Scutellarin alleviates complete freund’s adjuvant-induced rheumatoid arthritis in mice by regulating the Keap1/Nrf2/HO-1 pathway

Scutellarin(SCU)is a herbal flavonoid glucuronide with multiple pharmacological activities,including antioxidant,anti-inflammation,vascular relaxation,anti-platelet,and myocardial protection.However,the effect of SCU on complete Freund’s adjuvant(CFA)-induced rheumatoid arthritis(RA)had not been studied.In this study,we investigated the beneficial effects of SCU in the CFA-induced RA mice model and the anti-arthritic activity was evaluated by paw edema.Enzyme-linked immunosorbent assay(ELISA)was carried out to evaluate the plasma levels of immunoglobulin(Ig)G,IgE,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,receptor activator of nuclear factor-κB ligand(RANKL),and osteoprotegerin(OPG).Histological slides were prepared from the harvested paws of mice to determine the pathological changes in the joints.The proportions of T helper type 1(Th1)and T helper type 2(Th2)cells of CD4+T lymphocyte subsets were analyzed by flow cytometry.The expression of Kelch-like ECHassociated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)was analyzed using real-time quantitative PCR(RT-qPCR)and western blotting assays.The present study demonstrated that SCU prevented CFA-induced RA,and inhibited the expression of inflammation factors,IgG,IgE,TNF-α,IL-1β,and IL-6.While SCU also reduced the RANKL level,it increased OPG expression in RA mice.The Th1/Th2 ratio was significantly lower in mice treated with SCU.Additionally,HO-1 expression was reduced while the expression of Keap1 and Nrf2 was elevated following SCU treatment.Results provide preliminary evidence to employ SCU in arthritis treatment which might be related to the regulation of Th1/Th2 balance and the Keap1/Nrf2/HO-1 pathway.

Determination of Scutellarin in Rabbit Plasma after Oral Administration by HPLC-MS with Solid-Phase Extraction

Aim To establish a sensitive and specific liquid chromatography-massspeetrometry method for determination of scutellarin in rabbit plasma after oral administration.Methods For the quantitative analysis, rutin was used as an internal standard and solid-phaseextraction (SPE) was performed by using a Phenomenex C_8 cartridge. HPLC was carried out using aZorbax Extend-C_(18) column (150 mm x 2.1 mm ID, 5 μm) with a guard cartridge (Phenomenex) .Gradient elution was selected with the mobile phase of methanol 10 mmol·L^(-1) ammonium acetatesolution (pH adjusted to 8.0 with ammonia solution). The flow rate of mobile phase was 0.4mL·min^(-1) and the column temperature was 35 ℃ . Both scutellarin and the internal standard rutinin rabbit plasma extracts were detected by mass spectrometry using an ESI interface in the negativeion mode. Results The linear range was from 2 to 200 ng· mL^(-1), with acceptable accuracy andprecision (RSD) . Conclusion A sensitive, reliable and accurate method for the quantitation ofscutellarin in rabbit plasma has been established.

Pharmacokinetics of Scutellarin in Dogs

Aim To establish an RP-HPLC method for the determination of scutellarin in plasma and study its pharmacokinetics in dogs. Methods Scutellarin was given to dogs by intravenous injection and determined by RP-HPLC, the mean plasma concentration-time curve was plotted and pharmacokinetic parameters were calculated by program 3p87. Resu;ts The concentration-time curve of scutellarin could be fitted to three-compartment model with T1/2 pi, T1/2 α and T1/2 β being 1.05 ± 0.80 min, 6.99 + 2.76 min and 51.61 + 28.78 min, respectively, Vc being 880.1 + 508.3 mL, CL being 189.6 + 53.8 mL@ min- 1, and AUC0-90 and AUC0-∞ being 574.43 + 133.95 μg@ min@ mL - 1 and 599.34 ± 132.00μg@ min@mL- 1, respectively. Conclusion The fact that the concentrations of scutellarin in plasma declined rapidly after the medication suggested that the T1/2 of scutellarin should be taken into account in drug administration and preparation development.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2） was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco＇s modified Eagle＇s medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-＇reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine （8-OHdG）, 4-hydroxy-2-nonenal （4-HNE） and 3-nitrotyrosin （3-NT） in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

Formulation optimization of scutellarin-loaded HP-β-CD/chitosan nanoparticles using response surface methodology with Box–Behnken design

The aim of this paper is to investigate and optimize the preparation of scutellarin(SCU)-loaded HP-β-CD/chitosan(CS) nanoparticles(CD/CS-SCU-NPs). CD/CS-SCU-NPs were prepared by ionic cross-linking method and the process and formulation variables were optimized using response surface methodology(RSM) with a three-level, three factor Box–Behnken design(BBD).The independent variables were the added amounts of CS, sodium tripolyphosphate(TPP)and Pluronic F-68 during the preparation. Dependent variables(responses) were particle size and entrapment efficiency. Mathematical equations and respond surface plots were used to correlate independent and dependent variables.The preparation process and formulation variables were optimized to achieve minimum particle size and maximum entrapment efficiency by calculating the overall desirability value(OD). The optimized NP formulation was characterized for particle size, PDI, zeta potential, entrapment efficiency and in vitro drug release.According to the results, an optimized CD/CS-SCU-NP formulation was prepared. Results for particle size, PDI, zeta potential and entrapment efficiency were found to be around 200 nm,0.5, 25 mV, and 70% respectively. For in vitro study, the release of SCU from the NPs exhibited a biphasic release and was in accordance with Higuchi equation. The optimized preparation was simple with the probability for industrialization. The combination use of RSM, BBD and overall desirability values could provide a promising application for incorporating CD into CS nanoparticles as drug delivery carrier and help develop lab-scale procedures.

Synthesis and Characterization of PEG-scuteilarin Conjugates,a Potential PEG Ester Prodrug for the Oral Delivery of Scutellarin

Highly water soluble esters of scutellarin with variable molecular weight polyethylene glycol （PEG） were prepared via PEGylation. The physicochemical properties and the stabilities under different conditions were investigated. By PEG modification, the greatly increased water solubility and desirable partition coefficient of scuteUarin were obtained, and the results showed that these conjugates were potential prodrugs for the oral delivery of scuteUarin.

Application of the Reversed-phase Liquid Chromatography Coupled to Ion Trap Mass Spectrometry with Principal Components Analysis for Metabonomics Studies of Scutellarin in Rat Urine

Metabonomics, a novel systemic approach, was applied to studies of Traditional Chinese Medicine scutellarin in rat urine. The liquid chromatography coupled with ion trap mass spectrometry combined with PCA was used in this paper. With this methodology, two potential metabolites of scutellarin were detected and the nine ions responsible for the gender variation and one ion for the dosage variation were found.

Scutellarin Reduces Cerebral Ischemia Reperfusion Injury Involving in Vascular Endothelium Protection and PKG Signal

Flavonoid glycoside scutellarin(SCU)has been widely applied in the treatment of cerebral ischemic diseases in China.In this article,we conducted research on the working mechanisms of SCU in hypoxia reoxygenation(HR)injury of isolated cerebral basilar artery(BA)and erebral ischemia reperfusion(CIR)injury in rat models.In isolated rat BA rings,HR causes endothelial dysfunction(ED)and acetylcholine(ACh)induces endothelium-dependent vasodilation.The myography result showed that SCU(100μM)was able to significantly improve the endothelium-dependent vasodilation induced by Ach.However,SCU did not affect the ACh-induced relaxation in normal BA.Further studies suggested that SCU(10-1000μM)dose-dependently induced relaxation in isolated BA rings which were significantly blocked by the cGMP dependent protein kinase(PKG)inhibitor Rp-8-Br-cGMPs(PKGI-rp,4μM).Pre-incubation with SCU(500μM)reversed the impairment of endothelium-dependent vasodilation induced by HR,but the reversing effect was blocked if PKGI-rp(4μM)was added.The brain slice staining test in rats’model of middle cerebral artery occlusion(MCAO)induced CIR proved that the administration of SCU(45,90 mg/kg,iv)significantly reduced the area of cerebral infarction.The Western blot assay result showed that SCU(45 mg/kg,iv)increased brain PKG activity and PKG protein level after CIR surgery.In conclusion,our findings suggested that SCU possesses the ability of protecting brain cells against CIR injury through vascular endothelium protection and PKG signal.

Effects of scutellarin on PKG in HCMECs after hypoxia roxygenation

OBJECTIVE This study discusses the effects of scutellarin(SCU),which is one of effective component of Erigeron breviscapus(Vant.)Hand-Mazz,on cGMP dependent protein kinase(PKG)in human cardiac microvascular endothelial cells(HCMECs)after hypoxia roxygenation(HR).METHODS Based on a model of HCMECs cells with HR injury,cell viability is examined by MTT assay.Protein expression of PKG is analyzed by Western blotting and its enzyme activity is investigated by ELISA technology.RESULTS The results of MTT assay- indicate that SCU(1,10μmol·L1)could protect HCMECs against HR injury.SCU(0.1,1 and 10μmol·L-1)canenhance PKG-I expression in control and HR injury cells.Furthermore,SCU(1,10μmol·L-1 significantly increase PKG activity in control cells(P<0.01),and also SCU(100μmol·L-1)appears similar on enzyme activity in HR injury cells(P <0.05).CONCLUSION SCU appears protective effects on endothelial cells against HR damage.While its mechanisms may be related to the influence of SCU on PKG activity in HCMECs.

Protective effects of scutellarin on vascular dysfunction caused by hypertension in SHR rats

OBJECTIVE To investigate the effect of scutellarin(SCU),which is the main effective component of Erigeron breviscapus(Vant.)Hand-Mazz native to Yunnan in China,on vascular dysfunction(VD)of cardiac coronary artery(CA)and cerebral basilar artery(BA)caused by hypertension in SHR rats.METHODS 1.BA and CA vesselsrings from 40 weeks of SHR rats were isolated and equilibrated in organ bath with MOPS-PSS buffer and ring tension was recorded,comparing with a normal control of WKY rats.SCU was treated accumulatively after pre-contracted with vasoconstrictor U46619(1μmol·L-1).2.ACH and SNP were treated accumulatively after pre-incubation with SCU(300μmol·L-1,100μmol·L-1)and pre-contracted by U46619(1μmol·L-1),K+60mmol·L-1,respectively.While U46619 was added accumulatively to BA/CA rings pre-incubated with SCU(300and 100μmol·L-1).ACH-induced relaxation rate was to evaluate endothelium-dependent relaxation,and SNP-induced relaxation rate to evaluate the artery non-endothelium-dependent relaxation.RESULTS SCU significantly dilated both BA and CA rings pre-contracted by U46619 in old rats.EC50 value of SCU in WKY ratswas less than that in SHR(P<0.05),which showing VD of CA and BA in SHR rats.Compared with WKY group,ACH relaxation curve of SHR group shifted to the right,suggesting that hypertension induced VD.When SCU 300μmol·L-1 pre-treated CA groups and SCU 100μmol·L-1 pre-treated BA groups,EC50 to ACH was significantly lower(P<0.05).Likewise,the vasodilatation of CA/BA rings to SNP was also improved obviously when pre-treated with SCU,and Emax to SNP was decreased significantly(P<0.05).Moreover,EC50 to U46619was significantly lower when pre-treated by SCU.CONCLUSION In SHR rats,SCU antagonized U46619 on CA/BA rings in a noncompetitive manner.Furthermore,SCU should appear to protect VD induced by hypertension.

Inhibition of self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells by scutellarin via Hedgehog signaling pathway

OBJECTIVE To investigate the inhibitory effect of scutellarin on the self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells(HT-29CSC)in vitro and in vivo,and to explore its mechanism.METHODS The effect of scutellarin on the growth of HT-29CSC was determined by 3D Culture assay.The effect of scutellarin on growth and transformation of HT-29CSC was probed by soft agar colony formation assay.The effect of scutellarin on the differentiation of HT-29CSC was determined by serum induction differentiation assay in vitro.The effects of scutellarin on the expressions of marker gene Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog gene were measured by quantitative real-time RT-PCR.Investigate the effect of scutellarin on the expression of c-Myc,Gli1,and Lgr5 protein by Western blotting.A subcutaneous xenograft model of colon cancer in nude mice was established and administered by intraperitoneal injection.The change of body weight and tumor size of nude mice were observed every two days.Investi⁃gate the effects of scutellarin on the growth of xenograft tumors in nude mice.The expression of CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,Nanog gene in tumors were measured by quantitative real-time RT-PCR.The expression of c-Myc,Gli1,Lgr5,CD133,Ki67 protein were measured by Western blotting.RESULTS Scutellarin can inhibit the growth of HT-29CSC in 3D culture.Compared with the solvent control group,scutellarin can significantly inhibit the growth and transformation and differentiation of HT-29CSC in vitro(P<0.01).The expression levels of marker genes Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog in HT-29CSC were down-regulated by scutellarin.Scutellarin can reduce the expression of c-Myc,Gli1,and Lgr5 protein in HT-29CSC.Scutellarin can inhibit the growth of colon cancer xenografts,lower CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,and Nanog mRNA level of xenograft tumors,reduce the expression of c-Myc,Gli1,Lgr5,CD133,and Ki67 protein of xenograft tumors in nude mice.CONCLUSION Scutellarin,which is the main component of scutellaria barbata,can inhibit the differentiation of HT-29CSC and the mechanism is to inhibit the activity of Hedgehog signaling pathway.

Neuroprotective effect of Angiopep-2 peptide modified scutellarin-loaded PEGylated PAMAM dendrimer nanoparticles on ischemic stroke by modulating the Toll-like receptors-dependent MyD88/IKK/NF-κB signaling pathway

OBJECTIVE The greatest challenge in chemotherapy of ischemic stroke is the construction a suitable delivery system to overcome the poor physicochemical properties of drug and its low permeability across the blood brain barrier(BBB).METHODS In the present study,dendrimer,polyamidoamine(PAMAM),was synthesized as the nano-drug carriers.Angiopep-2,which has been proved excellent ability to cross the BBB,was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethylene glycol(PEG).Then scutellarin(STA)was encapsulated into the functionalized nanoparticles(NPs)to formulate Angiopep-2 modified STA-loaded PEG-PAMAM NPs.Ischemic stroke model was established to evaluate the treatment efficacy and protective mechanism of Angiopep-2-STA-PEG-PAMAM NPs.RESULTS The pharmacokinetics and biodistribu-tion demonstrated that Angiopep-2-STA-PEG-PAMAM NPs exhibited significantly higher plasma concentration from 1 h to 10 h after intravenous administration and improve accumulation in brain(4.7-fold)compared with STA solution.Moreover,prolonged elimination half-life(4.8-fold)and lower clearance(3.4-fold)were observed.The brain uptake study of 6-coumarin confirmed that Angiopep-2-PEG-PAMAM NPs possessed better brain targeting efficacy(3.2-fold)than PEG-PAMAM NPs.Angiopep-2-STA-PEG-PAMAM NPs obviously ameliorated infarct volume,neurological deficit,histopathological severity and neuronal apoptosis.In addition,Angiopep-2-STA-PEG-PAMAM NPs markedly inhibited the calcium content and the levels of IL-12p40,IL-13,IL-17 and IL-23.Furthermore,Angiopep-2-STA-PEG-PAMAM NPs significantly decreased the m RNA and protein expressions of HMGB1,TLR2,TLR4,TLR5,My D88,TRIF,TRAM,IRAK-4,TRAF6,IкBα,IKKβand NF-кBp65.CONCLUSION The results suggested that Angiopep-2modified scutellarin-loaded PEG-PAMAM nanocarriers possessed remarkable neuroprotective effects on ischemic stroke through modulation of inflammatory cascades and HMGB1/TLRs/MyD 88-induced NF-κB activation pathways.

Flavonoid scutellarin positively regulates root length through NUTCRACKER

Exploring approaches to regulate meristem is of special importance and broad interest.In this study,we found that the flavonoid scutellarin,which has a 6-hydroxyl and a 7-glucoside,increased root length through the transcription factor NUTCRACKER(NUC).This root lengthening disappeared in NUCknockout and reappeared in NUC-rescue plants.Scutellarin induced NUC expression and promoted the division of cortex/endodermal initials.In contrast,naringenin,which has same chemical backbone but without 6-hydroxyl and with 7-hydroxyl group,showed the opposite or no effects.Our results demonstrate that scutellarin promotes root length through NUC-mediated regulatory pathways and reveal that flavonoids with and without the 6-hydroxyl and 7-glucoside have positive and negative effects on meristem size,respectively。

Suppression of AOM/DSS-induced colorectal cancer by scutellarin through downregulation of Wnt signaling pathway activity

OBJECTIVE To investigate the therapeutic effect of scutellarin on colitis-associated cancer(CAC)and its underlying mechanism based on Wnt/β-catenin signaling pathway.METHODS The mouse model of CAC was established by azomethane oxide(AOM)and sodium dextran sulfate(DSS),followed by scutellarin treatment,with recording the body weight,diarrhea and hematochezia.After sacrificing the mice,the colorectal length and colorectal tumor were assessed.The levels of pro-inflammatory factors TNF-αand IL-6 in mice′s sera were measured by the enzyme-linked immunosorbent assay(ELISA).The colorectal lesions were appraised by hematoxylin and eosin(H&E)staining.Theβ-catenin level in CAC tissues was probed by immunofluorescent analysis.The apoptosis-related genes Bax and Bcl-2,and Wnt signaling pathway-related genesβ-catenin,GSK-3β,TCF4,c-Myc and cyclin D1 were detected by real-time quantitative RT-PCR(RT-qPCR).Finally,Western blotting analysis(WB)was employed to examine the expressions of the apoptosis and Wnt signaling pathway-related proteins.RESULTS Scutellarin significantly improved AOM/DSS-caused weight loss,colorectal length shortening,and tumor growth in mice(P<0.01).Meanwhile,colorectal lesions could be substantially alleviated by scutellarin.ELISA results showed that the levels of pro-inflammatory factors TNF-αand IL-6 were drastically lessened(P<0.01).Scutellarin also sharply inhibited the nuclear translocation ofβ-catenin,as evidenced by the reduction in the nuclear level ofβ-catenin protein.In addition,scutellarin attenuated the mRNA expression of Wnt signaling pathway-relatedβ-catenin,TCF4,c-Myc and cyclin D1,whereas it heightened GSK-3βmRNA level.These results were consolidated by WB analysis,which indicated that scutellarin could mitigate the protein levels of phospho-GSK-3β,β-catenin,TCF4,c-Myc and cyclin D1,with the increase in GSK-3βprotein in CAC tissue.Moreover,scutellarin could induce the apoptosis of CAC,demonstrated by enhanced expression of Bax and diminished expression of Bcl-2 in both mRNA and protein levels.CONCLUSION Scutellarin may ameliorate colitis-associated colorectal cancer by weakening Wnt/β-catenin signaling cascade.

Scutellarin induces apoptosis of colorectal cancer HCT116 cells via Hippo signaling pathway

OBJECTIVE To investigate the effect of scutellarin on the apoptosis of human colorectal cancer cells via the Hippo signaling pathway in vitro.METHODS MTT colorimetric method was used to detect the influence of scutellarin on the survival rate of HCT116 cells.And the effect of scutellarin at various concentrations on cell morphology was observed by microscopy.Cell scratch experiment was used to detect the influence of scutellarin on the migration of HCT116 cells.Hoechst33342/PI double staining method was used to detect the effect of scutellarin on the apoptosis of HCT116 cells.Western blotting method was used to assess the action of scutellarin on the expressions of Hippo signaling pathway-related proteins Mst1,Lats1,YAP1,p-YAP(Ser127),TAZ,and its downstream effector proteins c-Myc and cyclin D1,as well as apoptosis-related proteins Bcl-2 and Bax in HCT116 cells.RESULTS Scutellarin significantly affected the morphology of HCT116 cells and reduced the survival rate of HCT116 cells.Hoechst33342/PI double staining showed that scutellarin effectively induced the apoptosis of HCT116 cells.Western blotting analysis showed that the expression levels of Hippo signaling pathway-related proteins Mst1,Lats1,YAP1,TAZ and its downstream effector proteins c-Myc,cyclin D1 were down-regulated in a concentration-dependent manner by scutellarin,and the expression of p-YAP(ser127)was up-regulated.Moreover,scutellarin substantially lessened the expression level of apoptosis-related protein Bcl-2,and promoted the protein level of Bax.CONCLUSION Scutellarin may inhibit the proliferation and migration of HCT116 cells,while induce its apoptosis,potentially by activation of Hippo signaling pathway.

Scutellarin prevents acute alcohol-induced liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and inhibiting inflammation by regulating the AKT,p38 MAPK/NF-κB pathways

Alcoholic liver disease(ALD)is the most frequent liver disease worldwide,resulting in severe harm to personal health and posing a serious burden to public health.Based on the reported antioxidant and anti-inflammatory capacities of scutellarin(SCU),this study investigated its protective role in male BALB/c mice with acute alcoholic liver injury after oral administration(10,25,and 50 mg/kg).The results indicated that SCU could lessen serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels and improve the histopathological changes in acute alcoholic liver;it reduced alcohol-induced malondialdehyde(MDA)content and increased glutathione peroxidase(GSH-Px),catalase(CAT),and superoxide dismutase(SOD)activity.Furthermore,SCU decreased tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and IL-1βmessenger RNA(mRNA)expression levels,weakened inducible nitric oxide synthase(iNOS)activity,and inhibited nucleotide-binding oligomerization domain(NOD)-like receptor protein 3(NLRP3)inflammasome activation.Mechanistically,SCU suppressed cytochrome P450 family 2 subfamily E member 1(CYP2E1)upregulation triggered by alcohol,increased the expression of oxidative stress-related nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)pathways,and suppressed the inflammation-related degradation of inhibitor of nuclear factor-κB(NF-κB)-α(IκBα)as well as activation of NF-κB by mediating the protein kinase B(AKT)and p38 mitogen-activated protein kinase(MAPK)pathways.These findings demonstrate that SCU protects against acute alcoholic liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and suppressing inflammation by regulating the AKT,p38 MAPK/NF-κB pathways.

Scutellarin alleviates liver injury in type 2 diabetic mellitus by suppressing hepatocyte apoptosis in vitro and in vivo

Objective: Scutellarin is a primary active composition come from Erigeron breviscapus. It is well known that scutellarin has anti-infammatory and antioxidant physiological functions. In this study, we detected the effects of scutellarin on hepatocyte cell apoptosis in type 2 diabetes mellitus(T2DM) rats.Methods: Sprague Dawley(SD)(6–8 weeks, 160–180 g) rats were randomly divided into six groups: control, model, scutellarin low-dose, medium-dose, high-dose treatment, and rosiglitazone positive groups;with 10 SD rats in each group(n = 10). The changes of biochemical factors in serum were detected by automatic biochemical instrument, the pathological changes of liver tissue were detected by hematoxylin and eosin(HE) staining, the apoptosis of liver tissue and cells was detected by tissue staining and fow analyzer, and the expression of apoptosis-related factors were determined by q PCR, Western blot and immunohistochemistry in liver tissues or cells.Results: The results showed that scutellarin decreased the levels of fasting blood glucose, total cholesterol, triglyceride, and low-density lipoprotein and increased the levels of high-density lipoprotein.Meanwhile, scutellarin decreased the levels of alanine transaminase(ALT) and aspartate transaminase(AST) and improved liver function. In addition, scutellarin suppressed the secretion of interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-a(TNF-a) and reduced hepatocyte apoptosis.Furthermore, scutellarin inhibited the expression of cleaved Caspase-3, Bax, and cytochrome C(Cyt-C)and promoted the expression of Bcl-2.Conclusion: Scutellarin can inhibit the apoptotic pathway, thereby relieving T2DM.

一测多评法同时测定半枝莲配方颗粒中5种黄酮类成分含量

目的建立同时测定半枝莲配方颗粒中野黄芩苷、黄芩苷、野黄芩素、柚皮素、芹菜素5种黄酮类成分含量的一测多评法。方法半枝莲配方颗粒经80%甲醇超声提取,色谱柱为Agilent Eclipse XDB C_(18)柱(250 mm×4.6 mm,5µm),流动相为甲酸水溶液(pH 2.5)-甲醇(65∶35,V/V),流速为0.8 mL/min,柱温为30℃,检测波长为280 nm,进样量为10µL。以野黄芩苷为参照,建立与黄芩苷、野黄芩素、柚皮素、芹菜素的相对保留时间和相对校正因子,并测定9批半枝莲配方颗粒中5种黄酮类成分的含量。结果黄芩苷、野黄芩素、柚皮素、芹菜素、野黄芩苷的质量浓度分别在1.58~50.63µg/mL(r=0.999)、6.33~101.25µg/mL(r=0.999)、1.57~50.25µg/mL(r=0.998)、1.45~46.50µg/mL(r=0.998)、28.14~900.50µg/mL(r=0.997)范围内与峰面积线性关系良好;平均加样回收率分别为97.44%,96.78%,100.59%,96.01%,95.33%,RSD分别为1.79%,1.67%,2.86%,1.79%,1.35%(n=6);精密度、重复性、稳定性试验结果的RSD均小于3.00%(n=6)。不同高效液相色谱仪和色谱柱测定的黄芩苷、野黄芩素、柚皮素、芹菜素相对于野黄芩苷的相对保留时间、相对校正因子的RSD均小于3.00%(n=9)。外标法和一测多评法测定9批半枝莲配方颗粒中5种黄酮类成分的含量相对偏差均不超过1.48%(n=9)。结论该方法简单可靠、结果准确,可用于半枝莲配方颗粒中5种黄酮类成分的含量测定。

酶法制备野黄芩素的工艺研究

目的应用黄芩茎叶葡萄糖醛酸水解酶(Scutellaria baicalensis stems and leaves glucuronic hydrolase,sbsl GUS)酶解灯盏花中野黄芩苷制备野黄芩素,经分离纯化获得高纯度的野黄芩素提取物。方法采用正交试验优选灯盏花提取工艺参数;以野黄芩苷酶解转化率为指标,对酶用量、酶解pH值、温度、时间、抗氧化剂等进行考察,优选野黄芩素制备工艺参数;采用乙醇提取、活性炭脱色和分步结晶精制纯化粗提物。结果灯盏花提取工艺为灯盏花切段,加10倍水煎煮提取2次,每次1 h。野黄芩素制备工艺为sbsl GUS提取液和灯盏花水煎液加入量(以生药计)比为1∶10,加0.5%偏重亚硫酸钠,酶解pH值约6.0,温度约45℃,时间20 h,得到含量大于60%的野黄芩素粗提物。经分步结晶对粗提物进行纯化,用80%乙醇回流提取,活性炭脱色,浓缩、析晶,得到含量大于85%的野黄芩素提取物。结论该工艺应用sbsl GUS酶解技术制备野黄芩素,生产简便可行,为野黄芩素的生产制备提供了新的途径。

Scutellarin inhibits caspase-11 activation and pyroptosis in macrophages via regulating PKA signaling

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide(LPS)leading to pyroptosis that has critical role in defensing against bacterial infection,whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases.However,there are few known drugs that can control caspase-11 activation.We report here that scutellarin,a flavonoid from Erigeron breviscapus,acted as an inhibitor for caspase-11 activation in macrophages.Scutellarin dosedependently inhibited intracellular LPS-induced release of caspase-11 p26(indicative of caspase-11 activation)and generation of N-terminal fragment of gasdermin D(GSDMD-NT),leading to reduced pyroptosis.It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome as evidenced by reduced apoptosisassociated speck-like protein containing a CARD(ASC)speck formation and decreased interleukin-1 beta(IL-1 b)and caspase-1 p10 secretion,whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1 b and caspase-1 p10 release and ASC speck formation but not pyroptosis.Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression.Moreover,scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A(PKA)-specific sites,and its inhibitory action on caspase-11 activation was largely abrogated by PKAinhibitor H89 or by adenylyl cyclase inhibitor MDL12330 A.Collectively,our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.