Determination on Heavy Metals Content of Achyranthes bidentata Blume. through Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

[Objective] The inductively coupled plasma mass spectrometry(ICP-MS)was constructed to determine the contents of lead,cadmium,mercury and arsenic in Archyranthes bidentata Blume.[Method]Under the optimum operation condition of ICP-MS,the samples were digested by microwave.The element 114In was taken as an internal standard element to compensate body effect and ICP-MS method was used to determine the contents of lead,cadmium,mercury and arsenic.[Result]For the determined elements,the correlation coefficient(r)of standard curve was over 0.9995 and recovery rate was from 96.7% to 106.4% while RSD was less than 11.2%.The result of determination showed that the heavy metal content in Archyranthes bidentata Blume.beyond standard was serious.[Conclusion]The constructed ICP-MS method with simple operation,rapid response,accuracy and high sensitivity in this experiment could be used for quality control of Chinese medicinal materials by detecting heavy metal contents in different Chinese medicinal materials from original places.

Analysis of the Constituents and Antisenile Function of Achyranthes bidentata Polysaccharides

Achyranthes bidentata polysaccharides (ABPS), water_soluble polysaccharides, isolated from the roots of Achyranthes bidentata Bl. of Amaranthaceae family, was divided into four parts, named as Con.1, Con.2, Con.3 and Con.4, respectively, by chromatography on DEAE_Sepharose fast_flow column and Sephadex G_100 column in order. Con.1 was the constituent of high molecular weight and the other three were all of low molecular weight. Micro_Kjeldahl analysis showed that Con.1 contained 3.95% of nitrogen and neither did the other three parts. The antisenile effects of the four parts of ABPS were studied with Drosophila melanogaster. Results showed that Con.1 has no antisenile effect and all the others could significantly increase the average body weight by 3.85%-5.47% and significantly prolonged the average lifespan by 2.61%- 3.16% of D. melanogaster at the concentration of 2 or 5 mg/g (ABPS/medium).

Inhibiting Effects of Achyranthes Bidentata Polysaccharide and Lycium Barbarum Polysaccharide on Nonenzyme Glycation in D-galactose Induced Mouse Aging Model

Objective To investigate the inhibiting effects and mechanism of achyranthes bidentata polysaccharide (ABP) and lycium barbarum polysaccharide (LBP) on nonenzyme glycation in D-galactose induced mouse aging model. Methods Serum AGE levels were determined by AGE-ELISA, MTT method was used to determine lymphocyte proliferation, IL-2 activity was determined by a bioassay method. Spontaneous motor activity was used to detect mouse's neuromuscular movement, latency of step-through method was used to examine learning and memory abilities of mouse, colormetric assay was used to determine hydroxyproline concentration in mouse skin, pyrogallol autoxidation method was used to determine superoxide dismutase (SOD) activity of erythrocytes. Results Decreased levels of serum AGE, hydroxyproline concentration in mouse skin and spontaneous motor activity in D-galactose mouse aging model were detected after treated with ABP or LBP, while lymphocyte proliferation and IL-2 activity, learning and memory abilities, SOD activity of erythrocytes, were enhanced. Conclusions ABP and LBP could inhibit nonenzyme glycation in D-galactose induced mouse aging model in vivo and ABP has a better inhibiting effect than LBP.

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion.Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion,but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear.Therefore,in the current study,we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons.Hippocampal neurons were treated with Mg^2+-free extracellular solution containing glutamate(300μM)for 3 hours as a model of glutamate-mediated excitotoxicity(glutamate group).In the normal group,hippocampal neurons were incubated in Mg^2+-free extracellular solution.In the Achyranthes bidentata polypeptide group,hippocampal neurons were incubated in Mg^2+-free extracellular solution containing glutamate(300μM)and Achyranthes bidentata polypeptide at different concentrations.At 24 hours after exposure to the agents,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear m'orphology,respectively.Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay,respectively.At various time points after glutamate treatment,reactive oxygen species in cells were detected by H2 DCF-DA,and mitochondrial membrane potential was detected by rhodamine 123 staining.To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors,electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons.Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate.In addition,Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current.Furthermore,the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment.These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway.All animal studies were approved by the Animal Care and Use Committee,Nantong University,China(approval No.20120216-001)on February 16,2012.

The Achyranthes bidentata polypeptide k fraction enhances neuronal growth in vitro and promotes peripheral nerve regeneration after crush injury in vivo

We have previously shown that Achyranthes bidentata polypeptides （ABPP）, isolated from Achyranthes bidentata Blume （a medicinal herb）, exhibit neurotrophic and neuroprotective effects on the nervous system. To identify the major active component of ABPP, and thus optimize the use of ABPP, we used reverse-phase high performance liquid chromatography to separate ABPP. We obtained 12 fractions, among which the fraction of ABPPk demonstrated the strongest neuroactivity. Immunocytochemistry and western blot analysis showed that ABPPk promoted neurite growth in cultured dorsal root ganglion explant and dorsal root ganglion neurons, which might be associated with activation of Erk1/2. A combination of behavioral tests, electrophysiological assessment, and histomorphometric analysis indicated that ABPPk enhanced nerve regeneration and function restoration in a mouse model of crushed sciatic nerve. All the results suggest that ABPPk, as the key component of ABPP, can be used for peripheral nerve repair to yield better outcomes than ABPP.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective:To develop the reproducible in vitro propagation protocols for the medicinally important plants viz.,Achyranthes aspera(A.aspera)L.and Achyranthes bidentata(A.bidentata)Blume using nodal segments as explants.Methods:Young shoots of A.aspera and A.bidentata were harvested and washed with running tap water and treated with 0.1%bavistin and rinsed twice with distilled water.Then the explants were surface sterilized with 0.1%(w/v)HgCl_2 solutions for I min.After rinsing with sterile distilled water for 3-4 times,nodal segments were cut into smaller segments(1 cm)and used as the explants.The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog(MS)medium supplemented with 3%sucrose,0.6%(w/v)agar(HiMedia,Mumbai)and different concentration and combination of 6-benzyl amino purine(BAP),kinetin(Kin),naphthalene acetic acid(NAA)and indole acetic acid(IAA)for direct regeneration.Results:Adventitious proliferation was obtained from A.aspera and A.bidentata nodal segments inoculated on MS basal medium with 3%sucrose and augmented with BAP and Kin with varied frequency.MS medium augmented with 3.0 mg/L of BAP showed the highest percentage(93.60±0.71)of shootlets formation for A.aspera and(94.70±0.53)percentages for A.bidentata.Maximum number of shoots/explants(10.60±0.36)for A.aspera and(9.50±0.56)for A.bidentata was observed in MS medium fortified with 5.0 mg/L of BAP.For A.aspera,maximum mean length(5.50±0.34)of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A.bidentata(5.40±0.61)was observed in the very same concentration.The highest percentage,maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of 1BA.Seventy percentages of plants were successfully established in polycups.Sixty eight percentages of plants were well established in the green house condition.Sixty five percentages of plants were established in the field.Conclusions:The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A.aspera and A.bidentata.The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can he easily adopted for commercial large scale cultivation.

Chemical Components of Achyranthes bidentata Leaves by Ultra High Performance Liquid Chromatography-Mass Spectrometry

[Objectives] To study the chemical components and relative content of Achyranthes bidentata leaves and provide a scientific basis for further development and utilization of A. bidentata leaves.[Methods] The chemical components of A. bidentata leaves were rapidly analyzed using the ultra high performance liquid chromatography-time of flight-high resolution mass spectrometry (UHPLC-TOF-MS).[Results] Thirty eight chemical compounds were identified in samples of A. bidentata leaves collected from Wen County of Henan Province, in which seven chemical compounds had the relative content higher than 5%, linoleic acid reached 25.7% and inokosterone A reached 13.8%.[Conclusions] A. bidentata leaves contain many kinds of chemical compounds. This study is expected to provide a certain basis for further extraction of linoleic acid and inokosterone A.

Protective effects of Achyranthes bidentata polypeptides on retinal ganglion cells post-optic nerve crush in rats

Achyranthes bidentata polypeptides（ABPP） have been reported to inhibit apoptosis of retinal ganglion cells（RGCs）.The present study investigated the protective effects of ABPP on RGCs in a rat model of optic nerve injury.With prolonged injury time,RGC densities were gradually decreased.ABPP（5 μg） significantly increased RGC densities and upregulated growth associated protein 43 expression in rats with optic nerve injury.Results demonstrate that ABPP can protect RGCs and promote axonal growth after optic nerve crush.

Achyranthes bidentata polypeptide protects dopaminergic neurons from apoptosis induced by rotenone and 6-hydroxydopamine

It has been well documented that Achyranthes bidentata polypeptides（ABPPs） are potent neuroprotective agents in several types of neurons. However, whether ABPPs protect dopaminergic neurons from apoptosis induced by neurotoxins is still unknown. This study was designed to observe the effect of ABPPk, a purified fraction of ABPPs, on apoptosis of dopaminergic neurons. SH-5YHY cells and primary dopaminergic neurons were pre-treated with ABPPk（25, 50, or 100 ng/mL） for 12 hours. Cells were then exposed to 6-hydroxydopamine（50 or 150 μM） or rotenone（50 or 200 μM） for 36 hours to induce cell apoptosis. Our results demonstrate that ABPPk markedly increased viability in SH-SY5Y cells and primary dopaminergic neurons, decreased lactate dehydrogenase activity and number of apoptotic dopaminergic neurons, elevated mitochondrial membrane potential, and increased Bcl-2/Bax ratio. These findings suggest that ABPPk protects dopaminergic neurons from apoptosis, and that ABPPk treatment might be an effective intervention for treating dopaminergic neuronal loss associated with disorders such as Parkinson＇s disease.

Effects of Pb on Rhizosphere Soil Enzyme Activity and Chemical Constituents of Achyranthes bidentata Blume

[Objectives]The purpose of this study was to investigate the effects of Pb on rhizosphere soil enzyme activity and chemical constituents of Achyranthes bidentata Blume.[Methods]A.bidentata Blume plants were cultivated in self-made polyvinyl chloride(PVC)pots.The soil was added with different levels of Pb(0,200,400,600,800 and 1000 mg/kg air-dried soil)to investigate the effects of Pb on dry mass,active ingredients(oleanolic acid and ecdysterone)and rhizosphere soil enzyme activity of A.bidentata Blume.[Results]The root dry mass of A.bidentata Blume cultivated in the soil with Pb level above 400 mg/kg significantly reduced.The Pb residues in the A.bidentata Blume.plants growing in the soil with Pb level below 400 mg/kg complied with national standard.The contents of oleanolic acid and ecdysterone in A.bidentata Blume growing in the soil with Pb level above 600 mg/kg declined significantly.At the Pb level of 1000 mg/kg,the activity of urease was inhibited significantly.The activity of phosphatase was inhibited in the presence of Pb in the soil.The activity of sucrase was activated in the soil with Pb level below 400 mg/kg,and was inhibited in the soil with Pb level above 400 mg/kg.[Conclusions]This study has important guiding significance for the reasonable selection of planting base for A.bidentata Blume and the guarantee of its yield and quality.

Tissue-based metabolite profiling and qualitative comparison of two species of Achyranthes roots by use of UHPLC-QTOF MS and laser micro-dissection

Achyranthes bidentata and Achyranthes aspera are saponin and steroid rich medicinal plants, used extensively for therapeutic treatments in Traditional Chinese Medicine(TCM) and Ayurveda. A. bidentata is reported to be one of the rare and extensively exploited medicinal plant species that face the issue of being endangered.Finding qualitative substitute with identical phyto-constituents contributing to similar composition and pharmacological benefits will help in reducing the burden of exploitation of the natural habitats of such plants.In the present study, a comparative metabolite analysis of the whole drug and specific tissues isolated by laser micro-dissection(LMD) was carried out for both the selected species, by use of ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UHPLC-QTOF MS). The results of the study indicate that the cortex and the medullary ray tissues are rich in their content of steroidal and saponin constituents such as(25 S)-inokosterone-20,22-acetonide, ginsenoside Ro, bidentatoside II and achyranthoside B.Metabolite profiling of the whole tissues of both the species indicates presence of identical constituents. Thus,it is inferred that A. bidentata and A. aspera can be used as qualitative substitutes for each other.

牛膝化学成分和药理作用研究进展及其质量标志物(Q-Marker)预测分析

牛膝在《神农本草经》中被列为上品,近几年研究证实牛膝的化学成分主要包括甾体、三萜皂苷、多糖、挥发油等化合物,具有抗炎镇痛、调节免疫、抗骨质疏松、保护心血管等药效。本文从牛膝的植物亲缘性,牛膝特有性、有效性、可测性化学成分和炮制后化学成分等方面,对牛膝的质量标志物(quality marker,Q-Marker)进行预测分析,牛膝甾酮A、25 S-牛膝甾酮、25 R-牛膝甾酮、齐墩果酸-3-O-β-D-(6′-丁酯)-吡喃葡萄糖醛酸苷、齐墩果酸-3-O-β-D-(6′-甲酯)-吡喃葡萄糖醛酸苷、β-蜕皮甾酮等可作为牛膝质量标志物,为建立牛膝质量评价体系和深入研究其药理作用提供依据。

防控花生白绢病的根际放线菌分离鉴定及防效评价

花生白绢病是由齐整小核菌Sclerotium rolfsii Sacc.引起的发生在花生茎基部的真菌病害,严重制约花生的品质与产量。本研究利用稀释涂布法从怀牛膝根际土壤中分离纯化了116株放线菌,并从中筛选鉴定了能够防控花生白绢病的菌株。通过平板对峙试验筛选得到两株抑菌活性较好且稳定的菌株Soil-1-5和Soil-3-28,它们对齐整小核菌的抑制率分别为80.43%和92.34%。根据形态学观察、生理生化试验及16S rRNA基因序列分析鉴定,两株菌分别被鉴定为疮痂链霉菌Streptomyces scabiei和藤黄灰链霉菌Streptomyces luteogriseus。采用菌丝生长速率法测定了拮抗菌株无菌发酵滤液对植物病原菌的抑制作用。结果表明,菌株Soil-1-5和Soil-3-28的无菌发酵滤液稀释5倍后对齐整小核菌的抑制率分别为73.67%和57.11%,且对禾谷镰孢菌等6种植物病原菌均有不同程度的抑制作用。此外,两菌株的无菌发酵滤液对齐整小核菌菌核萌发和菌核形成也有较好拮抗作用。盆栽试验结果表明,菌株Soil-1-5和Soil-3-28对花生白绢病的防治效果分别为51.92%和31.74%,其中菌株Soil-3-28对花生生长有促进作用。综上,菌株Soil-1-5和Soil-3-28对花生白绢病有较好的防治效果,具有潜在应用价值。

牛膝多糖对雏鸡免疫功能的影响

文章旨在研究牛膝多糖对雏鸡免疫功能的影响。试验将120只1日龄雏鸡随机分为4组,每组3个重复,每个重复10只鸡(公母各半)。在常规饲料中分别添加200、150、100、0 mg/kg牛膝多糖。所用雏鸡连续饲喂至30日龄,每组取10只进行取样。计算动物机体免疫器官指数,MTT检测T细胞增殖活性,Griess检测巨噬细胞释放NO能力,ELISA检测血清总IgG水平。结果表明,在雏鸡饲料中添加中剂量(150 mg/kg)的牛膝多糖能显著提高雏鸡免疫器官指数、T细胞增殖活性、巨噬细胞释放NO能力和血清总IgG水平(P<0.05)。结论:牛膝多糖在一定程度上对雏鸡的免疫功能具有增强作用,从而提高机体抗病能力,饲料中牛膝多糖最适添加量为150 mg/kg。

牛膝-续断药对抗骨质疏松作用及活性成分初探

目的:通过维甲酸建立骨质疏松小鼠模型,评价牛膝-续断药对的抗骨质疏松作用,探索其抗骨质疏松活性成分。方法:采用HPLC法建立牛膝-续断药对提取物的指纹图谱;使用“中药色谱指纹图谱相似度指纹评价系统(2004 A版)”对不同组别进行相似度评价。维甲酸诱导小鼠骨质疏松模型,计算肝肾指数与股骨干湿比,应用酶标法检测不同提取物对维甲酸诱导骨质疏松模型小鼠血清ALP、ACP水平;应用Spss软件进行牛膝-续断提取物中各成分与模型小鼠股骨干湿比和血清中ALP和ACP酶含量的pearson相关性分析。结果:成功建立牛膝-续断药对提取物的HPLC指纹图谱,标定9个共有峰。肝肾指数结果显示模型组与给药组无显著差异,股骨干湿比结果显示大部分治疗组与模型组相比有显著性差异。ELISA结果显示,牛膝续断药对主要影响ACP水平。指纹峰与干湿比分析结果显示,峰1可能为活性成分;与ACP相关性分析结果表明峰1、2、3、7可能为活性成分。结论:牛膝-续断药对提取物对骨质疏松有治疗作用,其抗骨质疏松作用主要通过降低ACP水平实现,峰1可能为牛膝-续断抗骨质疏松活性成分。

牛膝酒对膝骨关节炎模型大鼠的作用研究

目的:探讨牛膝酒对膝骨关节炎(KOA)模型大鼠的作用。方法:SD大鼠通过膝关节腔内注射碘乙酸钠溶液(60 mg/mL)建立KOA模型。将36只造模成功的大鼠随机分为模型组、牛膝酒低剂量组、牛膝酒中剂量组、牛膝酒高剂量组、塞来昔布组及黄酒组,另设空白对照组,每组6只,给予相应药物干预21 d(每天灌胃1次),每7 d测定膝关节肿胀度。观察各组大鼠关节肿胀度变化、膝关节表面病变情况、关节软骨病理情况并采用改良Mankin's评分标准进行评分,检测血清中IL-1β、TNF-α、IL-6及基质金属蛋白酶(MMP)-3、超氧化物歧化酶(SOD)水平。结果:与空白对照组比较,模型组大鼠各阶段膝关节肿胀度均升高(P<0.01),关节软骨表面损伤严重,可见炎症细胞聚集、软骨细胞减少且分布不均匀,基质染色重度减少,潮线不完整,改良Mankin's评分升高(P<0.01);血清中IL-1β、TNF-α、IL-6、MMP-3水平升高(P<0.01),SOD水平降低(P<0.01)。与模型组比较,牛膝酒低、中、高剂量组大鼠各阶段关节肿胀度均降低(P<0.05),关节软骨表面损伤、病理形态均有不同程度的改善;牛膝酒高剂量组血清中IL-1β、TNF-α、IL-6、MMP-3水平降低(P<0.05或P<0.01),SOD水平升高(P<0.01)。结论:牛膝酒可以缓解KOA模型大鼠关节肿胀及关节软骨损伤。这可能与其抑制炎症反应、调节软骨细胞代谢、拮抗氧化应激反应有关。

药对何首乌-牛膝抗骨质疏松的作用机制研究

目的:利用网络药理学研究牛膝何首乌联用抗骨质疏松的作用机制。方法:运用中药系统药理学数据库与分析平台(TCMSP)与文献检索筛选何首乌、牛膝两味中药所含的活性成分。利用Disgent数据库搜索与骨质疏松相关的蛋白靶点;利用Pubchem平台,预测何首乌、牛膝所含活性成分所作用的人类蛋白靶点。分析牛膝何首乌药的交集靶点,并将交集靶点进行蛋白网络分析,构建药物-成分-靶点的网络图。通过微生信平台对关键基因作用的靶向细胞进行富集分析。结果:确定何首乌成分5个,牛膝成分10个,二者共有成分1个,获得药对与疾病交集基因35个;PPI蛋白分析获得10个主要作用成分;绘制GO分析可视图、KEGG分析可视图并对其进行解析,解析结果表明何首乌牛膝主要成分通过抑制骨吸收,诱导骨形成等通路来减轻骨质疏松症的发展。结论:牛膝何首乌药对抗骨质疏松的作用机制可能是通过靶点与共有通路完成的,并且该药对可通过多通路、多靶点、多成分、多层次发挥对骨质疏松症的治疗作用。

牛膝-菟丝子药对防治高血压肾损伤网络药理学机制研究

目的:通过使用网络药理学方法探讨怀牛膝-菟丝子药治疗高血压肾损伤的潜在作用机制。方法:使用TCMSP、Uniprot数据库分别获取怀牛膝、菟丝子的活性成分和靶点基因,高血压肾损伤的疾病相关靶点是通过GenenCards、Omim数据库获取。在Venny在线工具中获取药物与疾病的交集基因,使用STRING数据库、Cytoscape 3.9.1软件构建蛋白质相互作用网络。通过Metascape数据库和微生信网站获取的数据构建GO和KEGG通路富集分析,建立活性成分-靶点-关键通路网络。结果:在TCMSP数据库中共筛选出怀牛膝、菟丝子符合条件的有24种活性药物成分,活性成分靶点及疾病靶点分别为206、1762个。Venny在线工具中获得了药物和疾病的113个共同基因。PPI网络显示怀牛膝、菟丝子药对发挥重要作用的靶点有:丝裂原活化蛋白激酶1、肿瘤抑制蛋白P53、蛋白激酶B1、肿瘤坏死因子、原癌基因、白细胞介素6等。从GO和KEGG通路富集分析结果中可得到,药物和疾病的交集基因与无机物、氧化应激、化学应激、活性氧、金属离子等分子生物进程关系密切,相关信号通路包括有:癌症的途径、脂质与动脉粥样硬化、糖尿病并发症中的AGE信号通路等。结论:怀牛膝-菟丝子治疗高血压肾损伤具有基本药理活性,可能通过AGE-RAGE信号通路、肿瘤坏死因子信号通路等作用于肿瘤抑制蛋白P53、蛋白激酶B1等靶点改善高血压肾损伤。

红花-牛膝治疗口腔黏膜下纤维化作用机制的网络药理学分析

目的联合使用网络药理学和分子对接技术探究红花-牛膝的有效成分作用于口腔黏膜下纤维化(OSF)的治疗靶点。方法通过网络药理学方法获得“红花”“牛膝”的有效成分及作用靶点,以及OSF的疾病靶点。利用STRING平台构建蛋白互作PPI网络图;对关键活性成分与核心靶点进行分子对接分析,并对交集靶点进行了GO和KEGG富集分析。结果发现红花-牛膝作用于OSF的主要活性成分为木犀草素、黄芩苷、黄连素和黄连碱等;主要核心靶点为肿瘤坏死因子(TNF)、基质金属蛋白酶9(MMP9)、丝裂原活化蛋白激酶3(MAPK3)、低氧诱导因子1α(HIF-1α)等;主要涉及癌症中的蛋白多糖、丝裂原活化蛋白激酶(MAPK)、白介素-17(IL-17)、C型凝集素受体等信号通路。分子对接结果亦表明活性分子与靶点基因均具备良好的结合力。结论本研究结果提示红花-牛膝可能通过多活性成分、多靶点以及多种信号通路来治疗OSF。

牛膝含药血清对大鼠骨髓间充质干细胞增殖和成骨分化的影响及其作用机制

目的:探讨牛膝含药血清对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)增殖、成骨分化的影响及其作用机制。方法:取4周龄雌性SPF级SD大鼠20只,随机分为空白组和牛膝低、中、高剂量组,每组5只。牛膝低、中、高剂量组大鼠分别以相应浓度的牛膝药液灌胃,空白组大鼠以同等剂量生理盐水灌胃,每日1次,共灌胃14 d。最后一次灌胃干预2 h后,取大鼠腹主动脉血,制备空白血清和相应浓度的牛膝含药血清。另取大鼠4只,处死后取出大鼠股骨和胫骨骨髓,进行BMSCs培养。细胞传到第3代时,用流式细胞仪进行细胞表型鉴定。将大鼠BMSCs分为胎牛血清组、空白血清组和牛膝含药血清低、中、高剂量组,分别加入胎牛血清、空白血清和牛膝低、中、高剂量含药血清进行干预,检测各组大鼠BMSCs的增殖活性;分别加入含相应血清的成骨诱导液进行成骨诱导,采用茜素红染色观察大鼠BMSCs成骨分化情况,采用荧光定量PCR检测大鼠BMSCs中成骨相关因子碱性磷酸酶(alkaline phosphatase, ALP)、骨钙素(osteocalcin, OCN)、Runt相关转录因子2(runt-related transcription factor 2,Runx2)和Osterix的mRNA相对表达量,采用蛋白质印迹法检测BMSCs中Hedgehog信号通路相关蛋白音猬因子(sonic hedgehog, SHH)、Gli2的蛋白相对表达量。结果:①细胞鉴定结果。细胞表型鉴定结果显示,培养的细胞为BMSCs。②大鼠BMSCs增殖活性检测结果。干预24 h、48 h、72 h、96 h后,5组大鼠BMSCs增殖活性组间总体比较,差异均有统计学意义;干预24 h后,牛膝含药血清高剂量组BMSCs的增殖活性高于胎牛血清组、空白血清组(P=0.006,P=0.008);干预48 h、72 h、96 h后,牛膝含药血清高剂量组BMSCs的增殖活性均高于胎牛血清组、空白血清组和牛膝含药血清低、中剂量组(P=0.000,P=0.000,P=0.010,P=0.021;P=0.003,P=0.000,P=0.007,P=0.016;P=0.000,P=0.000,P=0.002,P=0.047)。③大鼠BMSCs成骨分化检测结果。茜素红染色显示,各组大鼠BMSCs均出现细胞外矿化结节形成与沉积,其中牛膝含药血清高剂量组阳性染色面积较大,矿化结节明显。牛膝含药血清高剂量组矿化结节面积大于胎牛血清组和空白血清组(P=0.039,P=0.015)。④大鼠BMSCs中成骨相关因子的mRNA相对表达量检测结果。牛膝含药血清低、中、高剂量组大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相对表达量均高于胎牛血清组(P=0.003,P=0.000,P=0.000;P=0.011,P=0.001,P=0.000;P=0.009,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000)和空白血清组(P=0.000,P=0.000,P=0.000;P=0.005,P=0.000,P=0.000;P=0.031,P=0.001,P=0.000;P=0.000,P=0.000,P=0.000)。牛膝含药血清中剂量组大鼠BMSCs中ALP的mRNA相对表达量高于牛膝含药血清低剂量组(P=0.044)。牛膝含药血清高剂量组大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相对表达量均高于牛膝含药血清低剂量组(P=0.002,P=0.006,P=0.002,P=0.008)。牛膝含药血清高剂量组大鼠BMSCs中Runx2的mRNA相对表达量高于牛膝含药血清中剂量组(P=0.047)。⑤大鼠BMSCs中Hedgehog信号通路相关蛋白的蛋白相对表达量检测结果。牛膝含药血清高剂量组大鼠BMSCs中SHH、Gli2高表达。牛膝含药血清低、中、高剂量组SHH、Gli2的蛋白相对表达量均高于胎牛血清组(P=0.000,P=0.000,P=0.000;P=0.026,P=0.016,P=0.000)和空白血清组(P=0.000,P=0.000,P=0.000;P=0.031,P=0.018,P=0.000)。牛膝含药血清中、高剂量组大鼠BMSCs中SHH的蛋白相对表达量均高于牛膝含药血清低剂量组(P=0.000,P=0.000),牛膝含药血清高剂量组大鼠BMSCs中SHH的蛋白相对表达量高于牛膝含药血清中剂量组(P=0.000)。牛膝含药血清高剂量组大鼠BMSCs中Gli2的蛋白相对表达量高于牛膝含药血清低剂量组(P=0.001)。结论:牛膝含药血清可能通过激活Hedgehog信号通路和上调成骨相关因子ALP、OCN、Runx2、Osterix的表达,促进大鼠BMSCs的增殖和成骨分化。