Integrated network pharmacology and experimental verification to explore the potential mechanism of San Ying decoction for treating triple-negative breast cancer.

Traditional Chinese medicine (TCM) has been used to treat triple-negative breast cancer (TNBC), a breast cancer subtype with poor prognosis. Clinical studies have verified that the Sanyingfang formula (SYF), a TCM prescription, has obvious effects on inhibiting breast cancer recurrence and metastasis, prolonging patient survival, and reducing clinical symptoms. However, its active ingredients and molecular mechanisms are still unclear. In this study, the active ingredients of each herbal medicine composing SYF and their target proteins are obtained from the Traditional Chinese Medicine Systems Pharmacology database. Breast cancer-related genes are obtained from the GeneCards database. Major targets and pathways related to SYF treatment in breast cancer are identified by analyzing the above data. By conducting molecular docking analysis, we find that the active ingredients quercetin and luteolin bind well to the key targets KDR1, PPARG, SOD1, and VCAM1. In vitro experiments verify that SYF can reduce the proliferation, migration, and invasion ability of TNBC cells. Using a TNBC xenograft mouse model, we show that SYF could delay tumor growth and effectively inhibit the occurrence of breast cancer lung metastasis in vivo. PPARG, SOD1, KDR1, and VCAM1 are all regulated by SYF and may play important roles in SYF-mediated inhibition of TNBC recurrence and metastasis.

Xianlinglianxiafang Inhibited the growth and metastasis of triple-negative breast cancer via activating PPARγ/AMPK signaling pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high invasion and metastasis rates. Xian-Ling-Lian-Xia formula (XLLX) is a traditional Chinese medicine prescription widely used in China for treating TNBC. Clinical studies have shown that XLLX significantly reduces the recurrence and metastasis rate of TNBC and improves disease-free survival. However, the potential molecular mechanisms of XLLX on TNBC are not clear yet. Here, we investigated the effects of XLLX on TNBC using a mouse model and tumor cell lines. The results showed that XLLX significantly inhibited the proliferation, migration, and invasion abilities of TNBC cell lines MDA-MB-231 and 4T1 in vitro, induced apoptosis, and regulated the expression of proliferation, apoptosis, and EMT marker proteins in tumor cells. In in vivo experiments, XLLX treatment significantly reduced the progression of TNBC tumors and lung metastasis. Transcriptomics reveals that XLLX treatment significantly enriched differentially expressed genes in the peroxisome proliferator-activated receptor gamma (PPARγ) and AMP-dependent protein kinase (AMPK) signaling pathways. The western blot results confirmed that XLLX significantly upregulated the protein expression of PPARγ and p-AMPK in TNBC cells, tumors, and lung tissues. It is noteworthy that GW9662 (a PPARγ inhibitor) and Compound C (an AMPK inhibitor) partially reversed the anti-proliferation and anti-metastasis effects of XLLX in TNBC cells. Therefore, XLLX may effectively inhibit the growth and metastasis of TNBC by activating the PPARγ/AMPK signaling pathway.

Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. herb pair suppresses breast cancer liver metastasis by targeting ECM1-mediated cholesterol biosynthesis pathway.

BACKGROUND: Liver metastasis is a frequent event in breast cancer that causes low survival rate and poor prognosis. Citri Reticulatae Pericarpium-Reynoutria japonica Houtt. (CR), a traditional Chinese herb pair, is used for the treatment of breast cancer liver metastasis or cholesterol gallstone disease in clinics. PURPOSE: This study attempted to investigate the potential therapeutic target and mechanism of CR herb pair on breast cancer liver metastasis. METHODS: The anti-metastatic and cholesterol-lowering activities of CR extract were evaluated in triple-negative breast cancer (TNBC) cell lines and an experimental liver metastasis model. The role of extracellular matrix protein 1 (ECM1) in the cholesterol biosynthesis pathway was determined by the knockdown and overexpression of ECM1 gene of TNBC cells. Changes in the gene and protein expression levels of ECM1 and the cholesterol biosynthesis pathway after CR treatment were detected in vitro and in vivo by real-time PCR and Western blot. RESULTS: The invasive and metastatic potentials and hypercholesterol levels of TNBC cells were positively associated with ECM1 expression. ECM1 knockdown reduced tumor cholesterol levels via downregulating cholesterol biosynthesis genes, including ACAT2, HMGCS1, HMGCR, MVK, and MVD, whereas ECM1 overexpression elicited the opposite effects. CR herb pair exerts the potential therapeutic effects on TNBC liver metastasis, which is partially mediated by disrupting ECM1-activated cholesterol biosynthesis process in TNBC cells. CONCLUSION: This study reveals that ECM1 is a novel target for the activation of cholesterol biosynthesis to promote TNBC liver metastasis occurrence. CR herb pair, an ECM1 inhibitor, maybe be considered to serve as an adjuvant therapeutic drug for liver metastasis in clinical practice.

Exploration of potential mechanism of Rougan formula against hepatic fibrosis by network analysis and experimental assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Rougan Formula (RG) has long been clinically applied to treat hepatic fibrosis in patients with different chronic liver diseases. However, the core active substances and the potential pharmacological mechanisms of RG remain unclear. AIM OF THE STUDY: The purpose of this study is to explore bioactive components, key targets, and potential mechanisms of RG by performing network pharmacological analyses and experimental model validation. MATERIALS AND METHODS: All chemical components in RG extract were identified using ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass technology. The candidate components and drug targets of RG, as well as disease-related genes, were extracted from TCMSP and GeneCards databases. The potential pathways related to genes were predicted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The core bioactive components, key targets, and signaling pathways were ultimately obtained by analyzing protein-protein interaction (PPI) and component-target-pathway (C-T-P) networks. Subsequently, the efficacy and underlying mechanisms of RG on hepatic fibrosis were experimentally validated in transforming growth factor-beta 1 (TGF-ß1)-induced hepatic stellate cell activation model and CCL4-induced hepatic fibrosis mouse model. RESULTS: A total of 52 components in RG extract were obtained, and 22 of them were selected as the core bioactive components. Five hundred and thirty-nine overlapped targets were determined by matching drug targets with disease-related targets. The results of PPI and C-T-P network analyses revealed 100 key targets and 19 signaling pathways associated with RG efficacy. In vitro and in vivo studies further verified that RG exerted a significant anti-hepatic fibrotic effect by suppressing the activation of hepatic stellate cells by downregulating the TGF-ß1/Smads signaling pathway. CONCLUSIONS: These results may provide some evidence for further clinical research and development of RG formula as an effective and safe drug for hepatic fibrosis treatment.

Osthole inhibits the migration and invasion of highly metastatic breast cancer cells by suppressing ITGα3/ITGß5 signaling.

Metastasis is the leading cause of death in breast cancer patients. Osthole, as an active compound detected in the traditional Chinese medicine Wenshen Zhuanggu Formula, has shown a promising anti-metastatic activity in human breast cancer cells, but the underlying mechanisms remain ambiguous. In this study we elucidated the anti-metastatic mechanisms of osthole in highly metastatic breast cancer cells and a zebrafish xenograft model. We showed that the expression of integrin α3 (ITGα3) and integrin ß5 (ITGß5) was upregulated in highly metastatic MDA-MB-231, MDA-MB-231BO breast cancer cell lines but was downregulated in poorly metastatic MCF-7 breast cancer cell line, which might be the key targets of osthole's anti-metastatic action. Furthermore, we showed that knockdown of ITGα3 and ITGß5 attenuated breast cancer cell migration and invasion possibly via suppression of FAK/Src/Rac1 pathway, whereas overexpression of ITGα3 and ITGß5 caused the opposite effects. Consistently, osthole significantly inhibited breast cancer metastasis by downregulating ITGα3/ITGß5 signaling in vitro and in vivo. These results provide new evidence that osthole may be developed as a candidate therapeutic drug for metastatic breast cancer.

Ruyiping extract reduces lung metastasis in triple negative breast cancer by regulating macrophage polarization.

Lung metastasis of Triple-negative breast cancer (TNBC) causes severe breath-related events and poor prognosis. Ruyiping (RYP), a traditional Chinese medicine prescription, is used to treat breast cancer lung metastasis in clinical practice. This study was to explore the anti-lung-metastatic activities and mechanism of RYP extract by regulating macrophage polarization. The results showed that RYP can inhibit the viability and induce the apoptosis of TNBC cells. In in vitro experiments, RYP significantly inhibited the invasion and migration ability of TNBC cells promoted by M2, the subtype of macrophage which increased TNBC metastasis related genes. In in vivo experiments, RYP reduced the TNBC progression and lung metastasis. M2/M1 ration in the lung and M2 in the tumor was reduced by RYP, as well as M2 master regulator Stat6. Therefore, RYP extract may exhibit anti-lung metastasis function by reducing M2 in both tumor and lung through reducing Stat6.

Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinfukang has long been used for the clinical treatment of lung cancer. Previous studies have shown that Jinfukang can induce the apoptosis of circulating tumor cells by intervening ROS-mediated DNA damage pathway. However, whether Jinfukang can inhibit the metastasis of circulating tumor cells and its mechanism are still unclear. AIM OF THE STUDY: To further investigate the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of tumor exosomes. MATERIALS AND METHODS: The invadopodia formation was determined with immunofluorescence. Invasion and migration were detected using the Transwell assay. Ultracentrifugation was used to isolate exosomes. Exosomes were characterized by electron microscopy, nanoparticle tracking analysis and immunoblotting, and the protein profile was evaluated by proteomic analysis. The molecular functions, biological processes and signaling pathway enrichment analysis were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Key differentially expressed proteins were verified by Western blot. RESULTS: Jinfukang can inhibit the expression of MMP14, cortactin, Tks5 and the formation of invadopodia of CTC-TJH-01 cells. Furthermore, Jinfukang can significantly inhibit the invasion and migration of CTC-TJH-01 cells. The diameter of exosomes extracted from the CTC-TJH-01 cells treated by Jinfukang was 30-100 nm, and the exosomal markers CD63, CD81 and TSG101 were expressed. We identified 680 deferentially expressed proteins. Gene oncology analysis indicated that exosomes were mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The ingenuity pathway analysis showed that the EGF pathway was significantly inhibited, whereas the GP6 signaling pathway was significantly activated. We also confirmed that Jinfukang inhibited the expression of EGF pathway-related proteins in CTC-TJH-01 cells. Besides, when EGF was used to activate EGF signaling pathway, the inhibition of Jinfukang on CTC cell metastasis was reversed. CONCLUSION: Jinfukang inhibits the metastasis of CTC-TJH-01 cells through the EGF pathway.

WSZG inhibits BMSC-induced EMT and bone metastasis in breast cancer by regulating TGF-ß1/Smads signaling.

Bone metastasis of breast cancer causes severe skeletal-related events and poor prognosis. Wensheng Zhuanggu Formula (WSZG), a traditional Chinese prescription, is used to adjunctively treat breast cancer bone metastases in clinical practice. This study was undertaken to investigate the antibone-metastatic activities and mechanisms of WSZG extract by evaluating the effect of this formula on the cross-talk between bone marrow-derived mesenchymal stem cells (BMSCs) and breast cancer cells in triggering epithelial-mesenchymal transition (EMT) in vivo and in vitro. The results demonstrated that BMSCs might enhance the invasive and metastatic potentials of breast cancer cells as a consequence of EMT induction through direct cell-to-cell contact. WSZG treatment remarkably suppressed motility, invasion, EMT-related gene, and protein markers in BMSC-conditioned breast cancer cells and ameliorated bone metastases and damages in nude mice following co-injection of BMSCs and MDA-MB-231BO breast cancer cells. Further investigation showed that the transforming growth factor-ß1 (TGF-ß1)/Smads pathway was an important mechanism enabling BMSCs to induce EMT occurrence of breast cancer cells. WSZG treatment reversed BMSC-induced EMT by downregulating TGF-ß1/Smads signaling. Thus, WSZG extracts may be regarded as a potential antibone-metastatic agent for breast cancer therapy.

Wenshen Zhuanggu formula mitigates breast cancer bone metastasis through the signaling crosstalk among the Jagged1/Notch, TGF-ß and IL-6 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Advanced breast cancer frequently metastasizes to the bone, resulting in patient morbidity and mortality. The interaction of tumor cells with osteoclasts and osteoblasts seriously affects the occurrence and development of bone metastasis in breast cancer. The signaling crosstalk among the Jagged1/Notch, TGF-ß and IL-6 signaling pathways plays a significant role in the context of bone metastasis. AIM OF THE STUDY: Although Wenshen Zhuanggu (WSZG) formula efficiently decreased the risk of bone metastases in tumor-bearing mice, it remains unclear how WSZG formula regulates the interaction of cancer cells with osteoclasts and osteoblasts in bone metastasis of breast cancer. MATERIALS AND METHODS: In this study, we investigated the role of WSZG formula in the progress of bone metastasis in breast cancer and focused on the cell-cell interactions of tumor cells with osteoclasts and osteoblasts. Western blotting and quantitative real-time PCR were utilized to evaluate the inhibitory activities of WSZG formula on Jagged1 expression both in vivo and in vitro. Osteoblast co-culture and osteoclastogenesis co-culture were applied to analyze the effects of WSZG formula on the interaction of tumor cells with osteoclasts and osteoblasts. A breast cancer xenograft model was also used to test the inhibitory effects of WSZG formula on bone metastasis in breast cancer. RESULTS: WSZG formula decreased Jagged1 expression in osteolytic lesions in the breast cancer xenograft model. Additionally, WSZG formula decreased Jagged1 expression in tumor cell culture alone or co-culture with pre-osteoclasts and osteoblasts. In addition, WSZG formula decreased Jagged1 expression in Jagged1-overexpressing tumor cells. CONCLUSION: The results of this study suggest that WSZG formula mitigates breast cancer bone metastasis through the Jagged1/Notch signaling pathway mediated by TGF-ß and IL-6.

Comparative pharmacokinetics of six coumarins in normal and breast cancer bone-metastatic mice after oral administration of Wenshen Zhuanggu Formula.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG) is a traditional Chinese medicine (TCM) prescription used in clinics for adjuvant treatment of breast cancer bone metastases in Longhua Hospital in China. WSZG has been reported to decrease the risk of bone metastases and alleviate the severity of bone lesions in a breast cancer xenograft model. AIM OF THE STUDY: The present study aimed at investigating the pharmacokinetic behaviors of six coumarins in normal and breast cancer bone-metastatic mice following oral administration of WSZG extract. MATERIALS AND METHODS: A bone-metastatic mouse model was established by intracardiac injection of MDA-MB-231BO breast cancer cells, and WSZG extract (1.60 g/kg) was given orally to the model and normal mice for 4 weeks. Then, the blood pharmacokinetic parameters of six bioactive components from WSZG (psoralen, isopsoralen, bergapten, xanthotoxin, osthole, and imperatorin) were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: There were significant differences in pharmacokinetic behaviors between normal and pathological states. Compared with normal mice, the model mice showed significantly increased AUC0-t and AUC0-∞ of the bioactive compounds (P < 0.05) and significantly decreased total blood clearance (CLZ/F) (P < 0.05). CONCLUSIONS: The different pharmacokinetic behaviors might be partly ascribed to intestinal functional disorders and imbalance of gastrointestinal microbiota under the morbid state. The findings provide some valuable information to evaluate the clinical efficacy and safety of this TCM formula.

Wenshen Zhuanggu formula effectively suppresses breast cancer bone metastases in a mouse Xenograft model.

Wenshen Zhuanggu formula (WSZG) is a traditional Chinese medicine used as an adjuvant for the prevention of bone metastases in breast cancer patients. In this study we investigated the efficacy of WSZG in preventing bone metastases and the potential mechanisms in a mouse xenograft model of breast cancer bone metastases. This model was established by injection of human MDA-MB-231BO-Luc breast cancer cells alone or a mixture of the cancer cells with bone marrow-derived mesenchymal stem cells (BMSCs) into left ventricle of the heart in female nude mice. Then the mice were treated with WSZG (3.25, 6.5 or 13.0 mg·kg-1·d-1, ig) for four weeks, whereas zoledronic acid (100 µg/kg per week, ig) was used as a positive control. The occurrence and development of bone metastases were monitored via bioluminescent imaging, and bone lesions were assessed using micro-CT. Intracardiac injection of the mixture of MDA-MB-231BO-Luc breast cancer cells with BMSCs significantly facilitated the bone metastatic capacity of the breast cancer cells, and aggravated bone lesions in the mouse xenograft model of breast cancer bone metastases. Administration of WSZG dose-dependently inhibited the incidence and intensity of bone metastases and protected against bone lesions by suppressing osteoclast formation and tumor cell infiltration. Furthermore, administration of WSZG caused a marked reduction in the expression of CCL5/CCR5 and IL-17B/IL-17BR in bone metastatic tissues. The results demonstrate that WSZG exerts potential therapeutic effects in a mouse xenograft model of breast cancer bone metastases, which are partially mediated by weakening the interaction between BMSCs and breast cancer cells in the tumor microenvironment.

Anti-metastatic effect and mechanisms of Wenshen Zhuanggu Formula in human breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG), a traditional Chinese medicine (TCM) empirical prescription, has been used to treat the patients with breast cancer bone metastasis as an adjuvant in clinical practice. To explore the anti-metastatic activity and potential mechanisms of WSZG-containing serum (WSZG-CS) on highly bone-metastatic human breast cancer MDA-MB-231BO cells. MATERIALS AND METHODS: MDA-MB-231BO cells were cultured alone or co-cultured with bone marrow-derived mesenchymal stem cells (BMSCs). Invasion assays were carried out in Matrigel-coated Transwell chambers. CC chemokine 5 (CCL5) and interleukin (IL)-17B secretion levels were detected by ELISA. CCR5 and IL-17BR protein expression levels were determined by immunocytochemistry and Western blot analysis. RESULTS: Compared with control serum, WSZG-CS significantly inhibited BMSC induced MDA-MB-231BO breast cancer cell invasion, reduced CCL5 and IL-17B levels in co-culture supernatants, and downregulated CCR5 and IL-17BR protein expression in breast cancer cells co-cultured with BMSCs. CONCLUSIONS: WSZG-CS exerts an anti-metastatic activity against MDA-MB-231BO breast cancer cells, due to its ability to mitigate the interaction between BMSCs and breast cancer cells mediated via the CCL5/CCR5 and IL-17B/IL-17BR signaling pathways.

Psoralen inhibits bone metastasis of breast cancer in mice.

Breast cancer is the most common female malignancy and it frequently metastasizes to bone. Metastatic breast cancer continues to be the primary cause of death for women in East and Southeast Asia. Psoralen is a furocoumarin that can be isolated from the seeds of Psoralea corylifolia L. Psoralen exhibits a wide range of biological properties and has been demonstrated as an antioxidant, antidepressant, anticancer, antibacterial, and antiviral agent. Additionally, it is involved in the formation and regulation of bone. This study investigated whether psoralen can inhibit metastasis of breast cancer to bone in vivo. Histological, molecular biological, and imaging analyses revealed that psoralen inhibits bone metastases in mice. Psoralen may function to inhibit breast cancer cell growth in the bone microenvironment and regulate the function of osteoblasts and osteoclasts in tumor-bearing mice. The results of this study suggest that psoralen is a bone-modifying agent and a potential therapeutic to treat patients with bone metastases.

Relationships between pharmacokinetics and efficacy of Xie-xin decoction in rats with experimental ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Xie-xin decoction (XXD) has been used as a classic formula in China for the treatment of gastrointestinal dysfunction such as ulcerative colitis (UC). However, no potential action mechanisms and active compounds had been systematically investigated. AIM OF THE STUDY: To explore the effectiveness and the material basis of XXD in trinitrobenzene sulfonic acid (TNBS)-induced UC rats. MATERIALS AND METHODS: XXD was administered orally for 8 days at a dosage of 2 or 4g/kg/day. Plasma pharmacokinetic properties and colon tissue concentrations of multiple compounds from XXD were detected. Tissue damage scores, production of interleukin (IL)-10 and myeloperoxidase (MPO), expression of tumor necrosis factor-alpha (TNF-α) and nuclear factor-kappa Bp65 (NF-κBp65) in colon tissues were examined. Canonical correlation analysis was performed to evaluate the relationships between pharmacokinetics and efficacy to elucidate significantly active compounds of XXD. RESULTS: XXD promoted the recovery of colitis and inhibited the colonic inflammation damage in UC rats by reducing the level of MPO and the expression of TNF-α and NF-κBp65, and increasing the production of IL-10 in colon tissues. Efficacy of XXD was positively related with AUC of five plasma compounds (baicalin, berberine, wogonoside, wogonin, and rhein) and concentrations of six colon tissue compounds (coptisine, jatrorrhizine, palmatine, berberine, baicalein and emodin), respectively. CONCLUSIONS: The multiple compounds in plasma and colon tissues from XXD might be the main material basis for therapeutic potentials in UC rats.

[Effects of platycodin D in combination with different active ingredients of Chinese herbs on proliferation and invasion of 4T1 and MDA-MB-231 breast cancer cell lines].

OBJECTIVE: To investigate the effects of platycodin D in combination with different active ingredients of Chinese herbs under different therapeutic principles on proliferation and invasion of 4T1 and MDA-MB-231 breast cancer cell lines. METHODS: The effective doses of platycodin D, Ophiopogon total saponins, curcumenol and osthole in inhibiting proliferation of breast cancer cell lines 4T1 and MDA-MB-231 were detected by methyl thiazolyl tetrazolium (MTT) assay, respectively. Optimized combinations of platycodin D with Ophiopogon total saponins, curcumenol, or osthole were determined by uniform design method. Effects of the optimized combinations of platycodin D with the three ingredients on proliferation and invasion of 4T1 and MDA-MB-231 cells were verified and evaluated by MTT assay and Transwell chamber test, respectively. RESULTS: Verifying study showed that the inhibitory effects of platycodin D in combination with curcumenol or osthole on proliferation of 4T1 and MDA-MB-231 cells were better than those of platycodin D in combination with Ophiopogon total saponins and each ingredient used alone (P<0.05 or P<0.01). The inhibitory effect of platycodin D in combination with Ophiopogon total saponins or osthole on invasion of 4T1 cells was significantly better than those of platycodin D in combination with curcumenol and each ingredient used alone (P<0.05 or P<0.01). Moreover, the inhibitory effect of platycodin D in combination with curcumenol or osthole on invasion of MDA-MB-231 cells was significantly better than that of platycodin D in combination with Ophiopogon total saponins (P<0.01). CONCLUSION: The optimized combinations of platycodin D with three different active ingredients of Chinese herbs under different therapeutic principles can significantly inhibit the proliferation and decrease the invasion of 4T1 and MDA-MB-231 cells. Different platycodin D combinations have different potency in suppressing breast cancer cell proliferation and invasion.

A sensitive method for determination of platycodin d in rat plasma using liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

Platycodin D (PD), a major component isolated from the root of Platycodon grandiflorum, is widely used in traditional Chinese medicine. A sensitive rapid analytical method was established and validated to determine the PD in rat plasma. This method was further applied to assess the pharmacokinetics of PD in rats following administration of a single dose. Liquid chromatography tandem mass spectrometry (LC/MS/MS) in multiple reaction monitoring mode (MRM) was used in the method, and tubeimoside I was used as the internal standard (IS). A simple protein precipitation based on methanol (MeOH) was employed. The combination of a simple sample cleanup and short chromatographic running time (4 min) increased the throughput of the method substantially. The method was validated over the range of 0.5-1000 ng/mL with a correlation coefficient > 0.99. The lower limit of quantification was 0.5 ng/mL for PD in plasma. Intra- and inter-day accuracies for PD were 90-115 % and 96-108 %, respectively, and the inter-day precision was less than 15 %. After a single oral dose of 10 mg/kg of PD, its mean peak plasma concentration ( CMAX) was 13.7 ± 4.5 ng/mL at 0.5 h. The area under the plasma concentration-time curve ( AUC0-24 H) was 35.4 ± 16.1 h·ng/mL, and the elimination half-life ( T1/2) was 1.48 ± 0.13 h. In case of intravenous administration of PD at a dosage of 0.5 mg/kg, the area under the plasma concentration-time curve ( AUC0-24 H) was 2203 ± 258 h ·â€Šng/mL, and the elimination half-life (T½) was 6.57 ± 0.70 h. Based on the results, the oral bioavailability of PD in rats at 10 mg/kg is 0.079 %.

[Inhibitory effects of osthole, psoralen and aconitine on invasive activities of breast cancer MDA-MB-231BO cell line and the mechanisms].

OBJECTIVE: To explore the inhibitory effects and to investigate the mechanisms of combined treatment of osthole, psoralen with aconitine on human breast cancer cell line MDA-MB-231BO. METHODS: The best inhibitory concentration of osthole, psoralen combined with aconitine on MDA-MB-231BO cells was obtained by stepwise regression analysis after adopting a uniform experiment design. The invasive activities were observed by transwell assays, and expressions of transforming growth factor-ß1 (TGF-ß1), Smad2, Smad3, Smad4, Smad7, nuclear factor-κB (NF-κB) and receptor activator of NF-κB ligand (RANK) mRNAs were analyzed by real-time quantitative polymerase chain reaction. RESULTS: The optimal combination concentrations of osthol, psoralen and aconitine were 6.44, 8.89 and 9.44 µg/mL, respectively. Cell invasion was significantly inhibited after 24 hours of treatment using the combination drugs and zoledronic acid. TGF-ß1, Smad2, Smad3, Smad4, Smad7, NF-κB and RANK mRNA expressions of the optimal combination group and zoledronic acid group were significantly reduced compared with the control group (P<0.01). Furthermore, TGF-ß1, Smad2, Smad3, Smad4, Smad7, NF-κB and RANK mRNA expressions of the optimal combination group were significantly lower than those of the weak combination group (P<0.01). CONCLUSION: Combination treatment of osthole, psoralen with aconitine can inhibit cancer cell invasion, which is a result of alteration of the TGF-ß/Smad signaling pathway and down-regulation of NF-κB and RANK expressions.

[Attenuation and mechanism of endoplasmic reticulum stress-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by qinggan huoxue recipe and its disassembled formulas].

OBJECTIVE: To explore attenuation and mechanism of endoplasmic reticulum stress (ERS)-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by Qinggan Huoxue Recipe (QGHXR) and its disassembled formulas (Qinggan Recipe and Huoxue Recipe respectively). METHODS: A rat model of chronic alcoholic liver injury was successfully established using a compound reagent of alcohol, corn oil, and pyrazol. The modeled rats were randomly divided into the model group, the QGHXR group, the Qinggan Recipe (QGR) group, and the Huoxue Recipe group (HXR). The CCl4 control group and the normal control group were also set up. There were ten rats in each group. All rats of modeled groups were gastrogavaged with alcohol compound reagent every morning. Rats in the QGHXR group (at the daily dose of 9. 5 g/kg, QGR group (at the daily dose of 3.0 g/kg), and HXR group (at the daily dose of 6.5 g/kg) were administered with corresponding medicines by gastrogavage every afternoon. Equal volume of normal saline was given to rats of the model group by gastrogavage. CCl4 was intraperitoneally injected at the dose of 0.3 mL/kg to rats in the CCl4 control group, once per week. Normal saline was given to rats in the normal control group by gastrogavage. The treatment was lasted for two weeks. Pathological changes of the liver were observed by histopathology. Serum total homocysteine (tHCY) level was detected by ELISA. The hepatocyte apoptosis rate was detected using flow cytometry. The gene and protein expressions of eukaryotic translation initiation factor 2 alpha (elF-2alpha), phosphorylation elF-2alpha (pelF-2alpha), glucose-regulated protein 78 (GRP78), and Caspase-3 in the liver were examined using Real-time PCR and Westen blot respectively. RESULTS: Compared with the normal control group, typical pathological changes of chronic alcoholic liver injury such as steatosis, inflammation, and even fibrosis occurred in model rats. The hepatocyte apoptosis obviously increased, with the apoptosis rate reaching the five-fold of that in normal rats. Besides, early apoptosis dominated. The serum tHCY level significantly increased. The expressions of p-elF-2alpha, GRP78, and Caspase-3 protein obviously increased (P < 0.01). Expressions of GRP78 and Caspase-3 mRNA significantly increased (P < 0.05, P < 0.01). Compared with the model group, the degrees of the liver injury and the hepatocyte apoptosis in the QGHXR group, the QGR group, and the HXR group were significantly alleviated. The serum tHCY level was significantly lowered. The protein expressions of p-elF-2a, GRP78, and Caspase-3 obviously decreased (P < 0.01). mRNA expressions of GRP78 and Caspase-3 obviously decreased in the QGHXR group (P < 0.05, P < 0.01). Only GRP78 mRNA expression obviously decreased in the QGR group (P < 0.05). CONCLUSION: QGHXR and its disassembled formulas could attenuate ERS-mediated hepatocyte apoptosis in alcohol-induced liver injury rats by lowering the serum tHCY level and expressions of ERS apoptosis correlated factors.

Influences of Fructus evodiae pretreatment on the pharmacokinetics of Rhizoma coptidis alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma coptidis is a traditional Chinese medicine with pharmacological properties. It is usually prescribed with Fructus evodiae as traditional Chinese medicine (TCM) formulas. Here we report the influences of Fructus evodiae on the pharmacokinetics of the Rhizoma coptidis alkaloids and propose possible mechanisms. MATERIALS AND METHODS: Pharmacokinetic experiments were performed in rats. In vitro absorption experiments were performed in everted rat gut sacs, while in vitro metabolism experiments and determination of hepatic UDP-glucuronosyltransferase (UGT) 1A1 mRNA expression were performed in rat liver microsomes. RESULTS: Pretreatment with Fructus evodiae extract for two weeks decreased the systemic exposure of the Rhizoma coptidis alkaloids. This effect was not due to inhibition of absorption or enhanced hepatic phase I metabolism of the Rhizoma coptidis alkaloids. However, Fructus evodiae pretreatment enhanced both the activity and expression of hepatic UGT1A1. CONCLUSIONS: The results showed that Fructus evodiae pretreatment decreased the systemic exposure of the Rhizoma coptidis alkaloids by inducing hepatic UGT1A1.

Astragaloside IV regulates expression of ATP-sensitive potassium channel subunits after ischemia-reperfusion in rat ventricular cardiomyocytes.

OBJECTIVE: Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the heart from myocardial ischemic injury, but the mechanisms of action are unknown. ATP-sensitive potassium (KATP) channels are activated during ischemia and exert a compensatory protective effect on cardiomyocytes. We therefore examined the effects of AsIV on KATP channel currents and channel expression in isolated rat ventricular cardiomyocytes after ischemia-reperfusion injury. METHODS: Forty Wistar rats were divided into five groups: control group, ischemia-reperfusion (IP) group, IP + glibenclamide group, IP + pinacidil group and IP + AsIV group. The ischemia-reperfusion injury model was established in enzymatically isolated ventricular cardiomyocytes by perfusion with calcium-free Tyrode solution for 10 min, arrest for 30 min, and reperfusion for 45 min. The different drugs were applied for 10-15 min, and the KATP channel current (I(KATP)) was recorded with voltage-clamp mode by whole-cell patch-clamp technique. Protein and mRNA expression of the KATP channel subunits Kir6.1, Kir6.2, SUR2A and SUR2B was quantified using western blotting and real-time PCR. RESULTS: The KATP current in IP group was significantly greater than that in control group (211.45 +/- 33.67 vs 83.51 +/- 23.67 pA; P < 0.01). Glibenclamide (10 micromol/L) blocked KATP currents, whereas both AsIV (1 mg/L) and the known channel opener pinacidil (50 micromol/L) significantly increased I(KATP) (P < 0.05). Consistent with this, AsIV significantly up-regulated protein and mRNA expression of Kir6.1, Kir6.2, SUR2A, SUR2B (P < 0.01 vs IP group). CONCLUSION: The protective effects of AsIV in ischemia-reperfusion injury may be related to the up-regulation of several KATP channel subunits and facilitation of KATP currents.