目的 优选逆萎康的最佳提取工艺.方法 采用L9(34)正交试验方法,以雪胆总皂苷、黄芪甲苷含量为指标,考察提取次数、提取时间及固液比因素对逆萎康提取效果的影响,优选提取工艺.结果 逆萎康的最佳工艺条件为提取2次,提取1 h,固液比12倍.结论 该提取工艺操作简便、准确,可为逆萎康颗粒剂的制备提供参考.

Objective:To evaluate the effect of mitochondrial CB1 receptor (mtCB1) on mitochondrial fission in a rat hippocampal neuron model of hypoxia/re-oxygenation (H/R) injury.Methods:Primarily cultured hippocampal neurons obtained from Wistar rats were divided into 5 groups (n=60) using a random number table:normal group (N group):were cultured in normal medium,without any administration;H/R group:the hippocampal neurons were subjected to oxygen-glucose deprivation (OGD) for 6h followedby re-oxygenation for 20h;H/R+ACEA+AM251 group:ACEA and AM251 were add with respectively final concentration 1 umol/L,10 umol/L during the 20h re-oxygenation;H/R+ACEA+ Hemopressin group:ACEA and Hemopressin were add into culture medium with respectively final concentration 1 umol/L,10 umoVL during the re-oxygenation for 20h;H/R + Vehicle group (H/R+V group):DMSO was add into culture medium with final concentration ＜0.1％ during the 20h re-oxygenation.Laser scanning confocal microscope was used to measure Ca2+ concentration in cytoplasm.The apoptosis rate was tested by flow cytometry.Western blot was adopted to examine the expression of apoptosis-inducing factor (AIF),dynamin-related protein 1 (Drp 1),fission 1 (Fis1),apoptosis-related protein cytochrome c (Cytc),and Rho-associated coiled-coil containing protein kinase (ROCK1).Results:Compared to the N group,the apoptosis rate,Ca2+concentration,the expression of AIF,Drp1,Fis1,Cytc and ROCK1were significantly increased in the other four groups (P＜0.05);Compared to the H/R group,those detection indexes mentioned above were significantly decreased in H/R+ ACEA+ Hemopressin group (P＜0.05),and there were no significant differences were observed in H/R+ACEA+AM251 group and H/R+ Vehicle group (P＞0.05);Conclusion:The reduction of ROS induced by mtCB1 can alleviate the expression of ROCK1 and Ca2+ concentration in cytoplasm,and inhibit the mitochondrial fission,and eventually attenuate the H/R injury ofhippocampal neurons.

Objective To investigate the effect of Cuyu Shengjing Prescription in the treatment of infertility in mice and its mechanism.Methods A total of 50 clean male Kunming mice were randomly divided into blank control group,model group,and low-,medium-,and high-dose Cuyu Shengjing Prescription groups.Cyclophosphamide was used to establish a mouse model of oligoasthenospermia,and the Cuyu Shengjing Prescription groups were given Cuyu Shengjing Prescription for 35 d.An optical microscope was used to observe sperm density and motility,RT-PCR was used to measure the mRNA expression of activator of cAMP-responsive element modulator in testis (ACT),cAMP-responsive element modulator (CREM),and cAMP response element-binding protein (CREB) in testicular tissue,and Western blot was used to measure the protein expression of ACT,CREM,and CREB.Results The model group had significantly lower sperm density and motility than the normal group (F =2.83-121.16,P＜0.05),suggesting that the mouse model of oligoasthenospermia was established successfully.The medium-and highdose Cuyu Shengjing Prescription groups had significantly higher sperm density than the model group (F=121.16,P＜0.05),while there was no significant difference between the low-dose Cuyu Shengjing Prescription group and the model group (P＞0.05).The low-and medium-dose Cuyu Shengjing Prescription groups had significantly higher sperm motility than the model group (F =2.83,18.94;P＜0.05),while there was no significant difference between the high-dose Cuyu Shengjing Prescription group and the model group (P＞0.05).The model group had significantly lower mRNA and protein expression of ACT,CREM,and CREB than the blank control group (F =16.28-154.32,P＜0.05).The medium-dose Cuyu Shengjing Prescription group had significant increases in the mRNA and protein expression of ACT compared with the model group (F =29.04,64.28;P ＜0.05),while there were no significant differences between the low-and high-dose Cuyu Shengjing Prescription groups and the model group (P＞0.05).Compared with the model group,the low-and medium-dose Cuyu Shengjing Prescription groups had significant increases in the mRNA and protein expression of CREM (F =17.77,41.83;P ＜ 0.05),while there were no significant differences between the high-dose Cuyu Shengjing Prescription group and the model group (P＞0.05).Compared with the model group,the low-dose Cuyu Shengjing Prescription group had significant increases in the mRNA and protein expression of CREB (F =16.89,154.32;P＜0.05),while there were no significant differences between the medium-and high-dose Cuyu Shengjing Prescription groups and the model group (P＞0.05).Conclusion Cuyu Shengjing Prescription can effectively improve the mRNA and protein expression of ACT,CREM,and CREB in testicular tissue in mice with oligoasthenospermia and improve sperm quality.

目的 为缺血性脑损伤模型的制备及研究建立一种更好的体外培养海马神经元的方法.方法 实验组取出生24 h内的Wistar新生鼠海马,用木瓜酶和DNA酶消化,以1×108/L的密度接种于含体积分数0.20胎牛血清的DMEM-F12培养液中,24 h后更换无血清培养液,每隔3d半量换液.对照组采用传统的培养方法培养细胞.培养期间用倒置显微镜观察细胞形态及数量变化.采用免疫荧光法测定神经元特异性烯醇化酶表达,判断海马神经元培养是否成功.结果实验组的海马神经元培养12h,可见大部分细胞贴壁;培养24 h,大部分细胞可见3～4个突起,突起长度为(25.0±2.5)μm;培养3d,神经元胞体增大,可见明显光晕;之后神经元分化逐渐成熟,胞体透亮,呈锥形或多极形,光晕明显,神经元之间的突起联系更加紧密,形成密集的神经细胞网络;培养13 d后,神经元细胞开始退化、变性,胞体萎缩,神经细胞网络开始老化.随着培养天数的增加,神经元可出现少量凋亡,但实验组培养1、3、5、8d时的神经元数量明显高于对照组(t=11.5～49.5,P＜0.05).免疫荧光法鉴定显示,实验组神经元细胞阳性率为(93.53±1.67)％.结论用上述改良方法培养得到的海马神经元细胞生长状态良好,可用于后续实验研究.

Objective To evaluate the role of mitochondrial fission in hypoxia-reoxygenation (H/ R) injury to hippocampal neurons of rats.Methods Primarily cultured hippocampal neurons obtained from newborn Wistar rats were randomly divided into 5 groups (n =60 each) using a random number table: normal group (N group), vehicle group (V group), H/R group, H/R + vehicle group (H/R + V group),and mitochondrial division inhibitor group (group M).The cells were cultured in normal culture medium in group N.Dimethyl sulfoxide (DMSO) was added to the culture medium with the final concentration ＜ 0.1％, and the cells were incubated for 40 min in group V.The cells were subjected to 6 h hypoxia, followed by 20 h reoxygenation in H/R, H/R+V and M groups.DMSO was added to the culture medium with the final concentration ＜0.1％ at 40 min before hypoxia in group H/R+V.In group M, mitochondrial division inhibitor Mdivi-1 50 mmol/L (dissolved in DMSO, DMSO concentration ＜0.1％) was added to the culture medium at 40 min prior to hypoxia.Mito Tracker staining was used to examine mitochondrial morphology.Western blot was used to measure the expression of mitochondrial dynamin-related protein 1 (Drp1), mitofusin 2 (Mfn2) , peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (TFAM).Multifunctional microplate reader and fluorescent microscope were used to detect the mitochondrial reactive oxygen species (ROS) level.The flow cytometer was used to detect the apoptosis in hippocampal neurons.Apoptosis rate was calculated.Results Compared with group N, the expression of Drp1, PGC-1α, NRF-1 and TFAM was significantly up-regulated, the ROS content and apoptosis rate were increased, and the expression of Mfn2 was down-regulated in group H/R (P＜0.05).Compared with group H/R, the expression of Drp1 was significantly down-regulated, the ROS content and apoptosis rate were decreased, and the expression of Mfn2, PGC-1α, NRF-1 and TFAM was up-regulated in group M (P＜0.05).Conclusion Mitochondrial fission is involved in H/R injury to hippocampal neurons of rats.

目的 优化青蒿素微囊的制备工艺,并对制备的青蒿素微囊进行质量评价研究.方法 以明胶为囊材,单凝聚法制备青蒿素微囊,通过星点设计-效应面法优化其制备工艺,并对包封率、载药量、微囊的粒径分布、体外溶出进行研究.结果 明胶制备青蒿素微囊的最佳工艺条件的明胶质量分数为5.06％,囊心囊材质量比为1∶3,搅拌速度为751 r/min.此最佳工艺制备的青蒿素微囊包封率为89.31％,平均粒径69.91 μm,载药量为22.25％,体外溶出度测定0.5h为25.8％,12 h累积释放达到95％以上.结论 以最佳工艺条件制备青蒿素微囊工艺稳定,包封率高,同时体外释放试验表明,该微囊具有较好的缓释作用.

目的:探讨不稳定心绞痛在方证对应与不对应干预下的证候动态变化特点.方法:入选88例经冠脉造影明确诊断的冠心病不稳定性心绞痛患者,血瘀证组、气阴两虚证组各44例,按照随机、双盲、双模拟的设计方法,以血府逐瘀胶囊和生脉胶囊分别给予方证对应与方证不对应干预治疗,收集治疗前、治疗后1周、2周、3周、4周的症状及证候信息,运用生存分析比较症状及证候在两种方法干预下的变化特点.结果:实验期间脱落6例,纳入统计分析的共82例.结果表明胸闷、心悸、气短、倦怠乏力、自汗和血瘀证、气虚证的起效时间方证对应早于不对应,而胸痛、畏寒肢冷、腰膝酸软、不寐和阴虚证的起效时间两种方法无差别;方证对应治疗时,2证组合逐渐增多,3证组合逐渐减少,而不对应治疗2证组合逐渐减少,3证组合逐渐增多.结论:方证对应治疗证候起效时间早于不对应治疗.

Objective To study the effect of mitochondrial calcium uniporter (MCU) activity on mitochondrial energy metabolism in cerebral ischemia/reperfusion injury (I/RI) in rats.Methods Fifty-two male rats were randomly divided into four groups:control group (sham),ischemia/reperfusion group (I/R),ischemia/reperfusion + ruthenium red group (I/R+RR) and ischemia/reperfusion +spermine group (I/R+Sper).The MCAO rats were generated by thread embolism of middle cerebral artery.Ischemia was induced for 2 h followed by 24 h of reperfusion,cerebral infarction area percentage,mitochondrial complex activity,mitochondrialmembrane potential (△Ψ m),intracellular adenosine triphosphate (ATP) level and reactive oxygen species (ROS) content weredetermined after reperfusion.Results Mitochondrial complex Ⅰ-Ⅳ activity [(17.67±0.21),(6.17±0.42),(22.29±1.40),(31.73±0.84) nmol·min1·mg-1],△Ψm and intracellular ATP level (5.2±60.19) nmol/mg protein were decreased in I/R group,I/R+RR group and I/R+Sper group compared with control group(P＜0.01),but cerebral infarction area percentage(35.43±0.74)％ and ROS content(1455.21±39.52) U/mg protein were increased in the three groups (P＜0.01).Ruthenium red treatment could improve the parameters above:compared with those in ischemia/reperfusion group,ruthenium red in ruthenium red group obviously enhanced mitochondrial complex activity [(22.1 ±70.55),(7.50 ±0.15),(36.92 ±2.13),(49.32 ±1.33) nmol ·min 1 ·mg-1],△Ψm and intracellular ATP level (7.85±0.17) nmol/mg protein (P＜0.01),with reducing cerebral infarction area (26.00±1.71)％,and decreasing ROS content(1 262.53±29.92) U/mg protein(P＜0.01).While all the parameters deteriorated in spermine group compared with those in I/R group.Conclusions Inhibition of the activity of MCU can obviously improve mitochondrial energy metabolism during cerebral I/RI.

Objective:ascertain the effective part of anti-tumor activity in Viscum coloratum(Kom.)Nakai f.Rubroaurantiacum Kitag(VCR).Method:We made sarcoma S180 mice as the animal model,the inhibition rate of tumor was used as investigation index.We carried out the screen assay of the anti-tumor for different solvent extraction part of the VCR extracts by 75% alcohol,including petroleum extraction part,chloroform extraction part,acetate extraction part,n-butanol extraction part and the rest.We randomly divided mice with inoculated tumor into 7 groups according to tumor size.The positive control group of mice were given ip cyclophosphamide of 100 mg·kg-1,the other experimental group at the same time were given ig of 14.2 g·kg-1·d-1 and the blank control group were given the same volume of the blank solvent,continuously administrated for 8 days.Result:The results showed that the extraction of VCR could obviously prevent the growing of the tumor in mice.The inhibition rate of tumor for the chloroform extraction part was 48.8%;the rate of the n-butanol extraction part was 44.1%.Conclusion:The chloroform extraction part and the n-butanol extraction part are the effective part of anti-tumor from VCR.