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Total flavonoids of Dracocephalum moldavica L. (TFDM) is an effective component extracted and isolated from the traditional Uighur medicinal herb Cymbidium fragrans. Cymbidium fragrans has the effects of tonifying the heart and brain, promoting blood circulation and resolving blood stasis, and has been widely used in the treatment of cardiovascular and cerebrovascular diseases for a long time. The purpose of this study was to determine the effect of total flavonoids from Cymbidium fragrans on hypoxia/re-oxygenation (H/R) injury in H9c2 (rat cardiomyocytes) cells and its mechanism. A model (H/R) of hypoxia/re-oxygenation injury in H9c2 cells was established using hypoxia and glucose deprivation for 9 h combined with re-oxygenation and rehydration for 2 h to simulate myocardial ischemia-reperfusion injury. The effects of total flavonoids from Cymbidium fragrans on cell viability, markers of myocardial cell damage, oxidative stress levels, and reactive oxygen radical (ROS) content were investigated, Western blot was used to detect the expression of vascular endothelial growth factor B (VEGF-B) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway related proteins. The results showed that the total flavonoids of Cymbidium fragrans significantly increased the viability of myocardial cells after H/R injury, and decreased the content of lactate dehydrogenase (LDH) and creatine kinase isozyme (CK-MB) in the cell supernatant. It significantly reduced malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and decreased intracellular ROS and nitric oxide (NO) content. Western blot analysis showed that the total flavonoids of Cymbidium fragrans decreased Bax levels in H9c2 cells damaged by H/R and increased Bcl-2 expression. Total flavones of Cymbidium fragrans upregulate VEGF-B/AMPK pathway related proteins VEGF-B, vascular endothelial growth factor receptor 1 (VEGFR-1), neuropilin 1 (NRP-1), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), phosphorylated adenosine monophosphate activated protein (p-AMPK) and phospho mechanistic target of rapamycin (p-MTOR) levels. The above research results indicate that the total flavonoids of Cymbidium can significantly reduce the H/R injury of myocardial cells, which may be related to the upregulation of VEGF-B/AMPK pathway and inhibition of oxidative stress response.
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Aim To investigate the protective effect of TFDM on doxorubicin-induced endothelial cell injury and its mechanism.Methods Cell viability was detected by CCK-8 assay.Cell morphology was observed by microscope.The changes of LDH, SOD and mitochondrial membrane potential were detected by kit method.Cell migration was detected by Transwell assay; Endothelial dysfunction and VEGF-B/AMPKa pathway related protein expression were detected by Western blot.Results Compared with model group, TFDM significantly increased cell viability, improved the morphologic changes of HUVEC induced by DOX, decreased LDH leakage, increased SOD activity, increased mitochondrial membrane potential, promoted endothelial cell migration, and inhibited endothelial cell injury.The results of Western blot showed that com pared with control group TFDM increased the expression levels of non-receptor tyrosine kinase ( Src) and focal adhesion kinase (FAK) .increased the phosphorylation level of eNOS, and decreased the expression level of ET-1 protein, thereby inhibiting endothelial dysfunction.The protein expression levels of VEGF-B, NRP1 , VEGFR1 and the ratio of p-AMPKa/AMPKa significantly increased in the administration group.Conclusion TFDM may inhibit doxorubicin-induced endothelial cell injury by activating VEGF-B/AMPKa pathway.
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We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.
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The gut microbiota takes part in many in vivo important physiological activities of host, such as the substance metabolism and energy exchange, etc. The interaction between the host and the intestinal microorganisms has attracted scholars' attention. Flavonoids are a group of polyphenol compounds widely found in natural plants, with the bioactive effect of regulation of glucose and lipid metabolism, anti-inflammation. However, their low bioavailability cause difficulty to clarify the effective substances and the mechanism of flavonoids. Apart from the metabolic effects of liver on flavonoids, recent studies have shown that the gut microbiota can interact with flavonoids. On the one hand, flavonoids can be metabolized by gut microbiota and subsequent metabolites can produce pharmacological activities different from the parent components. On the other hand, flavonoids and their metabolites can in turn regulate the composition and physiological activities of the intestinal flora, which seems to provide a new insight for the research on the effective substances of flavonoids. In this review, we introduced the metabolic characteristics of flavonoids under the actions of intestinal bacteria, and the regulation effects of flavonoids on gut microbiota was also summarized. Meanwhile, the therapeutic effect of flavonoids under the action of intestinal bacteria was discussed.
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Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase II (CaMKII), and Western blot results showed that compared with control group, the expression levels of p-CaMKII and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.
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The purpose of this research is to investigate the effects of acacetin on serum lipid metabolism and atherosclerosis in mice and explore its molecular mechanism. HepG2 cells were treated with different concentrations of acacetin. The expression of LDL receptor (LDLR) and sterol-regulatory element binding protein-2 (SREBP-2) were detected by RT-qPCR and/or Western blot. C57BL/6J mice were given acacetin (50 mg·kg-1) for 5 weeks by gavage. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) were analyzed by an automatic biochemical analyzer. The expression of LDLR or SREBP-2 was detected by Western blot. After 12 weeks of intragastric administration of acacetin (30 mg·kg-1) in apolipoprotein E knockout (ApoE KO) mice, the serum lipid levels were determined by an automatic biochemical analyzer. The lipid deposition in aortic plaque (en face) and aortic root plaque were stained with oil red O. The expression of LDLR and SREBP-2 were detected by RT-qPCR and/or Western blot. The intestinal content microflora was analyzed by 16S rDNA sequencing (All animal studies were approved by the Animal Experimentation Ethics Committee of Institute of Medicinal Biotechnology, CAMS & PUMC). In vitro results indicated that acacetin significantly up-regulated LDLR mRNA and protein levels, and stimulated LDLR transcription factor SREBP-2 protein expression. As indicated from in vivo studies, compared with control group, acacetin significantly decreased the serum levels of TC and LDL-C in C57BL/6J mice by 34% and 57% (P<0.01), respectively. Furthermore, mechanic study showed that acacetin significantly increased the protein expression of hepatic LDLR and SREBP-2. Although the results of serum lipid profiles, hepatic LDLR/SREBP-2 expression and area of atherosclerotic lesions in aorta and aortic root in ApoE KO mice showed differences between acacetin and high-fat diet group, the differences did not reach statistical significance. Nevertheless, acacetin exhibited a profound influence on the composition of the intestinal microbiota as indicated by 16s rDNA sequencing analysis. In conclusion, these results demonstrated that acacetin can decrease the serum lipid levels in C57BL/6J mice through up-regulation of hepatic LDLR and SREBP-2, and alter gut microflora in high-fat diet fed Apo KO mice. This study suggests the possibility that acacetin has a potential role in inhibiting the progression of atherosclerosis.
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Influenza caused by influenza virus,seriously threaten human life and health.Drug treatment is one of the effective measurement. However, there are only two classes of drugs, one class is M2 blockers and another is neuraminidase (NA)inhibitors. The recent antiviral surveillance studies reported a global significant increase in M2 blocker resistance among influenza viruses, and the resistant virus strains against NA inhibitor are also reported in clinical treatment.Therefore thediscovery of new medicines with low resistance has become very urgent.As all known,traditional medicines with multi-target features and network mechanism often possess low resistance. Compound Yizhihao, which consists of radix isatidis,folium isatidis,Artemisia rupestris,is one of the famous traditional medicine for influenza treatment in China, however its mechanism of action against influenza is unclear. In this study, the multiple targets related with influenza disease and the known chemical constituents from Compound Yizhihao were collected, and multi-target QSAR (mt-QSAR) classification models were developed by Na?ve Bayesian algorithm and verified by various datasets. Then the classification models were applied to predict the effective constituents and their drug targets.Finally,the constituent-target-pathway network was constructed,which revealed the effective constituents and their network mechanism in Compound Yizhihao. This study will lay important basis for the clinical uses for influenza treatment and for the further research and development of the effective constituents.
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Compound Yizhihao, consists of Radix isatidis, Folium isatidis, Artemisia rupestris, has a significant therapeutic effect on the treatment of influenza and fever. However, the mechanism of its action is still unclear. In this investigation, we collected the key target molecule of influenza disease and the chemical constituents of Compound Yizhihao, and developed Naïve Bayesian classification models based on the input molecular fingerprints and molecule descriptors. The built models were further applied to construct classifiers for predicting the effective constituents. We used the professional network-building software to build the constituent-target network and target-pathway network, which revealed the network pharmacology of the effective constituents in Compound Yizhihao. It will contribute to the further research of mechanism of Compound Yizhihao.
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<p><b>OBJECTIVE</b>To study the purification technology of Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis in Fufang Xuelian dropping pills by macroporous resin.</p><p><b>METHOD</b>Taking osthole, isomperatorin as index ingredients, the type of resin sampling amount and elution solvent were decided, and the influence of sample concentration pH of sample and ratio of diameter to height of column to adsorption were studied.</p><p><b>RESULT</b>HPD400A was chosen to purify, the suitable sampling ratio of resin volume to raw material was 1:2; pH 3.5 (crude drug) and ratio of diameter to height was 1:7; 95% ethanol of the elution solvent was satisfactory eluant for desorption.</p><p><b>CONCLUSION</b>HPD400A macroporous resin can be used to purify Rhizoma et Radix Notopterygii and Radix Angelicae Pubescentis.</p>
Subject(s)
Angelica , Chemistry , Apiaceae , Chemistry , Drugs, Chinese Herbal , Chemistry , Plants, Medicinal , Chemistry , Porosity , Resins, Synthetic , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To study the chemical constituents of Dracocephalum moldavica.</p><p><b>METHOD</b>The compounds were isolated by using RA polystyrene resin, polyamide and silica gel column chromatography, The structures of the compounds were elucidated on the basis of physic-chemical properties and spectra data.</p><p><b>RESULT</b>Six compounds were identified as syringaresinol4-O-beta-D-monoglucoside (I), sy-ringaresinol-4,4'-O-bis-beta-D-glucoside (II), kaempferol-3-O-beta-D-(6"-O-p-coumaroyl)-galactopyranoside (III), 2"-p-coumarylastragalin (IV), takakin-8-O-beta-D-glucopyranoside (V), beta-daucosterol (VI).</p><p><b>CONCLUSION</b>Compounds I-V were obtained from genus Dracocephalum for the first time.</p>