[Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7].

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

Shenlian extract attenuates myocardial ischaemia-reperfusion injury via inhibiting M1 macrophage polarization by silencing miR-155.

CONTEXT: Shenlian extract (SL) is a combination of Salvia miltiorrhiza Bge. (Labiatae) and Andrographis paniculata (Burm. F.) Wall. Ex Nees (Acanthaceae) extracts, which promote blood circulation and clear endogenous heat toxins. Myocardial ischaemia-reperfusion injury (MI/RI) is aggravated myocardial tissue damage induced by reperfusion therapy after myocardial infarction. OBJECTIVES: This study explores the effect of SL on MI/RI and the underlying mechanism. MATERIALS AND METHODS: Primary peritoneal macrophages (pMACs) were treated with LPS and SL (5, 10 or 20 µg/mL) for 24 h. The myocardial ischaemia-reperfusion (MI/R) model was established after administration of different doses of SL (90, 180 or 360 mg/kg). Myocardial tissue injury was assessed by methylthiazolyl tetrazolium (TTC) staining and levels of creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in mice. The double immunofluorescence staining of iNOS/F4/80 and CD86/F4/80 was used to detect macrophage M1 polarization. The levels of miR-155, inflammatory factors and chemokines were detected by qRT-PCR or ELISA. CD86, iNOS, SOCS3, JAK2, p-JAK2, STAT3 and p-STAT3 proteins expressions in macrophages were analyzed by western blotting. Conditioned medium transfer systems were designed to unite M1 macrophages with H/R cardiomyocytes, and cell apoptosis was detected by TUNEL staining, western blotting or immunohistochemistry. RESULTS: SL reduced apoptosis, diminished CK and LDH levels, raised SOD concentration and decreased infarct size in the MI/R model. Meanwhile, SL decreased miR-155 level, inhibited M1 macrophage polarization and inflammation. Furthermore, SL promoted SOCS3 expression and blocked JAK2/STAT3 pathway in vitro. CONCLUSIONS: SL may be a promising TCM candidate for MI/RI. The underlying mechanisms could be associated with inhibition of M1 macrophage polarization via down-regulating miR-155.

Network pharmacology analysis and experimental validation to explore the mechanism of Shenlian extract on myocardial ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian extract (SL), extracted from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm. f.) Nees, has been proved to be effective in the prevention and treatment of atherosclerosis. Recently, we have partially elucidated the mechanisms involved in the therapeutic effects of SL on myocardial ischemia (MI). However, the underlying mechanisms remain largely unclear. AIM OF THE STUDY: This study aims to explore the potential molecular mechanism of SL on MI on the basis of network pharmacology. MATERIALS AND METHODS: First, the main active ingredients of SL were screened in the Traditional Chinese Medicine Integrated Database, and the MI-associated targets were collected from the DisGeNET database. Then, we used compound-target and target-pathway networks to uncover the therapeutic mechanisms of SL. On the basis of network pharmacology analysis results, we assessed the effects of SL in MI rat model and oxygen glucose deprivation model of H9c2 cells and validated the possible molecular mechanisms of SL on myocardial injury in vivo and in vitro. RESULTS: The network pharmacology results showed that 37 potential targets were recognized, including TNF-α, Bcl-2, STAT3, PI3K and MMP2. These results revealed that the possible targets of SL were involved in the regulation of inflammation and apoptosis signaling pathway. Then, in vivo experiments indicated that SL significantly reduced the myocardial infarction size of MI rats. Serum CK-MB, cTnT, CK, LDH, and AST levels were significantly decreased by SL (P < 0.05 or P < 0.01). In vitro, SL significantly increased H9c2 cell viability. The levels of inflammation factors including TNF-α and MMP2 were significantly decreased by SL (P < 0.05 or P < 0.01). TUNEL and Annexin V/propidium iodide assays indicated that SL could significantly decrease the cell apoptotic rate in vivo and in vitro (P < 0.05 or P < 0.01). The remarkable upregulation of anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax protein level further confirmed this result. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the PI3K-AKT and JAK2-STAT3 pathways were significantly enriched in SL. Compared with the model group, SL treatment significantly activated the PI3K-AKT and JAK2-STAT3 pathways in vivo and in vitro according to Western blot analyses. CONCLUSION: SL could protect the myocardium from MI injury. The underlying mechanism may be related to the reduction of inflammation and apoptosis by activating the PI3K/AKT and JAK2/STAT3 pathways.

Muyin extract inhibits non-small-cell lung cancer growth by inducing autophagy and apoptosis in vitro and in vivo.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer with a higher mortality rate. Both apoptosis and autophagy are crucial processes in the pathophysiology of NSCLC. Muyin extract (MSE) is a combination of Momordica cochinchinensis (Lour.) Spreng seeds and Epimedium brevicornu Maxim extract, with an optimal ratio of 1:1. Our previous research has firstly shown that MSE exerts a good anti-tumor activity, especially for NSCLC. PURPOSE: This study aims to evaluate the inhibitory effect of MSE on NSCLC and explore the underlying mechanism. METHODS: In vitro, cell proliferation was examined by MTT and colony formation. Apoptosis was detected by annexin V-FITC/PI assay while autophagy was assessed by Acridine orange (AO) and Monodansylcadaverine (MDC) staining. In vivo, Lewis lung cancer cell transplanted mice model was established to measure the effect of MSE on tumor growth. Hematoxylin eosin (H & E) staining was used to observe the pathological changes of the tumor after MSE treatment. The apoptosis in tumor tissue was detected by TUNEL assay. Meanwhile, the cellular proliferation marker Ki67 and autophagy marker LC3Ⅱ were observed by immunohistochemistry staining. The IL-4 and IFN-γ concentrations in blood were tested by Elisa. The apoptosis related factors (Bcl-2, Bax Caspase-3, cleaved Caspase-3, Caspase-9 and p53), autophagy marker proteins (Atg-5, Becline-1, LC3Ⅱ/Ⅰand p62) as well as Akt/mTOR pathway were detected by western blotting. RESULTS: Present study showed that MSE greatly inhibited the proliferation of NSCLC in vitro and in vivo, together with apoptotic rate increasing. P53 and cleaved Caspase-3 levels were up-regulated while Bcl-2/Bax ratio, Caspase-3 and Caspase-9 levels were significantly down-regulated treated with MSE. Meanwhile, MSE activated autophagy, Atg-5, Becline-1 as well as the ratio of LC3Ⅱ/Ⅰ were notably up-regulated while p62 was down-regulated after MSE treatment. Importantly, MSE significantly blocked Akt/mTOR pathway, which is a common upstream signal triggered by autophagy and apoptosis. Furthermore, when co-treated with specific autophagy inhibitor, the inhibitory rate and anti-apoptotic Bcl-2 level were significantly reversed. Impressively, MSE remarkably increased IFN-γ/ IL-4 ratio while VP16 did not in animal model, and the inhibition rate in tumor weight after MSE treatment was higher than xiaojin pill. CONCLUSION: Taken together, it is proved that MSE may be a promising oral TCM candidate for NSCLC therapy with immunity improvement. The underlying mechanisms could be associated with the induction of apoptosis and autophagy through blocking Akt/mTOR pathway, meanwhile, it may promote crosstalk between autophagy and apoptosis.

Identification of ZEB2 as an Immune-Associated Gene in Endometrial Carcinoma and Associated with Macrophage Infiltration by Bioinformatic Analysis.

Endometrial cancer (EC) is the most common gynecological tumor arising from the endometrium. In this study, we use a published single-cell transcriptome profile of endometrial carcinoma (EC) to reveal the composition of immune cells and found an immunosuppressive environment since the presence of macrophage subtype M2 and exhausted CD8+ T cell markers. We focused on ZEB2 (Zinc finger E-box binding homeobox 2), a well-known player in epithelial to mesenchymal transition process, and we showed that ZEB2 is exclusively expressed in immune cells in single-cell transcriptome and, at the same time, downregulated in TCGA-UCEC (The Cancer Genome Atlas-Uterine Corpus Endometrial Carcinoma) bulk RNA-seq data and negatively associated with tumor purity. Loss of ZEB2 protein in EC in normal endometrium and EC samples was validated in samples using immunohistochemistry (IHC) from HPA (Human Protein Atlas) database. Furthermore, we found ZEB2 was associated with immune infiltrations especially for macrophage using TIMER 2.0. Interestingly, ZEB2 prognostic significance differed under various macrophage and Th2 helper cell content using Kaplan-Meier Plotter analysis. More importantly, we showed that over 11% EC patients have somatic mutations of ZEB2 in EC samples collected from cBioportal and they have a lower body weight, earlier diagnosis age, and better overall survival and disease-free survival status compared with the unaltered group. Analysis in TIMER2.0 suggested that ZEB2 mutation would possibly change the composition of tumor-infiltrating lymphocytes. Taken together, by combining the results from single-cell data, bulk TCGA RNA-seq, and other online bioinformatic tools, we provided evidence that ZEB2 might have a unique role in the immune environment of EC. These results would provide a better insight into the pathogenetic process and ZEB2 might further be used an immunotherapeutic target of EC.

[Shenlian extract attenuates TNF-α-induced ECV304 injury by regulating Nrf2/Keap1 signaling pathway].

This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1ß( IL-1ß) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 µg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( P<0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1ß( P<0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( P<0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1ß content( P<0. 01 or P<0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( P<0. 01 or P<0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.