黄芪注射液对大鼠脑缺血再灌注损伤的保护作用研究

目的 探讨黄芪注射液对大鼠脑缺血/再灌注损伤的保护作用和机制.方法 应用线栓法经左侧颈外-颈内动脉插线建立大鼠大脑中动脉阻塞再灌注（MCAO/R）模型,经腹腔注射黄芪注射液（3 ml/kg）干预治疗.Longa法评价大鼠神经行为功能,氯化三苯基四氮唑（TTC）染色观察脑梗死体积,苏木精-伊红（HE）染色观察海马神经元形态结构,TUNEL法检测细胞凋亡,免疫组化检测c-jun氨基末端激酶（JNK3）蛋白表达水平.结果 经黄芪注射液治疗后大鼠海马神经元JNK3蛋白表达水平显著降低,凋亡细胞数量显著减少,神经元形态恢复,脑梗死体积缩小,大鼠神经行为功能显著改善.结论 黄芪注射液可通过下调JNK3表达水平而抑制细胞凋亡,缩小脑梗死体积,改善大鼠神经行为功能.     

Comparative Anti-Inflammatory and Hepatoprotective Activities of Astragalus Gummifer Labill Herb and Roots in Rats

Background

The Astragalus gummifer (F. Fabaceae), herb and roots were studied for anti-inflammatory and hepatoprotective activities.

Materials and method

The alcoholic extracts of Astragalus gummifer (F. Fabaceae), herb (AGHE), and roots (AGRE), were used for anti-inflammatory and hepatoprotective activities in Wister rats. The effects of AGHE and AGRE were compared with the standard drugs Phenylbutazone and silymarin, for anti-inflammatory and hepatoprotective activities respectively.

Result

Both extracts showed significant anti-inflammatory activity (P< 0.001). AGRE showed comparatively more significant hepatoprotective activity (P< 0.001), than AGHE (P< 0.05); at doses of 250 and 500 mg/kg body weight as manifested by lowering the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and total bilirubin. The hepatoprotective activity was, also, supported by total protein (TP), malondialdehyde (MDA), nonprotein sulfhydryls (NP-SH), and histo-pathological studies of liver tissue.

Discussion

To the best of our knowledge, this is the first report of the anti-inflammatory and hepatoprotective activities of Astragalus gummifer. The results of present studies indicated that both AGHE and AGRE can be used in inflammatory conditions, while investigation supports the use of AGRE in cases that hepatoprotection are required in the hepatotoxic conditions. More supportive studies are required before clinical recommendation.     

Astragalus Mongholicus Regulate the Toll-Like-Receptor 4 Meditated Signal Transduction of Dendritic Cells to Restrain Stomach Cancer Cells

Background

According to the traditional view, we depend on three methods to treat tumors; surgery, chemotherapy and radiotherapy. However, these methods have its own limitations in application. Traditional Chinese Medicine (TCM) is one of the oldest healing systems. Astragalus mongholicus (AMs) that is the common herbal medicine, the biggest part of TCM, have been proved to be effective in treating cancers from lots of clinical cases. However, we have not fully understood the anti-tumor mechanism of AMs, and this has lead to some doubt for some Western-Medicine scholars and restricts its wide use. The main objective of this research is to discuss the effect and mechanism of AMs to human stomach cancer.

Materials and Methods

To observe the effect and mechanism of tumor treatment by AMs, we have done the research from three major aspects, the influence of DCs, the inhibition of tumor in vitro as well as the animal studies in vivo after treatment. First, we culture the mouse dendritic cells (DCs) from bone marrow of mouse hind legs according to the method using Interleukin-4(IL-4) and Granulocyte-macrophage colony stimulating factor (GM-CSF), which refer to the way established by Inaba (Inaba K, 1992). And then we investigate the growth-rate of the DCs co-cultured with AMs injection. We analyze the expression of the Toll-like-receptor 4 (TLR4), with SYBR-Green I Real-time PCR and the I-kappa-B-alpha (IκB-α) with Western-Blot, the main regulatory protein to control nuclear factor NFκB-p65 nuclear translocation. Second, we choose the human gastric cancer cell lines MKN 45 as the target cell, which was co-cultured with DCs, T cells from spleen of mouse and AMs injection, and use MTT assay to judge the amount of cell lines and Immnunoflurescene to analyze the expression of anti-active caspase 3 pAb anti-PARP P85 fragment pAb, the mark of apoptosis of cells. Third, we have conducted the animal studies beside the basic experiment in vitro. The nude mouse developed stomach cancer, due to intra-preritoneal injection with MKN45 have been divided into two groups: the treatment group challenged with AMs injection and the control group with saline injection. We took the average of the diameter of each group as the y axis and the days after administered with AMs as x axis. After 40 days, all animals were killed by detruncation, and the tumor were removed and measured. We compare the diameter (<40 days) and weight (>40 days) of the tumor as well as the survival days between different groups to investigate the effect of inhibition of cancer.

Results

All results show that AMs is effective in treating human stomach cancer and the mechanism might be regulated by TLR4 mediated signal transduction of DCs. The results are briefly introduced as follows: First, we succeed in culturing the DCs induced by IL-4 and GM-CSF and find the positive rate of CD11c expression, the mark of DCs, is beyond 90% (Fig-1). We detect AMs can precipitate DCs maturation by upregulating TLR4 in SYBR-Green I Real-time PCR (Fig-2) and suppressing I.B-aby Western-Blot (Fig-3). Second, after the MKN45 co-cultured with DCs, T cells and AMs injection, the result show that AMs can great reduce the amount of cell lines by MTT assay (Fig-4) and induce apoptosis with Immunofluorescence (Fig-5). Finally, we have conducted animal studies beside the experiment in vitro, and the result in vivo show that AMs can delay tumor development from the diameter and weight of the tumor (Fig-6, Fig-7), prolong life-span and improve life-quality.Open in a separate windowFigure 1the morphology and phenotypic identification of DCs.

1. The form of DCs observed by microscope with field 20*.
2. The isotype antibody control using FCM.
3. The positive rate of CD11c expression.

Open in a separate windowFigure 2the melting curve and the chart of TLR4 expressiona) the melting curve of beta-actin; b)the melting curve of TLR4;c)the TLR4 expression of DCs stimulated with AM at different dose. There is significant statistic difference between the 60ng/mL and 80ng/mL group and other group (P<0.05 by rank test)Open in a separate windowFigure 3the IκB-α expression of DCs with different dose of AMsL0: 0ng/mL; L1:20ng/mL; L2:40ng/mL; L3:60ng/mL; L4:80ng/mLOpen in a separate windowFigure 4MTT assay to analyse the viability and proliferation of the two cell lines (P<0.05 between the group with the dose of 60ng/mL and 80ng/mL and other group). the horizontal axis is the group treated with AM and saline at different dose, the vertical axis is the cell number.Open in a separate windowFigure 5the anti-active caspase-3 pAb (a) and anti-PARP P85 fragment pAb (b) actived by immunofluorescence.The cell mix were treated with 100uL anti-active caspase-3 pAb at a 1:250 dilution and anti-PARP P85 fragment pAb at a 1:100 dilution, and the secondary Ab was donkey anti-rabbit Cy®3 conjugate diluted 1:500 in PBS (Jackson Cat#711-165-152). From the photo, we find that anti-active caspase-3 pAb and anti-PARP P86 fragment pAb can express which is very important to indicate cell-apoptosis.Open in a separate windowFigure 6The difference of tumor between treatment group and control group.

Conclusion

Ams Can play a great role in treating human stomach cancers as a good Chinese herbal medicine by precipitating DCs maturation, which is probably due to its effects by regulating the TLR4 mediated signal transduction.     

黄芪、丹参注射液合用治疗急性脑梗死

目的：观察黄芪、丹参合用治疗急性脑梗死的疗效。方法：５０例脑梗死病人随机分为黄芪加丹参组 ３０例，用黄芪注射液 １６ ｍＬ（含黄芪 ３２ ｇ）和丹参注射液 １６ ｍＬ含丹参 ２４g）；丹参组 ２０例，用丹参注射液 １６ ｍＬ。均置 ５％葡萄糖注射液 ５００ ｍＬ中静脉滴注，ｑｄ。疗程均为 ２０ ｄ。于治疗前后作神经功能缺损评分并测定血浆依前列醇（ＰＧＩ_(2)）和凝血哑烷Ａ_(2)（ＴＸＡ_(2)）水平。结果：黄芪加丹参组临床疗效、血ＴＸＡ_(2)下降值与丹参组比较差异有显著意义（前者 ９０％和 ７５％， Ｐ＜ ０． ０５；后者 ５３ ｎｇ· Ｌ~(-1)±２４ ｎｇ·Ｌ~(-1)和 ３５ ｎｇ· Ｌ~(-1)± ２２ ｎｇ· Ｌ~(-1)， Ｐ＜ ０． ０１），但血ＰＧＩ_(2)增高值差异无显著意义。结论：黄芪与丹参合用治疗急性脑梗死的疗效优于单用丹参。     

黄芪注射液对人乳腺癌细胞中VEGF和CXCR4表达的影响

目的：研究黄芪对人乳腺癌细胞中MDA-MB-231系血管内皮生长因子（VEGF）及其受体（flk-1）和血小板源性因子受体-4（CXCR4）表达的影响。方法：在无酚红DMEM培养的人乳腺癌细胞中加0.5、1.0、2.0 g/L黄芪处理,对照组（Con）加等体积的蒸馏水。收集培养48 h的细胞,用免疫细胞化学和免疫印迹检测乳腺癌细胞中VEGF、flk-1和CXCR4蛋白的表达。结果：与Con组相比,黄芪组乳腺癌细胞中的VEGF、flk-1和CXCR4蛋白表达均下降,且高浓度组中表达明显降低。结论：高浓度黄芪能下调乳腺癌细胞中VEGF、flk-1和CXCR4蛋白的表达量,由此可能会减少肿瘤血管生成和降低肿瘤侵袭。     

黄芪注射液三阴交注射治疗动力性尿潴留的研究

目的探讨黄芪注射液三阴交穴位注射治疗动力性尿潴留的效果。方法将麻醉手术后的患者65例随机分成2组。实验组35例，在常规护理干预的基础上采用黄芪注射液于三阴交穴位注射；对照组30例，给予常规的护理干预。观察2组解除尿潴留的效果。结果实验组解除尿潴留的效果显著优于对照组，实验组干预后34例占97．1％患者于2h内自行排尿，其中27例为一次性排完，7例部分排尿，与对照组比较差异有显著性。结论黄芪注射液三阴交穴位注射治疗动力性尿潴留是一种解除尿潴留的有效方法，可减轻患者的痛苦，减少导尿及导尿所致的并寿痒．     

黄芪总黄酮对急性心肌梗死心室肌细胞钠电流的作用

目的探讨黄芪总黄酮（TFA）对急性心肌梗死（AMI）后大鼠心室肌细胞钠电流（ⅠNa）的影响。方法大鼠开胸左前降支结扎造成AMI，酶解分离心室肌细胞，采用全细胞膜片钳记录技术记录左前降支供血区心外膜细胞的ⅠNa。结果应用TFA（20mg／kg）后与AMI组比较，ⅠNa峰值从（-4．79±1．88）nA下降到（-2．74±1．67）nA，差异有统计学意义（P〈0．01，n=6）。结论TFA可显著降低AMI大鼠心室肌细胞ⅠNa的幅值。     

中药黄芪对实验性脊髓损伤的神经保护作用

目的探讨黄芪(AR)对脊髓继发性损伤的保护作用,并与甲基强地松龙(MP)进行对照.方法 Wistar大鼠60只,以改良Allen氏法制备脊髓打击伤模型,随机分为三组.测定不同药物处理后4 h、8 h、24 h脊髓组织线粒体SOD活性和MDA浓度以及血液流变学改变;光镜观察用药后1、2周黄芪对病理学改变的影响,同时进行联合行为学评分(CBS).结果黄芪处理后脊髓组织MDA浓度明显低于各时相点对照组,SOD活性显著升高(P＜0.01),与MP治疗组无明显差异(P＞0.05).血液流变学指标也有所改善.病理检查发现黄芪治疗组髓鞘受损轻微,组织赦免范围增大.结论黄芪治疗可以缓解脊髓损伤后的脂质过氧化反应,改善微循环,从而发挥脊髓保护作用.     

中药黄芪对脊髓星形胶质细胞单核细胞趋化蛋白-1分泌的影响

目的 :研究黄芪对体外培养的脊髓星形胶质细胞趋化蛋白 1(MCP 1)分泌的影响。方法 :自Wistar大鼠脊髓组织分离、纯化星形胶质细胞 ,体外培养 ,分别予以TNF α及TNF α 黄芪处理 ,ELISA方法检测培养上清液中MCP 1的表达。结果 :TNF α可以显著刺激星形胶质细胞合成、释放MCP 1,而黄芪可下调其刺激作用。结论 :受炎症因子刺激活化的星形胶质细胞可能是损伤脊髓局部MCP 1的来源之一 ,黄芪可以减少脊髓损伤后内源性MCP 1的产生 ,从而缓解继发性脊髓损伤 ,发挥脊髓保护作用。     

黄芪延缓衰老作用的研究

作者进行了黄芪的小白鼠试验及果蝇试验，并临床观察健康人３９例心，脑血管疾病患者９８例，均有明显的延缓衰老作用。