联用多步骤虚拟筛选方法发现具有新母核的GABAA受体正性变构调节剂

GABAA受体主要介导哺乳动物中枢神经系统的抑制性信号传递,是镇静催眠药的关键靶点。在寻找具有新母核的镇静催眠药的过程中,计算机辅助药物设计(CADD)方法显示出巨大的优势。在这项研究中,首先,我们通过机器学习模型、分子对接模型和分子力学广义玻恩比表面积(MMGBSA)方法筛选了来自于商业数据库中的41112种化合物。经过筛选,我们得到了16个化合物,然后我们通过全细胞膜片钳电生理学实验验证了4个结构新颖的化合物确实为有效的GABAA受体正性变构调节剂。其中,化合物GPR120在细胞水平和动物水平都得到了实验验证。在重组表达α1β2γ2型受体的皮层神经元中,在10和50µmol∙L^(−1)浓度下,GPR120可将GABA EC3-10电流分别增强71.5%和163.8%。通过全分解贡献分析和点突变实验,我们发现GPR120与GABAA受体结合的关键位点是H102,与阳性药物地西泮相似。为了进一步验证GPR120在动物水平上的功能,我们进行了运动活动测试和翻正反射消失(LORR)实验。GPR120对小鼠的运动活动有抑制作用,6 h后可恢复,说明GPR120是一种中度镇静剂。在戊巴比妥钠(PB)诱导的翻正反射消失实验中,与生理盐水组相比,GPR120(20 mg∙kg^(−1))可显著缩短开始LORR的时间并延长LORR的持续时间。综上所述,通过联用多种虚拟筛选方法,我们发现了GPR120是一种具有新型母核的中度强度镇静剂。

蒺藜皂苷抗衰老作用的实验研究

目的观察蒺藜皂苷对衰老动物学习记忆功能及脑衰老生化指标的影响.方法采用D-半乳糖所致小鼠亚急性衰老模型.结果蒺藜皂苷可明显提高衰老小鼠学习记忆功能,降低脑组织中老化代谢产物与其相关酶(LPO,LF,MAO-B)的活性,并能促进免疫器官的增长.结论提示了蒺藜皂苷具有抗衰老的作用.

益肾清对膜性肾病模型的治疗及其机理研究

目的 :观察益肾清对家兔膜性肾病的治疗作用及其机理研究。方法 :参考Border法 ,观察益肾清灌胃口服对阳离子化牛血清白蛋白诱导的家兔膜性肾病的治疗作用 ,并了解它对正常小鼠免疫功能、大鼠尿量和血液流变学的影响。结果 :益肾清能明显缓解家兔蛋白尿 ,保护肾功能 ,恢复肾小球的病理改变 ;还具有较强的免疫增强、利尿和活血化瘀功能。结论 :益肾清对治疗家兔膜性肾病具有较好的疗效 ,其作用机制可能与利尿、活血化瘀或免疫增强作用有关。

DJ-1融合蛋白的原核表达、纯化和鉴定

Nagakubo等[1]在1997年发现并报道了DJ-1之后,人们研究发现DJ-1基因突变与帕金森病（Parkinsons disease,PD）相关[2],此外,DJ-1还具有转录调控、抗氧化应激等功能[3-4]。目前DJ-1逐渐成为PD的研究热点,但国内尚未见有关DJ-1蛋白原核表达的报道。

Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity

Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 （DE3）. Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside （IPTG）, and the interest protein was identified by SDS-PAGE and purified using one-step nickel （Ni） affinity chromatography. Under the optimal expres- sion condition （0.05 mM IPTG for 6 h at 37 ℃）, the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.

Calf spleen extract alleviates cyclophosphamide-induced leucopenia in mice and inhibits the glycolysis of HL60 cells

Calf spleen extract(CSE) has been clinically used as an adjuvant agent in malignant tumor therapy.It can improve the physical status of patients.However,its mechanism of action remains relatively unclear.In this study,we investigated the effect of CSE on leucopenia in mice and on promyelocytic leukemia cells(HL60).CSE in 10 mL/kg(2.5 mg bioactive polypeptides and 190μg ribose/mL) significantly increased leukocyte numbers in leucopenia mice.The effect on neutrophil numbers,among all leukocytes,was the most evident.CSE stimulated the proliferation of bone marrow cells in vitro and decreased the GM-CSF level in serum.In addition,CSE significantly reduced the viability of HL60 cells,decreased the production of lactate and adenosine triphosphate(ATP) of these cells.CSE induced the apoptosis and S-phase arrest in HL60 cells.In conclusion,CSE can enhance leukocyte numbers,which may be attributed to its direct stimulatory effect on bone marrow cells.CSE is an inhibitor of promyelocytic leukemia cell viability,which may be attributed to the induction of the apoptosis,the arrest of cell cycle and the inhibition of the glycolysis of cells.

Cyclovirobuxine D inhibits blood coagulation and venous thrombosis

Cyclovirobuxine D （CVB-D） is a compound extracted from Chinese traditional plant Buxus microphylla, which has been used for treating arrhythmia and myocardial ischemia in China. In this study, we investigated its effect on blood coagulation and thrombotic formation in mouse and rat models. The doses of CVB-D used in this study （5-20 mg/kg） prolonged clotting time （CT） in a dose-dependent manner （P〈0.01）. It also significantly prolonged thrombin time （TT）, prothrombin time （PT） and activated partial thromboplast time （aPTT） （P〈0.05 or P〈0.01） at the doses of 10-20 mg/kg. CVB-D did not affect the bleeding time （BT） compared with the control group, while warfarin significantly prolonged the bleeding time. CVB-D at the doses of 5-20 mg/kg reduced wet weight of thrombosis （P〈0.01）. This study demonstrated the anti-coagulation effect and anti-thrombosis effect of orally administered CVB-D without substantially increasing bleeding. These findings suggest that CVB-D probably can be used as an oral anti-coagulant in addition to its current applications.

Methoxyl methyl ether isoamylene quercetin, a quercetin derivative, protects rat aorta endothelial cells against oxidation and apoptosis

Methoxyl methyl ether isoamylene quercetin （MIAQ） is one of the newly synthesized quercetin derivatives. The present study investigated the effect of MIAQ on rat aorta endothelial cells （RAECs） injured by hydrogen peroxide （H2O2）, as well as the potential mechanisms. We observed that MIAQ at 2.5-10μmol/L significantly enhanced the viability of injured RAECs, and the effect was more potent than quercetin and ct-tocopherol. However, M1AQ at the same concentration failed to show anti-oxidant activity in a cell-free system. In H2O2-injured endothelial cells treated with MIAQ （5-10μmol/L）, the level of nitric oxide （NO） and malondialdehyde was decreased, and the activities of superoxide dismutase and glutathione peroxidase was enhanced. In addition, RAECs treated with MIAQ （2.5-10 μmol/L） exhibited significant inhibiting apoptosis. In conclusion, MIAQ had protective effect on RAECs, possibly through increasing NO production and antioxidases activities, as well as inhibiting apoptosis. These findings suggest that MIAQ is possibly beneficial in the prevention of atherosclerosis and other diseases related to endothelial injury.

Neuroprotective effects of xanthone extract from Swertia punicea Hemsl against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson's disease

As a widely used traditional Chinese medicine （TCM）, Swertia punicea Hemsl has exhibited effects on anti-hepatitis B virus （HBV）, liver protection, hypoglycemic activity and cholecystitis. In this study, we confirmed that xanthone extract from Swertia punicea Hemsl （XSPH） improved the motor deficit, increased the levels of striatal dopamine （DA） and homovanilic acid （HVA）, and alleviated the loss of tyrosine hydroxylase （TH）-positive neurons located in substantia nigra pars compacta （SNpc） in MPTP-induced mouse model of Parkinson＇s disease （PD）. In conclusion, the present results indicated that XSPH offered neuroprotective effects against the neurotoxicity of MPTP and it might be a potential treatment for PD.

Intervention of multiple pathways by multiple active components provides potent protection against cerebral ischemia injury

To explore the effect of multiple pathway intervention in acute cerebral ischemia injury, we prepared a medicine formula （formula 2） consisting of ginsenosides, pueraria flavonoids, ophiopogonis and borneol as a tool medicine. The effects of formula 2 and its components on PC12 cell viability and potential pathway were investigated, and the influence of this formula on venous thrombosis and platelet aggregation was also assessed, then the effect of formula 2 on middle cerebral artery occlusion （MCAO） reperfusion was observed in rats. Formula 2 markedly enhanced the cell viability, which was stronger than that of each individual component. Formula 2 significantly inhibited the NO production in PC12 cells induced by H202, and this effect was also stronger than that of each individual component. Moreover, formula 2 enhanced the SOD activity, and the effect was stronger than that of ginsenosides. In addition, formula 2 reduced the MDA content, and this effect was stronger than that of ophiopogonins. In vivo, formula 2 showed potent inhibitory effects on platelet aggregation and venous thrombosis. Furthermore, formula 2 （single dose, s.c.） significantly reduced the infarct volume and neurobehavioral scores in MCAO reperfusion rats. Take together, our results suggests that formula 2 has powerful ability of inhibiting the ischemia/reperfusion injury, and this effect might be attributed to its simultaneous intervention in the cascade reaction of neuronal injury via multiple pathways contributed by multiple components during cerebral ischemia/reperfusion.

rAcAP5: high-yield strain screening, expression, purification and thrombolytic effect evaluation in rat embolic middle cerebral artery occlusion model

Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.

Protective effects of orally administered honokiol on cerebral ischemia reperfusion in rats and on stroke in SHRsp

Honokiol is a protective agent for cerebral ischemia injury when administered intravenously. In the present study, we aimed to investigate the oral effect of honokiol microemulsion on cerebral isehemia-reperfusion （I-R） injury in rats and stroke in SHRsp. Both tMCAO and SHRsp models in rats were used to evaluate the efficacy of the microemulsion. Rat aortic segment contraction test, primary rat aortic endothelial cells and primary brain microvascular endothelial cells （BMECs） injured by OGD-R were used to explore its potential action mechanism. Oral honokiol microemulsion significantly reduced infarct volume, neurological score and brain water content in tMCAO model, and it evidently reduced neurological score and increased the survival rate of SHRsp. Moreover, honokiol significantly inhibited aortic contraction induced by KC1 and phenylephrine, and L-NAME suppressed these inhibitory effects. On the other side, honokiol increased NO and p-eNOS levels in rat endothelial cells. In addition, it also protects BMECs against OGD-R injury and increased eNOS expression in BMECs. In conclusion, oral honokiol administration has protective effects in tMCAO and in SHRsp rats, and its action mechanism is likely to be associated with its vasodilative effect produced by eNOS activation and with its protective effect on BMECs.

A new compound W026B alleviates ischemic brain injury through inhibiting the production of inflammatory cytokines in pMCAO and tMCAO, and enhances the beneficial effect of tPA

Neruoprotection is considered as one of important therapeutic approaches for ischemic stroke.Inflammation plays an important role in the pathogenesis of ischemic stroke,and the inhibition of inflammation in the ischemic brain tissue may provide neuroprotective effect.In this study,we observed the influence of permanent middle cerebral artery occlussion（pMCAO） and transient MCAO（tMCAO） on NF-κB level and production of several inflammatory cytokines in injured hemisphere in mice,investigated the regulative effect of a new compound W026 B on these influences in the two MCAO models.In pMCAO model,10 μg/kg and 100 μg/kg of W026 B（i.v.） significantly reduced infarct volumes,100 μg/kg of W026 B significantly decreased neurologic deficit scores and brain water contents,and 10 μg/kg and 100 μg/kg of W026 B reduced Evans blue exudation from ischemic brain tissue.The level of NF-κB was elevated by 17.6 times in injured hemisphere,and the levels of TNF-α,IL-1β and IL-17 were elevated by 2.3 times,2.2 times and 3.8 times compared with the sham operation group,respectively,100 μg/kg of W026 B significantly reduced these inflammatory cytokines.In tMCAO model,the elevation of NF-κB,TNF-α,IL-1β and IL-17 was 2.3 times,1.4 times,1.5 times and 1.4 times compared with the sham operation group,respectively.Moreover,100 μg/kg of W026 B significantly decreased the levels of these inflammatory cytokines.In embolic MCAO mice model,W026 B alone significantly reduced infarct volumes,and combined application with tPA further reduced infarct volume.In conclusion,W026 B displayed significant protecive effect on three brain ischemia models.It could protect brain against injury induced by ischmia and ischemia-reperfusion through inhibiting the production of NF-κB,TNF-α,IL-1β and IL-17.These results suggest that W026 B has a value for further study.

Design, preparation and activity evaluation of novel recombinant thrombolytic proteins

Recombinant tissue plasminogen activator （rPA） has been used as a thrombolytic agent. However, considerable improvements have been done to prolong its plasma half-life （tl/2） and reduce its side effects, such as intracranial hemorrhage. Based on these improvements, a mutant ofrPA, mrPA, was designed by mutating its PAI-1 binding site to extend its tl/2. Furthermore, a fusion protein conjugating mrPA with NR3 was designed, which was a rAcAp5 mutant with a platelet GPIIb/IIIa-binding RGD motif, to enhance the ability of targeted-thrombus and thrombolysis. The synthesized DNA sequences coding the two proteins were amplified by PCR, cloned into pET30a to construct recombinant plasmids pET30a-mrPA and pET30a-mrPA-NR3, and transformed into E. coli BL21 （DE3）. The two proteins were expressed in inclusion bodies induced by isopropy113-D-1-thiogalactopyranoside. After purified to qualified purity using one-step Ni affinity chromatography, the denatured proteins were refolded by dialysis. Their thrombolytic effects in vitro and in vivo were evaluated. In vitro 3.5 and 7 pmol/L of mrPA significantly reduced thrombus weight; 1.75, 3.5 and 7 ~tmol/L of mrPA-NR3 also significantly reduced the thrombus weight, and mrPA-NR3 displayed stronger thrombolytic effects than mrPA at 7 lamol/L. In vivo both mrPA and mrPA-NR3 showed significantly thrombolytic effect at 60-240 ktmol/kg in thrombolytic model of inferior vena cava. Importantly, mrPA-NR3 exhibited more potent thrombolytic effect than both mrPA and rhM-tPA （positive control） at 240 p.mol/kg. In addition, these two novel proteins did not increase bleeding time while they exerted thrombolytic effect. In conclusion, we engineered two novel proteins and proved that fusion protein had better thrombolytic effect than non-fusion protein, and the results suggest that dual thrombolytic mechanism or thrombus-target potentiated the thrombolytic effect ofrPA and alleviated hemorrhage side reaction. This study may shed light on the development of novel thrombolytic agents with targeted thrombolysis and reduced side effects.

002C-3 protects the brain against ischemia-reperfusion injury by inhibiting autophagy and stimulating CaMKK/CaMKIV/HDAC4 pathways in mice

This study was designed to investigate the effect of 002C-3, a derivative of magnolol, on transient cerebral middle occlusion （tMCAO） in a mice model and to identify the underlying mechanisms. 002C-3 （100 and 150 pg/kg, i.v. after ending occlusion） significantly reduced neurological deficit scores, infarct volumes, and brain water contents after 1.5 h MCAO and 24 h reperfusion. 002C-3 （75 150μg/kg） decreased the exudation of Evans blue from brain capillaries. 002C-3 （100 μg/kg） significantly inhibited the activity of MMP-9 and MMP-2 in the injured hemisphere. 002C-3 decreased the expression of autophagy-associated proteins, Beclin-1 and LC3B-Ⅱ, and increased the level of p62 in injured hemisphere. 002C-3 （100 pg/kg） significantly increased the expression of p-CaMKIV and p-HDAC4 in injured hemisphere. In conclusion, 002C-3 shows a neuroprotective effect on tMCAO injury in mice, and its mechanisms may be associated with alleviation of blood-brain barrier damage caused by the activation of MMPs, inhibition of autophagy, and stimulation of calcium signals related to cell survival. These findings suggest that 002C-3 is a neuroprotective agent that acts on multiple pathways.

The proteomic study and the target discovery of W026B, a new compound with brain protective effect

W026B is a new compound that has a protective effect on cerebral ischemia reperfusion(I-R)injury in mice,while its specific mechanism is still unknown.In this study,proteomics was used to observe the effect of W026B on protein expression in brain I-R tissue,and to reveal its potential target.A total of 42 significantly altered proteins were identified in both brain I-R model and W026B treatment from 4852 proteins detected by proteomics,and most of these proteins were related to immunity and inflammation,metabolism,neuroprotection as well as cell proliferation and cell structure.Western blotting analysis showed that three out of five selected proteins showed consistent alteration with the proteomics.Regulator of G protein signaling 17(RGS17)was selected for further study,and its knockdown by siRNA RGS17 aggravated brain injury and abolished the protective effect of W026B.W026B could bind with RGS17(KD:6.04×10–6 mol/L).The knockdown of RGS17 aggravated Neuro-2 a cell damage induced by group I metabotropic glutamate receptors(mGluRs)agonist,and abolished the protective effect of W026B.In conclusion,W026B protected brain against I-R injury by affecting diverse proteins.RGS17 might be one of its targets and a potential therapeutic target of brain I-R injury.The upstream receptor of G protein,which was regulated by RGS17 and affected by W026B,might be group I m GluRs.This study provided useful evidence for the further R&D and the potential clinical application of W026B.

Preparation and characterization of drop pills of effective part from safflower for anti-Parkinson's disease

Parkinson’s disease(PD) is a common degenerative disease of the central nervous system, and the pathologic features are mainly degeneration of substantianigra and dopamine neurons. Studies have shown that safflower flavonoid extract(SAFE) exhibits the neuroprotective effect. In this study, the safflower flavonoid extract drop pills(SAFE-DPs) for anti-PD were prepared by the heating and melting method using SAFE and matrix PEG6000. The performances of the pills were evaluated with powder X-ray diffraction(PXRD), differential scanning calorimetry(DSC), Fourier transform infrared spectroscopy(FT-IR), scanning electron microscopy(SEM) and dissolution testing. The analysis results demonstrated an amorphous state for SAFE dispersion in the matrix PEG6000 without any chemical reaction. The SAFE-DPs demonstrated acceptable chemical and physical stability irrespective of the manufacturing process and the storage period. Dissolution testing in three dissolution media(pH 1.0, pH 6.8 and pH 7.5) indicated that SAFE-DPs had excellent dissolution property. The transport of Kaempferol-3-rutinoside(K3 R) on the Caco-2 monolayer and the absorption of K3 R in situ intestinal perfusion revealed that the principal component of SAFE had a good transport and absorption capacity. Therefore, the drop pills had better release and absorption in the gastrointestinal tract, corresponding with the pharmacological and pharmacodynamic results for PD in vivo.

The influence of B55γ on the neuroprotective effect of W026B in t-MCAO mice

As a newly synthesized lignan derivative,W026B has been proved to be a neuroprotective agent,and it can significantly reduce cerebral infarct volume,improve behavioral scores and protect blood brain barrier in different models.However,its exact mechanism is still unclear.In the present study,a protein named B55γ,one of candidate targets of W026 B screened by proteomic study,was investigated to explore its influence on the effect of W026B in t-MCAO mice.siRNA PPP2R2C was used to knockdown the expression of protein B55γin t-MCAO mice.The results showed that the knockdown of B55γsignificantly suppressed the neuroprotective effect of W026B.Further results showed that the knockdown of PPP2R2C,B55γgene,abolished the effect of W026B on reducing the level of NF-κB p65 in ischemic brain tissue,and knockdown of PPP2R2C also reversed the effect of W026B on decreasing the activity of caspase-3 in ischemic brain tissue.In conclusion,protein B55γmight be an important mediator of the protective effect of W026B.B55γactively participated in the brain protective effect of W026B by affecting the inflammatory response and apoptotic pathway during ischemia reperfusion.These results revealed a new mechanism underlying the neuroprotective effect of W026B.

The design, synthesis and α7 nicotinic acetylcholine receptors positive allosteric modulative evaluation of 3H-quinazolin-4-one derivatives

A series of new 6-substituted 3 H-quinazolin-4-ones（3 a-3 d） were designed, synthesized and evaluated as the type I positive allosteric modulators（PAMs） of human α7 n ACh R expressed in Xenopus ooctyes by two-electrode voltage clamp. However, no compound showed a better efficacious PAM than lead compound 2 in the presence of acetylcholine（100 μM）. The structure-activity relationship（SAR） analysis suggested that thiazolo[4,5-d]pyrimidin-7（6 H）-one was the key biological skeleton.

Electrophysiological characterization of furo[3,2-b]pyridine derivatives as negative allosteric modulator of a7 nicotinic acetylcholine receptor

A series of lH-pyrrolo[3,2-b]pyridine (3a-3f) and fUroP,2-b]pyridine derivatives (4a-4g) were evaluated on human a7 nicotinic acetylcholine receptors (nAChRs) using two-electrode voltage clamp (TEVC) recording. A representative 2-(2-methoxyphenyl)- furo[3,2-b]pyridine 4f as negative allosteric modulator (NAM) selectively inhibited alpha7 nAChR over a3p4, a4p2 nAChRs and 5-HT3a receptor, with a potency of IC50 of 5.51 |1M and a maximum inhibition rate of 87.8%. The preliminary analysis of structure-activity relationship (SAR) suggested that compound 4f could serve as a basis for further discovery of potent and selective a7 nAChR NAMs.