Synergism of calycosin and bone marrow-derived mesenchymal stem cells to combat podocyte apoptosis to alleviate adriamycininduced focal segmental glomerulosclerosis

BACKGROUND Bone marrow-derived mesenchymal stem cells(MSCs)show podocyte-protective effects in chronic kidney disease.Calycosin(CA),a phytoestrogen,is isolated from Astragalus membranaceus with a kidney-tonifying effect.CA preconditioning enhances the protective effect of MSCs against renal fibrosis in mice with unilateral ureteral occlusion.However,the protective effect and underlying mechanism of CA-pretreated MSCs(MSCsCA)on podocytes in adriamycin(ADR)-induced focal segmental glomerulosclerosis(FSGS)mice remain unclear.AIM To investigate whether CA enhances the role of MSCs in protecting against podocyte injury induced by ADR and the possible mechanism involved.METHODS ADR was used to induce FSGS in mice,and MSCs,CA,or MSCsCA were administered to mice.Their protective effect and possible mechanism of action on podocytes were observed by Western blot,immunohistochemistry,immunofluorescence,and real-time polymerase chain reaction.In vitro,ADR was used to stimulate mouse podocytes(MPC5)to induce injury,and the supernatants from MSC-,CA-,or MSCsCA-treated cells were collected to observe their protective effects on podocytes.Subsequently,the apoptosis of podocytes was detected in vivo and in vitro by Western blot,TUNEL assay,and immunofluorescence.Overexpression of Smad3,which is involved in apoptosis,was then induced to evaluate whether the MSCsCA-mediated podocyte protective effect is associated with Smad3 inhibition in MPC5 cells.RESULTS CA-pretreated MSCs enhanced the protective effect of MSCs against podocyte injury and the ability to inhibit podocyte apoptosis in ADR-induced FSGS mice and MPC5 cells.Expression of p-Smad3 was upregulated in mice with ADR-induced FSGS and MPC5 cells,which was reversed by MSCCA treatment more significantly than by MSCs or CA alone.When Smad3 was overexpressed in MPC5 cells,MSCsCA could not fulfill their potential to inhibit podocyte apoptosis.CONCLUSION MSCsCA enhance the protection of MSCs against ADR-induced podocyte apoptosis.The underlying mechanism may be related to MSCsCA-targeted inhibition of p-Smad3 in podocytes.

Calycosin improves cognitive function in a transgenic mouse model of Alzheimer's disease by activating the protein kinase C pathway

The major pathological changes in Alzheimer＇s disease are beta amyloid deposits and cognitive impairment. Calycosin is a typical phy- toestrogen derived from radix astragali that binds to estrogen receptors to produce estrogen-like effects. Radix astragali Calycosin has been shown to relieve cognitive impairment induced by diabetes mellitus, suggesting calycosin may improve the cognitive function of Alzhei- mer＇s disease patients. The protein kinase C pathway is upstream of the mitogen-activated protein kinase pathway and exerts a neuropro- tective effect by regulating Alzheimer＇s disease-related beta amyloid degradation. We hypothesized that calycosin improves the cognitive function of a transgenic mouse model of Alzheimer＇s disease by activating the protein kinase C pathway. Various doses of calycosin （10, 20 and 40 mg/kg） were intraperitoneally injected into APP/PS1 transgenic mice that model Alzheimer＇s disease. Calycosin diminished hippocampal beta amyloid, Tau protein, interleukin-lbeta, tumor necrosis factor-alpha, acetylcholinesterase and malondialdehyde levels in a dose-dependent manner, and increased acetylcholine and glutathione activities. The administration of a protein kinase C inhibitor, cal- phostin C, abolished the neuroprotective effects of calycosin including improving cognitive ability, and anti-oxidative and anti-inflammato- ry effects. Our data demonstrated that calycosin mitigated oxidative stress and inflammatory responses in the hippocampus of Alzheimer＇s disease model mice by activating the protein kinase C pathway, and thereby improving cognitive function.

Calycosin attenuates severe acute pancreatitis-associated acute lung injury by curtailing high mobility group box 1-induced inflammation

BACKGROUND Acute lung injury(ALI)is a common and life-threatening complication of severe acute pancreatitis(SAP).There are currently limited effective treatment options for SAP and associated ALI.Calycosin(Cal),a bioactive constituent extracted from the medicinal herb Radix Astragali exhibits potent anti-inflammatory properties,but its effect on SAP and associated ALI has yet to be determined.AIM To identify the roles of Cal in SAP-ALI and the underlying mechanism.METHODS SAP was induced via two intraperitoneal injections of L-arg(4 g/kg)and Cal(25 or 50 mg/kg)were injected 1 h prior to the first L-arg challenge.Mice were sacrificed 72 h after the induction of SAP and associated ALI was examined histologically and biochemically.An in vitro model of lipopolysaccharide(LPS)-induced ALI was established using A549 cells.Immunofluorescence analysis and western blot were evaluated in cells.Molecular docking analyses were conducted to examine the interaction of Cal with HMGB1.RESULTS Cal treatment substantially reduced the serum amylase levels and alleviated histopathological injury associated with SAP and ALI.Neutrophil infiltration and lung tissue levels of neutrophil mediator myeloperoxidase were reduced in line with protective effects of Cal against ALI in SAP.Cal treatment also attenuated the serum levels and mRNA expression of pro-inflammatory cytokines tumor necrosis factor-α,interleukin-6,IL-1β,HMGB1 and chemokine(CXC motif)ligand 1 in lung tissue.Immunofluorescence and western blot analyses showed that Cal treatment markedly suppressed the expression of HMGB1 and phosphorylated nuclear factor-kappa B(NF-κB)p65 in lung tissues and an in vitro model of LPSinduced ALI in A549 cells suggesting a role for HGMB1 in the pathogenesis of ALI.Furthermore,molecular docking analysis provided evidence for the direct interaction of Cal with HGMB1.CONCLUSION Cal protects mice against L-arg-induced SAP and associated ALI by attenuating local and systemic neutrophil infiltration and inflammatory response via inhibition of HGMB1 and the NF-κB signaling pathway.

Study on Pharmacological Effects of Calycosin in Astragali Radix

Calycosin is an important isoflavone compound in traditional Chinese medicine Astragali Radix.It not only has remarkable anti-oxidation,anti-inflammatory and anti-tumor functions,but also has the characteristics of small adverse reactions and low toxicity,so it has attracted wide attention of scholars and researchers.This paper reviewed the pharmacological effects and mechanisms of calycosin in recent years,in the hope of providing a theoretical basis for its clinical application.

Anti-hepatic carcinoma mechanisms of calycosin through targeting ferroptosis

Background Ferroptosis,a pathologic state induced by lipid-driven oxidative stress,is associated with the development of human cancers.Calycosin,a natural compound with antioxidant and anti-inflammatory activities,has promising antitumor effects.However,the ferroptosis-related mechanism of calycosin in the treatment of hepatic carcinoma has not been reported.Methods This study applied network pharmacology and bioinformatic approaches(including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis)to investigate the pharmacologic targets and mechanism of action of calycosin in the treatment of hepatic carcinoma through targeting ferroptosis.By searching online databases including The Cancer Genome Atlas,FerrDb,GeneCards,SwissTargetPrediction,SuperPred,BindingDB,TargetNet,BATMAN-TCM,and Drugbank,we identified 13 ferroptosis-related putative target genes of calycosin against hepatic carcinoma including IL-6,PTGS2,SRC,HRAS,NQO1,NOX4,PGK1,G6PD,GPI,MIF,NOS2,ALDOA,and SQSTM1.Results Molecular docking analysis revealed that calycosin potentially binded directly with the target proteins IL-6,PTGS2,and SRC.Functional enrichment analysis of these proteins indicated that they were involved in gluconeogenesis and apoptosis through regulation of ERK1,ERK2,and MAPK activities(P<0.05).Conclusion Calycosin exerts antitumor effects in hepatic carcinoma by targeting ferroptosis through regulation of IL-6,PTGS2,and SRC.

基于UPLC-MS/MS和网络药理学、分子动力学探讨红芪免疫调节机制

目的基于UPLC-MS/MS、网络药理学方法预测红芪免疫调节的核心靶点及作用通路,通过分子对接、分子动力学技术对网络药理学结果进行验证,探讨红芪入血成分免疫调节的作用机制。方法基于UPLC-MS/MS技术定性定量红芪入血成分;通过TCMSP、本草组鉴数据库筛选红芪入血成分对应靶点;以DisGeNET、OMIM、TTD、MalaCards数据库获取免疫相关疾病靶点;构建“红芪入血成分-免疫相关疾病”网络;进行GO、KEGG富集分析,绘制PPI网络;应用分子对接、分子动力学技术进行验证。结果UPLC-MS/MS法共鉴定8个原型入血成分,协同作用于101个靶点,参与免疫反应、基因表达的正调控、受体结合、细胞因子活性等538个生物学过程,涉及HIF-1、Toll样受体、JAK-STAT、T细胞受体、PI3K-Akt、FoxO等116条信号通路。核心靶点为MAPK14、PTGS2、MMP9、PPARG、CCND1等。分子对接结果显示,芒柄花素、毛蕊异黄酮与MAPK14的对接结合活性较高,分子动力学模拟进一步验证了芒柄花素、毛蕊异黄酮与MAPK14的结合具有较好的结构稳定性及结合亲和力。结论通过血清药物化学、网络药理学及分子动力学相互印证,揭示了红芪调节免疫的物质基础及机制,可为其作用机制研究提供科学依据。

不同产地黄芪的多指标质量评价研究

目的:对12个不同产地的黄芪样品的质量进行评价。方法:以总多糖、总黄酮、总皂苷、毛蕊异黄酮葡萄糖苷、毛蕊异黄酮、芒柄花素、芒柄花苷及黄芪甲苷8个指标对甘肃、山西、内蒙古及宁夏等产地的12个黄芪样品进行多维分析,并考察8个指标的相关性。结果:12个样品中有3个样品的毛蕊异黄酮葡萄糖苷含量以及8个样品的黄芪甲苷含量不符合《中华人民共和国药典》(2020年版)规定要求。12个不同产地的黄芪样品中总多糖、总黄酮、总皂苷、毛蕊异黄酮葡萄糖苷、毛蕊异黄酮、芒柄花素、芒柄花苷及黄芪甲苷等8个指标的含量(mg/g)分别为13.389~68.777、3.132~4.408、6.761~18.284、0.143~0.374、0.023~0.169、0.020~0.119、0.044~0.110、0.051~1.399 mg/g。芒柄花苷含量与毛蕊异黄酮葡萄糖苷含量呈正相关(r=0.838),芒柄花素含量与毛蕊异黄酮含量呈正相关(r=0.973),黄芪甲苷含量与其他7个指标含量无相关性。结论:不同产地黄芪的化学成分含量差异显著,质量差异明显,选购黄芪药材需注意其直径的大小。

毛蕊异黄酮治疗脊髓损伤大鼠的作用机制

目的:探究毛蕊异黄酮治疗脊髓损伤的潜在作用机制。方法:30只大鼠随机分为假手术组、模型组、毛蕊异黄酮组,每组10只。模型组和毛蕊异黄酮组采用改良Allen重物打击法建立大鼠脊髓损伤模型后,毛蕊异黄酮组大鼠给予20 mg/kg毛蕊异黄酮灌胃。造模5周后,测定改良Tarlov评分、皮层体感诱发电位波潜伏期时长和斜板试验,测定SCI大鼠脊髓神经功能。观察HE染色后脊髓损伤的情况;蛋白质印迹法检测脊髓组织中p-Akt、gp130、IL-6蛋白表达。结果:与假手术组比较,模型组中改良Tarlov评分及倾斜平面临界角度显著降低(P<0.05),皮层体感诱发电位波潜伏期时长和p-Akt、gp130、IL-6蛋白表达均显著上调(P<0.05);与模型组比较,毛蕊异黄酮治疗后改良Tarlov评分及斜板试验角度显著上升(P<0.05),皮层体感诱发电位波潜伏期时长和p-Akt、gp130、IL-6蛋白表达均显著下调(P<0.05)。结论:毛蕊异黄酮促进大鼠脊髓损伤后神经功能的恢复,通过抑制gp130、IL-6的蛋白表达和PI3K/Akt信号通路磷酸化起作用。

毛蕊异黄酮对人诱导多能干细胞内皮分化的影响及机制

背景:内皮损伤是心血管疾病的诱因之一,人诱导多能干细胞易于获得、分化能力强、排异性较小,其内皮分化细胞可被用作心血管疾病研究的理想细胞。目的:探讨毛蕊异黄酮对人诱导多能干细胞定向内皮分化的作用及机制,为微血管再生提供技术支持。方法:将人诱导多能干细胞分为对照组与毛蕊异黄酮1.25,2.5μg/mL组,进行内皮定向诱导分化。诱导分化8 d后,采用流式细胞术检测各组细胞内皮细胞标志物CD144阳性率,免疫荧光技术检测CD144、CD31荧光表达。利用慢病毒RNAi-GFP puromycin沉默人诱导多能干细胞Piezo1 mRNA,再进行内皮定向诱导分化,诱导分化8 d后,采用流式细胞术检测分化细胞CD144阳性率,qPCR检测CD144、Piezo1、MEK的mRNA表达水平。结果与结论:①与对照组相比,毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率显著升高(P<0.05);毛蕊异黄酮2.5μg/mL组CD144、Piezo1、MEK mRNA表达水平提高(P<0.05);毛蕊异黄酮2.5μg/mL组CD144(P<0.01)和CD31(P<0.001)荧光表达显著升高;②与shNT组相比,shNT+毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率和CD144、Piezo1、MEK mRNA表达显著升高(P<0.05),与shPiezo1组相比,shPiezo1+毛蕊异黄酮1.25,2.5μg/mL组CD144阳性率和CD144、Piezo1、MEK mRNA表达无显著变化(P>0.05);③实验结果提示2.5μg/mL毛蕊异黄酮能够促进人诱导多能干细胞内皮分化,毛蕊异黄酮可以通过靶向调节Piezo1表达水平,促进下游MEK表达,从而促进人诱导多能干细胞内皮分化。

运用根形态及黄酮类成分鉴定不同年份黄芪品质

为了给黄芪的规范化栽培和合理采收提供依据,对3~8年生6个不同年份黄芪根的形态进行比较,并对根中总黄酮和毛蕊异黄酮葡萄糖苷积累规律进行研究.运用游标卡尺对黄芪根的最粗直径予以测量,通过显微切片染色方法观测其解剖结构并定位黄酮类成分主要位置,利用紫外可见分光光度法和高效液相色谱法对不同年份黄芪总黄酮和毛蕊异黄酮葡萄糖苷含量进行测定.6个不同年份黄芪根直径差异显著,韧皮部是黄芪根黄酮类成分主要储存部位,且5年生黄芪根韧皮部所占比例最高,5年生黄芪根中总黄酮和毛蕊异黄酮葡萄糖苷的含量最高.综合分析认为,黄芪药材的合理采收应以5年生黄芪为宜.

黄芪健骨胶囊质量标准研究

目的建立黄芪健骨胶囊的质量标准。方法采用薄层色谱(TLC)法对制剂中黄芪、山茱萸药材进行定性鉴别;采用高效液相色谱(HPLC)法测定制剂中马钱苷、毛蕊异黄酮苷、柚皮苷的含量,色谱柱为XSelect HSS T_(3)柱(250 mm×4.6 mm,3.5μm),流动相为甲醇-0.3%磷酸水溶液(梯度洗脱),流速为0.8 mL/min,检测波长为240 nm,柱温为25℃,进样量为10μL。结果黄芪、山茱萸的TLC图特征斑点清晰,分离度好,且阴性对照无干扰。马钱苷、毛蕊异黄酮苷、柚皮苷质量浓度分别在21.54~430.80μg/mL(R^(2)=0.9993)、5.02~100.40μg/mL(R^(2)=0.9999)、20.28~405.60μg/mL(R^(2)=0.9997)范围内与峰面积线性关系良好;精密度、稳定性、重复性试验结果的RSD均小于3.0%;平均加样回收率分别为100.66%,98.58%,100.17%,RSD分别为1.70%,2.36%,1.98%(n=6)。结论该方法操作简便,结果准确,重复性好,可用于黄芪健骨胶囊的质量控制。

兽用中药玉屏风口服液质量标准提升研究

建建立一种可同时测定玉屏风口服液中升麻素苷、5-0-甲基维斯阿米醇苷、毛蕊异黄酮葡萄糖苷含量的高效液相色谱法(HPLC)。采用以十八烷基硅烷键合硅胶为填充剂的色谱柱(5μm 4.6×250mm),以乙睛和0.2%磷酸溶液为流动相进行梯度洗脱,流速1.0mL·min^(-1),柱温30℃,检测波长254nm,进样体积10μL。检测的三种成分在测定的质量浓度范围内线性关系良好,r值均为0.999,平均加样回收率均在96%~102%之间,相对标准偏差均小于2.0%。试验精密度、重复性、准确性和稳定性良好。新建立的检测方法简单、准确、高效,为进一步提升玉屏风口服液的质量控制提供了依据。

毛蕊异黄酮对氧糖剥夺再灌注BV2小胶质细胞极化的影响

目的探讨毛蕊异黄酮对氧糖剥夺再灌注BV2小胶质细胞极化的影响。方法将对数生长期BV2细胞随机分为6组:正常组、模型组、尼莫地平组(5μg·mL^(-1))、毛蕊异黄酮高剂量组(20μg·mL^(-1))、毛蕊异黄酮中剂量组(10μg·mL^(-1))、毛蕊异黄酮低剂量组(5μg·mL^(-1))。氧糖剥夺3 h后,各给药组换成含药的完全培养基,复氧6 h。采用CCK-8法检测细胞活力;测定细胞上清液中的一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-10含量;免疫荧光双染法检测BV2细胞极化;WesternBlot法检测BV2细胞的iNOS、CD206蛋白表达水平。结果与正常组比较,模型组的BV2细胞活力显著降低(P<0.01),NO、TNF-α、IL-1β水平明显升高(P<0.05),IL-10水平明显降低(P<0.05);免疫荧光双染检测中M1型小胶质细胞标记物iNOS表达显著增强(P<0.01);Western Blot检测中iNOS蛋白表达明显上调(P<0.05),M2型细胞标志物CD206蛋白表达明显下调(P<0.05)。与模型组比较,毛蕊异黄酮低、中、高剂量组及尼莫地平组的BV2细胞活力显著提高(P<0.01),NO、TNF-α、IL-1β水平明显降低(P<0.05),IL-10水平明显升高(P<0.05);免疫荧光双染检测中毛蕊异黄酮(10μg·mL^(-1))组BV2细胞的iNOS表达显著减弱(P<0.01);Western Blot检测中毛蕊异黄酮(10μg·mL^(-1))组BV2细胞的iNOS蛋白表达明显下调(P<0.05),CD206蛋白表达明显上调(P<0.05)。结论毛蕊异黄酮能促进活化的BV2小胶质细胞向M2型极化,抑制其向M1型极化,减少炎性介质的产生,降低氧化损伤,对缺血后脑组织损伤具有保护作用。

HPLC-MS/MS测定当归六黄汤中4种不同成分的含量

目的建立HPLC-MS/MS的分析方法同时测定当归六黄汤中4种主要成分黄柏碱、巴马汀、毛蕊异黄酮、阿魏酸的含量,为当归六黄汤的质量控制提供方法学参考和借鉴。方法基于HPLC-MS/MS的分析方法,质谱检测采用ESI源MRM的正离子数据采集模式。色谱柱为Agilent Extend-C_(18)(5μm,4.6 mm×250 mm),甲醇和含0.5%的甲酸水溶液进行梯度洗脱。结果黄柏碱的线性范围为2~200 nmol/ml,巴马汀、毛蕊异黄酮、阿魏酸的线性范围均为20~2000 nmol/ml,当归六黄汤7批样品中4种成分的含量相对稳定,其中,阿魏酸主要存在于黄柏和黄连中;黄柏碱只在黄柏中测到;毛蕊异黄酮在黄芩和黄芪中含量较高;巴马汀在黄柏和黄芪中都能检测到。结论该方法灵敏度高,专属性好,样品的稳定性良好,能够满足中药复方定量分析的要求,可用于当归六黄汤的质量控制和评价研究。

Calycosin-7-O-β-D-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNT signaling pathways

The iso fl avone calycosin-7-O-β-D-glucopyranoside(CG) is a principal constituent of Astragalus membranaceus(AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin(Ocal) m RNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2(BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family(WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.

The cardiovascular protective effect and mechanism of calycosin and its derivatives

Cardiovascular disease is the main cause of mortality and morbidity in the world,especially in developing countries.Drug therapy is one of the main ways to treat cardiovascular diseases.Among them,great progress has been made in the treatment of cardiovascular diseases with traditional Chinese medicine.In terms of experimental research,the mechanism of traditional Chinese medicine in the treatment of cardiovascular diseases has been thoroughly discussed in vitro and in vivo.In terms of clinical treatment,traditional Chinese medicine with flavonoids,saponins and alkaloids as the main effective components has a definite effect on the treatment of cardiovascular diseases such as arrhythmia,myocardial ischemia,angina pectoris and myocardial infarction,with high safety and good application prospects.With the further research on the effective ingredients,mechanism and adverse reactions of traditional Chinese medicine,it will be beneficial to the effectiveness of traditional Chinese medicine,reduce side effects and promote the modernization of traditional Chinese medicine.Calycosin and its derivatives,the main bioactive flavonoids in Astragalus membranaceus have multiple biological effects,such as antioxidant,pro-angiogenesis,anti-tumour,and anti-inflammatory effects.Based on the above biological effects,calycosin has been shown to have good potential for cardiovascular protection.The potent antioxidant effect of calycosin may play an important role in the cardiovascular protective potential.For injured cardiac myocytes,calycosin and its derivatives can alleviate the cell damage mainly marked by the release of myocardial enzymes and reduce the death level of cardiac myocytes mainly characterized by apoptosis through various mechanisms.For vascular endothelial cells,calycosin also has multiple effects and multiple mechanisms,such as promoting vascular endothelial cell proliferation,exerting vasodilating effect and directly affecting the synthesis function of endothelial cells.The present review will address the bioactivity of calycosin in cardiovascular diseases such as protective effects on cardiac myocytes and vascular endothelial cells and elucidate main mechanism of calycosin and its derivatives to exert the above biological effects.

HPLC-UV法测定经典名方当归补血汤中5种化学成分含量

本研究旨在建立高效液相色谱-紫外检测法,同时测定当归补血汤中阿魏酸、洋川芎内酯Ⅰ、毛蕊异黄酮苷、毛蕊异黄酮及芒柄花苷的含量。采用Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇(B):0.4%甲酸水(A)梯度洗脱,检测波长280 nm。阿魏酸、洋川芎内酯Ⅰ、毛蕊异黄酮苷、毛蕊异黄酮和芒柄花苷分别在0.091~1.824、0.015~0.308、0.092~1.848、0.036~0.728、0.082~1.656μg/mL质量范围内线性关系良好,r> 0.999;定量限分别为0.091、0.015、0.092、0.036、0.082μg/mL;检测限分别为0.027、0.005、0.028、0.011、0.025μg/mL。该方法精密度、重复性、稳定性、加样回收试验的RSD均小于5%;回收率分别为99.68%、99.40%、100.24%、101.04%和100.88%。所建立的测定方法操作简单,分析灵敏快速,结果准确,可用于当归补血汤的含量测定及质量控制。

毛蕊异黄酮对脑缺血再灌注损伤大鼠的保护作用

目的探讨毛蕊异黄酮对脑缺血再灌注损伤大鼠的保护作用。方法将SPF级雄性SD大鼠随机分为4组:假手术组、模型组、毛蕊异黄酮组、阳性药(依达拉奉)组。酶联免疫吸附法(ELISA)检测ANG-1、iNOS和VEGF水平;蛋白免疫印迹法(Western Blot)检测HIF-1α、EGFR和VEGFR1的蛋白表达水平;免疫组化法观察GFAP、VWF和GLUT1阳性细胞;实时荧光定量法(PCR)检测HIF-1α、VEGFA和RCAN2的mRNA表达水平。结果毛蕊异黄酮和依达拉奉均能够增加SD大鼠血清中VEGF、ANG-1和iNOS含量(P<0.05或P<0.01),增加SD大鼠脑组织中HIF-1α、VEGFR1、EGFR和GLUT1蛋白表达水平(P<0.05或P<0.01),增加SD大鼠脑组织中HIF-1α、VEGFA和RCAN2 mRNA表达量(P<0.05或P<0.01)。结论毛蕊异黄酮能够通过改善血管生成对脑缺血再灌注大鼠具有一定的保护作用,且与阳性对照药疗效相当。

毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响

目的探讨毛蕊异黄酮(CAL)调控PI3K/Akt信号通路对脊髓损伤(SCI)大鼠神经元自噬及凋亡的影响及其作用机制。方法32只成年SD大鼠随机分为假手术组(Sham组),模型组(SCI组)和CAL低、高剂量组,每组8只。采用改良Allen’s法建立大鼠中度SCI模型,术后CAL低、高剂量组立即分别予以20、40 mg/kg CAL腹腔注射,1次/d,连续7 d;Sham组和SCI组给予等量生理盐水。术后1、3和7 d,采用行为学评分(BBB)评估大鼠运动功能的恢复情况;术后7 d,尼氏染色检测大鼠脊髓前角运动神经元存活数量,Western blot检测p62、Beclin-1、微管相关蛋白1轻链3(LC3B)、磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)和活化型胱天蛋白酶-3(Cleaved-Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)表达,免疫荧光染色检测大鼠脊髓前角神经元LC3B的表达。结果与Sham组相比,SCI组大鼠BBB评分、神经元存活数量、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平降低,p62、Beclin-1、LC3BⅡ、LC3BⅡ/Ⅰ、Cleaved-Caspase-3、Bax水平升高(P<0.05);与SCI组相比,CAL低、高剂量组BBB评分、神经元存活数量、LC3BⅡ、LC3BⅡ/Ⅰ、Bcl-2、Bcl-2/Bax、p-PI3K、p-PI3K/PI3K、p-Akt、p-Akt/Akt水平升高,p62、Cleaved-Caspase-3、Bax水平降低(P<0.05)。结论CAL可通过激活PI3K/Akt信号通路促进SCI大鼠神经元自噬,抑制其凋亡,进而发挥神经保护作用。

当归补血泡腾片的制备及质量分析

优选当归补血泡腾片的处方工艺,并对其质量进行分析。通过Box-Behnken响应面法优化制剂处方。通过TLC法对样品中阿魏酸、藁本内酯进行薄层鉴别。通过HPLC-CAD法对黄芪甲苷进行含量测定,通过HPLC-DAD法对样品中阿魏酸、毛蕊异黄酮葡萄糖苷进行含量测定。在当归黄芪提取物用量15%、柠檬酸用量22%、碳酸氢钠用量19%、聚乙二醇6000用量5%、可溶性淀粉用量39%时,制剂的综合评分最佳,实测值与预测值偏差为1.43%。崩解时限、重量差异、硬度、pH等检查符合药典规定。在TLC检测中,阿魏酸、藁本内酯主斑点清晰。黄芪甲苷、阿魏酸、毛蕊异黄酮葡萄糖苷的含量分别为448.38、242.31、526.02μg/g。实验确立的当归补血泡腾片处方科学合理,质量分析方法方便可靠。