Xiao Xianxiongtang Regulates Ca2+ Load and Inhibits Epithelial-mesenchymal Transition， Invasion， and Migration of MGC-803 Cells： Based on Wnt5a/ Ca2+/NFAT Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of Xiao Xianxiongtang （XXXT） on the transforming growth factor （TGF）-β1-induced invasion， metastasis， and epithelial-mesenchymal transition （EMT） of gastric cancer MGC-803 cells and the underlying mechanism. MethodThe molecular docking between XXXT and nuclear factor of activated T cells （NFAT） was performed by CB-DOCK （http：//clab.labshare.cn/cb-dock/）. The invasion and metastasis model of MGC-803 cells was established with 10 μg·L-1 TGF-β1. MGC-803 cells were classified into blank group， model group， 0.1 g·L-1 XXXT group， 0.2 g·L-1 XXXT group， and 0.4 g·L-1 XXXT group. For further clarifying the key role of Wnt5a/Ca2+/NFAT signaling pathway in the inhibition of XXXT on gastric cancer， MGC-803 cells were transfected with Wnt5a overexpression plasmid， and then the cells were classified into blank plasmid group， Wnt5a-OE group， blank plasmid + XXXT （0.4 g·L-1） group， and Wnt5a-OE + XXXT （0.4 g·L-1） group. Cell viability was determined by cell counting kit-8 （CCK-8） assay， cell invasion and migration ability by Transwell invasion assay and wound healing assay， expression of EMT-related proteins （E-cadherin， N-cadherin， Vimentin， Snail） and Wnt5a/Ca2+/NFAT signaling pathway-related key proteins ［Wnt5a， calcineurin （CaN）， NFAT1， and p-NFAT1］ by Western blot， and changes in intracellular Ca2+ concentration by immunofluorescence assay. ResultMolecular docking suggested that XXXT acted on Wnt5a/Ca2+/NFAT signaling pathway. XXXT （0.1， 0.2， 0.4 g·L-1） significantly promoted the loss of MGC-803 cell viability （P<0.05，P<0.01）. It inhibited cells from invading the transwell lower chamber and slowed down the healing of cell wounds in a dose-dependent manner （P<0.05， P<0.01）. Moreover， it promoted the expression of E-cadherin while suppressed the expression of N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）. Further experiments showed that XXXT could inhibit the expression of Wnt5a， CaN， NFAT1， and p-NFAT1， and reduce the nuclear expression of NFAT1 and the transcription activity mediated by NFAT1， so as to reduce the cellular Ca2+ concentration （P<0.05， P<0.01）. XXXT can reverse the effect of Wnt5a （P<0.05， P<0.01）. ConclusionXXXT can attenuate the invasion， metastasis， and EMT of MGC-803 cells via the Wnt5a/Ca2+/NFAT pathway， thereby weakening the tumor-promoting effect of TGF-β1. In summary， XXXT may exert therapeutic effect on gastric cancer by regulating the invasion， metastasis， and EMT of gastric cancer cells.

Modified Xiao Xianxiongtang Inhibits Epithelial-Mesenchymal Transition of Human Gastric Cancer MGC803 Cells via Wnt/β-catenin Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the inhibitory effect of modified Xiao Xianxiongtang on epithelial-mesenchymal transition （EMT） of human gastric cancer MGC803 cells and its relationship with secretory glycoprotein Wnt/β-catenin pathway. MethodThe BALB/c nude mice were implanted with human gastric cancer MGC803 cell suspension in the heterotopic subcutaneous position for inducing tumor. After successful modeling， they were randomly divided into the model group， low-， medium-， and high-dose （16.0，32.0，and 64.0 g·kg-1） groups of modified Xiao Xianxiongtang， and capecitabine （400 mg·kg-1） group， with eight mice in each group， and gavaged with the corresponding drugs， once per day， for 28 consecutive days. Those in the capecitabine group received one-week discontinuation after every two weeks of treatment. The general state and body weight of the nude mice were observed， and the transplanted tumor volume was measured. After being killed， they were weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was carried out for observing the pathological changes in transplanted tumor tissues. The gene and protein expression levels of Wnt1 and β-catenin were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， followed by the determination of matrix metalloproteinase-9 （MMP-9）， vascular endothelial growth factor （VEGF）， N-cadherin， E-cadherin， Vimentin， and Snail protein expression by Western blot. The expression levels of cyclooxygenase 2 （COX2） and prostaglandin E2 （PGE2） were detected by enzyme-linked immunosorbent assay （ELISA）. ResultIt was found that the transplanted tumor in each group showed different growth trends with time， with the most obvious growth observed in the model group. Compared with the model group， the low-， medium-， and high-dose modified Xiao Xianxiongtang groups exhibited reduced tumor volume and slowed growth to varying degrees over time. After medication for days 7，14，21，and 28， the tumor volumes in the low- and high-dose modified Xiao Xianxiongtang groups and capecitabine group declined （P<0.05， P<0.01）， and that in the medium-dose Xiao Xianxiongtang group was also remarkably reduced after medication for days 14，21，and 28 （P<0.01）. Compared with the model group， the high-dose modified Xiao Xianxiongtang group and capecitabine group showed a significant reduction in the relative tumor volume after treatment for days 7，14，21，28 （P<0.01）， and the low- and medium-dose modified Xiao Xianxiongtang groups also presented with decreased relative tumor volume after treatment for days 14，21，28 （P<0.05， P<0.01）. Compared with the model group， the modified Xiao Xianxiongtang at low， medium， and high doses and capecitabine all increased the tumor inhibition rate to varying degrees （P<0.01）， down-regulated the mRNA and protein expression levels of Wnt1 and β-catenin in tumor tissue （P<0.05， P<0.01） and protein expression levels of MMP-9， VEGF， N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）， up-regulated E-cadherin protein expression （P<0.05， P<0.01）， and reduced COX2 and PGE2 contents （P<0.05， P<0.01）. ConclusionModified Xiao Xianxiongtang inhibits the EMT of human gastric cancer MGC803 cell-transplanted tumor， which may be related to Wnt/β-catenin pathway.

Extract from Modified Xiao Xianxiongtang Inhibits Epithelial-mesenchymal Transition and Invasion and Migration Mediated by TGF-β1 of Human Gastric Cancer MGC-803 Cells via Wnt5a/Ca2+/NFAT Signaling Pathway / 中国实验方剂学杂志

Objective:This studu aims to investigate the effect of aqueous extract of modified Xiao Xianxiongtang on the epithelial mesenchymal transition（EMT） and the change of its invasion and migration ability of human gastric cancer MGC-803 cells mediated by transforming growth factor-<italic>β</italic>₁（TGF-<italic>β</italic>₁），and to explore the mechanism of regulating Wnt5a/Ca²⁺/ activated T-cell nuclear factor（NFAT） signaling pathway to inhibit EMT and invasion and metastasis of MGC-803 cells. Method:TGF-<italic>β</italic>₁（10 μg·L^-1）was used to induce EMT and the invasion and metastasis model of human gastric cancer MGC-803 cells. Transwell chamber experiment， scratchhealing experiment， Western blot and immunofluorescence assay were used to detect cell invasion and migration ability， expression of EMT marker protein and key protein expression of Wnt5a/Ca²⁺/NFAT signaling pathway， and intracellular Ca²⁺ concentration. Result:Compared with the blank group， TGF-<italic>β</italic>₁ could significantly enhance the invasion and migration ability of MGC-803 cells（<italic>P</italic><0.01）， down-regulate the level of E-cadherin（<italic>P</italic><0.01）， up-regulate protein expressions of N-cadherin， Snail and Vimentin（<italic>P</italic><0.01）， and induce cell Wnt5a， calcineurin （CaN）， total protein of activated T-cell nuclear factor 1（NFAT1）， up-regulation of phosphorylated proteins p-NFAT1 and NFAT1 nucleoprotein and intracellular accumulation of Ca²⁺（<italic>P</italic><0.01）. Compared with the TGF-<italic>β</italic>₁ group， modified Xiao Xianxiongtang （10， 20， 40 mg·L^-1） could significantly inhibit this phenomenon，and 40 mg·L^-1 had the best effect（<italic>P</italic><0.05，<italic>P</italic><0.01）.The specific inhibitors of Wnt5a/Ca²⁺/NFAT signaling pathway （<italic>R</italic>）-（+）-Bay-K-8644 and modified Xiao Xianxiongtang （40 mg·L^-1） could significantly inhibit theinvasion and migration of MGC-803 cells mediated by TGF-<italic>β</italic>₁， up-regulate the level of E-cadherin， and down-regulate expressions of N-cadherin， Snail， Vimentin， Wnt5a， CaN and NFAT1 proteins and reduce the intracellular accumulation of Ca²⁺（<italic>P</italic><0.05，<italic>P</italic><0.01）.Moreover， （<italic>R</italic>）-（+）-Bay-K-8644 combined with modified Xiao Xianxiongtang （40 mg·L^-1） had stronger inhibitory effect（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:These results suggest that modified Xiao Xianxiongtang can inhibit the EMT mediated by TGF-<italic>β</italic>₁ via Wnt5a/Ca²⁺/NFAT signaling pathway，thereby reducing the invasion and migration ability of MGC-803 cells.

Pharmacological Mechanism of Xiao Xianxiongtang in Treatment of Type 2 Diabetes Mellitus Based on Network Pharmacology / 中国实验方剂学杂志

Objective::To explore the pharmacological mechanism of Xiao Xianxiongtang in treating type 2 diabetes mellitus (T2DM) by network pharmacology. Method::The main active ingredients, corresponding targets and target genes of Xiao Xianxiongtang were searched on Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) website. Relevant target genes of T2DM were obtained through Gene Cards. The targets of drug active ingredients were mapped to the targets of T2DM, and the intersection targets were obtained as the predictive targets of Xiao Xianxiongtang on T2DM. Cytoscape 3.7.1 software was used to construct the drug active ingredient-intersection target network model and select the key active ingredients. Interactive protein-protein interaction network (PPI) was constructed by STRING website, and key target genes were selected. Gene function analysis (GO) and enrichment analysis based on the Kyoto encyclopedia of genes and genomes (KEGG) pathway were performed on the intersecting targets using DAVID6.8 online tool. Result::Xiao Xianxiongtang had 30 active ingredients, 156 relevant targets, 14 key active ingredients and 18 key target genes on T2DM. GO analysis showed that the biological functions of Xiao Xianxiongtang in the treatment of potential genes of T2DM mainly involved transcriptional regulation, oxidative stress, protein binding and inflammatory reaction. KEGG pathway enrichment showed that the main pathways of Xiao Xianxiongtang in the treatment of T2DM were hypoxia inducible factor-1 (HIF-1) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway and thyroid hormone signaling pathway, phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway, hepatitis B, hepatitis C, tyrosine kinase receptor2(ErbB) signaling pathway, calcium signaling pathway and nuclear factor-kappa B (NF-κB) signaling pathway. Conclusion: Xiao Xianxiongtang is a multi-component, multi-target and multi-pathway process in the treatment of T2DM. It plays an important role in the treatment of T2DM by regulating transcription, oxidative stress, protein binding and inflammatory reaction. Conclusion::The mechanism of Xiao Xianxiongtang in treating T2DM may alleviate insulin resistance, increase insulin sensitivity and reduce blood sugar by inhibiting the secretion of inflammatory factors, participating in anti-inflammatory response, reducing oxidative stress, increasing intracellular calcium concentration, blocking glucagon signaling pathway and activating PI3K/Akt pathway.

Network Pharmacology-based Mechanism of Xiao Xianxiongtang in Treatment of Coronary Heart Disease / 中国实验方剂学杂志

Objective::To predict Xiao Xianxiongtang's treatment of coronary heart disease (CHD) targets and analyze their function by the network pharmacology method, and build ingredients-targets-channel network pharmacological model, in order to reveal potential pathways and mechanisms of Xiao Xianxiongtang for CHD treatment. Method::Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to obtain components, and CHD targets over Xiao Xianxiongtang were predicted by using Swiss Target Prediction reverse pharmacophore matching method. CHD targets which Food and Drug Administration (FDA) approved were collected from Therapeutic Target Database (TTD), Drugbank and Disease-gene Net databases (DisGeNET). Wenn diagram was used to obtain the correlation intersection.Target characteristics were analyzed with GEO2R online, Reactome FI was used to analyze the enrichment of target pathways, and Cytoscape software was used to construct the " component-target-pathway" network. Result::Network analysis showed that Xiao Xianxiongtang treated CHD by regulating 24 target proteins through 25 therapeutic components, and acting on 21 specific pathways and 4 biological processes.According to the multiple gene chip analysis of GEO2R online, there were up-down-regulated differences in the targets, including 11 up targets and 13 down targets. Conclusion::Xiao Xianxiongtang treats CHD by involving the biological processes through berberine and flavonoid groups of Coptidis Rhizoma, nucleosides and organic acids of Arum ternatum Thunb, stigmasterols and flavonoids of Trichosanthes kirilowii Maxim, such as gene expression, metabolism and protein metabolism, adjusting the gene expressions of relevant target proteins, regulating gene transcription pathways, such as biological oxidation reaction and lipid and lipoprotein metabolism, insulin-like growth factor binding protein (IGFBPs) of insulin-like growth factor (IGF) transshipment and intake, and the degradation of extracellular matrix signaling pathways.

Research Progress on Antitumor Active Components and Mechanism of Xiao Xianxiongtang / 中国实验方剂学杂志

Tumor is one of the diseases that seriously endanger human health， and how to treat tumor effectively is still one of the important problems in the field of medicine. At present， most of the radiotherapies and chemical drugs for cancer have serious side effect despite of an obvious efficacy. With a unique syndrome differentiation treatment system and overall concept， traditional Chinese medicine has become the key research and development object of antitumor drugs due to many advantages， such as multiple channels， multiple levels， multiple links， multiple targets and less toxicity， and could can fully mobilize the immune and epidemic prevention mechanism of the body. A large number of studies have shown that Xiao Xianxiongtang and its effective ingredients have obvious antitumor effect. Many doctors have applied Xiao Xianxiongtang and modified formulas in clinical treatment of tumors， and relevant pharmacological studies have also confirmed the effectiveness of this formula， but with a lack of systematic summary of its effective ingredients and its mechanism of action. Now， with alkaloids， ketones， sterols and phenols in Xiao Xianxiongtang as the starting point， this study mainly focuses on inhibition of tumor cell proliferation， invasion and migration， induction of tumor cell apoptosis and autophagy， inhibition of tumor cell cycle， enhancement of tumor cell sensitivity， inhibition of tumor angiogenesis and regulation of immunosuppressive tumor microenvironment from two ways to sort out composition， function and mechanism of drugs. In this paper， effective components， main targets and mechanism of intervention in the tumor development of Xiao Xianxiongtang were reviewed， in order to provide a new idea for subsequent antitumor research and development of this prescription.