Effects of hesperetin on memory function and apoptosis-related protein expression in senile model rats / 中华行为医学与脑科学杂志

Objective:To investigate the effect and mechanism of different doses of luteolin on memory function and apoptosis-related proteins of aging rats induced by D-galactose.Methods:Forty-eight SPF-grade male Wistar rats aged 6-8 weeks were randomly divided into control group, model group, luteolin low-dose group (25 mg/kg), medium-dose group (50 mg/kg), high-dose group (100 mg/kg), and vitamin C group (100 mg/kg), with 8 rats in each group. D-galactose (1 000 mg/kg) was subcutaneously injected to establish the aging rat model, while luteolin was used for preventive treatment. The Morris water maze test was used to evaluate the learning and memory abilities of the rats.Transmission electron microscopy was used to detect the morphology of hippocampal neurons in rats.Spectrophotometry was used to detect the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), malondialdehyde (MDA), and the total antioxidant capacity (T-AOC). RT-PCR was used to detect miR-34a mRNA expression.Western blot technique was used to detect the expression levels of silent regulator protein 1 (SIRT1), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, p53, and p21. Statistical analysis was performed using SPSS 22.0, and one-way ANOVA was used for multi-group comparison, followed by LSD- t test for further pairwise comparisons. Results:(1) The differences in escape latency among the 6 groups of rats were statistically significant ( F=120.93, P<0.001). The latency of first finding the platform location of the model group rats ((54.61±3.60) s) was higher than that of the control group ((10.54±4.27) s) ( P<0.05). The latency of first finding the platform location of rats in the low, medium and high dosage groups of luteolin ((45.50±3.81)s, (37.46±2.94) s, (32.32±3.14) s) was lower than that of the model group ((54.61±3.60) s) (all P<0.05). (2) The differences of SOD, MDA, T-AOC, TNF-α, IL-1β, and IL-6 levels in the cerebral cortex of the 6 groups of rats were all statistically significant ( F=281.636, 75.119, 208.228, 38.999, 28.428, 52.767, all P<0.001). Compared with the control group, the model group showed abnormal levels of inflammatory factors and antioxidant indexes. In the medium and high dosage groups of luteolin, the SOD and T-AOC contents in the cerebral cortex of rats were higher than those in the model group (all P<0.05), while the levels of MDA, TNF-α, IL-1β, and IL-6 were lower than those in the model group (all P<0.05). (3) The differences in relative expression levels of miR-34a mRNA among the 6 groups of rats were statistically significant ( F=81.439, P<0.001). The expression levels of miR-34a mRNA in the hippocampal tissues of rats in the luteolin treatment group were lower than those in the model group ( P<0.05). (4) The differences in protein expression levels of SIRT1, p53, and p21 in the hippocampal tissues of the 6 groups of rats were statistically significant ( F=159.946, 38.342, 123.608, all P<0.001). The expression levels of p53 and p21 in the medium and high dosage groups of luteolin were lower than those in the model group (all P<0.05), while the expression level of SIRT1 protein was higher than that in the model group ( P<0.05). (5) The differences in protein expression levels of Bcl-2 and cleaved caspase-3 in the hippocampal tissues of the 6 groups of rats were statistically significant ( F=112.659, 43.296, both P<0.05). The expression levels of Bcl-2 in the low, medium, and high dosage groups of luteolin ((0.24±0.04), (0.40±0.03), (0.48±0.05) pg/μg) were higher than those in the model group ((0.09±0.06) μg) ( P<0.05), while the expression levels of cleaved caspase-3 in the low, medium, and high dosage groups of luteolin ((0.62±0.04), (0.61±0.09), (0.51±0.10) μg) were lower than those in the model group ((0.75±0.05) μg) ( P<0.05). Conclusions:Luteolin can alleviate cellular oxidative damage through downregulating the miR-34a SIRT1/p53 signaling pathway and reducing cell apoptosis.

Study on the Inhibitory of Pyruvate-Ferredoxin Oxidoreductase by Luteolin and Its Anti-Clostridium Difficile Effect / 中药新药与临床药理

Objective To investigate the inhibitory effects of pyruvate-ferredoxin oxidoreductase(PFOR)by luteolin and its anti-Clostridium difficile effect.Methods The PFOR encoding sequence of Clostridium difficile was cloned into the expression vector pET-2a and transformed into competent Escherichia coli.The crude enzyme was prepared after induction with IPTG(Isopropyl β-D-Thiogalactoside).The inhibitory rate of the test compounds on PFOR was determined after an 8-hour anaerobic reaction between PFOR and 40 μmol·L-1 of test compounds at 25℃.The minimum inhibitory concentration(MIC)of PFOR inhibitors against C.difficile strains(ATCC BAA 1382 and ATCC BAA 1870)was determined by monitoring the OD600 of the bacterial culture.Molecular docking was performed to investigate the possible interaction mechanisms between PFOR and inhibitors.Results Among the tested compounds,the luteolin showed the strongest inhibitory activity against PFOR,with a single-point inhibition rate of approximately 33%,which is comparable to that observed with the positive inhibitor nitazoxanide(40%).Molecular docking revealed that luteolin could form hydrogen bonds with Asp428,Val431,Gly429,Asp456,Lys458,Lys459,and other residues in the PFOR domain.The MIC of luteolin against C.difficile was approximately 32 μg·mL-1.Conclusion Luteolin exhibits good activity against C.difficile,and PFOR may be a target for its antibacterial action.

Study on in vitro dissolution and in vivo pharmacokinetics of Luteolin solid dispersion / 中国药房

OBJECTIVE To explore in vitro dissolution and in vivo pharmacokinetics of Luteolin solid dispersion in Beagle dogs. METHODS The dissolution of Luteolin solid dispersion was investigated according to the second method （paddle method） of the “dissolution determination method” in the 2020 edition of Chinese Pharmacopoeia （Part Ⅳ）. UPLC-MS/MS method was established to determine the concentration of luteolin in the plasma of Beagle dogs. Twelve Beagle dogs were randomly divided into luteolin group and Luteolin solid dispersion group， with 6 dogs in each group. They were given relevant medicine orally at the dose of 10 mg/kg luteolin. Blood was collected before medication （0 h）， at 5， 10， 15， 30， 45 min and 1， 2， 4， 6， 8， 10， 12， 24， 48 h after administration. After protein precipitation with acetonitrile， the blood concentration of luteolin in Beagle dogs was determined by UPLC-MS/MS and the major pharmacokinetic parameters were calculated with non-compartmental models by using DAS 3.2.8 pharmacokinetic software. RESULTS The dissolutions of Luteolin solid dispersion in purified water and 0.1% sodium dodecyl sulfate solution was significantly higher than those of luteolin； the dissolution rate reached 95% in 0.1% sodium dodecyl sulfate solution for 120 minutes. The peak concentration （cmax） of luteolin in the Luteolin solid dispersion group of Beagle dogs was 5.62 times higher than the luteolin group， and the relative bioavailability was 348%. Compared with luteolin group， cmax and the area under the drug time curve of luteolin in the Luteolin solid dispersion group of Beagle dogs were significantly increased， while the apparent distribution volume was significantly reduced （P＜0.05）. CONCLUSIONS Luteolin solid dispersion can improve in vitro dissolution and bioavailability of luteolin in Beagle dogs.

Pueraria decoction in the treatment of osteoarthritis:network pharmacology analysis and animal model validation / 中国组织工程研究

BACKGROUND:Pueraria decoction is a famous prescription of traditional Chinese medicine,which has good anti-inflammatory and analgesic effects.The mechanism of Pueraria decoction in osteoarthritis was analyzed using network pharmacology to obtain the main therapeutic components of Pueraria decoction. OBJECTIVE:To analyze the mechanism of Pueraria decoction in the treatment of osteoarthritis through network pharmacology and animal experiments. METHODS:First,the active ingredients of Pueraria decoction were screened through the Chinese Herbal Medicine Analysis platform(TCMSP)and the genes related to osteoarthritis were collected in the GeneCards database.Second,Cytoscape software was used to construct the"active ingredient-target-disease"network diagram,explore hub genes and analyze gene expression differences.Subsequently,the therapeutic effect of luteolin,one of the main components of Pueraria decoction,was verified in a mouse model of osteoarthritis.Finally,the Kyoto Encyclopedia of Genes and Genomes(KEGG)and the gene ontology(GO)enrichment analyses of the target genes were conducted to further explore the relevant mechanisms. RESULTS AND CONCLUSION:115 active ingredients and 147 target genes related to osteoarthritis were identified.GO and KEGG analyses found that Pueraria decoction could affect osteoarthritis through a variety of reaction mechanisms and metabolic pathways.Six hub genes and compounds acting on these genes were determined.Luteolin,the main component of Pueraria decoction,could better promote cartilage repair,accelerate the decrease of typy II collagen and inhibit the expression of matrix metalloproteinase 1 and cyclooxygenase 2 in animal experiments.To conclude,Pueraria decoction contains various active ingredients to prevent the progression of osteoarthritis through oxidative stress and metabolic pathways.

Luteolin induces ferroptosis in adriamycin resistant K562/ADR cells through Nrf2/HO-1 signaling pathway / 肿瘤

Objective:To investigate the effect of luteolin(Lut)on the proliferation and ferroptosis of human adriamycin(ADR)resistant chronic myeloid leukemia(CML)K562/ADR cells and the underlying molecular mechanism. Methods:K562 and K562/ADR cells were treated with different concentrations of ADR.The sensitivity of K562 and K562/ADR cells to ADR was evaluated by CCK-8 assay;the effect of different concentrations of Lut on the proliferation of K562/ADR cells was assessed by CCK-8 assay,and the half inhibitory concentration(IC50)was calculated for subsequent experiments;the morphological changes of the cells were observed by an inverted microscope;CCK-8 assay was used to examine the sensitizing effect of Lut on ADR;FCM assay was used to study the effect of Lut on the apoptosis of K562/ADR cells;fluorescence probe DCFH-DA,Fe2+colorimetric assay and glutathione(GSH)kit were used to detect the content of reactive oxygen species(ROS),Fe2+and GSH in K562/ADR cells,respectively;Western blotting was used to examine the expression levels of glutathione peroxidase 4(GPX4),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)in K562/ADR cells;the effects of Lut on the proliferation of K562/ADR cells,ROS content,GSH content,Fe2+content and GPX4 expression were studied after treatment with ferroptosis inhibitor Ferrostatin-1(Fer-1). Results:Compared with control group(cells treated with 0 μmol/L Lut),Lut significantly inhibited the proliferation of K562/ADR cells(P＜0.001),improved the chemosensitivity of K562/ADR cells to ADR(P＜0.05),increased apoptosis rate(P＜0.001)and ROS level(P＜0.05)of K562/ADR cells,reduced the GSH level(P＜0.001),and increase Fe2+content(P＜0.01).Compared with control group(cells treated with 0 μmol/L Lut),the protein expressions of GPX4,Nrf2 and HO-1 decreased with the increase of Lut concentration(P＜0.05).Compared with the Lut treatment alone,the inhibitory effect on the proliferation of K562/ADR cells induced by Lut was partially restored by Fer-1 intervention(P＜0.05),and intracellular ROS level was decreased(P＜0.001),GSH level was increased(P＜0.001),Fe2+content was decreased(P＜0.001)and the expression of GPX4 was increased(P＜0.01)in K562/ADR cells. Conclusion:Lut can inhibit the proliferation of K562/ADR cells through ferroptosis pathway,improve the chemosensitivity to ADR,and the potential mechanism may be related to the Nrf2/HO-1 signaling pathway,which provides experimental basis for the treatment of leukemia by ferroptosis.

Protective effect of luteolin on acute lung injury induced by extracorporeal circulation and its mechanism / 中国胸心血管外科临床杂志

@#Objective    To investigate the protective effect of atomized inhalation of nano-luteolin preparation on acute lung injury caused by extracorporeal circulation, and to explore the anti-inflammatory mechanism of luteolin, so as to provide study basis for clinical application. Methods    Thirty male SD rats aged 5-6 weeks and weighting 160-190 g, were randomly divided into a preoperative baseline (BL) group, arteriovenous partial diversion (ECC) group, luteolin atomization pretreatment for 1 h group, 2 h group, and 3 h group by random number method, with 6 rats in each group. In the BL group, lung tissue samples were collected directly without any treatment. The ECC group received mechanical ventilation, and the whole body was heparinized after the jugular arteriovenous intubation. The flow was transferred for 30 minutes, followed by observation for 60 minutes, then lung tissue samples were collected. Subjects in the 1 h, 2 h and 3 h groups were placed in a small animal atomizer 1 h, 2 h and 3 h before flow transfer respectively, and the subsequent operation was the same as that in the ECC group. The inflammatory level of lung tissue was detected to evaluate the degree of pathological injury of lung tissue. Western blotting (WB) was used to detect the contents of p65, IKKα, IKKβ and IKKγ in the cytoplasm of lung tissue samples of each group. Results    Compared with the ECC group, the levels of IL-6 and TNF-α in lung tissues and the degree of pathological injury in the 1 h, 2 h and 3 h groups decreased, and the difference between the 3 h group and the ECC group was statistically different (P<0.05). WB results showed that compared with the ECC group, the levels of p65 in lung tissue of the 1 h, 2 h and 3 h groups decreased; the levels of IKKβ in the lung tissue increased in the 1 h, 2 h and 3 h groups, and the difference of the 3 h group was statistically different from the ECC group (P<0.05). Conclusion    Luteolin has a protective effect on acute lung injury induced by ECC, and atomization 3 h in advance has the best protective effect on lung. The mechanism plays a protective role in ECC-induced acute lung injury, may be through inhibition of IKKβ phosphorylation, thereby inhibiting the classical NF-κB signaling pathway.

Luteolin inhibits the invasion and migration of endometrial stromal cells by regulating HPGD expression / 安徽医科大学学报

Objective @#To investigate the effects of luteolin on invasion and migration of endometriosis stromal cells and whether its mechanism is related to the regulation of 15-hydroxyprostaglandin dehydrogenase (HPGD) expres- sion．@*Methods @#The endometrial stromal cells HEM15A were divided into control group (cells were cultured in nor- mal medium) ，luteolin group ( cells were treated with different concentrations of luteolin) ，si-HPGD group ( cells were transfected with si-HPGD) ，si-NC group ( cells were transfected with si-NC ) ，luteolin + si-HPGD Group (cells were transfected with si-HPGD and treated with 20 μmol / L luteolin) ，Luteolin + si-NC Group ( cells were transfected with si-NC and treated with 20 μmol / L luteolin) . Real-time quantitative PCR was used to detect mRNA level of HPGD．Cell proliferation was detected by CCK-8 assay，Transwell and scratch assays were used to detect cell invasion and migration．The protein expression of proliferating cell nuclear antigen (PCNA) ，matrix metallopro- teinase (MMP-2) ，MMP-9 and HPGD were detected by Western blot，and the level of prostaglandin E2 ( PGE2) was detected by ELISA. @*Results @#Compared with 0 μmol / L luteolin，luteolin at 20，50 and 100 μmol / L significantly inhibited the proliferation activity of hEM15A cells，and reduced PCNA expression ( all P＜0. 05) ．Compared with the control group，20 μmol / L of luteolin significantly inhibited cell invasion and migration (P ＜0. 05) ，decreased the expressions of MMP-2 and MMP-9 (P＜0. 05) ，and up-regulated the mRNA and protein levels of HPGD (P < 0. 05) ，while inhibited cellular PGE2 level (P＜0. 05) ．Compared with the luteolin group，the luteolin + si-HPGD group increased cell invasion and migration (P ＜0. 05 ) ，increased the expressions of MMP-2 and MMP-9 (P < 0. 05) ．@*Conclusion @#Luteolin regulates HPGD / PGE2 signaling pathway to inhibit the invasion and migration of en- dometrial stromal cells.

Study on the mechanism of luteolin reversing multidrug resistance in leukemia K562/ADR cells / 天津医药

Objective To investigate the mechanism of luteolin's(Lut)reversal effect on multidrug resistance of chronic myeloid leukemia K562/ADR cells.Methods CCK-8 assay was used to detect drug resistance in K562 and K562/ADR cells 24 hours after treatment with different doses of adriamycin(ADR).CCK-8 assay was used to assess the cytotoxicity and sensitizing effect of Lut on ADR after K562/ADR cells were treated with Lut alone or in combination with ADR for 24 hours.K562/ADR cells in logarithmic growth phase were separated into three group:0μmol/L Lut,2μmol/L and 4μmol/L Lut groups.ADR accumulation in cells was measured using flow cytometry.Nuclear factor erythroid-2-related factor 2(Nrf2),multidrug resistance associated protein 1(MRP1),P-glycoprotein(P-gp)and glutathione-S-transferase-PI(GST-pi)mRNA and protein expressions were identified using RT-PCR and Western blot assay.Glutathione(GSH)kit was used to detect intracellular GSH content.Results Compared with K562 cells,K562/ADR cell line was significantly resistant to ADR,and the drug resistance was 53.69 times.K562/ADR cell proliferation was decreased to variable degrees by different doses of Lut when compared to the 0μmol/L Lut group(P＜0.05).The proliferation inhibition rates of K562/ADR cells treated with 2 and 4μmol/L Lut were less than 10%,indicating that the concentration of Lut was non-toxic.Compared with the 0 μmol/L Lut group,the 2 μmol/L Lut group and the 4 μmol/L Lut group showed significantly increased ADR growth inhibition rate on K562/ADR and increased accumulation of ADR in cells,improved the reversal resistance fold,and decreased GSH content in cells.MRP1,P-gp,GST-pi and Nrf2 mRNA and protein expression were reduced in cells(P＜0.05).The effect of 4 mol/L Lut was greater than that of 2 mol/L Lut.Conclusion Lut may decrease K562/ADR cell proliferation and reverse ADR medication resistance.The mechanism could be connected to the downregulation of Nrf2,MRP1,P-gp and GST-pi expression,which leads to an increase in ADR accumulation in K562/ADR cells.

Study on the effects and mechanism of luteolin on osteogenic repair of bone defects / 中国药房

OBJECTIVE To investigate the effects and mechanism of luteolin on osteogenic repair of bone defects. METHODS The targets and potential pathways of luteolin in the treatment of bone defects were screened by network pharmacology method， and then the top 2 targets were selected by Hub gene screening for molecular docking verification， with binding energy as the evaluation standard. In vitro experiments were conducted on rat bone mesenchymal stem cells （BMSC） and rat umbilical vein endothelial cells （RUVEC）. Phenotypic validation was performed using alkaline phosphatase staining， alizarin red S staining， and in vitro angiogenesis experiments. Western blot assay was used to detect the protein expressions of phosphatidylinositol 3 kinase （PI3K） and protein kinase 1 （Akt1）， so as to validate the mechanism of luteolin on osteogenic differentiation of BMSC and angiogenesis of RUVEC in vitro. RESULTS The results of network pharmacology showed that the effects of luteolin on vascular formation and bone repair in bone defects were mainly related to Akt1， SRC， estrogen receptor 1， epidermal growth factor receptor， cyclooxygenase 2， matrix metalloproteinase 9 targets， and were closely related to PI3K-Akt signaling pathway. The results of molecular docking showed that luteolin binding to Akt1 and SRC proteins was stable. The results of in vitro experiments showed that luteolin could significantly improve the expressions and activities of alkaline phosphatase in BMSC， increased the number of calcium salt deposits and calcified nodules， and promoted calcification of BMSC. Compared with luteolin 0 μmol/L group， the angiogenesis ability of RUVEC was enhanced significantly in luteolin 1， 10 μmol/L groups， the length of blood vessels and the protein expressions of PI3K and Akt1 were significantly increased （P＜0.05 or P＜0.01）； the higherthe concentration， the better the effect. CONCLUSIONS Luteolin may play a role in promoting angiogenesis and bone repair at the fracture site by activating PI3K/Akt signaling pathway and promoting the protein expressions of PI3K and Akt1.

Luteolin regulates M1 macrophage polarization by inhibiting glycolytic pathway induced by HIF-1 / 中国药理学通报

Aim To investigate the effect of luteolin on M1 macrophages polarization through HIF-1α-mediated glycolytic pathway. Methods RAW264.7 cells were divided into control groups(M0)and LPS+IFN-γ groups(M1). M1 groups were further divided into luteolin group, 2-DG(glycolysis inhibitor)group, luteolin+2-DG group,luteolin+DMOG(HIF-1α agonist)group. The protein expression levels of iNOS, Arg-1 and HIF-1α were detected by Western blot. Macrophage phenotype was detected by flow cytometry. In addition, the expression levels of IL-6 and IL-10 were measured by ELISA. The gene expression levels of GLUT1, HK2, PFK1, PK and HIF-1α were quantified by quantitative real-time PCR. Results Compared with M1 groups, luteolin and luteolin+2-DG treatment groups decreased the expression levels of GLUT1, HK2, PFK1, PK and HIF-1α related to glycolysis. In addition, luteolin and luteolin+2-DG treatment group significantly inhibited the expression of M1 macrophage markers such as iNOS, CD86 and IL-6, whereas up-regulated M2 macrophage markers Arg-1, CD206 and IL-10. Notably, the inhibitory effects of luteolin on M1 macrophages were restored by DMOG. Conclusion Luteolin regulates M1 macrophage polarization by inhibiting the glycolytic pathway induced by HIF-1α.

Active Ingredients of Reduning Injection Maintain High Potency against SARS-CoV-2 Variants / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-coronavirus potential and the corresponding mechanisms of the two ingredients of Reduning Injection: quercetin and luteolin.@*METHODS@#A pseudovirus system was designed to test the efficacy of quercetin and luteolin to inhibit SARS-CoV-2 infection and the corresponding cellular toxicity. Luteolin was tested for its activities against the pseudoviruses of SARS-CoV-2 and its variants. Virtual screening was performed to predict the binding sites by Autodock Vina 1.1.230 and PyMol. To validate docking results, surface plasmon resonance (SPR) was used to measure the binding affinity of the compounds with various proteins of the coronaviruses. Quercetin and luteolin were further tested for their inhibitory effects on other coronaviruses by indirect immunofluorescence assay on rhabdomyosarcoma cells infected with HCoV-OC43.@*RESULTS@#The inhibition of SARS-CoV-2 pseudovirus by luteolin and quercetin were strongly dose-dependent, with concentration for 50% of maximal effect (EC50) of 8.817 and 52.98 µmol/L, respectively. Their cytotoxicity to BHK21-hACE2 were 177.6 and 405.1 µmol/L, respectively. In addition, luetolin significantly blocked the entry of 4 pseudoviruses of SARS-CoV-2 variants, with EC50 lower than 7 µmol/L. Virtual screening and SPR confirmed that luteolin binds to the S-proteins and quercetin binds to the active center of the 3CLpro, PLpro, and helicase proteins. Quercetin and luteolin showed over 99% inhibition against HCoV-OC43.@*CONCLUSIONS@#The mechanisms were revealed of quercetin and luteolin inhibiting the infection of SARS-CoV-2 and its variants. Reduning Injection is a promising drug for COVID-19.

Discovery of the radio-protecting effect of Ecliptae Herba, its constituents and targeting p53-mediated apoptosis in vitro and in vivo

Radiation protection drugs are often accompanied by toxicity, even amifostine, which has been the dominant radio-protecting drug for nearly 30 years. Furthermore, there is no therapeutic drug for radiation-induced intestinal injury (RIII). This paper intends to find a safe and effective radio-protecting ingredient from natural sources. The radio-protecting effect of Ecliptae Herba (EHE) was discovered preliminarily by antioxidant experiments and the mouse survival rate after 137Cs irradiation. EHE components and blood substances in vivo were identified through UPLC‒Q-TOF. The correlation network of "natural components in EHE-constituents migrating to blood-targets-pathways" was established to predict the active components and pathways. The binding force between potential active components and targets was studied by molecular docking, and the mechanism was further analyzed by Western blotting, cellular thermal shift assay (CETSA), and ChIP. Additionally, the expression levels of Lgr5, Axin2, Ki67, lysozyme, caspase-3, caspase-8,8-OHdG, and p53 in the small intestine of mice were detected. It was found for the first time that EHE is active in radiation protection and that luteolin is the material basis of this protection. Luteolin is a promising candidate for RⅢ. Luteolin can inhibit the p53 signaling pathway and regulate the BAX/BCL2 ratio in the process of apoptosis. Luteolin could also regulate the expression of multitarget proteins related to the same cell cycle.

Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS / 中国中药杂志

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

Luteolin ameliorates loperamide-induced functional constipation in mice

Functional constipation (FC) is one of the most common gastrointestinal disorders characterized by hard stools and infrequent bowel movements, which is associated with dysfunction of the enteric nervous system and intestinal motility. Luteolin, a naturally occurring flavone, was reported to possess potential pharmacological activities on intestinal inflammation and nerve injury. This study aimed to explore the role of luteolin and its functional mechanism in loperamide-induced FC mice. Our results showed that luteolin treatment reversed the reduction in defecation frequency, fecal water content, and intestinal transit ratio, and the elevation in transit time of FC models. Consistently, luteolin increased the thickness of the muscular layer and lessened colonic histopathological injury induced by loperamide. Furthermore, we revealed that luteolin treatment increased the expression of neuronal protein HuC/D and the levels of intestinal motility-related biomarkers, including substance P (SP), vasoactive intestinal polypeptide (VIP), and acetylcholine (ACh), as well as interstitial cells of Cajal (ICC) biomarker KIT proto-oncogene, receptor tyrosine kinase (C-Kit), and anoctamin-1 (ANO1), implying that luteolin mediated enhancement of colonic function and contributed to the anti-intestinal dysmotility against loperamide-induced FC. Additionally, luteolin decreased the upregulation of aquaporin (AQP)-3, AQP-4, and AQP-8 in the colon of FC mice. In summary, our data showed that luteolin might be an attractive option for developing FC-relieving medications.

Anti-hyperglycemic fraction from Alternanthera sessilis L. leaves gets elucidated following bioassay-guided isolation and mass spectrometry

Abstract The anecdotal use of Alternanthera sessilis L. as a relief for diabetes has been known in the Philippines for generations, and antidiabetic activity of similar varieties in other countries is likewise documented. However, the compounds responsible for this activity remain unclear. This study aims to isolate the anti-hyperglycemic fraction of local A. sessilis leaves and identify the compounds in this fraction. Methanol extract of A. sessilis leaves and its hexane, ethyl acetate (ASE), and water fractions were administered to alloxan-induced diabetic mice. ASE (250mg/kg) had the highest anti-hyperglycemic activity at 6-h post-treatment (25.81%±12.72%), with almost similar blood glucose reduction rate as metformin (30.13±3.75%, p=0.767). Repeated fractionation employing chromatographic separation techniques followed by in vivo anti-hyperglycemic assay yielded partially purified subfractions. A. sessilis ethyl acetate subfraction 4-2 (100mg/kg) displayed remarkable suppression of blood glucose rise in diabetic mice at 6-h post-treatment (26.45±3.75%, p<0.0001), with comparable activity with metformin (100mg/kg, 27.87±5.65%, p=0.652). Liquid chromatography/mass spectrometry showed eight distinct peaks, with four peaks annotated via the Traditional Chinese Medicine library and custom library for A. sessilis. Among these, luteolin, apigenin, ononin, and sophorabioside were identified as putative compounds responsible for the anti-hyperglycemic activity. This result provided basis for the reported anecdotal claims and potential utility of the local variety of A. sessilis leaves as sources of anti-hyperglycemic agents

Role of three drugs in traumatic proliferative vitreoretinopathy in rabbits / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of triamcinolone acetonide(TA), artesunate(ART), and luteolin(LU)on the prevention and treatment of traumatic proliferative vitreoretinopathy(TPVR). <p>METHODS: Forty-eight cyanotic blue rabbits were selected to prepare TPVR animal models by making a penetrating eye injury and intravitreal injection of 0.3mL platelet-rich plasma, and were randomly divided into four groups(<i>n</i>=12), in which the vitreous cavity of the control group was injected with 0.1mL saline; The vitreous cavity of the TA group was injected with 0.1mL(1mg/mL)triamcinolone acetonide; The vitreous cavity of the ART group was injected with 0.1mL(20μg/mL)artesunate; 0.1mL(10μg/mL)luteolin was injected into the vitreous cavity of the LU group. The vitreous and retinal proliferation were observed by fundus photography and ocular ultrasound at 1, 2, 3 and 4wk postoperatively. The expression levels of α-SMA and VIM protein in the vitreous fluid of each group of rabbit eyes were detected by Western Blot at 28d postoperatively, and the retinal tissue structure of each group was observed by retinal HE staining. <p>RESULTS: At 28d postoperatively, the TPVR grading of rabbit eyes in the TA, ART and LU groups were significantly lower than that in the control group(<i>P</i><0.05), and the TPVR grading of rabbit eyes in the TA group was significantly lower than that in the ART and LU groups(<i>P</i><0.05). The expression levels of α-SMA and VIM proteins in the vitreous fluid of the rabbit eyes in the TA, ART and LU groups were significantly lower than those in the control group at 28d after surgery(<i>P</i><0.01). The results of HE staining showed that the arrangement of retinal layers in rabbit eyesin the control group were disordered, severely distorted or locally broken, the structure of each layer were unclear, the anterior membrane was obviously thickened, and the retina was obviously detached; The arrangement of retinal layersin rabbit eyes in the LU group were slightly distorted, inflammatory exudation was visible in front of the retina, and the retina was superficially detached; The structure of retina in rabbit eyes in the ART group were clear, with mild edema and superficial detachment; The structure of retinal layers in rabbit eyes in the TA group were clear, the arrangement was still neat, the retinal folds were locally visible, and there was no retinal detachment.<p>CONCLUSION: Intravitreal injection of triamcinolone acetonide, artesunate and luteolin were all effective in preventing and treating traumatic TPVR, among which triamcinolone acetonide has the most obvious effect.

Recent progress of neuroprotective mechanisms of luteolin in Alzheimer's disease / 中国药理学通报

Luteolin is a widely distributed type of flavone herbsand vegetables, which has diverse pharmacological activities against human ailments including Alzheimer's disease(AD). Recently, luteolin has been the most widely studied phytochemicals for their neuroprotective effects against experimental models of AD. Luteolin also improves brain insulin sensitivity and neuroinflammation, which attenuates the phosphorylation of tau and the formation of tangles, and the tendency of Aβ to form deposits. Furthermore, luteolin has anti-oxidative stress and anti-apoptosis effect in AD. The present study aims at reviewing experimental studies and describing the possible underlying molecular mechanisms by which luteolin and the related compounds protect against AD.

Study on the inhibition of compound salvia miltiorrhiza on the proliferation of ovarian cancer cells and its preliminary mechanism / 中国临床药理学与治疗学

AIM: To explore the inhibitory effect and mechanism of compound salvia miltiorrhiza on ovarian cancer by using network pharmacology and molecular docking knowledge. METHODS: The core components of compound salvia miltiorrhiza and the inhibitory effect of compound salvia miltiorrhiza on ovarian cancer cell cycle were studied by combining the methods of MTT cell cycle inhibition and MTT signal network of compound salvia miltiorrhiza. RESULTS: Based on network pharmacology, the core components of compound salvia miltiorrhiza were luteolin and quercetin, and the core target of the disease was VEGFA, SRC, EGFR, hsp90aa1. The docking mode between the core component and the core target EGFR was verified and analyzed by molecular docking. Finally, MTT colorimetry was used to verify that luteolin, one of the core components, significantly inhibited the proliferation of ovarian cancer cells. The results of flow cytometry showed that luteolin induced ovarian cancer A2780 cell cycle arrest in G1/S phase.CONCLUSION: Compound salvia miltiorrhiza preparation can inhibit the proliferation of ovarian cancer cells, which may be related to PI3K Akt signal pathway mediated by EGFR; Network pharmacology and molecular docking technology have important predictability and possibility for the treatment of tumor by compound salvia miltiorrhiza, and have guiding significance for the role and mechanism of compound salvia miltiorrhiza against ovarian cancer cells.

Protective effect of Luteolin on oxidative damage of retinal pigment epithelium cells by regulating the Nrf2/HO-1 pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the protective effect and mechanism of luteolin on H2O2-induced oxidative damage of retinal pigment epithelium(RPE)cells. <p>METHODS:ARPE-19 cells were divided into the control group, H2O2 group, different doses of luteolin groups and Nrf2 inhibitor group, and the oxidative damage model of RPE was prepared by 100μmol/L H2O2, except for the control group. Cell activity was detected by Methyl thiazolyl tetrazolium(MTT)assay and proper experimental concentration of luteolin was determined. The cell morphology and activity was observed in each group. Cell apoptosis rate and reactive oxygen species(ROS)were detected by flow cytometry, malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by kit method, and the expression of caspase-3, poly adeno-sine diphosphate ribose polymerase(PARP), B cell lymphoma-2(Bcl-2), nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)proteins were detected by Western blot. <p>RESULTS: 100μmol/L luteolin has toxic effects on ARPE-19 cells, so 25μmol/L and 50μmol/L luteolin were selected for subsequent experiments. The cell activity, SOD activity and the protein expression levels of Bcl-2, Nrf2, HO-1 in 25μmol/L and 50μmol/L luteolin groups were significantly higher than the H2O2 group(<i>P</i><0.05). The apoptosis rate, ROS, MDA content and the protein expression levels of Caspase-3 and PARP in 25μmol/L and 50μmol/L luteolin groups were significantly lower than the H2O2 group(<i>P</i><0.05). The cell activity, SOD activity \〖(13.83±1.49)U/mL <i>vs</i>(22.69±1.83)U/mL\〗 and the protein expression levels of Bcl-2, Nrf2 and HO-1 protein expression in the Nrf2 inhibitor group were significantly lower than the 50μmol/L luteolin group(<i>P</i><0.05). The apoptosis rate, ROS, MDA content \〖(654.96±26.99)<i>vs</i>(446.52±29.42),(3.89±0.29)nmol/mL <i>vs</i>(2.06±0.19)nmol/mL\〗 and the protein expression levels of Caspase-3 and PARP in the Nrf2 inhibitor group were significantly higher than the 50μmol/L luteolin group(<i>P</i><0.05).<p>CONCLUSION: Luteolin can improve the oxidative damage of RPE cells induced by H2O2, and its mechanism may be related to the activation of the Nrf2/HO-1 pathway.

Isomeric discrimination of kaempferol versus luteolin by online energy-resolved mass spectrometry / 中国中药杂志

Isomers are widely distributed in Chinese herbal medicines,and can be discriminated by energy-resolved mass spectrometry( ER-MS). However,ER-MS was performed through direct injection of reference compounds with syringe pump,which encountered a significant technical barrier for high-throughput and automated measurements. Herein,online ER-MS was conducted using LC-MS platform,and a pair of isomers,kaempferol vs luteolin,were employed as a case study to illustrate and assess the utility of online ER-MS for isomeric discrimination. High-resolution tandem mass spectrometry data of both flavonoids were acquired on LC-QE-Orbitrap-MS,and the fragmentation pathways responsible for the primary fragment ions were proposed. The primary signal in MS1 occurred at m/z 285( [M-H]-),and the primary signals of either compound generated by retro-Diels-Alder fragmentation were observed at m/z 151 and 133. The spectral information was subsequently transferred onto LC-Qtrap-MS platform to carry out online ER-MS. Two precursor-to-product ion transition candidates were constructed as m/z 285>151 and 285>133,and either afterward derived a set of pseudo-ion transitions( PITs) and so forth,exactly corresponding to a series of progressive collision energies( eg-5,-8,-11 e V,and so on). All PITs were typed into the monitoring list of multiple reaction monitoring program to generate the peak area datasets. Either dataset was normalized using the highest values in the set and imported into Graph Pad Prism software to plot the Gaus-sian-shaped curve that was termed as the break-down graph. The apex of the regressive curve was termed as optimal collision energy( OCE). The OCE values corresponding to m/z 285>151 were calculated as-29. 06 e V and-35. 71 e V for kaempferol and luteolin,respectively. In the case of m/z 285>133,the OCEs were yielded as-44. 15 e V for kaempferol and-49. 01 e V for luteolin. With re-ference to their chemical structures,the location of hydroxyl group was regarded to be responsible for the differences of either m/z 285>151 or 285>133 between the isomers,attributing to their different bond properties. Above all,online ER-MS offers an eligible tool for isomeric discrimination,and provides meaningful information for the accurate chemical composition characterization based on LC-MS,which is not limited to Chinese herbal medicines.