant increases in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1ß, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). Compared with the model group, the breviscapine administration groups showed alleviated pathological injury of the uterus and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, significant increases in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and HO-1 in the brain tissue of the offspring rats (P<0.05), and significant reductions in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1ß, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). The high-dose breviscapine group had a significantly better effect than the low-dose breviscapine (P<0.05). ML385 significantly inhibited the intervention effect of high-dose breviscapine (P<0.05). CONCLUSIONS: Breviscapine can inhibit inflammatory response in brain tissue of preterm rats caused by intrauterine inflammation by activating the Nrf2 pathway, and it can also inhibit microglial pyroptosis and alleviate brain injury.

Effects of breviscapine on cerebral ischemia-reperfusion injury and intestinal flora imbalance by regulating the TLR4/MyD88/NF-κB signaling pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Erigeron breviscapus (Vant.) Hand.-Mazz.,a Chinese herbal medicine with multiple pharmacological effects and clinical applications, has been traditionally used in the treatment of paralysis caused by stroke and joint pain from rheumatism by the Yi minority people of Southwest China for generations.However, its mechanism involves many factors and has not been fully clarified. AIM OF THE STUDY: Taking intestinal flora as the target, the protective effect of extract(breviscapine) of E. breviscapus on cerebral ischemia and its possible mechanism were discussed from the perspective of brain inflammatory pathway and intestinal CYP3A4, which depends on intestinal flora. MATERIALS AND METHODS: In this study, we first verified the binding ability between major active ingredient of Erigeron breviscapus and the core target TLR4 protein by molecular docking using Vina software.We established a rat model of cerebral ischemia-reperfusion injury in vivo.The neurological function of rats was scored by Bederson score table, the cerebral infarction volume was detected by TTC staining, and the serum NSE level was detected by ELASA. 16S rRNA sequencing was used to detect the intestinal flora of rats in each group.The expression levels of cerebral TLR4/MyD88/NF-κB and CYP3A4 mRNA and protein in different intestinal segments were detected by qRT-PCR and Western blot. RESULTS: Compared with the model group, the neurological injury score, infarct volume and serum NSE concentration of breviscapine low, medium and high dose groups and nimodipine groups decreased significantly. Meanwhile, breviscapine could significantly reduce the expression level of the TLR4/MyD88/NF-κB in brain tissue and CYP3A4 in different intestinal segments of rats with cerebral ischemia-reperfusion injury. In addition, breviscapine also significantly ameliorated intestinal flora dysbiosis of rats with cerebral ischemia-reperfusion injury. CONCLUSIONS: Breviscapine can protect rats from cerebral ischemia-reperfusion injury by regulating intestinal flora, inhibiting brain TLR4/MyD88/NF-κB inflammatory pathway and intestinal CYP3A4 expression.

Breviscapine Combined with BMSCs Reduces Aß Deposition in Rat with Alzheimer's Disease by Regulating Circular RNA ciRS-7.

AIMS: This study aimed to clarify that breviscapine combined with bone marrow mesenchymal stem cells (BMSCs) treatment can reduce Aß deposition in Alzheimer's disease (AD) patients. BACKGROUND: AD is a common degenerative disease of the central nervous system. Aß protein deposition in the cerebral cortex and hippocampus causes neuronal peroxidation damage, synaptic dysfunction, neuroinflammation, and nerve cell apoptosis, and ultimately leads to AD. OBJECTIVE: To investigate whether breviscapine combined with BMSCs treatment can reduce Aß deposition in AD. METHODS: The AD rat model was successfully induced by Aß1-42. The expression of protein and mRNA was detected by western blot and reverse transcription-quantitative PCR (RT-qPCR), respectively. RESULTS: In AD rat brain tissue, the expression of circular RNA ciRS-7 (ciRS-7), ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), and NF-kappaB p65 was significantly downregulated, and the expression of ß-amyloid precursor protein (APP), ß-site APPcleaving enzyme 1 (BAEC1), and Aß was upregulated. The expression of ciRS-7, UCHL1, and p65 was significantly upregulated after breviscapine or BMSCs treatment, and there was increased APP and BAEC1 degradation. Notably, breviscapine combined with BMSCs treatment was more effective than either treatment alone. In SH-SY5Y cells, overexpression of ciRS-7 reduced Aß deposition by upregulating UCHL1 to degrade APP and BAEC1, but these effects were reversed with inhibition of NF-kB signaling. Finally, knockdown of ciRS-7 elevated Aß, APP, and BAEC1 expression in each group of rats compared with the control. CONCLUSION: Breviscapine combined with BMSCs treatment can reduce Aß deposition in AD rats and promote the degradation of APP and BAEC1 by activating NF-kB to promote UCHL1 expression.

Scutellarin Mediates Cytochrome P450 3A4 and 2C19 Expression via Pregnane X Receptor and Constitutive Androstane Receptor.

BACKGROUND: Breviscapine is a flavonoid extracted from Erigeron breviscapus (Vant.) Hand.-Mazz., and mainly contains scutellarin. Nuclear receptors play important roles in regulating transporter and drug metabolic enzymes. OBJECTIVE: To investigate the regulatory effects of scutellarin on CYP3A4 and 2C19 in HepG2 and Caco-2 cells based on nuclear receptors PXR and CAR. METHODS: The proteins and mRNA levels of CYP3A4 and CYP2C19 treated with scutellarin were detected by Western Blot and RT-qPCR. Using assays of the dual-luciferase reporter, promoter sequences containing hPXR and hCAR protein recognition and binding regulatory elements CYP3A4 and CYP2C19 were inserted upstream of the reporter gene, and the expression vector and the reporter vector were cotransfected into HepG2 and Caco-2 cells. RESULTS: Scutellarin inhibited mRNA of CYP3A4 and PXR, and promoted mRNA expression of CYP2C19 and CAR in RT-qPCR results. Western-blot results showed scutellarin inhibited the expression of CYP3A4 and promoted the expression of CYP2C19. The dual-luciferase reporter genes showed that scutellarin enhanced the expression level of CYP2C19, and when its concentration was 40 and 80µmol/L, CYP3A4 was significantly increased. CONCLUSION: Scutellarin down-regulates CYP3A4 through PXR, and its mechanism may work by up-regulating CAR, binding to PXR to inhibit PXR-mediated expression of CYP3A4. Scutellarin up-regulates CYP2C19 through CAR.

Protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism / 中国当代儿科杂志

OBJECTIVES@#To study the protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism.@*METHODS@#A preterm rat model of brain injury caused by intrauterine inflammation was prepared by intraperitoneal injections of lipopolysaccharide in pregnant rats. The pregnant rats and preterm rats were respectively randomly divided into 5 groups: control, model, low-dose breviscapine (45 mg/kg), high-dose breviscapine (90 mg/kg), and high-dose breviscapine (90 mg/kg)+ML385 [a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg] (n=10 each). The number and body weight of the live offspring rats were measured for each group. Hematoxylin-eosin staining was used to observe the pathological morphology of the uterus and placenta of pregnant rats and the pathological morphology of the brain tissue of offspring rats. Immunofluorescent staining was used to measure the co-expression of ionized calcium binding adaptor molecule-1 (IBA-1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex of offspring rats. ELISA was used to measure the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) in the brain tissue of offspring rats. Western blotting was used to measure the expression of Nrf2 pathway-related proteins in the brain tissue of offspring rats.@*RESULTS@#Pathological injury was found in the uterus, and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, and severe microglia pyroptosis occurred in the cerebral cortex of the offspring rats in the model group. Compared with the control group, the model group had significant reductions in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain tissue of the offspring rats (P<0.05), but significant increases in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). Compared with the model group, the breviscapine administration groups showed alleviated pathological injury of the uterus and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, significant increases in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and HO-1 in the brain tissue of the offspring rats (P<0.05), and significant reductions in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). The high-dose breviscapine group had a significantly better effect than the low-dose breviscapine (P<0.05). ML385 significantly inhibited the intervention effect of high-dose breviscapine (P<0.05).@*CONCLUSIONS@#Breviscapine can inhibit inflammatory response in brain tissue of preterm rats caused by intrauterine inflammation by activating the Nrf2 pathway, and it can also inhibit microglial pyroptosis and alleviate brain injury.

Breviscapine remodels myocardial glucose and lipid metabolism by regulating serotonin to alleviate doxorubicin-induced cardiotoxicity.

Aims: The broad-spectrum anticancer drug doxorubicin (Dox) is associated with a high incidence of cardiotoxicity, which severely affects the clinical application of the drug and patients' quality of life. Here, we assess how Dox modulates myocardial energy and contractile function and this could aid the development of relevant protective drugs. Methods: Mice were subjected to doxorubicin and breviscapine treatment. Cardiac function was analyzed by echocardiography, and Dox-mediated signaling was assessed in isolated cardiomyocytes. The dual cardio-protective and anti-tumor actions of breviscapine were assessed in mouse breast tumor models. Results: We found that Dox disrupts myocardial energy metabolism by decreasing glucose uptake and increasing fatty acid oxidation, leading to a decrease in ATP production rate, an increase in oxygen consumption rate and oxidative stress, and further energy deficits to enhance myocardial fatty acid uptake and drive DIC development. Interestingly, breviscapine increases the efficiency of ATP production and restores myocardial energy homeostasis by modulating the serotonin-glucose-myocardial PI3K/AKT loop, increasing glucose utilization by the heart and reducing lipid oxidation. It enhances mitochondrial autophagy via the PINK1/Parkin pathway, eliminates damaged mitochondrial accumulation caused by Dox, reduces the degree of cardiac fibrosis and inflammation, and restores cardiac micro-environmental homeostasis. Importantly, its low inflammation levels reduce myeloid immunosuppressive cell infiltration, and this effect is synergistic with the anti-tumor effect of Dox. Conclusion: Our findings suggest that disruption of the cardiac metabolic network by Dox is an important driver of its cardiotoxicity and that serotonin is an important regulator of myocardial glucose and lipid metabolism. Myocardial energy homeostasis and timely clearance of damaged mitochondria synergistically contribute to the prevention of anthracycline-induced cardiotoxicity and improve the efficiency of tumor treatment.

Preparation, Characterization, and Evaluation of Breviscapine Nanosuspension and Its Freeze-Dried Powder.

As a biopharmaceutics classification system (BCS) class IV drug, breviscapine (Bre) has low solubility in water, poor chemical stability, a short biological half-life and rapid removal from plasma. This paper prepared a Bre nanosuspension (Bre-NS) by an ultrasound-assisted anti-solvent precipitation method. Characterization of Bre-NS was studied using a Box-Behnken design concerning drug concentration in DMSO, an anti-solvent-to-solvent ratio, and sonication time. Under the optimized conditions of 170 mg/mL for the drug concentration, a 1:60 solvent-to-anti-solvent ratio, and a 9 min sonication time, the particle size of Bre-NS was 303.7 ± 7.3 nm, the polydispersity index was 0.178 ± 0.015, and the zeta potential was -31.10 ± 0.26 mV. Combined with the results from differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform-infrared spectroscopy (FT-IR), the findings indicated that the crystal form and chemical structure of Bre-NS did not change during the entire process. The optimized formulation displayed good stability, increased solubility, and better in vitro release. Therefore, the results of this study can be a reference for the delivery system design of insoluble active components and effective parts in traditional Chinese medicine.

Advances in Chemical Constituents, Clinical Applications, Pharmacology, Pharmacokinetics and Toxicology of Erigeron breviscapus.

Dengzhanxixin (DZXX), the dried whole plant of Erigeron breviscapus (Vaniot) Hand.-Mazz., belonging to Compositae and first published in Materia Medica of South Yunnan by Lan Mao in the Ming Dynasty (1368 AD-1644 AD), is included in Medicinal Materials and Decoction Pieces of the 2020 edition of the Pharmacopeia of the People's Republic of China. Its main chemical components are flavonoids that mainly include flavonoid, flavonols, dihydroflavones, flavonol glycosides, flavonoid glycosides, coffee acyl compounds, and other substances, such as volatile oil compounds, coumarins, aromatic acids, pentacyclic terpenoids, phytosterols, and xanthones. Among them, scutellarin and 1,5-dicoffeoylquininic acid are the main active components of DZXX. DZXX has pharmacological effects, such as improving cerebral and cerebrovascular ischemia, increasing blood flow, inhibiting platelet aggregation, promoting antithrombotic formation, improving microcirculation, reducing blood viscosity, protecting optic nerves, exhibiting anti-inflammatory properties, scavenging free radicals, and eliciting antioxidant activities. It is widely used in the treatment of cardiovascular and cerebrovascular ischemic diseases, kidney diseases, liver diseases, diabetic complications, and glaucoma. Pharmacokinetic studies have shown that the active components of DZXX have a low bioavailability and a high elimination rate in vivo. Nevertheless, its utilization can be improved through liposome preparation and combination with other drugs. Acute and subacute toxicity studies have shown that DZXX is a safe medicinal material widely used in clinical settings. However, its target and drug action mechanism are unclear because of the complexity of its composition. In this paper, the clinical application and pharmacological toxicology of DZXX are reviewed to provide a reference for further studying its active components and action mechanism.

Breviscapine: A Review on its Phytochemistry, Pharmacokinetics and Therapeutic Effects.

Breviscapine is one of the extracts of several flavonoids of Erigeron breviscapus. Scutellarin is the main active component of breviscapine, and the qualitative or quantitative criteria as well. Scutellarin and its analogs share a similar skeleton of the flavonoids. Breviscapine has been widely used in the treatment of cerebral infarction and its sequelae, cerebral thrombus, coronary heart disease (CHD), and angina pectoris. Breviscapine has a broad spectrum of pharmacological activities, such as increasing blood flow, improving microcirculation, dilating blood vessels, decreasing blood viscosity, promoting fibrinolysis, inhibiting platelet aggregation, and thrombosis formation, etc. In addition, breviscapine and its analogs have significant value for drug research and development because of the superiority of those significant bioactivities. Furthermore, an increasing number of pharmacokinetic studies have explored the mechanism of scutellarin and its analogs. To provide a comprehensive understanding of the current research on breviscapine, scutellarin, and the analogs, the structural features, distribution situation, preparation method, content determination method, clinical applications, pharmacological action as well as pharmacokinetics are summarized in the present review.

Anticancer mechanism of breviscapine in non-small cell lung cancer A549 cells acts via ROS-mediated upregulation of IGFBP4.

BACKGROUND: The overall 5-year survival rate of non-small cell lung cancer (NSCLC) is less than 15% because of multiple drug resistance to chemotherapy and the limitations of early diagnosis. Thus, safe and effective drugs to treat NSCLC are required. The present study aimed to investigate the effects of breviscapine (BVP) on NSCLC cell apoptosis and proliferation, and to study its possible mechanisms. METHODS: Using the NSCLC A549 cell line and BVP (0, 25, 50, and 100 µM), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect A549 cell proliferation, and flow cytometry was used to assess cell apoptosis. Insulin-like growth factor binding protein 4 (IGFBP4) levels was assessed using enzyme-linked immunosorbent assays and western blotting. Flow cytometry of hydrogen peroxide and superoxide was used to assess intracellular reactive oxygen species (ROS) generation. Western blotting was used to assess the levels of BCL2-associated X, apoptosis regulator (BAX) and B-cell CLL/lymphoma 2 (BCL2). Quantitative real-time reverse transcription PCR (qRT-PCR) was used to assess IGFBP4 mRNA expression. RESULTS: BVP induced apoptosis, inhibited cell proliferation, and increased ROS in A549 cells. Western blotting and qRT-PCR showed that BVP increased IGFBP4 protein and mRNA expressions in A549 cells. Compared with BVP treatment alone, IGFBP4 expression decreased in A549 cells treated with BVP and the ROS scavenger N-acetylcysteine. IGFBP4 overexpression increased BVP-induced proliferation inhibition, while increasing BAX expression and decreasing BCL2 expression. Silencing IGFBP4 had the opposite effects. CONCLUSIONS: BVP could inhibit the growth of NSCLC A549 cells by promoting apoptosis via ROS-mediated upregulation of IGFBP4.