ABSTRACT
ObjectiveTo investigate the effects of Yanghetang （YHT） on breast cancer 4T1 cells and their mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodThe YHT-containing serum was prepared from SD rats. The rats were randomly assigned into a blank group （normal saline） and low-， medium-， and high-dose （5.8， 11.6， 23.2 g·kg-1， respectively） YHT groups. The serum containing 10% YHT in each group was mixed with 90% RMPI 1640 complete medium， and the mixture was used to interfere with the cells. Cell counting kit-8 （CCK-8） method was used to detect the proliferation of the 4T1 cells treated with YHT for 24， 48， 72 h. The apoptosis， migration， and invasion of 4T1 cells were detected by flow cytometry， scratch test， and Transwell assay， respectively. Western blot was employed to determine the expression levels of MEK1/2， phosphorylation （p）-MEK1/2， ERK1/2， p-ERK1/2， and rat sarcoma virus （RAS） protein. ResultCompared with the blank group， the intervention with YHT-containing serum for 24， 48， and 72 h had significant inhibitory effect on 4T1 cell proliferation （P<0.05， P<0.01）. After intervention with YHT-containing serum for 48 h， the apoptosis rate of cells increased （P<0.01）. Compared with the blank group， the intervention with YHT for 24 h and 48 h decreased the healing ability of cells in the scratch test （P<0.01）. The invasive ability of cells treated with the low， medium， and high-dose YHT containing serum showed a decreasing trend （P<0.01）. Compared with the blank group， YHT-containing serum did not change the expression of MEK1/2 and ERK1/2 while down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein （P<0.01）. ConclusionYHT can inhibit the proliferation， migration， and invasion and promote the apoptosis of breast cancer 4T1 cells. In may promote the apoptosis by inhibiting the MEK/ERK signaling pathway and down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein.
ABSTRACT
ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.
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Objective::To investigate the effect of ancient recipe Yanghetang and its drug-contained serum on apoptosis of triple negative breast cancer cell line MDA-MB-231 based on the signal pathway of early growth response gene 1 (Egr1)/cyclin dependent inhibitor (p21). Method::The drug-containing serum of Yanghetang was prepared from rats, and MDA-MB-231 cells were cultured in vitro. The blank control group was set, and MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were intervened for 24, 48, 72 h, respectively. After that, the cell counting kit-8(CCK-8) method was used to detect the cell proliferation of each group. The blank control group was set, while MDA-MB-231 cells in Yanghetang high, middle, and low dose groups (23.4, 11.7, 5.85 g·kg-1) were treated for 48 h, and then flow cytometry was used to detect the apoptosis of each group and the distribution of cell cycle. The expression of Egr1 and p21 mRNA in each group was detected by quantificational Real-time polymerase chain reaction (Real-time PCR), while the expression of Egr1 and p21 protein in each group was detected by Western blot. Result::After MDA-MB-231 cells were intervened by Yanghetang for 24, 48, 72 h, MDA-MB-231 cell proliferation was significantly inhibited in Yanghetang high and middle dose groups as compared with the blank control group (P<0.01). After MDA-MB-231 cells were intervened by Yanghetang for 48 h, the apoptosis was significantly increased in Yanghetang high and middle dose groups as compared with the blank control group (P<0.05, P<0.01). In the Yanghetang high, middle dose groups, the proportion of cell cycle G0/G1 phase decreased, and the proportion of G2/M phase increased (P<0.05, P<0.01). The mRNA and protein expressions of Egr1, p21 were increased in Yanghetang high and middle groups (P<0.05, P<0.01). Conclusion::Yanghetang can activate Egr1/p21 signaling pathway in MDA-MB-231 cells, increase the expression of Egr1 and p21, and cause G2/M cell cycle arrest, thereby inducing apoptosis of MDA-MB-231 cells.
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Objective::To investigate the effect of serum containing Yanghetang (YHT) on the apoptosis of MCF-7 cells in breast cancer based on the mitogen-activated protein kinase (p38)/signal transduction and transcriptional activator 3 (STAT3) signal pathway. Method::YHT liquid with crude drug 1 g·mL-1 was prepared. Female SD rats were randomly divided into control group (distilled water), and high, medium and low-dose YHT groups (24, 12, 6 g·kg-1). YHT-medicated serum was prepared, and 10%medicated serum was used to intervene MCF-7 cells. Cell proliferation and cytotoxicity assay (CCK-8) was used to detect the effect of serum containing YHT on MCF-7 cell proliferation, apoptosis of MCF-7 cells was detected by flow cytometry protein expressions of p38 and STAT3 were detected by Western blot, Quantitative Real-time PCR (Real-time PCR) was used to detect the expressions of B-cell lymphoma/leukemia-xl(Bcl-xl) and Survivin mRNA. Result::CCK-8 assay showed that YHT serum inhibited the proliferation of MCF-7 cells in a time and dose-dependent manner compared with the blank group. The inhibitory effect was most obvious in the high-dose group, with the inhibition rates of 38%, 45%and 54%at different time points (P<0.01). Flow cytometry showed that, compared with the blank group, the apoptosis rate in the medium and high-dose groups increased significantly in a dose-dependent manner, with the apoptosis rates at 11.6%and 16.5%respectively (P<0.05, P<0.01). Western blot analysis showed that, compared with the blank group, the expressions of p38 and STAT3 protein was decreased in high, medium-dose YHT groups (P<0.01) in a dose-dependent manner. Compared with the blank group, the expressions of Bcl-xl and Survivin mRNA were decreased in high, medium-dose YHT groups (P<0.05, P<0.01) in a dose-dependent manner. Conclusion::YHT serum can promote the apoptosis of MCF-7 cells in breast cancer, which may be related to the p38/ STAT3 signaling pathway.
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Objective:To explore the mechanism of mitochondrial apoptotic pathway in rat degenerative intervertebral disc cells in improving intervertebral disc degeneration under the action of Bushen Zhuangdu recipe. Method:The 100 SD male rats were randomly divided into blank group, model group, low, medium and high dose Bushen Zhuangdu recipe group (0.38,0.77,1.53 g·kg-1).Histopathological changes of rat intervertebral discs were observed by hematoxylin-eosin(HE) staining after 4 weeks of continuous administration of Chinese medicine. The apoptotic rate of nucleus pulposus cells in degenerative intervertebral discs was detected by TUNEL(terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), and the levels of active Cysteinyl aspartate-specific proteinase-3(active Caspase-3), B cell leukemia-2(Bcl-2), cytochrome C (cytC) and Bcl-2-associated X protein(Bax) protein in intervertebral discs were detected by Western blot. Result:Compared with blank group, the histopathological score of intervertebral disc in the model group was significantly increased, the apoptosis rate of nucleus pulposus was significantly increased (PPPPPPConclusion:Bushen Zhuangdu recipe may improve the degeneration of intervertebral disc by reducing the expression of active Caspase-3, cytC and Bax, increasing the expression of Bcl-2 and inhibiting the apoptotic pathway of mitochondria in a dose-dependent manner.