Analysis of the Factors Affecting the Configuration of the Anti-parallel G-quadruplex Formed by 22-nt Telomere RNA Fragments / 中国生物化学与分子生物学报

G-quadruplexes with different conformations often exhibit different binding abilities to proteins and play different physiological functions. It is of great significance to analyze the factors affecting the configuration of G-quadruplex to explore its physiological function. Different from DNA, RNA sequences have shown parallel G-quadruplex structures in previous reports. However, we found that the only RNA sequence, the 22-nt telomere RNA fragment (ORN-N, A

GPER inhibitor increases tamoxifen-induced apoptosis in T-47DTR-resistant cells / 中国药理学通报

Aim To study the effect of G protein-coupled estrogen receptor(GPER)inhibitor G15 on the sensitivity of breast cancer tamoxifen-resistant cells to T-47DTR. Methods Experiments were carried out with 4-hydroxytamoxifen(4-OHT),the active form of tamoxifen in vivo. The sensitivity of tamoxifen-resistant breast cancer cell line T-47DTR and its parental cell line T-47D to tamoxifen was detected by MTT assay; the expression of GPER protein was analyzed by plasma separation of inhibitor G15; the effect of 4-OHT combined with G15 on the apoptosis of T-47DTR cells was analyzed by flow cytometry AnnexinV-FITC/PI double staining; the expression levels of apoptosis-related proteins Bax,Bcl-2,caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9 were analysed by Western blot. Results(1)Compared with the parental cell T-47D,the resistance of T-47DTR-resistant cells to 4-OHT was significantly enhanced.(2)When 4-OHT(2 μmol·L-1)was administered,the membrane distribution of GPER increased,indicating that GPER was activated in T-47DTR-resistant cells compared with the control group; Compared with OHT,the use of G15(5 μmol·L-1)and OHT significantly reduced the expression of GPER.(3)GPER inhibitor G15 could increase the apoptotic rate of T-47DTR-resistant cells while down-regulating the anti-apoptotic protein Bcl-2 and up-regulating the expression of pro-apoptotic proteins Bax,cleaved caspase-3,cleaved caspase-9. Conclusions The GPER inhibitor G15 increases the apoptosis of T-47DTR cells and restores the sensitivity of drug-resistant cells to tamoxifen.

The protective effect of terpinen-4-ol on inflammatory injury of VSMCs induced by high glucose through improving autophagic flux disorder / 中国药理学通报

Aim To investigate the effect of terpinen-4-ol (T40) on inflammatory injury of vascular smooth muscle cells (VSMCs) induced by high glucose based on the improvement of autophagic flow disorder and involved molecular signals. Methods The scratch test was used to analyze the migration ability of VSMCs, the levels of IL-1β and IL-6 in cell culture supernatant were measured by ELISA, the expression levels of inflammation-related proteins NF-κb p65, p-NF-κb p65, IL-1β, IL-18 and autophagy-related proteins p62, LC3-HYLC3-I, Beclinl, p-Beclinl were de-tected by Western blot. Results T40 inhibited migration of VSMCs induced by high glucose, reduced the secretion and release of pro-inflammatory factors IL-1β and IL-6, inhibited the expression of p-NF-κb p65/ NF-κb p65, IL-1β, IL-18, downregulated the expression of p62, LC3-TJ/LC3- I and p-Beclinl at same time. After interfering the autophagic flux of VSMCs with autophagy inhibitor chloroquine (CQ) , T40 pre-treatment significantly inhibited the protein expression levels of the above inflammatory factors and autophagy-related signals which mediated by CQ. Conclusion T40 inhibits the inflammatory injury of VSMCs induced by high glucose through improving the autophagic flow disorder.

Terpine-4-ol on inhibiting vascular smooth muscle cell calcification by improving mitochondrial dynamics / 药学学报

The aim of this study was to investigate the role and mechanism of terpinen-4-ol (T4O) on high glucose (HG) -induced calcification in vascular smooth muscle cell (VSMC). To investigate the role of T4O on HG-induced calcium deposition, osteogenic phenotypic transformation and mitochondrial dynamics in VSMC, Mdivi-1, a mitochondrial dynamin-related protein 1 (Drp-1) inhibitor, was used to analyze the correlation between mitochondrial dynamics and VSMC calcification and the role of T4O. Alizarin red S staining was used to observe calcium salt deposition and flow cytometry to detect intracellular Ca2+ content; Western blot and immunofluorescence were used to detect the expression of phenotypic switching-related markers α-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2) and Runt related transcription factor 2 (Runx2), and mitochondrial dynamics-related markers mitofusin 1 (MFN1), mitofusin 2 (MFN2) and Drp-1. The results showed that low and high doses of T4O could inhibit HG-induced down-regulation of α-SMA, MFN1 and MFN2 expression levels, and up-regulation of BMP2, Runx2 and Drp-1 expression levels, reduce intracellular Ca2+ content and calcium salt deposition, and effectively inhibit HG-induced VSMC calcification and mitochondrial dynamics disorders. The T4O group, Mdivi-1 group and T4O+Mdivi-1 group were able to up-regulate the expression levels of HG-induced α-SMA, MFN1 and MFN2, down-regulate the protein expression levels of BMP2, Runx2 and Drp-1, and inhibit calcium salt deposition, and there was no significant difference between the above indexes in the T4O and T4O+Mdivi-1 groups. The above findings suggest that T4O can inhibit the expression level of Drp-1, regulate the disturbance of mitochondrial dynamics, and suppress HG-induced VSMC calcification.

Quantitative Pulmonary Administration of Essential Oil from Alpiniae Zerumbet Fructus for Treatment of Emphysema / 中国实验方剂学杂志

ObjectiveTo investigate the effect of quantitative pulmonary administration of the essential oil from Alpiniae Zerumbet Fructus （EOAZF） on porcine pancreatic elastase （PPE）-induced emphysema in mice and explore its action mechanism. MethodC57BL/6J mice were randomly divided into five group， namely the control group， model group， low- （2 mg·kg-1） and high-dose （20 mg·kg-1） EOFAZ groups， and positive control dexamethasone （DEX，1 mg·kg-1） group. The mice were treated with pulmonary administration of PPE using a microsprayer aerosolizer， once every seven days， for four times in total， for inducing emphysema. During this period， EOFAZ were administered with a quantitative microsprayer aerosolizer once every other day， for 14 times. The lung tissues were then sampled and stained with hematoxylin-eosin （HE） for observing the morphological changes and calculating the pulmonary mean linear intercept （MLI）. The concentrations of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in the plasma were determined by enzyme-linked immunosorbent assay （ELISA）. The activities of superoxide dismutase （SOD） and catalase （CAT） and the content of malondialdehyde （MDA） in the lung tissues were measured using the biochemical assay kits. The protein expression levels of nuclear factor E2-related factor 2 （Nrf2）， quinone oxidoreductase1 （NQO1）， B cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， and Bcl-2 in lung tissues were detected by Western blot. ResultThe results of lung morphological observation and MLI detection showed that compared with the control group， the model group showed obvious inflammatory infiltration， alveolar enlargement and fusion， and increased MLI （P<0.05）. Compared with the model group， EOFAZ effectively alleviated the pathological changes such as alveolar dilatation， pulmonary inflammatory cell infiltration， and lung cell apoptosis caused by PPE， and decreased the MLI （P<0.05）. As revealed by ELISA， the inflammatory level of mice in the model group increased significantly （P<0.01）， while the TNF-α， IL-1β， and IL-6 levels in the plasma were decreased after quantitative administration of EOFAZ （P<0.01）. Compared with the control group， the model group exhibited significantly enhanced oxidative stress （P<0.01）. After treatment with EOFAZ by quantitative administration， the activities of SOD and CAT in the lung tissue were increased （P<0.01） and the content of MDA was decreased （P<0.01）. Western blot results demonstrated that the apoptosis-related protein expression in the model group was increased significantly as compared with that in the control group （P<0.01）， whereas the expression levels of antioxidant stress proteins Nrf2 and NQO1 declined （P<0.05）. The relative protein expression of apoptosis-related proteins Bax/Bcl-2 in the EOFAZ groups was lower than that in the model group （P<0.01）， while the expression of antioxidant stress proteins Nrf2 and NQO1 was higher （P<0.05）. ConclusionQuantitative pulmonary administration of EOFAZ effectively alleviates the inflammation and oxidative stress， reduces lung cell apoptosis， and hinders the occurrence and development of emphysema. Its antioxidant mechanism is closely related to the up-regulation of Nrf2 and its downstream NQO1.

The protective effect of essential oil from fructus alpiniae zerumbet on T2DM induced pancreatic injury in mice based on p62/Keapl/Nrf2 signaling pathway / 中国药理学通报

Aim To investigate the protective effect of essential oil from Fructus Alpiniae Zerumbet (EOFAZ ) on type 2 diabetes-induced pancreatic injury in mice and its mechanism.Methods After C57 BL/6 mice fed with high-sugar and high-fat ( HFS) feed developed insulin resistance, streptozotocin ( STZ, 120 mg • kg 1 ) was injected intraperitoneal

Scutellarin improves LPS + ATP induced inflammation and pyroptosis of endothelial cells by inhibiting NLRP3/caspase-l signaling pathway / 中国药理学通报

Aim To investigate whether Scutellarin ( Seu ) could alleviate LPS + ATP induced inflammation and pvrolysis and the underlying mechanisms.Meth¬ods Primary human umbilical vein endothelial cells (HUVCEs) were pretreated with different concentra¬tions of Scu for 1 h, 2.5 mg • L 1 LPS treatment for 5.5 h, and then 2.5 mmol • L 1 ATP was activated for 0.5 h.The damage rate and activity of endothelial cells were detected by MTT method and determination of LDH content in cell supernatant.ELISA was used to detect the contents of IL-ip,lL-18 and HMGB-1 in su-pernatant.The expression levels of inflammation and pyroptosis related proteins including NLRP3 , ASC , pro- caspase-1 , caspase-1 p20 and GSDMD-NT were detec¬ted by WB method.The expression of pro-caspase-1 , caspase-1 p20 and GSDMD-NT protein and the contents of IL-ip,IL-18 and HMGB-1 in supernatant were de¬tected by Scu and pan caspase receptor inhibitor ( Z-YVAD-FMK).Results Compared with LPS + ATP group, Scu concentration-dependently improved ATP- induced cytotoxic damage, increased cellular activity, reduced the release of 1L-1(3, IL-18, HMGB-1 , down- regulated NLRP3, ASC, pro-caspase-1 , GSDMD-NT protein level to inhibit the activation of pro-caspase-1 , and the effect was comparable to Z-YVAD-FMK.The inhibitory effect of Scu on pro-caspase-1 activation was not significantly different from that of Scu/Z-YVAD- FMK combined group, but significantly inhibited the production of pro-caspase-1.Conclusions Scutellarin can reduce the formation of NLRP3 inflammasome com¬positions and inhibit pro-caspase-1 activation to im¬prove LPS + ATP induced endothelial cell inflammation injury and pyrolysis, which provides a new basis for the clinical prevention and treatment of atherosclerosis.

Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway / 中国实验方剂学杂志

Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L^-1 glucose）， positive control group （100 mg·L^-1 vitamin C）， and the low- （0.25 μg·L^-1）， medium- （1 μg·L^-1）， and high-dose （4 μg·L^-1） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L^-1， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.

Salidroside inhibits endothelial-mesenchymal transition by regulating KLF4/eNOS signaling pathway / 中国药理学通报

Aim To investigate the effect and mechanism of salidroside (SAL) on homocysteine (Hcy)-induced endothelial-mesenchymal transition (EndMT) based on the KLF4/eNOS signaling pathway. Methods (1) Salidroside inhibited Hcy-induced EndMT. HUVECs were pretreated with different concentrations of SAL for 2 h, then followed by Hcy (1 mmol · L-l) co-incubation for 48 hours to induce EndMT. The expression levels of VE-cadherin, α-SMA, KLF4 and eNOS were detected by Western blot, scratch repair experiment was used to determin cell migration ability, the level of NO in cells was determined by nitrate reductase method, and the expression and location of KLF4 were observed by immunofluorescence technology. (2) The signal mechanism of SAL inhibiting End-MT through KLF4/eNOS signal was studied. siRNA mediated knockdown of KLF4 in HUVECs, and the protein expression levels of VE-cadherin, α-SMA, KLF4 and eNOS in each group were determined by Western blot. Results Western blotting demonstrated that SAL significantly reversed the Hcy-induced increase in α-SMA and KLF4 expression and decrease in VE-cadherin and eNOS expression. The immunofluorescence analysis suggested that SAL inhibited the translocation of KLF4 from cytoplasm to nucleus. (2) Silencing KLF4 down-regulated the expression of α-SMA and KLF4, and up-regulated the expression of VE-cadherin and eNOS. Compared with the group of SAL + siKLF4, there was no significant difference in the effect of SAL group and siKLF4 group on phenotypic markers. Conclusions Salidroside inhibits EndMT induced by Hcy, which may be related to the regulation of KLF4/eNOS signaling pathway.

Cyclovirobuxine D ameliorates aldosterone-induced primary neonatal rat cardiac myocyte injury via Ca / 中国药理学通报

Aim To investigate the protective effect of cyclovirobuxine D(CVB-D) on aldosterone (ALD)-induced primary neonatal rat cardiac myocytes (PNRC-Ms) injury and the possible mechanism. Methods PNRCMs were extracted by trypsin, and the PNRCMs injury model was established by ALD (10 μmol · L

1, 8-CineoIe improves endothelial cell injury induced by high glucose via regulating autophagy / 中国药理学通报

Aim To investigate the protective effect of 1, 8-Cineole on the injury of human aortic endothelial cells (HAECs) induced by high glucose (HG) via regulating autophagy. Methods Cells were incubated with different doses of 1, 8-Cineole followed by exposing to HG for 60 h, and MTT assay was used to analyse cell viability, lactate dehydrogenase (LDH) was used to detect cytotoxicity, and Western blot was used to detect Beclin1, LC3-II/I, p62, caspase-3 and caspase-9 expressions. Autophagy inhibitor (chloroquine, CQ) was treated on HAECs, and the expressions of Beclinl, LC3-II/I, p62, caspase-3 and caspase-9 were measured by Western blot. Results 1, 8-Cineole increased cell viability, reduced the content of LDH, activated autophagy and inhibited apoptosis. Compared with control group, the expression of Beclinl, LC3-II/I, p62, caspase-3 and caspase-9 in CQ group increased. Simultaneously, the expression of above-mentioned between CQ + HG group and CQ + HG + CH group. Conclusions 1, 8-Cineole has protective effect on the injury of HAECs induced by high glucose, and its effect is related to improving autophagy flux.

Effect of Oxymatrine on Oxidative Stress Injury of H9C2 Cells Induced by High Glucose / 中国实验方剂学杂志

Objective:To study the protective mechanism of oxymatrine on oxidative stress induced by high glucose in H9C2 cells. Method:H9C2 cardiomyocytes were cultured in groups and divided into normal group, high glucose (HG) group, low-dose oxymatrine (OMT) group (50 mg·L-1), high-dose OMT group (100 mg·L-1), positive drug vitamin E (VE) group (1×10-4 mol·L-1) and mannitol (M) wasotonic control group. Cell damage was detected by lactate dehydrogenase leakage, changes in cell superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected, intracellular reactive oxygen species (ROS) content and cellular mitochondria and functional integrity were detected by fluorescent probes, and Western blotting was used to detect the expressions of Bcl family proteins. Result:Compared with the normal group, the content of malondialdehyde and reactive oxygen species and the expression level of pro-apoptotic protein in the high glucose group were significantly increased (P<0.01), while the activity of superoxide dismutase and the expression levels of mitochondrial membrane potential (MMP) and anti-apoptotic protein were significantly decreased (P<0.01). Compared with the high glucose group, oxymatrine significantly reduced the leakage of lactate dehydrogenase, significantly inhibited the production of intracellular ROS (P<0.01), reduced the amount of malondialdehyde and down-regulated the expression of pro-apoptotic protein (P<0.05), increased the activity of superoxide dismutase, regulated MMP and improved the expression of anti-apoptotic protein (P<0.01). Conclusion:Oxymatrine can regulate oxidative stress by improving mitochondrial function, so as to inhibit the apoptosis of H9C2 cardiomyocytes induced by high glucose.

Essential Oil from Alpinia zerumbet Rhizome Inhibits Macrophage-derived Foam Cell Formation Via Modulating Expression of CD36 and ABCA1 / 中国实验方剂学杂志

Objective::To clarify the inhibitory effect of essential oil from Alpinia zerumbet rhizome (EOFAZ) on oxidized low-density lipoprotein (ox-LDL)-induced transformation of macrophage into foam cell and explore its possible mechanism. Method::THP-1 monocyte was incubated with 100 μg·L-1 phorbol myristate acetate (PMA) to grow into macrophage, experiment was divided into 4 groups as follows, control group, model group (80 mg·L-1 ox-LDL), EOFAZ at low dose (80 mg·L-1 ox-LDL+ 4 μg·L-1 EOFAZ)and EOFAZ at high dose (80 g·L-1 ox-LDL+ 20 μg·L-1 EOFAZ). Mathye thiazolye telrazliurn (MTT) method was employed to examine the influence of EOFAZ on macrophage viability. Western blot was used to analyze the expression level of cluster of differentiation 36(CD36) and ATP-binding cassette transporter A1(ABCA1) protein in macrophage. Enzyme-linked immunosorbent assay (ELISA) was used to detect cholesteryl ester contents in macrophage. Oil red O staining was applied to determine the accumulation of lipids in macrophage. Result::EOFAZ showed non-toxic effect on macrophage. Compared to control group, macrophage in model group displayed higher level of cholesteryl ester and lipid droplet(P<0.01), as well as significant increasing of CD36 expression (P<0.01), but no effect on ABCA1 expression. EOFAZ notably reduced the contents of lipids and cholesteryl ester(P<0.01), down-regulated expression of CD36 and up-regulated expression of ABCA1 in macrophage in comparison with the model group(P<0.01), indicating that EOFAZ inhibited transformation of macrophage into foam cell. Conclusion::EOFAZ could inhibit ox-LDL-induced transformation of macrophage into foam cell, the underlying mechanism may involves its ability to increase CD36 expression and decrease ABCA1 expression in macrophage.

Effect of Ultrafine Powder of Gastrodiae Rhizoma in Improving Learning and Memory Ability of Vascular Dementia Rats by Regulating Cholinergic System / 中国实验方剂学杂志

Objective:To investigate whether ultrafine powder of Gastrodiae Rhizoma （UPG） can alleviate the learning and memory impairment of vascular dementia rats and delay the process of VD， and whether this effect is related to the release of acetylcholine （Ach） through the regulation with acetylcholinesterase （AChE） and choline acetyltransferase （ChAT） and control of cholinergic system. Method:SD rats were randomly divided into sham operation group， UPG low dose group （0.45 g·kg-1）， UPG high dose group （1.8 g·kg-1） and Huperzine A group （80 μg·kg-1）， with 12 rats in each group. The drug administration groups were given orally drugs once a day for 8 weeks， and sham group and model group were given orally the same amount of distilled water. The learning and memory ability of the rats with VD were evaluated by the Morris water maze. Htoxylin eosin（HE） staining was used for pathomorphological observation of hippocampus CA1 area of the rats. The content of Ach was determined by enzyme-linked immunosorbent assay（ELISA）， AChE and ChAT protein expressions were detected by Western blot， and expression of ChAT in hippocampus CA1 area was observed by immunohistochemistry. Result:Compared with the sham operation group， the escape latency of the model group was significantly increased （P<0.01）， and the frequency of crossings platform and the time of staying in the target quadrant were reduced significantly （P<0.01）. HE staining of hippocampal tissues from VD rat showed neuron disorders， loss and degeneration and necrosis， pyknosis of the nucleus and light coloration of the cytoplasm. The level of acetylcholine in the hippocampus was significantly decreased by ELISA （P<0.05）， the expression level of AChE protein was significantly up-regulated， and the expression level of ChAT protein was significantly down-regulated （P<0.01）. Compared with model group， each administration group could significantly reduce the escape latency of the model rats， and significantly increase the frequency of crossing platform and the time of staying in the target quadrant （P<0.01）， the content of Ach was significantly increased （P<0.05）， the expression of AChE protein was significantly down-regulated （P<0.01）， while the expression of ChAT protein was significantly up-regulated （P<0.01）. Conclusion:UPG improves the learning and memory ability of vascular dementia rats， and its mechanism may be related to the increase of Ach， ChAT level and the decrease of AChE level.

Effect of Gastrodiae Rhizoma Powder on Prevention of Dementia and Oxidation Resistance in Mice / 中国实验方剂学杂志

Objective::To study whether long-term administration of Gastrodiae Rhizoma powder can improve the learning and memory ability of APPswe/PSldE9 double transgenic (APP/PS1) Alzheimer' s disease(AD) model mice and delay the progress of AD whether these effects are related to the regulation of antioxidant stress pathway in Kelch-like epoxylopropylamine-related protein 1(Keap1)-nuclear factor E2 related factor 2 (Nrf2)/heme oxygenase(HO)-1, and further explore the neuroprotective mechanism of Gastrodiae Rhizoma powder and its role in the prevention and treatment of AD. Method::APP/PS1 double transgenic mice model, the mice consisted of five groups: normal, normal administration group, model group, Gastrodiae Rhizoma powder prevention group, Gastrodiae Rhizoma powder treatment group.The mice in the normal administration group and the Gastrodiae Rhizoma powder prevention group were given the same dose of Gastrodiae Rhizoma powder (1.5 g·kg-1) daily at the age of 8 weeks.The normal group and model group were given the same amount of normal saline at the same time, until 24 weeks old, Morris water maze was used to test the learning and memory ability of mice, and the treatment group was treated with Gastrodiae Rhizoma powder at 22 weeks old.The mice were given the same dose of Gastrodiae Rhizoma powder (1.5 g·kg-1) every day for 2 weeks.The number of crossing platform, escape latency and platform residence time of mice were detected by Morris water maze from 24 weeks old to 24 weeks old.RNA, Real-time PCR was extracted from mouse hippocampus to detect the mRNA level of Nrf2, HO-1, Keap1, and Western blot was used to detect the expression of Nrf2, HO-1, Keap1 protein in mouse hippocampus. Result::Compared with normal group, the water maze test showed that the learning and memory ability of model group was lower than that of the model group (P<0.01), and the learning and memory ability of Gastrodiae Rhizoma powder prevention group and Gastrodiae Rhizoma powder treatment group was significantly higher than that of model group (P<0.01). Compared with normal group, the levels of Nrf2, HO-1 and protein in the hippocampus in model group decreased in varying degrees (P<0.05). Compared with model group, Gastrodiae Rhizoma powder prevented Nrf2, in the hippocampus of mice in model group.The level of HO-1 in mRNA and protein increased in different degrees (P<0.05, P<0.01). Levels of Nrf2, HO-1 mRNA in Gastrodiae Rhizoma powder treatment group was significantly higher than that in Gastrodiae Rhizoma powder group (P<0.05). There was no significant difference in the expression of Nrf2, HO-1 protein.There was no significant difference in mRNA and protein levels of Keap1 among different groups. Conclusion::Morris water maze test and other results showed that Gastrodiae Rhizoma powder could improve the learning and memory ability of APP/PS1 mice, and it may enhance the expression of downstream antioxidant genes by regulating Keap1-Nrf2/HO-1 pathway.And then improve the learning and memory ability of APP/PS1 mice.

Investigation of Mechanism of Tryptanthrin Inhibiting Breast Cancer in Mice Based on Proteomics / 中国实验方剂学杂志

Objective:A systematical study on the anti-breast cancer mechanism of tryptanthrin in breast cancer-bearing mice was done by Label-free proteomics. Method:UPLC-MS was used to detect the expressed-proteins of tryptanthrin inhibiting breast cancer in mice， chromatographic separation was achieved on the Ionoptics nano UPLC C18 column （0.075 mm×250 mm， 1.6 μm）， and gradient elution was performed with 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution as mobile phase. Data acquisition was carried out in electrospray ionization （ESI） under the positive ion mode， the scanning range was m/z 100-1 700， MaxQuant 1.6.5.0 was used for database retrieval. Label-free proteomics with high resolution mass spectrometry was used to screen differentially expressed proteins between the model group of 4T1 breast cancer mice and oral administration group of tryptanthrin （100 mg·kg-1）. The proteomics of tryptanthrin against breast cancer was carried out. Result:A total of 3 997 proteins were identified in this proteomics research， and 2 911 proteins were quantifiable. A total of 750 differentially expressed proteins were identified between the model group and the tryptanthrin group， 286 proteins were up-regulated and 464 proteins were down-regulated. Gene ontology analysis showed that these differentially expressed proteins were mainly involved in biological processes of proliferation， cell migration， apoptosis， immunity， angiogenesis， inflammatory regulation， etc. Kyoto encyclopedia of genes and genomes pathway analysis further indicated that these proteins were mainly concentrated in T cell receptors， B cell receptors， Toll-like receptors， nuclear transcription factor-κB （NF-κB）， Ras proteins， interleukin-17， tumor necrosis factor， phosphatidylinositol 3-kinase/protein kinase B （PI3K-Akt）， mitogen-activated protein kinase （MAPK） and other signaling pathways. Conclusion:The differentially expressed proteins closely related to anti-breast cancer effect of tryptanthrin on 4T1 breast cancer mice are effectively screened out， including up-regulating proteins of leukocyte differentiation antigen 14 （CD14）， prostaglandin G/H synthase 2 （PTGS2）， E3 ubiquitin-protein ligase and down-regulating proteins of CD44， heat shock 70 kDa protein 1A （HSPA1A）， macrophage migration inhibitory factor （MIF）， NF-κB， ribosomal protein S6 kinase alpha-4 （RPS6KA4） and high mobility group protein B1 （HMGB1）. These findings suggest that tryptanthrin can inhibit breast cancer in mice mainly through regulating tumor inflammatory microenvironment.

Inhibitory Effect of Essential Oil from Fructus Alpinia zerumbet on Endothelial-to-mesenchymal Transition Induced by High Glucose via Nrf2/Notch1 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet （EOFAZ） on endothelial-to-mesenchymal transition （EndMT） induced by high glucose （HG）. Method:Human umbilical vein endothelial cells （HUVECs） was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group， HG group （35 mmol·L-1）， EOFAZ low dose group （1 μg·L-1） and EOFAZ high dose group （4 μg·L-1）. After EOFAZ intervention for 2 h， HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule （CD31）. Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species （ROS） levels were detected by 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe and the contents of malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2 （Nrf2） and Notch1. The overexpression of Nrf2 was achieved by adenovirus （AD） transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group， HG group （35 mmol·L-1）， AD-Nrf2 group， EOFAZ group （4 μg·L-1）， AD-Nrf2+EOFAZ group （4 μg·L-1）. The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h， then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h， HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2， CD31， vimentin， Notch1 and Snail. Result:Compared with the HG group， after treatment with EOFAZ， the protein expression of CD31 was significantly up-regulated （P<0.05）， the protein expression of vimentin was significantly down-regulated （P<0.01）， the ability of cell migration was decreased （P<0.01）， and the contents of ROS and MDA were decreased （P<0.05， P<0.01）， the levels of CAT and SOD were increased （P<0.01）. In addition， EOFAZ could significantly up-regulate the protein expression of antioxidant signal Nrf2 （P<0.01） and down-regulate the protein expression of Notch1 （P<0.01）. High expression of Nrf2 was achieved by stable AD transfection into HUVECs. The results of Western blot showed that， compared with the HG group， the protein expression levels of Nrf2 and CD31 in each treatment group were significantly increased （P<0.01）， while the protein expression levels of vimentin， Notch1 and Snail were down-regulated （P<0.01）. At the same time， compared with the AD-Nrf2 group， the AD-Nrf2+EOFAZ group could further up-regulate the protein expressions of Nrf2 and CD31 （P<0.05， P<0.01）， while decrease the protein expression levels of vimentin， Notch1 and Snail （P<0.01）. Conclusion:EOFAZ ameliorates oxidative stress injury of vascular endothelial cells induced by HG and inhibits EndMT， which is related to Nrf2/Notch1 signaling pathway.

Preparation of Oxymatrine Phospholipid Complex Solid Lipid Nanoparticles Lyophilized Powder and Evaluation of Its Quality / 中国实验方剂学杂志

Objective: To prepare oxymatrine phospholipid complex solid lipid nanoparticles(OMT-PC-SLN) lyophilized powder and evaluate its pharmaceutical properties. Method: Pseudo-ternary phase diagram was employed to optimize the formula of microemulsion;single factor experiments were adopted to optimize the formulation process of OMT-PC-SLN lyophilized powder with encapsulation efficiency as index;the morphology of this preparation was observed by transmission electron microscope(TEM).The particle size was measured by particle size analyzer and the in vitro release performance of OMT-PC-SLN lyophilized powder was examined. Result: Optimal formulation process was as following:taking soybean phospholipid and polyethylene glycol 15-hydroxystearate(Kolliphor HS 15) as the emulsifier,ethanol as co-emulsifier,ratio of emulsifier to co-emulsifier(Km)=3:2,oil phase:(emulsifier+co-emulsifier)=1:9,oxymatrine phospholipid complex-stearic acid-soybean phospholipid-Kolliphor HS 15-ethanol(30:100:180:360:360);taking 50 mL of 4%mannitol solution as the external aqueous phase,ice bath stirring at 1 000 r·min-1 and solidifying for 1 h,precooled at -20℃ for 24 h,took out and dried for 24 h.OMT-PC-SLN lyophilized powder was spherical in appearance with encapsulation efficiency of (38.09±1.24)%,average particle size of 785.5 nm,polydispersity coefficient(PDI) of 0.456 and the Zeta potential of -24.82 mV.The cumulative release rates of OMT-PC-SLN lyophilized powder were 72.63%at 2 h and 98.42%at 12 h;the cumulative release rate of oxymatrine(crude drug) was 98.60%at 2 h. Conclusion: This optimized formulation process of OMT-PC-SLN lyophilized powder is stable with good repeatability;compared with oxymatrine,OMT-PC-SLN lyophilized powder has a certain sustained-release effect.

Analysis of Pharmacokinetic Behavior of Five Components in Qingkailing (Lyophilized) for Injection in Normal Rats and Cerebral Ischemia Rats by UPLC-MS/MS / 中国实验方剂学杂志

Objective: To establish a UPLC-MS/MS analysis method for determination of baicalin, geniposide, chlorogenic acid, cholic acid and hyodeoxycholic acid in Qingkailing (lyophilized) for injection in rat plasma, and to investigate the pharmacokinetic behavior of this preparation in normal and cerebral ischemic rats. Method: Rats were randomly divided into normal group and cerebral ischemia model group. The rat model of cerebral ischemia was established by suture embolization. The rats were given by intraperitoneal injection, and normal saline was used as the solvent. Blood samples were taken at the corresponding time points. After treatment, UPLC-MS/MS was used to determine the blood concentration of five components. The main detection conditions were mobile phase of 0.1%formic acid aqueous solution-acetonitrile for gradient elution (0-0.25 min, 90%A; 0.25-1 min, 90%-75%A; 1-2 min, 75%-50%A; 2-2.6 min, 50%-45%A; 2.6-2.65 min, 45%-90%A; 2.65-4.0 min, 90%A), the flow rate of 0.4 mL·min-1, the column temperature at 40℃, electrospray ionization under negative ion mode. The pharmacokinetic parameters were fitted and the bioavailability was calculated, the differences of treatment process of five components from Qingkailing (lyophilized) for injection in normal and cerebral ischemic rats were analyzed. Result: Compared with the normal group, the area under the curve (AUC0-t) of geniposide in rats from cerebral ischemia model group decreased significantly after intraperitoneal injection of Qingkailing (lyophilized) for injection (PTmax) of chlorogenic acid in rats from cerebral ischemia model group was significantly earlier than that in the normal group (PConclusion: Qingkailing (lyophilized) for injection has a certain difference in the treatment process between normal and cerebral ischemic rats, which has certain guiding significance for the clinical treatment of cerebral ischemic diseases with this preparation.

Effect of Oxymatrine in Inhibiting Proliferation and Migration of HT-29 Cells Induced by Insulin / 中国实验方剂学杂志

Objective:To investigate the effect of oxymatrine (OMT) on the proliferation and migration of human colon cancer cell line HT-29 under Type Ⅱ diabetes environment by co-culturing HT-29 with insulin to simulate hyperinsulinemia. Method:The effect of OMT (2, 4, 8 mmol·L-1) on insulin-induced proliferation of HT-29 was detected by methyl thiazolyl tetrazolium (MTT) assay and cloning assay. The morphology change and cell migration were evaluated under microscope and by wound healing assay. The Annexin V/propidium iodide(PI) assay was used to detect the change of insulin-induced HT-29 cell cycle and apoptosis. Western blot was performed to validate the expression of cell cycle-related protein and cell migration protein. Result:Insulin significantly increased growth of HT-29 (P-1 showed a significant inhibitory effect in this model (P0/G1 phase (P1, CDK4 and the up-regulation of p27 by OMT might involve the growth inhibition mechanism. Furthermore, OMT reduced the migration of insulin-induced HT-29 according to wound healing assay(PPPConclusion:OMT can inhibit the proliferation and migration of insulin-induced HT-29 cells. The changes of cell cycle and migration related proteins may be correlated with the mechanism.