Adenophorone, An Unprecedented Sesquiterpene from Eupatorium adenophorum: Structural Elucidation, Bioinspired Total Synthesis and Neuroprotective Activity Evaluation.

(-)-Adenophorone (1), a caged polycyclic sesquiterpene featuring an unprecedented tricyclo[4.3.1.05,9 ]decane skeleton, was isolated from Eupatorium adenopharum Spreng. The structure of 1 was unambiguously established by a combination of spectroscopic analysis, X-ray crystallography, and bioinspired total synthesis. Key synthetic features include a sequential Reformatsky/oxidation/regio- and stereoselective hydrogenation, and subsequent merged MBH-Tsuji-Trost cyclization. The concise synthetic sequence efficiently constructs the bicyclic skeleton of cadinene sesquiterpene (+)-euptox A (2) in 8 steps from commercially available monoterpene (-)-carvone (6), with outstanding performance on diastereocontrol. The bioinspired synthesis of 1 was achieved from 2, a plausible biogenetic precursor, via transannular Michael addition. This work provides experimental evidence of our proposed biosynthetic hypothesis of 1. Additionally, compound 1 showed potent neuroprotective activity in H2 O2 -treated SH-SY5Y and PC12 cells.

[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 protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. AIM OF THE STUDY: This study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. METHODS: The ApoE-/- atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. RESULTS: Our study reported that, in ApoE-/- atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. CONCLUSIONS: By highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

Shenlian extract decreases mitochondrial autophagy to regulate mitochondrial function in microvascular to alleviate coronary artery no-reflow.

Shenlian (SL) extract has been proven to be effective in the prevention and treatment of atherosclerosis and myocardial ischemia. However, the function and molecular mechanisms of SL on coronary artery no-reflow have not been fully elucidated. This study was designed to investigate the contribution of SL extract in repressing excessive mitochondrial autophagy to protect the mitochondrial function and prevent coronary artery no-reflow. The improvement of SL on coronary artery no-reflow was observed in vivo experiments and the molecular mechanisms were further explored through vitro experiments. First, a coronary artery no-reflow rat model was built by ligating the left anterior descending coronary artery for 2 hr of ischemia, followed by 24 hr of reperfusion. Thioflavin S (6%, 1 ml/kg) was injected into the inferior vena cava to mark the no-reflow area. Transmission electron microscopy was performed to observe the cellular structure, mitochondrial structure, and mitochondrial autophagy of the endothelial cells. Immunofluorescence was used to observe the microvascular barrier function and microvascular inflammation. Cardiac microvascular endothelial cells (CMECs) were isolated from rats. The CMECs were deprived of oxygen-glucose deprivation (OGD) for 2 hr and reoxygenated for 4 hr to mimic the Myocardial ischemia-reperfusion (MI/R) injury-induced coronary artery no-reflow in vitro. Mitochondrial membrane potential was assessed using JC-1 dye. Intracellular adenosine triphosphate (ATP) levels were determined using an ATP assay kit. The cell total reactive oxygen species (ROS) levels and cell apoptosis rate were analyzed by flow cytometry. Colocalization of mitochondria and lysosomes indirectly indicated mitophagy. The representative ultrastructural morphologies of the autophagosomes and autolysosomes were also observed under transmission electron microscopy. The mitochondrial autophagy-related proteins (LC3II/I, P62, PINK, and Parkin) were analyzed using Western blot analysis. In vivo, results showed that, compared with the model group, SL could reduce the no-reflow area from 37.04 ± 9.67% to 18.31 ± 4.01% (1.08 g·kg-1 SL), 13.79 ± 4.77% (2.16 g·kg-1 SL), and 12.67 ± 2.47% (4.32 g·kg-1 SL). The extract also significantly increased the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) (p < 0.05 or p < 0.01). The fluorescence intensities of VE-cadherin, which is a junctional protein that preserves the microvascular barrier function, decreased to ~74.05% of the baseline levels in the no-reflow rats and increased to 89.87%(1.08 g·kg-1 SL), 82.23% (2.16 g·kg-1 SL), and 89.69% (4.32 g·kg-1 SL) of the baseline levels by SL treatment. SL administration repressed the neutrophil migration into the myocardium. The oxygen-glucose deprivation/reoxygenation (OGD/R) model was induced in vitro to mimic microvascular ischemia-reperfusion injury. The impaired mitochondrial function after OGD/R injury led to decreased ATP production, calcium overload, the excessive opening of the Mitochondrial Permeability Transition Pore, decreased mitochondrial membrane potential, and reduced ROS scavenging ability (p < 0.05 or p < 0.01). The normal autophagosomes (double-membrane vacuoles with autophagic content) in the sham group were rarely found. The large morphology and autophagosomes were frequently observed in the model group. By contrast, SL inhibited the excessive activation of mitochondrial autophagy. The mitochondrial autophagy regulated by the PINK/Parkin pathway was excessively activated. However, administration of SL prevented the activation of the PINK/Parkin pathway and inhibited excessive mitochondrial autophagy to regulate mitochondrial dysfunction. Results also demonstrated that mitochondrial dysfunction stimulated endothelial cell barrier dysfunction, but Evans blue transmission was significantly decreased and transmembrane resistance was increased significantly by SL treatment (p < 0.05 or p < 0.01). Carbonylcyanide-3-chlorophenylhydrazone (CCCP) could activate the PINK/Parkin pathway. CCCP reversed the regulation of SL on mitochondrial autophagy and mitochondrial function. SL could alleviate coronary artery no-reflow by protecting the microvasculature by regulating mitochondrial function. The underlying mechanism was related to decreased mitochondrial autophagy by the PINK/Parkin pathway.

[Pharmacodynamic substances and therapeutic potential of Wuji Pills:based on UPLC-Q-TOF-MS/MS and network pharmacology].

As a classic prescription, Wuji Pills is composed of Coptidis Rhizoma, Euodiae Fructus Preparata, and stir-fried Paeo-niae Radix Alba at the ratio of 6∶1∶6. The practical application of it is limited compared with other famous Chinese medicine prescriptions. Only one company produces Wuji Pills in China. In this study, ultra-performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to analyze and identify 26 identical compounds from Wuji Pills and drug-containing plasma of rats. Based on these components, 46 potential targets were screened out with network pharmacology methods, followed by the component-target network construction, Gene Ontology(GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and disease prediction. It was concluded that Wuji Pills acted on core targets such as PTGS2, PTSG1, NCOA2, HSP9 OAD1, and RXRA through magnoflorine, hydroxyevodiamine, daucosterol, and berberine and exerted pharmacodynamic effects through various pathways such as calcium ion signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3 K-Akt) signaling pathway, and vascular endothelial growth factor(VEGF) signaling pathway. Thus, Wuji Pills has therapeutic potential for Alzheimer's disease, diabetes mellitus, myocardial ischemia, and other diseases in addition to the conventional disease(irritable bowel syndrome, IBS). The above research results can provide a reference for the comprehensive interpretation of the pharmacodynamic basis of Wuji Pills and the expansion of clinical application. At the same time, a lot of components in serum and the in vivo transformed and metabolized components of Wuji Pills have similar structure and relative molecular weight. In theory, these components may show additive effects and the competitive/antagonistic effects on the same target. According to the hypothesis of "additive effect of multiple components for a single target" in traditional Chinese medicine, multiple similar components may exert the additive effects on local targets. This study can partly prove the scientificity of this hypothesis and provide laboratory evidence.

[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.

[Screening combination ratio and exploring mechanism of Momordicae Semen and Epimedii Folium].

The aim of this paper was to obtain low toxicity and high efficiency anti-tumor Chinese medicine through screening the combination ratios of Momordicae Semen and Epimedii Folium, and to explore the anti-tumor mechanism of the combination of two drugs by observing their effect on apoptosis-related proteins in cancer cells. Methyl thiazolyl tetrazolium(MTT) assay was used to observe the effect of drug combination on the proliferation of tumor cells from different tissue sources. The effects of the combination of the two drugs on tumor cells were analyzed by Compusyn software. Plate cloning assay was used to observe the effect of combination of these two drugs on the proliferation of A549 cells in vitro. The expression of reactive oxygen species(ROS) and apoptotic proteins p53, Bcl-2 and Bax were compared by using ROS kit and Western blot. Lewis lung cancer model was used to observe the anti-tumor effect of drugs in vivo. The results showed that the anti-tumor effect of their ethanol extract was more significant than that of water extract, and the anti-proliferation effect was strongest when the ratio was 1∶1(P<0.05). Compusyn analysis showed that the combination of the two drugs had synergistic effect. Further studies showed that after combined use, the number of clonogen formation in A549 cells was significantly reduced(P<0.01); ROS production was increased; the expression of apoptosis-related protein p53 was up-regulated, and the ratio of Bcl-2/Bax was decreased. In vivo animal study showed that the tumor inhibition rate was 53.06%(P<0.05) in the high dose group. As compared with the single use of the two drugs, the combination of the two drugs had more significant anti-proliferative effect on tumors, and the optimum ratio was 1∶1. The combination of the two drugs at a ratio of 1∶1 inhibited the proliferation of various tumor cells, and had no significant effect on normal liver cells LO2 when compared with other ratios. Therefore, it can be preliminarily inferred that the combination of the two drugs may have the effect of synergism and detoxification. Further studies showed that the combination of the two drugs can significantly inhibit the proliferation of A549 cells, and its mechanism may be related to the activation of endogenous apoptotic pathway. In vivo experiments also showed that the tumor inhibition rate increased with the increase of drug concentration.

[Research progress of effect of artemisinin family drugs on T lymphocytes immunomodulation].

Artemisinin was isolated from traditional Chinese herb Artemisia annua for treating malaria. A series of derivatives,like dihydroartemisinin,artesunate,artemether,artether,had the same core chemical structure,and sesquiterpene lactone containing peroxide bridge constitute the basic chemical structure. Besides anti-malaria,artemisinin family drugs were found to ameliorate many different diseases,which have attracted wide attention in recent years. Among different diseases,artemisinin family drugs were found to have T lymphocytes immunomodulation effects,including activation,proliferation,differentiation,apoptosis and subsets function. Because T cell immunologic response is the key point of many diseases,and impact the pathogenic process,therapeutic effect and prognosis,the drug studies with it as the target have become hotspots in recent years. Studies of artemisinin family drug on T cell immunomodulation were still at the initial stage and involved in different disease; furthermore,T cell immune process involves complicated molecular mechanism,it is imperative to summarize the advance of current studies for further systematic explanation and exploration of their characteristics and mechanisms. This article will summarize the research progress of artemisinin family drugs for malaria,autoimmune disease,hypersensitivity reaction,tumor,schistosomiasis and AIDS relating to T cell immune modulation,so as to provide basic and professional reference for related research and application.

[Efficacy and mechanism of Lianhua Qingwen Capsules(LHQW) on chemotaxis of macrophages in acute lung injury (ALI) animal model].

This paper was mainly to discuss the potential role and mechanism of Lianhua Qingwen Capsules(LHQW) in inhibiting pathological inflammation in the model of acute lung injury caused by bacterial infection. For in vitro study, the mRNA expression of MCP-1 in RAW264.7 cells and THP-1 cells, the content of MCP-1 in cell supernatant, as well as the effect of LHQW on chemotaxis of macrophages were detected. For in vivo study, mice were randomly divided into 7 groups, including normal group, model group(LPS 5 mg·kg~(-1)), LHQW 300, 600 and 1 200 mg·kg~(-1)(low, middle and high dose) groups, dexamethasone 5 mg·kg~(-1) group and penicillin-streptomycin group. Then, the anal temperature was detected two hours later. Dry weight and wet weight of lung tissues in mice were determined; TNF-α and MCP-1 levels in alveolar lavage fluid and MCP-1 in serum were detected. In addition, the infiltration of alveolar macrophages was also observed and the infiltration count of alveolar macrophages was measured by CCK-8 method. HE staining was also used to observe the inflammatory infiltration of lung tissues in mice. Both of the in vitro and in vivo data consistently have confirmed that: by down-regulating the expression of MCP-1, LHWQ could efficiently decrease the chemotaxis of monocytes toward the pulmonary infection foci, thus blocking the disease development in ALI animal model.

[Research advancement in natural anti-cancer product].

Human health has been severely threatened by malignant tumors continuously.Rational and effective drug use provides an effective means for the treatment of malignant tumors,and is expected to become an important way to solve the problem of tumor treatment in the future.In recent years,with the escalation of new cancer theories and the emergence of clinical drug resistance,innovative research and development of anti-cancer drugs has always been a hot spot and focus in cancer research.Among them,the discovery of novel anti-cancer drugs from natural compound is of top priority due to its strong anti-cancer efficacy and the abundant drug resources.Therefore,it is imperative to systematically summarize the cutting-edge advancements of the natural products and their potential pharmacological mechanisms according to the characteristics of tumor progression,and put forward the new directions and trends for further development of anti-cancer natural products in the future.Specifically,the research advancements on anti-cancer effect of natural products were reviewed,focusing on both the traditional and innovative application.We hope this review could bring the light on the research path of the natural anti-cancer products clearly and comprehensively,and also provide inspirations for innovative,safer and more effective anti-cancer drug development and exploration.

[Study and opinion on toxicity of aristolochic acid].

On October 18th, 2017, a research article named "Aristolochic acids and their derivatives are widely implicated in liver cancers in Taiwan and throughout Asia" was published on Science Translational Medicine. This article pointed out that herbs containing aristolochic acids could cause liver cancer by inducing the specific "aristolochic acids mutational signature". The public was also suggested to avoid the intake of herbs containing aristolochic acids. Since 2000, CFDA has gradually abolished the medicinal standards for herbs containing aristolochic acids such as caulis aristolochiae manshuriensis, aristolochia heterophylla and radix aristolochiae. Related drugs have been strengthened supervision since then. Chinese Pharmacopoeia has also removed the records of a series of related herbs. State Administration of Traditional Chinese Medicine held a conference on the "toxicity" of aristolochic acids as soon as the article was published. After a discussion of the studies on the toxicity of aristolochic acids, experts attending the meeting discovered several problems, including the unclearness of exposure history, tumor-producing dose and latent period, the absence of some key factors such as hepatitis B, the small sample size, miscellaneous factors, incomplete evidence chains, the missing of analyses between data with huge differences, the insufficiency of fundamental research arguments, etc. In order to understand the toxicity of aristolochic acids and the carcinogenic risks, as well as guide clinical safe medication, the experts suggested that：①Complete the systematical evaluation of aristolochic acids carcinogenicity as soon as possible. Scientifically elucidate the relationship between aristolochic acids and the genesis of liver cancer. ②Establish medication risk warnings of aristolochic acids and strengthen the supervision. ③Make an in-depth study of the toxicity of traditional Chinese medicine. Find out the adverse effects of all traditional Chinese medicine step by step.

[Protective effect of Shuyu Yiban decoction on atherosclerosis in rats induced by chronic unpredictable mild stress].

Shuyu Yiban decoction(SYYB) has significant effect in treating the patients with coronary heart disease combined with chronic psychological stress. In this study, in order to observe the effects of SYYB on early formation of atherosclerosis(As) and inflammation response induced by chronic psychological stress, high-fat diet+intraperitoneal injections of Vitamin D3 were given to establish As early lesion models, and based on these models, chronic unpredictable mild stress(CUMS) was used to observe whether the chronic psychological stress could increase coronary atherosclerotic lesions investigate the intervention effect of SYYB(6.6, 13.2, 26.4 mg•kg⁻¹). The tail suspension test and novelty-feeding test were adopted to detectadrenocortico-tropic hormone(ACTH), cortisol(Cor) andnoradrenaline(NE) in serum and weigh thymus and adrenal gland, in order to assess the effects of SYYB on CUMS model rats. The pathological changes of vascular tissues in aortic arch were observed by using hematoxylin and eosin(HE) staining, and then the levels of triglycerides(TG), total cholesterol(TC) and high density lipoprotein(HDL-C) in serum were determined to assess effects of SYYB on As lesions. The effects of SYYB on the inflammatory response in As rats were assessed by detecting high-sensitivity C-reactive protein(hsCRP), interleukin-1ß(IL-1ß) and interleukin-6(IL-6) in serum. The results showed that as compared with the blank control group, TC and TG levels in As group were increased while HDL-C was markedly decreased; furthermore, the aortic wall was thickened in HE staining. Meanwhile, foam cells were formed, and the behavioral assessment and serum stress hormone test showed that there was a chronic stress response, indicating successful establishment of As+CUMS models in this study. The experiment demonstrated that SYYB could lower the levels of serum TC and TG, reduce foam cells, calcification and inflammatory cells infiltration. Moreover, SYYB could obviously lower levels of ACTH, Cor and NE and the As related inflammatory indicatorhs-CRP, IL-1ß and IL-6 in serum.These results indicated that SYYB had protective effect on chronic psychological stress induced in As rats, and the mechanism was associated with balancing the neuroendocrine-immune network system and regulating inflammation response.

Extract of Caulis Spatholobi, a novel blocker targeting tumor cell­induced platelet aggregation, inhibits breast cancer metastasis.

Metastasis of breast cancer is the vital step for malignant progression. During such a process, hematogenous metastasis is an indispensable approach for the dissemination of cancer cells. A platelet, contributes to hypercoagulable state, and is also identified the crucial factor in the coagulation system for supporting metastasis. Therefore, the relationship of a platelet and a tumor cell plays a critical role in tumor cell metastasis. Consequently, inhibiting tumor cell­induced platelet aggregation (TCIPA) is recongnized as a crucial target on suppression of tumor metastasis such as aspirin (ASA). Under such circumstance, here we report that, through dissociating the tumor­platelet (T­P) complex, 80% ethanol extracts of Caulis Spatholobi (SET) successfully alleviated the hypercoagulation state, thereby reducing tumor metastasis and improving the prospects of survival in breast cancer cell model. Through MTT and anti­aggregation assay stimulated by ADP, we detected the optimum treatment time and the optimum dose of SET. By using confocal microscopy, we observed that SET can strongly block the formation of T­P complex in vitro. The result was further quantified and confirmed by the FACS analysis. The fluorescent value of T­P complex was obviously decreased in the drug­treated groups. In vivo, 4T1 cells were injected through the mouse tail vein for dynamic visualization by small animal imaging system. The metastatic intensity was quantified and the survival curve was analyzed. Additionally, general observation and hematoxylin and eosin (H&E) staining of lung tissue was performed. SET exerted an obvious effect on the inhibition of metastasis and increasing the survival rate of mice. For the molecular mechanism study of anti­TCIPA, zymography and RT­PCR assay preliminarily revealed the molecular mechanism of SET in the regulation of P­T interaction. Collectively, through drug efficacy identification and pharmacological revealing, we have obtained a promising candidate for the interference of breast metastasis by suppressing TCIPA, which will be beneficial for clinical cancer treatment.

A novel cell cycle blocker extracted from Stellera chamaejasme L. inhibits the proliferation of hepatocarcinoma cells.

Currently, liver cancer is the sixth most prevalent cancer and the third most common cause of cancer-related death. However, effective chemotherapeutic drugs with low drug resistance and few side-effects for the clinical treatment of liver cancer are lacking. Therefore, the search for novel drugs to compensate for the defects of existing drugs is urgently needed. Herein, we successfully screened an extract named from Stellera chamaejasme L. (SCL), a historically confimed antitumor plant, through a novel extraction platform. In the present study, we firstly screened the anticancer effect of ESC by the sulforhodamine B (SRB) cell proliferation assay in a wide range of malignant cell lines, including A549, NCI-H157, NCI-H460, SK-HEP-1 and HepG2. With the highest inhibitory rate in hepatocarcinoma cells, we further identified the tumor-suppressive efficacy and the safety of ESC in an H22 hepatocarcinoma xenograft model in vivo. In a mechanistic study, flow cytometry and western blot analysis were performed to evaluate the effects of ESC on the induction of cell apoptosis, intervention of cell cycle distribution and its influence on key G2/M-phase regulators. The results showed that ESC significantly inhibited the cell growth of liver cancer cell lines. Accordingly, the tumor inhibition rate was also increased following ESC administration with little systemic toxicity in H22-transplanted mice. Mechanistically, ESC caused obvious G2/M-phase arrest in both the SK-HEP-1 and HepG2 cell lines without cell apoptosis. Furthermore, cyclin B1 was downregulated, while the phosphorylation level of CDK1 was increased in response to ESC treatment. All these data confirmed that ESC possesses potent anti-proliferative efficacy for hepatocarcinoma through the induction of cyclin-mediated cell cycle arrest. Thus, ESC is a promising candidate for hepatocarcinoma treatment in the future.

[Protective effect and mechanism of compound Ginkgo biloba granules on oxidative stress injury of HUVEC].

To reveal the protective and anti-apoptosis effect of compound Ginkgo biloba granules on oxidative stress injury of human umbilical vein endothelial cells (HUVEC). Negative control group, H2O2 model group and 4 drug pretreatment groups (80, 160, 320, 640 mg• L⁻¹) were established. The cell proliferation, morphological changes in each group after oxidative stress injury was detected by MTT assay and through microscope observation respectively. The content of LDH, MDA, SOD and NO and SOD activity in supernatant were detected to judge the protection effect of the drugs on endothelial cells. The protective effect on HUVEC apoptosis was analyzed by Caspase-3 activity test and Annexin V-FITC/PI staining. Western blot was used to observe the expression of apoptosis-related proteins Bcl-2 and Bax. Results showed that 1 200 µmol• L⁻¹ H2O2 can induce oxidative stress injury in endothelial cells and reduce the cell survival rate; cell proliferation inhibition degree is positively correlated with the effect time of H2O2. Besides, 80, 160, 320 640 mg•L⁻¹ compound Ginkgo biloba granules can protect HUVEC from oxidative stress injury, recover the normal proliferation level of cells, improve their state, prohibit cell apoptosis, and can up-regulate and down-regulate the expression level of Bcl-2 and Bax respectively. In conclusion, compound G. biloba granules can protect HUVEC from the oxidative stress injury induced by H2O2, its mechanism may be correlated with inhibition of the mitochondrial apoptotic pathway in HUVEC.

ESC reverses epithelial mesenchymal transition induced by transforming growth factor-ß via inhibition of Smad signal pathway in HepG2 liver cancer cells.

BACKGROUND: Epithelial mesenchymal transition (EMT) mediated by TGF-ß pays an important role in malignant tumor acquired abilities of migration and invasion. Our previous study showed that the extract of Stellera chamaejasme L. (ESC) was against proliferation of a variety of tumor cells, but there were no studies in the effects of ESC on EMT in tumor cells. In this study, TGF-ß was adopted to induce EMT in HepG2 cells and the influence of ESC on EMT was observed. METHODS: MTT assay was used to observe the cell viability. Wound healing assay and transwell assay were used to observe the migration and invasion activities. Western blot and immunofluorescence methods were used to observe the expression of proteins. RESULTS: We found that HepG2 cells induced by TGF-ß showed mesenchymal morphology, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker Vimentin, indicating that TGF-ß could mediate epithelial mesenchymal induction in HepG2 cells. ESC could reverse the mesenchymal morphology and regulate expressions of marker proteins in HepG2 induced by TGF-ß and significantly inhibit TGF-ß induced HepG2 cell migration and invasion. We further found that ESC could also significantly depress Smad2 phosphorylation and nuclear translocation, and ESC had coordination with SB432542, a specific inhibitor of TßRI kinases. CONCLUSIONS: These results suggested that the ESC could reverse epithelial mesenchymal transition induced by TGF-ß via inhibition Smad2 signaling pathway.

Chamaejasmin B exerts anti-MDR effect in vitro and in vivo via initiating mitochondria-dependant intrinsic apoptosis pathway.

Multidrug resistance (MDR) is the main obstacle limiting the efficacy of cancer chemotherapy. Looking for novel anti-MDR agents is an important way to conquer cancer drug resistance. We recently established that chamaejasmin B (CHB), a natural biflavone from Stellera chamaejasme L., is the major active component. However, its anti-MDR activity is still unknown. This study investigated the anti-MDR effect of CHB and the underlying mechanisms. First, it was found that CHB inhibited the growth of both sensitive and resistant cell lines in vitro, and the average resistant factor (RF) of CHB was only 1.26. Furthermore, CHB also displayed favorable anti-MDR activity in KB and KBV200 cancer cells xenograft mice. Subsequent study showed that CHB induced G0/G1 cell cycle arrest as well as apoptosis both in KB and in resistant KBV200 cancer cells. Further studies showed that CHB had no influence on the level of Fas/FasL and activation of procaspase 8. However, CHB-induced apoptosis was dependent on the activation of caspase 9 and caspase 3. Moreover, CHB treatment resulted in the elevation of the Bax/Bcl-2 ratio, attenuation of mitochondrial membrane potential (ΔΨm), and release of cytochrome c and apoptosis-inducing factor from mitochondria into cytoplasm both in KB and KBV200 cells. In conclusion, CHB exhibited good anti-MDR activity in vitro and in vivo, and the underlying mechanisms may be related to the activation of mitochondrial-dependant intrinsic apoptosis pathway. These findings provide a new leading compound for MDR therapy and supply a new evidence for the potential of CHB to be employed in clinical trial of MDR therapy in cancers.

[Protection of Shenlian extracts to PM2.5 infected RAW 264.7 cell damage].

The aim of this research is to investigate the protection of PM2.5 infected RAW264.7 cell by traditional Chinese medicine (TCM)--Shenlian(SL) extracts and to establish the damage model. We use cell growth, cell damage and oxidative stress related markers, and inflammatory cytokines as observation index to evaluate the protection of PM2.5 infected RAW264.7 by SL extract. The results showed that 50 mg x L(-1) PM2.5 could cause cell particle deposition, inhibit the growth of cells, and significantly increase the cell supernatant of LDH, NO release quantity and intracellular reactive oxygen species (ROS) level during 4 h and 24 h. In the intervention of SL extract 50, 25, 10 mg x L(-1), the particle deposition of RAW264.7 cells, cell supernatant of LDH, NO, IL(-1) beta release, MCP-1 was significantly decreased, the SOD activity increased significantly. It shows that SL extracts of PM2.5 infected RAW264.7 cell damage has obvious protective effect, the effect may be related to the direct protection of cells, reduce oxidative stress and inflammatory injury.

Cytotoxic biflavones from Stellera chamaejasme.

Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2″ and 3″ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 µM, respectively. Moreover, some structure-function relationships of these bioflavones for cytotoxic activities were explored and summarized.

[Effect on M1 macrophages of shenlian extracts].

This study discusses the effects of Shenlian extracts (SL) on M1 macrophages in atherosclerosis. The MTT assay was used to detect the growth inhibition rates of RAW264.7 cells. RAW264.7 cells were stimulated with murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) to induce M1 macrophages. The different concentrations of SL extracts (high-dose 50 mg x L(-1), moderate-dose 25 mg x L(-1), low-dose 12.5 mg x L(-1)) were added. The CD86 of M1 macrophages in cell membrane was measured by flow cytometry. The mRNA expression of iNOS and TNF-alpha gene was detected by reverse transcription PCR (RT-PCR). And the supernatants were collected, the content of IL-6 and TNF-alpha were detected with ELISA kits. The results of this experiment show that the expression of the cell membrane molecule CD86, iNOS and TNF-alpha gene, the content of IL-6 and TNF-alpha was obviously increased in M1 macrophages by IFN-gamma and LPS. The different doses of SL extract could reduce the expression of the above indicators. The above experimental results demonstrate that IFN-gamma combined LPS can induce RAW264.7 cell to type into M1 macrophages, and SL extracts can inhibit M1 macrophages.