ABSTRACT
ObjectiveTo explore the therapeutic effect and possible mechanism of Banxia Xiexintang and its disassembled prescriptions in regulating the flora disorder induced by mixed antibiotics in young rats. MethodSeventy male BALB/C young rats were randomly assigned into 7 groups： blank group， model group， Bifidobacterium tetralogy viable tablets （0.68 g·kg-1） group， Banxia Xiexintang （9.1 g·kg-1） group， Xinkai （3.19 g·kg-1） group， Kujiang （1.82 g·kg-1） group， and Ganbu （4.1 g·kg-1） group， with 10 rats in each group. Except the blank group， the other groups were given mixed antibiotics by gavage to induce intestinal flora disorder. After 14 days， the rats in different drug groups were administrated with corresponding drugs by gavage， and those in the blank group and model group with the same amount of normal saline once a day for 14 days. After that， fecal samples were collected aseptically for 16S rDNA sequencing of intestinal flora， and lipopolysaccharide （LPS， 10 mg·kg-1） was injected intraperitoneally to induce inflammatory reaction. The tissue morphology of colonic mucosa was observed via hematoxylin-eosin （HE） staining， and the macrophage infiltration of colonic mucosa was observed via toluidine blue staining and immunohistochemistry. The expression of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， interleukin-8 （IL-8）， tumor necrosis factor-α （TNF-α）， and interleukin-10 （IL-10） mRNA were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the modeling changed the intestinal flora structure of the young rats （P<0.01）， damaged the colonic mucosa， reduced the macrophage infiltration， and down-regulated the mRNA levels of IL-1β， IL-6， IL-8， TNF-α， and IL-10 （P<0.01）. Compared with the model group， bifidobacterium quadruple viable tablets， Banxia Xiexintang and its disassembled prescriptions increased the diversity of intestinal flora and the relative abundance of beneficial bacteria such as Bacteroidetes and Firmicutes （P<0.01）. At the same time， they ameliorated colonic mucosal injury （P<0.05， P<0.01）， increased macrophage infiltration （P<0.05， P<0.01）， and up-regulated the mRNA levels of IL-6， IL-8， and TNF-α （P<0.01）. The mRNA level of IL-1β was up-regulated in Bifidobacterium tetralogy viable tablets， Banxia Xiexintang， Kujiang， and Ganbu groups （P<0.01）， and that of IL-10 was up-regulated in Bifidobacterium tetralogy viable tablets， Banxia Xiexintang， Xinkai， and Ganbu groups （P<0.01）. ConclusionBanxia Xiexintang and the disassembled prescriptions can adjust the intestinal flora of young rats exposed to antibiotics and protect the immune barrier of colonic mucosa after intestinal flora disorder. In particularly， the whole prescription of Banxia Xiexintang demonstrates the best performance.
ABSTRACT
Objective:To investigate the effect of Banxia Xiexintang on the epithelial-mesenchymal transition （EMT） of human peritoneal mesothelial cell line （HMrSV5） induced by gastric cancer-derived exosomes （Exo）. Method:Banxia Xiexintang-containing serum was prepared and the human gastric cancer NCI-N87-derived exosomes （NCI-N87-Exo） were extracted， followed by their identification by transmission electron microscopy and Western blotting and labeling with 1，1-dioctadecyl-3，3，3，3- tetramethylindocarbocyanine perchlorate （Dil）. The cells were divided into the blank group， model group， and low-， medium-， and high-dose （13.5，27，54 g·kg<sup>-1</sup>） Banxia Xiexintang groups. HMrSV5 cells in the blank group were cultured alone， the ones in the model group with 100 mg·L<sup>-1</sup> NCI-N87-Exo， and those in the low-， medium-， and high-dose Banxia Xiexintang groups with 100 mg·L<sup>-1</sup> NCI-N87-Exo plus low-， medium-， and high-dose 10% Banxia Xiexintang-containing serum， respectively. Confocal laser microscope was used to observe the uptake of NCI-N87-Exo by HMrSV5 cells at 24 h， 48 h and 72 h. Seventy-two hours later， the morphological changes in HMrSV5 cells were observed. The protein expression levels of E-cadherin， cytokeratin 19 （CK19）， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， elastin， and transforming growth factor-<italic>β</italic><sub>1</sub> （TGF-<italic>β</italic><sub>1</sub>）， Smad2/3， and p-Smad2/3 were assayed by Western blot. Result:It was observed under the transmission electron microscope that NCI-N87-Exo showed an oval or dish-shaped vesicle structure with a particle size ranging from 40 to 80 nm. Exo marker proteins CD9 and CD63 were highly expressed while calreticulin was not expressed， implying that the NCI-N87-Exo was confirmed. After 24 h， 48 h， 72 h of co-culture， it was observed under the fluorescence microscope that NCI-N87-Exo were taken up by HMrSV5 cells， which was positively correlated with time. Compared with the blank group， Banxia Xiexintang significantly inhibited the uptake of NCI-N87-Exo by HMrSV5 cells， with better effect noticed in the middle- and high-dose Banxia Xiexintang groups（<italic>P</italic><0.05，<italic>P</italic><0.01）. After intervention with Banxia Xiexintang-containing serum， the HMrSV5 cells were arranged densely， and the intercellular space was significantly reduced， with the most obvious changes present in the high-dose Banxia Xiexintang group. Western blot revealed that the protein expression levels of E-cadherin and CK19 in HMrSV5 cells after being intervened with the medium- and high-dose Banxia Xiexintang-containing serum were increased significantly as compared with those in the blank group， whereas the levels of <italic>α</italic>-SMA and Elastin were decreased significantly （<italic>P</italic><0.01）. Banxia Xiexintang-containing serum at the low， medium， and high doses remarkably down-regulated TGF-<italic>β</italic><sub>1</sub> and p-Smad2/3 protein expression（<italic>P</italic><0.05，<italic>P</italic><0.01）. However， there was no significant change in Smad2/3. Conclusion:NCI-N87-Exo can be taken up by HMrSV5 cells to induce EMT. Banxia Xiexintang can inhibit the uptake of NCI-N87-Exo by HMrSV5 cells and the resulting EMT induced by NCI-N87-Exo， which is related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway.
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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.