新思路·新技术——中药复方新药研发相关重大科学和技术问题

"第十二届全国中药与天然药物药理学术会议"于2019年10月11-13日在天津市社会山国际会议中心酒店举行。会议期间,中国药理学会中药与天然药物药理专业委员会召开了"中药复方新药研发相关重大科学和技术问题"专题研讨会。研讨会分为主题报告和综合讨论2部分。主题报告由中国药理学会中药与天然药物药理专业委员会副主任委员吴春福教授主持,刘建勋、孙晓波、段大跃和吕圭源教授作主题报告。中国药理学会理事长兼中药与天然药物药理专业委员会主任委员张永祥研究员主持综合讨论,国家卫生健康委员会科技教育司重大专项处顾金辉处长全程参加。与会专家围绕中药复方新药研发相关重大科学和技术问题进行了广泛、深入的讨论。本文整理了专家们的主要观点和建议,希望能为中药复方新药研发提供新思路和新技术,为提高我国中药复方新药的研发水平发挥积极的推动作用。

IcarisideⅡ alleviates oxygen-glucose deprivation and reoxygenation-induced PC12 celloxidative injury by activating Nrf2 / SIRT3signaling pathway

OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.

Targeting MAPK pathways by naringenin modulates microglia M1 / M2 polarization in lipopolysaccharide-stimulated cultures

OBJECTIVE Neuroinflammation is considered to be an important and inevitable pathological process associated with all types of damages to the central nervous system.The hallmark of neuroinflammation is the microglia activation.In response to different micro-environmental disturbances,microglia could polarize into either an M1 pro-inflammatory phenotype,exacerbating neurotoxicity,or an M2 anti-inflammatory phenotype,exerting neuroprotection.Therefore,shifting the polarization of microglia toward the M2 phenotype could possess a more viable strategy for the neuroinflammatory disorders treatment.Naringenin(NAR) is natural y a grapefruit flavonoid and possesses various kinds of pharmacological activities,such as anti-inflammatory and neuroprotective activities.In the present study,we aimed to investigate the potential effects of NAR on microglial M1/M2 polarization and further reveal the underlying mechanisms of actions.METHODS BV-2 cells were pretreated with NAR(100 μmol·L^(-1)) for 1 h and then incubated with LPS(1 mg·L^(-1)) for 24 h.The effects of NAR on LPS-induced microglia activation,microglial M1/M2 polarization and MAPK pathways were detected.In addition,BV-2 cells were incubated with or without anisomycin(ANI,a selective agonist of JNK) to evaluate the role of JNK on microglia activation and microglia M1/M2 polarization.RESULTS First,NAR inhibited LPS-induced microglial activation.Then,NAR shifted the M1 pro-inflammatory microglia phenotype to the M2 anti-inflammatory M2 microglia state as demonstrated by the decreased expression of M1 markers,ie,inducible tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and the elevated expression of M2 markers(ie,arginase 1,IL-4 and IL-10).In addition,the effects of NAR on microglial polarization was dependent on MAPK signaling,particularly JNK inactivation,as evidenced by the fact that the selective activator of JNK abolished NAR-promoted M2 polarization and further NAR-inhibited microglial activation.CONCLUSION NAR promotes microglia M1/M2 polarization,thus conferring anti-neuroinflammatory effects via the inhibition of MAPK signaling activation.These findings might provide new alternative avenues for neuroinflammation-related disorders treatment.

Icariin ameliorates learning and memory function via improving cerebral glucose metabolism disorder in APP/PS1/Tau triple transgenic Alzheimer disease mice

OBJECTIVE To investigate the protective effect of icariin(ICA) on learning and memory function in APP/PS1/Tau triple transgenic Alzheimer disease mice(3×Tg-AD mice),and then to explore whether its mechanism is related to the improvement of brain glucose metabolism disorder.METHODS Three-month-old male 3 ×Tg-AD mice were randomly divided into three groups(n=10):3×Tg group,3×Tg+ICA low-dose group(30 mg·kg-1) and 3×Tg + ICA high-dose group(60 mg·kg-1).Age-matched male wild type(WT) mice were randomly divided into two groups(n=10):WT control group and WT+ICA60 mg·kg-1 group.ICA in vehicle(0.5% Tween-80 in distilled water) was given orally once a day for five months in the 3×Tg+ICA groups.3×Tg and WT control group were given an equal volume vehicle.Morris water maze was used to detect the learning and memory function of mice.Brain glucose metabolism in 3×Tg mice was observed by 18 F-FDG microPET imaging technique.Nissl staining and HE staining were used to evaluate the survival neurons in hippocampus of mice.Glucose oxidase assay was used to detect glucose contents in cortex of mice.The protein expression of APP,Aβ1-40,Aβ1-42 and glucose transporter 1(GLUT1),and the phosphorylation level of tau protein at multiple sites in hippocampus were detected by Western blotting.RESULTS Behavioral examination revealed a profound decrease learning and memory function,accompanied by a decrease in number of neuronal cells in 3×Tg-AD mice.Moreover,the cerebral18 F-FDG uptake rate per gram tissue was reduced and the glucose contents in the cortex were increased in 3×Tg-AD mice.In addition,Western blotting analysis showed that the expression of APP,Aβ1-40,Aβ1-42 proteins and the levels of tau protein phosphorylation at Ser199/202 and PHF-1(Ser396/404) sites were increased significantly,followed by a decrease of GLUT1 expression in hippocampus of 3×Tg-AD mice.All of these changes in behavioral functions,neuronal loss and related protein expression were reversed when mice were treated with ICA.CONCLUSION ICA can improve the learning and memory ability of AD model mice,the mechanism may be related to the improvement of cerebral glucose metabolism dysfunction by increasing the expression of GLUT1.

Cyclin-dependent kinase 5 is required for suppressing D1-dependent signaling mediated through muscarinic 4 in isolated medium spiny neurons

OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.

Pharmacological effects of icariin and icariinriside on central nervous system

Epimedium Brevicornum is a traditional Chinese medicinal plant possessing properties of sweet, warm, tonifying kidney, strong bones and rheumatism. Icariin, a flavonoid compound, is one of the main active ingredients of Epimedium. Icariinriside(ICS) is the main metabolite of icariin. Icariinand ICS have multiple pharmacological effects such as anti-tumor, anti-oxidative stress, improvement of cardiovascular and cerebrovascular, and regulation of endocrine. We have conducted a series of studies on the neuroprotection and mechanisms of action of icariin and ICS for many years. The main findings are reported as follows.(1) Effect on Alzheimer disease(AD) model animals: Icariin significantly attenuated learning and memory loss, hippocampal neuron loss and senile plaque formation in APP/PS1 transgenic AD model mice, which may be related to inhibition of Aβ production and reduction of PDE5(phosphodiesterase 5).In addition, icariin significantly attenuated Aβ25-35-induced learning and memory decline and hippocampal neuronal apoptosis in rats, which may be related to lowering PDE5 content and up-regulating BDNF/Trkb/CREB signaling pathway, inhibiting MAPK and NF-κB signaling pathways, and increasing expression of acetylcholinesterase(ACHE) and choline acetyltransferase(CHAT) in the hippocampus. At the same time, icariin can significantly improve the learning and memory dysfunction induced by amanita proline in rats, which may be related to the inhibition of hippocampal neuronal apoptosis, antiexcitatory amino acid toxicity and regulation of MAPK and NF-κB signaling pathways.(2) Effects on Parkinson disease(PD) model animals: The study found that in LPS-induced dopaminergic neuron injury animal models and cell models, icariin can inhibit microglia by inhibiting the expression of inflammatory factors such as TNF-α, IL-1β, NO and COX-2. Activation of glial cells increases the expression of neurotrophic factors such as BDNF and GDNF, increases the content of dopamine(DA) and its metabolites 3, 4-dihydroxyphenylacetic acid(DOPAC) and homovanillic acid(HVA), inhibits MAPK and the NF-κB signaling pathway, protecting dopaminergic neurons. In addition, icariin significantly attenuated6-OHDA-induced dopaminergic neuronal damage. In Nrf2 knockout mice, the neuroprotective effect of icariin disappeared, suggesting that Nrf2 may be one of the targets of icariin to play neuroprotective effects.(3) Effects on vascular dementia(VD) model animals: Icarin can improve the learning and memory ability and memory function of chronic hypoperfusion rats, and its mechanism may be related to increase the level of VEGF/VEGFR2 protein in the brain and activate multiple downstream signaling pathways to promote angiogenesis to play an indirect protective effect on neurons;The level of BDNF/Trk B protein in the brain increases the phosphorylation level of CREB and exerts direct neuroprotective effects.(4)Effect on cerebral ischemia: In a model of ischemic brain injury, icariin acts to up-regulate Sirt1 by activating p38, thereby exerting an anti-ischemic injury and protecting neuronal cells. In addition, icariin has neuroprotective effects on cerebral ischemia-reperfusion injury in rats, which may increase GSH-Px,SOD activity, decrease MDA content, inhibit free radical damage, reduce NO content, NOS activity,and inhibit neurotoxic damage. Reduction of MPO activity, TNF-α, IL-1β content is associated with inhibition of inflammatory response.(5) Cell protection: Icariin has a protective effect on 6-OHDA-induced oxidative damage in PC12 cells, which may be related to inhibition of apoptosis and regulation of Keap1/Nrf2/ARE signaling pathway, while ICS can attenuate oxygen-glucose deprivation/reoxygenation-induced cellular damage in PC12 cells. The mechanism of cellular oxidative damage may be related to inhibition of apoptosis and regulation of Nrf2/SIRT3 signaling pathway.Icariin and ICS have good preventive and therapeutic effects on central nervous system diseases such as AD, PD, VD, etc. However, due to the complexity of the molecular mechanisms of icariin and ICS, the molecular mechanisms of the central nervous system are still worthy of further study.

Dendrobium nobile Lindl. alkaloids alleviate Mn-induced neurotoxicity via PINK1/Parkinmediated mitophagy in PC12 cells

OBJECTIVE Activation of the PINK1/Parkin-mediated autophagy pathway has been proposed to play a protective role in the development of neurological disorders through the elimination of damaged macromolecules or organelles.Exposure to excessive manganese(Mn) causes neurotoxicity and can produce a Parkinson disease(PD)-like neurological disorder.Mitochondrial dysfunction and oxidative stress are implicated in the mechanism of Mn induced neurotoxicity.The present study was designed to determine whether Dendrobium nobile Lindl.alkaloids(DNLA),a Chinese medicinal herb extract,confers protective function over Mn-induced cell toxicity,and to investigate whether the modulation of PINK1/Parkin-mediated autophagy is involved in the mechanism of DNLA-mediated cell protection over Mn toxicity.METHODS AND RESULTS Rat adrenal pheochromocytoma PC12 cells were utilized as an in vitro model of Mn cell toxicity.It was found that the treatment of the PC12 cells with Mn resulted in concentration-dependent cell death,accompanied by a decrease in mitochondrial respiration capacity and an increase in ROS generation,whereas pretreatment of cells with DNLA significantly alleviated cell toxicity induced by Mn and improved mitochondrial function and oxidative status.Mn treatment enhanced apoptotic cells along with a marked increase in the protein expression of Bax and a decrease in the expression of Bcl-2 protein.On the contrary,DNLA increased Bcl-2 expression,and concomitantly dramatically decreased the Bax/Bcl-2 ratio.Analysis of the expression of PINK1 and Parkin revealed that pretreatment of cells with DNLA significantly alleviated the decrease in protein levels of both PINK1 and Parkin caused by Mn.Furthermore,cells treated with Mn exhibited increased expression of LC3-Ⅱ and a decrease in accumulation of P62,which was noticeably reversed by the pretreatment of cells with DNLA.CONCLUSION DNLA inhibits Mn induced cytotoxicity,which may be mediated through modulating PINK1/Parkin-mediated autophagic flux and improving mitochondrial function.

Icariside Ⅱ, a PDE5 inhibitor, attenuates cerebral ischemia/reperfusion injury through activating BDNF/TrkB/CREB signaling pathway

OBJECTIVE To explore the effects and mechanism of icariside Ⅱ(ICS Ⅱ),a pharmacologically active compound derived from herbal Epimedii with previous study-proved phosphodiesterase 5(PDE5) inhibitors,was investigated in vivo using a middle cerebral artery occlusion/reperfusion(MCAO/R) model in rats and in vitro using an oxygen-glucose deprivation/reperfusion(OGD/R) model in primary hippocampal neurons.METHODS Laser Doppler flowmeter was introduced to examine the cerebral blood flow of MCAO/R rats.The neurological deficits scores,brain water content and infarction volume were assessed after MCAO/R.OGD/R-induced primary hippocampal neuronal injury and apoptosis were examined by MTT,lactate dehydrogenase(LDH) release,TUNEL staining and flow cytometry,respectively.Expressions of PDE5 A and memory-related signaling pathways were measured using Western blotting analysis.The direct interaction between ICS Ⅱand PDE5 was further evaluated by molecular docking.RESULTS ICS Ⅱ significantly decreased the infraction volume in MCAO/R rats.Furthermore,ICS Ⅱ significantly abrogated OGD/R-induced hippocampal neuronal death.Moreover,ICSⅡ not only effectively restored the 3′ 5′-cyclic guanosine monophosphate(cGMP) level and protein kinase G(PKG) activity both in vivo and in vitro,but also increased brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrkB) and cAMP response element-binding protein(CREB) expressions,thereby inhibited hippocampal neuronal apoptosis.Mechanistically,the beneficial effects of ICS Ⅱ was attributed to its activation of the PKG/TrkB/BDNF via increasing BDNF expression,evidenced by that the inhibition effects of ICSⅡ was abrogated by Rp-8-BrcGMPS,a PKG inhibitor,or ANA-12,a TrkB inhibitor.ICSⅡ also decreased both protein level and activity of PDE5.Notably,ICSⅡ might effectively bind and inhibite PDE5 as demonstrated by relatively high binding score.CONCLUSION ICSⅡ significantly protect against cerebral ischemia/reperfusion injury in rats and rescues OGD/Rinduced hippocampal neuronal injury,and the underling mechanisms are,at least partly,due to inhibition of PDE5 and activation of BDNF/TrkB/CREB signaling pathway.Hence ICS Ⅱ may be an effective agent for combating cerebral ischemia/reperfusion injury.

Ellagic acid supports neurons by regulating astroglia nuclear factor erythroid 2-related factor 2

OBJECTIVE Astroglia support neurons by providing substrates for neuronal metabolism, glutamate clearance and anti-oxidant protection. Nuclear factor erythroid 2-related factor 2(Nrf2) is the main switch of intracellular antioxidant defense system. Also, Nrf2 signaling is recognized to activate the neurotrophic pathway to replace/protect damaged organelles. Ellagic acid(EA), an excretion component of fruits and nuts, displays anti-oxidant, cardioprotective and antiinflammatory activities. However, few studies have been focused on the neurotrophic properties of EA. Our study investigated whether EA could enhance astroglia neurotrophic effects to support neurons and the underlying mechanisms as well. METHODS Primary neuron-enriched cultures, primary astroglia-enriched cultures and primary neuron-astroglia co-cultures were applied to detect whether pharmacological regulation of astroglia function by EA could be utilized to overcome neuronal death. RESULTS This study indicated that EA promoted neuronal survival. Furtherly, astroglia Nrf2 participate in EA-elicited neuronal survival with the following scenarios. First, EA induced astroglia proliferation, neurotrophic factors release and Nrf2 activation. Second, astroglia-targeted Nrf2 si RNA inhibited EA-mediated astrogliosis,neurotrophic factors excretion and neuronal survival. CONCLUSION EA mediated astroglia Nrf2 activation to enhanced neurotrophic effects on neurons, and these findings might provide new strategies for neurotrophic factor-based treatment of neurodegenerative disease.

左旋盐酸去甲基苯环壬酯对MPTP诱导的SH-SY5Y细胞损伤的保护作用(英文)

目的探究M4受体选择性拮抗剂左旋盐酸去甲基苯环壬酯(R-DM8021)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导人多巴胺能神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用。方法预先给予不同浓度R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))孵育SH-SY5Y细胞1 h后,加入MPTP1 mmol·L^(-1)共孵育24 h,MTT比色法检测细胞存活率,流式细胞仪检测细胞内活性氧(ROS)含量,TUNEL法观察细胞凋亡形态,Western蛋白印迹法检测细胞凋亡相关蛋白Bax和胱天蛋白酶3前体蛋白表达水平。结果 MTT结果显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,细胞存活率显著降低至(44.5±2.7)%(P<0.01);与MPTP组相比,预先给予R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))均可逆转MPTP对SH-SY5Y细胞的损伤作用,细胞存活率分别提高至(80.5±4.7)%,(71.5±8.1)%,(74.4±6.5)%和(61.3±3.2)%(P<0.01)。流式细胞检测结果显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,细胞内ROS含量显著升高至129±22(P<0.01);与MPTP组相比,预先给予R-DM8021(1×10^(-9),1×10^(-8),1×10^(-7),1×10^(-6)mol·L^(-1))显著降低MPTP诱导的胞内ROS释放,细胞内ROS含量降低至84±12,86±15,86±6和88±8(P<0.05)。TUNEL检测显示,与细胞对照组相比,MPTP 1 mmol·L^(-1)作用SH-SY5Y细胞24 h,贴壁细胞染色质皱缩,核膜裂解,并产生大量凋亡小体;与MPTP组相比,预先给予R-DM8021 1 nmol·L^(-1)可显著改善MPTP诱导的细胞凋亡,凋亡小体显著减少。Western蛋白印迹结果显示,与MPTP组相比,R-DM8021 1 nmol·L^(-1)可显著抑制MPTP诱导SH-SY5Y细胞凋亡,降低Bax和胱天蛋白酶3前体蛋白的表达(P<0.01)。结论 R-DM8021能有效对抗MPTP诱导的SH-SY5Y细胞损伤,其机制与提高细胞存活率,降低胞内ROS含量,抑制细胞凋亡有关。

Protective effect of sodium ferulate on cardiac hypertrophy in spontaneously hypertensive rats

OBJECTIVE To investigate the inhibitory effect and mechanism of sodium ferulate(SF)on myocardial hypertrophy in spontaneously hypertensive(SHR).METHODS Forty 14-week-old SHR male rats were randomly divided into model group(SHR,receive distilled water)and SF treatment groups(SF 20,40 and 80 mg·kg^-1 per day,respectively).Age-matched male Wistar-Kyoto(WKY)rats gavaged with distilled water served as controls.After 12 weeks of treatment,the effects of SF on cardiac hypertrophy were evaluated using echocardiographic measurement,pathological analysis and the expression of atrial natriuretic peptide(ANP),myosin heavy chainβ(β-MHC)-a gene related to myocardial hypertrophy.In order to explore the mechanism of SF on myocardial hypertrophy,the calcium-sensing receptor(CaSR),calcineurin(CaN),nuclear factor of activated T cell 3(NFAT3),phosphorylation NFAT3(p-NFAT3),zinc finger transcription factor(GATA4),phosphorylation GATA4(p-GATA4),protein kinase Cβ(PKC-β),Raf-1,extracellular regulated protein kinase 1/2(ERK 1/2),phosphorylation ERK1/2(p-ERK 1/2)and mitogen-activated protein kinase phosphatase-1(MKP-1)were detected.RESULTS The myocardial hypertrophy parameters,myocardial cell cross section area,left ventricular wall thickness and expression of ANP and β-MHC,CaSR,CaN,NFAT3,p-GATA4,PKC-β,Raf-1,and p-ERK 1/2 were significantly increased,while the left ventricular cavity was significantly smaller,expression of p-NFAT3 and MKP-1 were significantly decreased,meanwhile,the ultra⁃structure of cardiomyocytes was significantly damaged in 26-week-old SHR rats.Notably,SF significantly ameliorated myocardial hyper⁃trophy in 26-week-old SHR rats;suppressed the overexpression of ANP,β-MHC,CaSR,CaN,NFAT3,p-GATA4,PKC-β,Raf-1,and p-ERK 1/2 and increased the expression of p-NFAT3 and MKP-1.CONCLUSION SF can inhibit cardiac hypertrophy in SHR rats,and the mechanism may be related to the inhibition of CaSR mediated signaling pathway.

多叶斑叶兰繁殖体系建立及基于转录组的发育调控途径功能基因研究

多叶斑叶兰,是兰科斑叶兰属濒危野生植物、国家二级保护植物,具有极高的观赏和药用价值。多叶斑叶兰由于分布种群小,传播扩散能力弱,自然繁殖受到极大限制。以野生多叶斑叶兰茎段作为外植体,建立高效直接的植株再生体系。结合高通量转录组测序技术与生物信息学分析技术,深入挖掘参与多叶斑叶兰器官发育过程的功能基因。结果表明,Morel+2.0mg/L6-BA+0.5mg/LKT+1.0mg/LNAA+1g/L蛋白胨+25g/L蔗糖+7.0g/LAgar+1.0g/L活性炭+30g/L香蕉+50g/L土豆为最佳的芽诱导培养基;Morel+3mg/L6-BA+0.5mg/LNAA+0.5mg/LKT+0.01mg/LTDZ+2g/L蛋白胨+25g/L蔗糖+7.0g/LAgar+1.0g/L活性炭30g/L香蕉+50g/L土豆为最佳芽增殖培养基;1/2Morel+1.0mg/LIBA+0.1mg/LNAA+1g/L+花宝2号+25g/L蔗糖+7.0g/LAgar+1.0g/L活性炭+1g·L-1蛋白胨为最佳的生根培养基。转录组测序分析组装获得170688个Unigene,平均长度为584bp,N50为833bp。17352个Unigene比对注释到NR、Swiss-Prot、KOG、GO、KEGG数据库。野生苗与组培苗差异表达UnigeneGO与KEGG功能富集分析显示,Unigene主要参与调控植物激素信号转导、植物形态发育、次生代谢过程以及能量代谢过程等生物学功能。进一步分析获得511个参与植物器官发育调控相关的转录因子。成功建立了多叶斑叶兰种质资源保存与高效离体再生体系,结合高通量转录组学技术,获得全面完整的多叶斑叶兰转录组信息特征,为后期多叶斑叶兰快速扩繁、遗传转化以及功能基因鉴定、遗传发育及其调控机制研究奠定基础。

Protective effects of icariin on neurons injured by cerebral ischemia/reperfusion

Background It is very important to search for novel anti-ischemia/reperfusion neuroprotective drugs for prevention or treatment of cerebrovascular diseases. Icariin, the major active component of traditional Chinese herb Yinyanghuo, may have a beneficial role for neurons in cerebral ischemia/reperfusion caused by accident. However, it was not clear yet. In this study, we observed the protective effects of icariin on neurons injured by ischemia/reperfusion in vitro and in vivo and investigated its protective mechanism. Methods Cerebral cortical neurons of Wistar rats in primary culture were studied during the different periods of oxygen-glucose deprivation and reperfusion with oxygen and glucose. Cell viability was determined by methyl thiazoleterazolium （MTY） assay. The activity of lactate dehydrogenase （LDH） leaked from neurons, cell apoptosis and the concentration of intracellular free calcium were measured respectively. On the other hand, the mice model of transient cerebral ischemia/reperfusion was made by bilateral occlusion of common carotid arteries and ischemic hypotension/reperfusion. The mice were divided into several groups at random： sham operated group, model group and icariin preventive treatment group. The changes of mice behavioral, activities of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） were measured, respectively. Results Treatment with icariin （ final concentration 0. 25, 0. 5, and 1 mg/L） during ischemia./reperfusion- mimetic incubation in vitro concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing LDH release, decreasing cell apoptosis, and blunting elevation of intracellular calcium concentration. And in vivo the learning and memory abilities significantly decreased, activities of SOD were diminished and MDA level increased obviously in model group, compared with that in sham operated group. But pre-treatment of model mice with icariin （ 10, 30 and 100 mg/kg, i.g. ） significantly blunted the decrease of mice learning, memory ability and SOD activity, and markedly decreased MDA level. Conclusions Icariin has protective effects on cerebral ischemia./reperfusion injured neurons. And decreasing cell apoptosis, preventing intracellular calcium concentration elevation and enhancing anti-oxidant capacity may contribute to its protective effects.

金钗石斛生物碱成分分析及其对Aβ_(1-42)所致PC12细胞凋亡的抑制作用

目的用Aβ_(1-42)处理高分化PC12细胞建立体外阿尔茨海默病(AD)细胞模型,研究金钗石斛生物碱(DNLA)对Aβ_(1-42)所致PC12细胞凋亡的抑制作用及机制。方法用UPLC-ESI-HRMS技术分析DNLA成分。将PC12细胞分为对照组、DNLA正常给药组(0.03、0.3、3μg·mL^(-1))、30μmol·L^(-1) Aβ_(1-42)模型组、DNLA低浓度组(Aβ_(1-42)+0.03μg·mL^(-1) DNLA)、DNLA中浓度组(Aβ_(1-42)+0.3μg·mL^(-1) DNLA)、DNLA高浓度组(Aβ_(1-42)+3μg·mL^(-1) DNLA)和阳性对照组(Aβ_(1-42)+10μmol·L^(-1)美金刚),观察DNLA对Aβ_(1-42)所致细胞毒性的保护作用,探讨DNLA对钙/钙调蛋白依赖性蛋白激酶激酶2磷酸化,caspase-3和Cleaved caspase-3表达的调控作用。结果DNLA主要由石斛碱、石斛胺碱、石斛碱氮氧化物和石斛星碱等生物碱组成。DNLA不显著影响PC12细胞存活率;0.3、3μg·mL^(-1) DNLA预给药显著抑制Aβ_(1-42)诱导PC12细胞毒性,改善损伤细胞形态,减少凋亡细胞,下调钙/钙调蛋白依赖性蛋白激酶激酶2的Ser511位点磷酸化水平和caspase-3、Cleaved caspase-3蛋白表达(P<0.05)。结论金钗石斛生物碱抑制Aβ_(1-42)诱导PC12细胞凋亡,其机制与负向调控钙/钙调蛋白依赖性蛋白激酶激酶2的Ser511位点磷酸化,下调caspase-3蛋白表达、减少caspase-3蛋白活化有关。

淫羊藿苷调节海马BDNF通路减轻血管性痴呆大鼠的学习记忆障碍

目的观察淫羊藿苷(Ica)对血管性痴呆(VD)大鼠海马组织脑源性神经营养因子(BDNF)信号通路的影响。方法将大鼠随机分为假手术组,Ica正常对照(60 mg·kg^-1)组,模型组,Ica低、中、高剂量(15、30、60 mg·kg^-1)组(均n=8)。采用双侧颈总动脉结扎制备慢性脑低灌注诱导VD模型,仅分离颈总动脉而不结扎为假手术组,术后第9日每日灌胃给予相应剂量的Ica,连续28 d,假手术组和模型组大鼠灌胃等体积蒸馏水。Morris水迷宫实验检测大鼠空间学习记忆功能;Nissl染色观察海马神经元形态和数量;Western blot法检测BDNF信号通路关键蛋白的表达及激活。结果与假手术组大鼠相比,模型组大鼠逃避潜伏期明显延长(P<0.01),目标象限的停留时间显著减少(P<0.01),Ica中、高剂量组大鼠停留时间较模型组显著延长(P<0.05)。模型组大鼠海马CA1区神经元排列紊乱、松散,正常细胞数量较假手术组显著减少(P<0.01),Ica各剂量组神经元形态改善,正常神经元数量显著多于模型组(P<0.05)。模型组海马组织BDNF和酪氨酸激酶B(TrkB)蛋白表达量及磷酸化蛋白激酶B(p-Akt)水平均显著下降(P<0.01),细胞外调节蛋白激酶(ERK)和cAMP应答元件结合蛋白(CREB)磷酸化水平降低(P<0.05);与模型组比较,Ica中、高剂量组BDNF和TrkB蛋白表达水平、Akt磷酸化水平均显著增高(P<0.05),Ica高剂量组ERK和CREB磷酸化水平显著增高(P<0.05)。结论淫羊藿苷改善VD大鼠的学习记忆功能可能与调节海马BDNF信号通路相关。

金钗石斛生物碱对四氯化碳诱导急性肝损伤小鼠的作用

目的观察金钗石斛生物碱(ADNL)对四氯化碳(CCl4)诱导急性肝损伤小鼠的作用。方法50只C57小鼠随机分为正常对照组,正常给药(ADNL 20 mg·kg^(-1)·d^(-1))组,模型组和ADNL低、高剂量(10、 20 mg·kg^(-1)·d^(-1))组,每组10只,连续灌胃7 d。末次给药后2 h,除正常对照组和正常给药组外,其余各组腹腔注射0.2%CCl4玉米油溶液(10 mL·kg^(-1))建立急性肝损伤模型。采用全自动生化仪检测小鼠血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量, HE染色观察肝组织病理学变化, RT-PCR检测肝组织中肿瘤坏死因子(TNF)-α、白细胞介素1β(IL-1β)和白细胞介素6 (IL-6) mRNA表达水平, Western blot检测TNF-α、 IL-6、 p-核转录因子(NF)-κB p65蛋白表达水平。结果与正常对照组比较,正常给药组ALT、 AST及肝组织病理学无明显变化,模型组小鼠血清ALT、 AST的含量均明显升高(P <0.05),肝脏组织可见明显病理损伤, TNF-α、 IL-1β和IL-6 mRNA和IL-6、 TNF-α和p-NF-κB p65蛋白表达显著增加(P <0.05)。与模型组比较, ADNL低、高剂量组血清ALT和AST含量显著降低(P <0.05),肝组织病理损伤明显改善, TNF-α和IL-1βmRNA表达显著下调(P <0.05), TNF-α和p-NF-κB p65蛋白表达显著减少(P <0.05), ADNL高剂量组IL-6蛋白和mRNA表达也显著下调(P <0.05)。结论 ADNL对CCl4所致急性肝损伤具有显著改善作用,可能与抑制炎症反应有关。

以问题为基础的教学方法在药理学教学中应用及效果分析

目的分析以问题为基础的教学方法在药理学教学中应用及效果。方法随机选取2015~2018年遵义医科大学2015级临床医学系四个班级学生作为研究对象,其中两个班级分为PBL教学班(实验班)98人和传统教学班(普通班)96人进行研究。课程结束后,比较实验班和普通班理论考试成绩,采用自制教学满意度问卷调查表调查学生对教学的满意率并比较。结果实验班平均成绩为(78.47±11.46)分,高于同级普通班的(73.59±11.78)分(P<0.05);实验班考试获高分(>80分)高于普通班,考试不及格人数少于普通;共发放问卷194份,收回190份,回收率97.94%。实验班学生对各项调查指标的满意率均优于普通班(P<0.05)。结论应用PBL教学法,可以提高药理学考试成绩,能够激发学生的自主学习兴趣,有益于提升学生独立思考、提出问题和解决问题的能力,对提高药理学教学大有裨益。