ABSTRACT
ObjectiveTo study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells. MethodNon-small cell lung cancer NCI-H460 cells were divided into a blank group and S. tuberosa alkaloids groups （50， 100， 150， 200， and 250 mg·L-1）. The effect of S. tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide （MTT） assay and colony formation assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Real-time fluorescence-based polymerase chain reaction （Real-time PCR） was used to detect the effect of S. tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and epidermal growth factor receptor （EGFR）. The protein expression levels of Caspase-3， Bax， Bcl-2， protein kinase B （Akt）， phosphorylated （p-）Akt， EGFR， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （p38 MAPK）， and p-p38 MAPK were measured by Western blot. ResultCompared with the blank group， the S. tuberosa alkaloids groups showed increased inhibition rate on cell proliferation （P<0.01）， reduced number of cell clones formed and the rate of cell clonal formation （P<0.05， P<0.01）， and increased karyopyknosis， cytoplasmic aggregation， and cell apoptosis rate （P<0.01）. The S. tuberosa alkaloids groups at 100， 150， 200， and 250 mg·L-1 showed increased Caspase-3 mRNA expression （P<0.05）， decreased EGFR mRNA expression （P<0.05， P<0.01）， up-regulated protein expression of Caspase-3 and p-JNK （P<0.01）， and down-regulated protein expression of EGFR and p-Akt （P<0.05， P<0.01）. Additionally， compared with the blank group， the S. tuberosa alkaloids groups showed increased expression of Bax mRNA （P<0.01）， decreased expression of Bcl-2 mRNA （P<0.01）， up-regulated protein expression of Bax and p-p38 MAPK （P<0.01）， and down-regulated protein expression of Bcl-2 （P<0.01）. ConclusionsS. tuberosa alkaloids can inhibit proliferation and induce apoptosis of human non-small cell lung cancer NCI-H460 cells， and the mechanism may be related to the inhibition of EGFR protein expression and phosphorylation of Akt protein， as well as the activation of the JNK/p38 MAPK signaling pathway.
ABSTRACT
ObjectiveTo investigate the effect of Stemona tuberosa alkaloids （STA） on apoptosis and phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase （JNK/p38 MAPK） signaling pathways in human lung cancer A549 cells. MethodA549 cells were classified into blank group and STA groups （100， 150， 200， 250， 300 mg⋅L-1）. Thiazole blue （MTT） assay and colony formation assay were used to evaluate the proliferation of A549 cells. Apoptosis was observed based on Hoechst 33258 staining， flow cytometry， and Annexin V-FITC/PI staining. Western blot was employed to detect the expression of apoptosis-related proteins cysteine-aspartic acid protease-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， and Bcl-2， and the expression of PI3K， phosphorylated （p）-PI3K， Akt， p-Akt， JNK， p-JNK， p38 MAPK， and p-p38 MAPK. ResultCompared with the blank group， STA groups （150， 200， 250， 300 mg⋅L-1） demonstrated the increase in inhibition rate of cell proliferation （P<0.01） and cell clone inhibition rate， and decrease in cell clone formation rate （P<0.01）. In comparison with the blank group， STA groups showed typical characteristics of apoptosis， such as chromatin condensation and enhanced fluorescence reaction. The apoptosis rate of STA groups was significantly higher than that of the blank group （P<0.01）. Compared with the blank group， STA （150， 200， 250， 300 mg⋅L-1） significantly up-regulated the protein expression of Caspase-3 and Bax （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 protein （P<0.01）. Compared with the blank group， STA had no significant influence on the total protein expression of PI3K， Akt， JNK， and p38 MAPK. However， STA （150， 200， 250， 300 mg⋅L-1） significantly decreased the levels of p-PI3K and p-Akt （P<0.05， P<0.01） and increased the level of p-p38 MAPK （P<0.05， P<0.01）. Compared with the blank group， STA （200， 250， 300 mg⋅L-1） significantly raised the level of p-JNK （P<0.05， P<0.01）. ConclusionSTA can inhibit the proliferation and induce the apoptosis of A549 cells by inhibiting PI3K/Akt signaling pathway and activating JNK/p38 MAPK signaling pathway.
ABSTRACT
OBJECTIVE To establish the fingerprint of Zhuang medicine Jinmu granules and carry out chemometric analysis ， and determine the contents of three components . METHODS High performance liquid chromatography （HPLC） method was adopted. Using rutin as the reference ，HPLC fingerprints of 10 batches of Jinmu granules were drawn and similarity evaluation was performed by Similarity Evaluation of Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）；the common peak was determined by comparing with mixed control ；SPSS 21.0 software was used for cluster analysis ，and SIMCA 14.1 software was used for principal component analysis and orthogonal partial least squares-discriminant analysis. The differential components affecting the quality of Jinmu granules were screened by taking the variable importance in projection （VIP）value ＞1 as the standard ；the HPLC method was used to determine the contents of astilbin ，polydatin and berberine hydrochloride in Jinmu granules. RESULTS There were 22 common peaks in 10 batches of Jinmu granules ，and the similarities were 0.962-0.997；five common peaks were identified ，namely gallic acid （peak 2），polydatin（peak 9），rutin（peak 11），astilbin（peak 13）and kaempferol （peak 20）. The results of cluster analysis showed that 10 batches of Jinmu granules could be clustered into 3 categories：S1 and S 3-S4 were grouped into one category ；S5-S6 and S 9 were grouped into one category ；S2，S7-S8 and S 10 were grouped into one category. The results of principal component analysis showed that the parameter of model interpretation was 0.951 and that of prediction ability was 0.723. The classification results were basically consistent with cluster analysis. The classifica tion results of orthogonal partial least squares- com discriminant analysis were also ba sically consistent with clus- ter analysis. The common peaks with VIP value ＞1 in the order were peak 7＞peak 11（rutin）＞peak 17＞peak 13（astilbin）＞ peak 3＞peak 8＞peak 6＞peak 16 respectively. The linear ranges of astilbin ，polydatin and berberine hydrochloride were 0.012 6- 1.225 0，0.010 8-1.052 5 and 0.020 0-1.562 5 mg/mL，respectively（all R 2＝0.999 9）. RSDs of precision ，stability（24 h）and repeatability tests were all less than 3％. The average recoveries were 99.48％（RSD＝2.67％，n＝9），98.57％（RSD＝1.77％，n＝ 9）and 100.84％（RSD＝2.49％，n＝9）. The contents were 1.221 0-7.011 6，2.251 1-4.462 9，1.252 4-3.328 7 mg/g，respectively. CONCLUSIONS Established fingerprint and the method of content determination are accurate ，stable and simple. Combined with chemometric analysis ，it can be used for the quality control and evaluation of Zhuang medicine Jinmu granules.
ABSTRACT
OBJECTIVE： To establish HPLC fingerprint of Zhuang and Yao medicine Lespedeza formosa， and to provide reference for quality control of L. formosa from different producing areas. METHODS： Totally 10 batches of samples were collected from 5 producing areas as Guangxi Nanning， Guilin， Wuzhou and so on. HPLC fingerprints was established and similarity analysis was carried out by using “Similarity evaluation system of TCM chromatographic fingerprint” （2012 edition） software. The determination was performed on Inertsil ODS-3 column with mobile phase consisted of acetonitrile-0.2％ phosphoric acid solution （gradient elution） at the flow rate of 0.8 mL/min. The detection wavelength was set at 350 nm， and the column temperature was 35 ℃. The sample size was 10 μL. Common peaks were identified by comparing substance control. Cluster analysis and principal compoent analysis were performed by using IBM SPSS 21.0 statistical software. RESULTS： HPLC fingerprint was established， and 12 common peaks were calibrated. 3 common peaks were identified （common peak 8， 10， 11 were chafotalin， vitexin and isovitexin）. The similarity of 10 batches of samples were all higher than 0.9. Through cluster analysis， 10 batches of medicinal materials could be clutered into 2 groups. According to the principal component analysis， the cumulative variance contribution rate of the two principal component factors was 86.108％ （contribution rates of first principal components and second principal components were 66.891％ and 19.217％）. CONCLUSIONS： HPLC fingerprint of L. formosa is established successfully. The method is simple and easy to use， provides a reliable evalution method for quality control of L. formosa.
ABSTRACT
OBJECTIVE:To compare genetic diversity of Spatolobi caulis from different areas of Guangxi by random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR). METHODS:Through using POPGENE 32 software,Ntsys software and SPSS 17.0 software,RAPD and ISSR methods were used to study genetic diversity of 9 samples of S. caulis from dif-ferent areas of Guangxi. RESULTS:After amplification of screened 3 RAPD primers and 4 ISSR primers,and there were 198 and 315 locus,and 37 and 80 polymorphism locus. Rates of polymorphism locus were 18.7% and 25.4%;the number of effective al-leles were 1.416 8 and 1.584 0;genetic diversity index were 0.269 4 and 0.351 3;Shannon diversity index were 0.431 6 and 0.529 9. All the values of ISSR marker were higher than RAPD marker. The average genetic similarity coefficient of ISSR and RAPD were 0.757 64 and 0.683 80,indicating ISSR was more sensitive for the detection of genetic diversity. The clustering result of them was close to each other. The correlation coefficient of them were 0.847,indicating very significant positive correlation at the level of 0.001. CONCLUSIONS:ISSR could reflect more information of genetic diversity than RAPD,and is more suitable for research of genetic diversity of S. caulis from different areas of Guangxi.
ABSTRACT
@#Objective To study the effect of Du meridian electroacupuncture on growth associated protein-43 (GAP-43) expression in rats after spinal cord injury (SCI). Methods The SCI models were established with MASCIS Impactor at T11 segment in Sprague-Dawley rats. They were equally divided into control group (group A, n=18) and Du meridian electroacupuncture group (group B, n=18). Group B received electroacupuncture once a day since 1 week after SCI. They were evaluated with the Basso-Beattie-Bresnahan (BBB) score 1, 2, 4, 8 weeks after SCI. The mRNA and protein of GAP-43 was detected with RT-PCR and immunohistochemistry 2, 4, 8 weeks after SCI. Results Compared with group A, the BBB score, the expression of GAP-43 mRNA and protein increased in group B after SCI (P<0.01). Conclusion Du meridian electroacupuncture can promote the expression of GAP-43 after spinal cord injury.
ABSTRACT
AIM: To evaluate the role of human angiotensin Ⅱ(AngⅡ) type Ⅰ receptor (AT1R) antisense cDNA (ahAT1) on migration of cultured artery smooth muscle cells (VSMCs). METHODS: Two recombinant adenoviral vectors, Ad/CMV. ahAT1 containing full length antisense cDNA targeting to human AT1R mRNA, and Ad/CMV.LacZ containing LacZ called report gene, were constructed by orientation clone technology and homologous recombination, and then were used to transfect VSMCs in vitro. AT1R expression detected by RT-PCR and immunohistochemistry, and migration of VSMCs measured by Boyden's Chamer methods, were compared between transfected and non- transfected VSMCs. RESULTS: Forty-eight hours after Ad/CMV. ahAT1 transfection, the level of AT1R mRNA decreased markedly (50% of control group), and AT1R protein expression was significantly less (P