Exploring the potential molecular mechanism of Gualou Guizhi decoction in the treatment of rheumatoid arthritis based on network pharmacology and molecular docking.

BACKGROUND: Traditional Chinese medicine (TCM) has been used in China for a long time and is gradually gaining more and more recognition worldwide. Gualou Guizhi Decoction (GGD) has long been used as a folk medicine for the treatment of rheumatic diseases, but its bioactive components and therapeutic mechanisms are still unclear. METHODS: An integrated approach using network pharmacology and molecular docking and using methotrexate as a positive control drug. RESULTS: We obtained 157 active ingredients of GGD, 7542 RA disease targets and 49 intersecting targets. GO and KEGG enrichment analysis revealed that their functions were mainly related to cytokine active metal ion binding, enzyme binding and DNA binding, and enriched in TNF signaling pathway, T cell receptor signaling pathway, Toll-like receptor signaling pathway, RA pathway and other signaling pathways that are closely related to RA. The molecular docking results show that the effector components of GGD bind better to the core targets of RA, and some are even better than methotrexate. CONCLUSION: The therapeutic effect of GGD for RA is achieved by affecting the core targets such as VEGFA, IL-1ß, IL6, CXCL8, CCL2, and JUN, which together interfere with the tumor necrosis factor signaling pathway and RA pathway to treat RA. The above study provides new ideas for further exploration of this classic formula in the future.

[Mechanism of Chaenomelis Fructus in treatment of rheumatoid arthritis based on network pharmacology and experimental verification].

The material basis and mechanism of Chaenomelis Fructus in the treatment of rheumatoid arthritis(RA) were explored by network pharmacology, and the potential anti-RA targets of Chaenomelis Fructus were verified by molecular docking and animal experiments. The active components and targets of Chaenomelis Fructus were searched against the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform. GeneCards, DisGeNET, and OMIM were used to obtain RA-related targets. The common targets shared by Chaenomelis Fructus and RA were considered as the potential targets of Chaenomelis Fructus in the treatment of RA. Cytoscape 3.9.0 was employed to establish a "traditional Chinese medicine-active component-common target-disease" network. The protein-protein interaction(PPI) network was established by STRING, and the core genes were visualized by RStudio 4.1.0. DAVID was used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment to predict and visualize the involved signaling pathways. Molecular docking was carried out with the active components screened out as ligands and RA core genes as the targets. Finally, the prediction results were verified by animal experiments. Four main active components of Chaenomelis Fructus were obtained, which corresponded to 137 targets. Chaenomelis Fructus and RA shared 37 common targets. GO annotation yielded 239 terms(P<0.05), and KEGG pathway enrichment analysis screened out 94 signaling pathways(P<0.05), mainly involving interleukin-17(IL-17), tumor necrosis factor, Toll-like receptor, and nuclear factor-kappa B(NF-κB) signaling pathways. Molecular docking results showed that the main active components of Chaenomelis Fructus bound well with the core targets of RA. The results of animal experiments proved that Chaenomelis Fructus can alleviate joint swelling in the mice with RA. The results of ELISA showed that Chaenomelis Fructus lowered the levels of interleukin-6(IL-6) and interleukin-1ß(IL-1ß). Western blot showed that Chaenomelis Fructus down-regulated the protein level of vascular endothelial growth factor A(VEGFA). Chaenomelis Fructus exerts anti-inflammatory effect and reduces pannus formation by regulating the core targets such as VEGFA, IL-1ß, and IL6 in the treatment of RA. The findings of this study provide new ideas for the future treatment of RA with Chaenomelis Fructus.

Exploring the chondroprotective effect of Chaenomeles speciosa on Glucose-6-Phosphate Isomerase model mice using an integrated approach of network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has been used in China for a long time and is gradually gaining more and more recognition worldwide. Chaenomeles speciosa (CSP) (Chinese Pinyin: mugua) is a medicinal and food herb that has long been used as a folk medicine for rheumatic diseases, yet its bioactive components and therapeutic mechanisms are not clear. AIM OF THE STUDY: Exploring anti-inflammatory and chondroprotective effects of CSP on rheumatoid arthritis (RA) and its possible targets of action. MATERIALS AND METHODS: In this study, we performed an integrated approach of network pharmacology, molecular docking and experimental studies to explore the potential mechanism of action of CSP in the treatment of cartilage damage in RA. RESULTS: Studies have shown that Quercetin, ent-Epicatechin and Mairin may be the main active compounds of CSP in the treatment of RA, while AKT1, VEGFA, IL-1ß, IL-6, MMP9 etc. are considered as core target proteins to which the main active compounds in CSP bind, as further confirmed by molecular docking. In addition, the potential molecular mechanism of CSP for the treatment of cartilage damage in RA predicted by network pharmacology analysis was validated by in vivo experiments. CSP was found to downregulate the expression of AKT1, VEGFA, IL-1ß, IL-6, MMP9, ICAM1, VCAM1, MMP3, MMP13 and TNF-α and increase the expression of COL-2 in the joint tissue of Glucose-6-Phosphate Isomerase (G6PI) model mice. Thus CSP contributes to the treatment of rheumatoid arthritis cartilage destruction. CONCLUSION: This study showed that CSP has multi-component, multi-target and multi-pathway characteristics in treating cartilage damage in RA, which can achieve the effect of treating RA by inhibiting the expression of inflammatory factors, reducing neovascularization and alleviating the damage to cartilage caused by the diffusion of synovial vascular opacities, and reducing the degradation of cartilage by MMPs to play a protective role in RA cartilage damage. In conclusion, this study indicates that CSP is a candidate Chinese medicine for further research in treating cartilage damage in RA.

In Vitro Toxicological Investigation and Risk Assessment of E-Cigarette Aerosols Based on a Novel Solvent-Free Extraction Method.

Cigarettes, potentially safer alternatives to combustible cigarettes, have been reported to increase the health risk for long-term users, so accumulating information about their potential toxicity is of great concern. However, toxicological evaluations of e-cigarette aerosols are limited, which may be attributed to the lack of a simple and efficient extraction method. Here, we developed a high-speed centrifugal method for extracting e-cigarette aerosol collected mass (ACM) and prepared ACM samples of 26 representative e-cigarettes, and 10 samples were further selected based on their cytotoxicity for systematic toxicological assessments. The average extraction efficiency of ACM, primary aerosol components, and typical carbonyls exceeded 85%. The toxicological evaluation showed that the IC50 value range of e-cigarettes for cytotoxicity was 2-52 mg/mL ACM, all e-cigarettes can induce the risk of DNA damage, mitochondrial depolarization, and c-Jun-related signal disturbances; most e-cigarettes significantly caused disturbance of oxidative stress balance. E-cigarettes with higher cytotoxicity appeared to cause a higher degree of damage, while no e-cigarette promoted mutagenicity and cytochrome c release. The toxicity difference among e-cigarettes using nicotine equivalent was significantly lower than that of ACM. This study provides a novel extraction method and a comprehensive in vitro toxicity risk profile of e-cigarette aerosols.

Novel Solvent-Free Extraction Method for Analyzing Tobacco Heating Product Aerosols: An Analytical and In Vitro Toxicological Five-Way Product Comparison.

Harmful and potentially harmful constituents (HPHCs) in tobacco smoke are thought to be responsible for the increased health risks. Tobacco heating products (THPs) heat tobacco instead of burning it to achieve significantly fewer toxicants than conventional cigarettes. To assess the toxicity of THP aerosols, it is often desirable to extract the main constituents using a solvent method. In this study, we developed a high-speed centrifugal method for extracting the total particulate matter (TPM) from THPs to quantitatively compare the toxicity of different THPs and conventional cigarettes. Its TPM extraction efficiency exceeded 85%, and the primary aerosol components and typical HPHCs were comparable to those of the solvent method. The TPMs extracted from five THPs were subjected to 14 in vitro toxicology assessments, and the results were compared with those of a 3R4F reference cigarette. Physical separation can improve biases from solvent selectivity and potential interactions between solvent and aerosol constituents. By eliminating solvent influence, the extraction method could achieve high-dose exposures, enabling the toxicity comparison of different THPs. The relative toxicity of the THPs differed under different dosage units, including the TPM concentration, nicotine equivalent, and puff number.

In vitro toxicological evaluation of a tobacco heating product THP COO and 3R4F research reference cigarette on human lung cancer cells.

Cigarette smoking increases health risks, such as respiratory diseases and heart diseases. Despite the decline in smoking rates in some countries, millions of adults still choose to smoke cigarettes. The use of next-generation nicotine delivery devices, such as tobacco heating products (THPs), may become a potentially safer alternative to smoking. Here, we report on the development of an electrically heated THP, coded as THP COO, with three different flavored tobacco sticks. The purpose of the study was to measure the levels of a list of harmful and potentially harmful constituents (HPHCs) in the total particulate matter (TPM) generated and to conduct a set of toxicological assessments of THP COO as compared with 3R4F reference cigarette. For all 55 HPHCs identified, the levels generated by the THP tobacco sticks were significantly lower in comparison to those in 3R4F TPM. The rate of reduction of HPHCs was between 68.6% and 99.9% under Health Canada Intense (HCI) smoking regimen. Human lung cancer cells (NCI-H292) exposed to 3R4F TPM showed dose-dependent responses for most of the 15 in vitro toxicity endpoints, whereas those exposed to comparable doses of THP COO TPMs did not. Therefore, exclusive use of the THP COO products may reduce the exposure of those tested HPHCs and thus potentially reduce health risk of smoking.

[Shikimic acid extracted from Chaenomeles speciosa reduces chondrocyte differentiation by inhibiting RBL-2H3 cell degranulation].

Objective To investigate the reducing effects of shikimic acid from the total extract of Chaenomeles speciose on the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation. Methods The chondrocytes were identified by toluidine blue staining and tryptase immunohistochemical staining. The chondrocytes were divided into normal chondrocytes control group, C48/80 activated RBL-2H3 cell culture supernatant treatment group, 3, 10 and 30 µg/mL SA activated RBL-2H3 cell culture supernatant treatment groups. The toxicity of SA and RBL-2H3 cell supernatant were detected by MTT assay. Western blotting was used to detect the expression of collagen type II (Col2) and collagen type X (Col10) in chondrocytes. The levels of matrix metalloproteinase 13 (MMP13), soluble nuclear factor B receptor activated protein ligand (sRANKL) and bone protective factor (OPG) were determined by ELISA, and glycosaminoglycan polysaccharide (GAG) were tested by dimethylmethylene blue (DMB) colorimetry. Results (0~30) µg/mL SA had no significant effects on the growth of chondrocytes. Compared with the C48/80 activated RBI-2H3 cell supernatant treatment group, the expression of Col2 and GAG proteins increased significantly, while the expression of Col10 and MMP13 and the ratio of sRANKL/OPG decreased significantly in the SA treatment groups in a dose-dependent manner. Conclusion SA can effectively reduce the differentiation of chondrocytes into hypertrophic chondrocytes by inhibiting RBL-2H3 cell degranulation.

Activation of NLRP3 inflammasome promotes the proliferation and migration of esophageal squamous cell carcinoma.

Inflammasomes can identify endogenous danger signals as an inflammatory immune response. As the most common inflammasome, the NLR pyrin family domain containing 3 (NLRP3) inflammasome is associated with the pathogenesis of different tumors. However, the function of the NLRP3 inflammasome in esophageal cancer (EC) has rarely been reported. Herein, the expression levels of the components of NLRP3 inflammasome and Ki­67 were analyzed by immunohistochemistry. Furthermore, correlations between the NLRP3 inflammasome and Ki­67 along with the clinicopathological features of EC patients were evaluated. The components of the NLRP3 inflammasome were also assessed by western blot analysis and quantitative PCR. NLRP3 was silenced or overexpressed in different esophageal squamous cell carcinoma (ESCC) cell lines, and cell viability, migration and invasion were assessed by CCK­8 and Transwell assays. The present results showed that high NLRP3 expression in the tumor specimens was significantly associated with TNM stage and T category. Spearman's correlation analysis revealed a positive correlation between NLRP3 and the Ki­67 proliferation index. The mRNA and protein levels of NLRP3, apoptosis­associated speck­like protein containing a CARD (ASC), cleaved caspase­1, and interleukin (IL)­1ß in tumor tissues were higher than those in non­cancerous tissues. The level of secreted IL­1ß in tumor tissues was also increased, as compared to that in normal tissues. Silencing of NLRP3 in KYSE­70 and TE13 cells strongly attenuated cell viability, decreased cell mobility in wound­healing assays and greatly diminished the ability of cell migration and invasion in the Transwell system. Overexpression of NLRP3 in KYSE­510 and EC9706 cells markedly promoted the proliferation, migration and invasion. Collectively, these results revealed that the the NLRP3 inflammasome is upregulated in human ESCC tissues and promotes ESCC progression. Hence, NLRP3 could be a promising new candidate diagnostic and prognostic target.

Characterization of cluster of differentiation 47 expression and its potential as a therapeutic target in esophageal squamous cell cancer.

The increased expression of cluster of differentiation (CD)47 has been identified in a number of different tumor types and is recognized as an adverse prognostic factor that indicates an increased risk of mortality in patients. The binding of CD47 to signal regulatory protein α (SIRPα) inhibits the macrophage phagocytosis of tumor cells by triggering an inhibitory 'do not eat me' signal. This is one of the mechanisms used by tumor cells to evade immune surveillance. In the present study, CD47 levels and macrophage infiltration were assessed in patients with esophageal squamous cell cancer (ESCC). CD47-overexpressing ESCC cell lines were selected and human M2 macrophage phagocytic activity was measured. The results revealed that CD47 is highly expressed and macrophages are markedly infiltrated in cancerous tissue compared with non-cancerous tissue. High CD47 expression was detected in ESCC cell lines and the results of a phagocytosis assay indicated that human M2 macrophages phagocytized tumor cells in a dose-dependent manner following the blocking of CD47-SIRPα signaling by anti-CD47 antibodies. The results of the present study therefore support the use of anti-CD47 immunotherapy to treat patients with ESCC.

[Shikimic acid inhibits the degranulation and histamine release in RBL-2H3 cells].

Objective To study the effects of shikimic acid on the proliferation of rat RBL-2H3 cells and the degranulation of the cells induced by C48/80 and its mechanism. Methods MTT assay was performed to measure the proliferation of RBL-2H3 cells treated with 3, 10, 30 µg/mL shikimic acid. Toluidine blue staining was used to observe the degranulation of RBL-2H3 cells. The release of ß-hexosaminidase from RBL-2H3 cells treated with 0, 12.5, 25, 50, 80, 100 µg/mL C48/80 was determined by substrate assay. ELISA was used to detect the histamine content in the supernatant of each treated group. Results Shikimic acid at 3, 10, 300 µg/mL had no obvious inhibitory effect on the proliferation of RBL-2H3 cells. There was a dose-effect relationship between the degranulation of RBL-2H3 cells and C48/80 concentration. Shikimic acid inhibited the degranulation of RBL-2H3 cells compared with the positive control group, the ß-hexosaminidase release rate and histamine release were significantly reduced in RBL-2H3 cells treated with shikimic acid and C48/80. Conclusion Shikimic acid can inhibit the degranulation of RBL-2H3 cells and reduce histamine release.

[Effects of pargyline on histone methylation in promoter and enhancer regions and transcription of CYP3A4/3A7].

This study was designed to investigate effects of pargyline on histone methylation in the promoter and enhancer regions and transcription of cytochrome P450 3A4/3A7 (CYP3A4/3A7) gene. Human primary fetal liver cells were isolated, cultured and randomly divided into several groups including control, solvent, pargyline low, middle, high dose (treated with 0.6, 1.2, 2.4 mmol·L(−1)). HepG2 cells were cultured and treated with 0.03, 0.3, 3 mmol·L(−1) pargyline. After 48 hours, total RNAs were prepared from the cells to determine the expression of CYP3A m RNA in primary fetal cells and HepG2 cells with real-time quantative PCR (qPCR). HepG2 cells were cultured and then treated with 3 mmol·L(−1) pargyline for 48 hours. The chromatin immunoprecipitation (ChIP) assay was performed with dimethylation of histone H3 at lysine 4 (H3K4me2), and IgG antibodies respectively. The precipitated DNA was resuspended and used for qPCR. Primers were used to detect different regions of CYP3A4/3A7 promoter and enhancer. Occupancy of H3K4me2 was shown as percent of input DNA relative to control cells. The results suggested that pargyline has an effect on primary fetal liver cells and HepG2 cells proliferation. The level of CYP3A7 was markedly enhanced in human primary fetal liver cells by treatment with 1.2, 2.4 mmol·L(−1) of pargyline (P < 0.05, P < 0.01) and the levels of CYP3A4/3A7 were remarkably enhanced by treatment with 3 mmol·L(−1) of pargyline in HepG2 cells (P < 0.001) compared with solvent control. Occupancy of H3K4me2 on human CYP3A4 promoter (−362 to +53) and enhancer segment (−7 836 to −6 093) harbored the overlapping hepatocyte nuclear factors 4A (HNF4A) binding site compared with a negative control. Occupancy of H3K4me2 on human CYP3A7 promoter (−163 to +103) and enhancer segment (−4 054 to −3 421, −6 265 to −6 247) overlapped with glucocorticoid receptor (GR) binding site. In conclusion, the enriched H3K4me2 in the promoter and enhancer regions was induced by pargyline with HNF4A or GR binding site in CYP3A4/3A7 gene to activate the corresponding genes.

[Total saponins of Chaenomeles speciosa inhibit the degranulation of primary mouse bone marrow-derived mast cells in vitro].

OBJECTIVE: To study the effects of total saponins of Chaenomeles speciosa on the degranulation of mouse bone marrow-derived mast cells (BMMCs). METHODS: Bone marrow cells were isolated from C57BL/6 mice and were cultured in RPMI1640 medium containing 100 mL/L fetal bovine serum, 20 ng/mL IL-3 and 40 ng/mL stem cell factor (SCF). After four-week culture, flow cytometry was used to identify the purity of double-positive (CD117(+)FcEpsilonRIα(+)) mast cells; toluidine blue was used to detect the maturity of mast cells. After total saponins of Chaenomeles speciosa was added into the medium of BMMCs, CCK-8 assay was performed to assess the toxic effects of total saponins of Chaenomeles speciosa on BMMCs; the amount of ß-hexosaminidase release was detected by fluorescence spectrophotometry; the content of tryptase in cell supernatants was detected by ELISA. RESULTS: After cultured for four weeks, the purity of double-positive cells was more than 95%, and the cells presented the features of mature mast cells, with blue nuclear and purple cytoplasm by toluidine blue staining. After BMMCs were cultured in the presence of total saponins of Chaenomeles speciosa (0.01, 0.03, 0.10 mg/mL) for 12 hours, CCK-8 assay indicated that the total saponins did not exert the toxic effects on the BMMCs. Fluorescence spectrophotometry revealed that the release amount and release rate of ß-hexosaminidase decreased in the cells treated with the total saponins of Chaenomeles speciosa, DNP-BSA and A23187 compared with the controls, and ELISA assay showed that the amount of tryptase release in the supernatants was reduced as well. CONCLUSION: Total saponins of Chaenomeles speciosa can inhibit the degranulation of primary mouse BMMCs stimulated by different antigens with a clear dose-effect relationship.

Two new asymmetric sesquiterpene dimers from the rhizomes of Ligularia muliensis.

Two new asymmetric eremophilane-type sesquiterpene dimers, ligulamulienin A (1) and B (2), were isolated from the rhizomes of Ligularia muliensis. Their structures were determined based on their spectroscopic data, including IR, EI-MS, HR-electrospray ionization (ESI)-MS, 1D and 2D-NMR spectroscopy. The cytotoxicity of compounds 1 and 2 was measured in in vitro human carcinoma (MGC-803), human hepatoma (HEP-G2), and murine sarcoma (S-180) cell lines.

TGF-beta1 signal pathway may contribute to rhabdomyosarcoma development by inhibiting differentiation.

Overexpression of transforming growth factor-beta1 (TGF-beta1) and its downstream molecules in the rhabdomyosarcoma (RMS) RD cell line has been reported previously, but the regulatory role of TGF-beta1 on RMS has not been studied extensively. In the present study, we showed that expression of TGF-beta1 and its downstream molecules type II TGF-beta receptor (TbetaRII) and Smad4 was significantly higher in RMS than in normal skeletal muscle, and there was a significant relationship between TGF-beta1 expression and histological grade. Gene silencing with TGF-beta1 short-hairpin RNA (shRNA)-expressing vectors significantly decreased the growth of RD cells, which was confirmed by caspase-3 (in vitro) and TUNEL (in vivo) assays. Moreover, a proportion of treated rhabdomyosarcoma (RD) cells changed to a round shape from the normal fusiform or polygonal shape and expressed myofilaments. Myogenin is one of the myogenic differentiation genes (MyoD) family of myogenic regulators, and was obviously higher in TGF-beta1-shRNA-treated tumors than it in control at the mRNA and protein level. Immunohistochemical staining with myogenic differentiation markers such as myosin and desmin in subcutaneous RMS tissue showed that TGF-beta1 shRNA increased staining for myosin. These results provide new insight into the biological function of TGF-beta1 in malignant tumors, and imply that the TGF-beta1 signal pathway is a potential therapeutic target for drugs that induce differentiation of RMS.

Prostate stem cell antigen enhancer and uroplakin II promoter based bladder cancer targeted tissue-specific vector.

PURPOSE: To construct a dual specific vector which contains prostate stem cell antigen enhancer (PSCAE) and uroplakin II (UPII) promoter targeted bladder cancer. METHODS: UPII promoter and PSCAE were amplified by polymerase chain reaction (PCR). Luciferase gene (LUC) was obtained from plasmid pBK-CMV-LUC. PSCAE, UPII promoter and LUC were inserted into shuttle plasmid to create Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. Rp-UPII-LUC and Rp-PSCAE-UPII-LUC were cotransfected with pCMV-beta-gal into various cell lines at the presence or absence of androgen receptor agonist R1881 and androgen receptor antagonist flutamide. Luminescence was detected with luciferase assay kit and counted on liquid scintillation counter. RESULTS: Bladder cancer cells showed higher LUC activity than non-bladder cancer cells after transfected with plasmids Rp-UPII-LUC and Rp-PSCAE-UPII-LUC. PSCAE could improve the LUC activity in both AR positive and AR negative bladder cancer cells but not in non-bladder cancer cells and normal human urothelial (NHU) cells. R1881 could increase the LUC activity in AR positive bladder cancer cells but not in AR negative bladder cancer cells and non-bladder cancer cells. Flutamide could not inactivate PSCAE in bladder cancer cells. CONCLUSIONS: PSCAE can improve target gene expression in bladder cancer cells but not in non-bladder cancer cells and NHU cells. PSCAE maintains a certain level of androgen independent activity in bladder cancer cells. PSCAE is active in both AR positive and AR negative bladder cancer cells. The results suggest that combination of PSCAE with UPII promoter is feasible in constructing bladder cancer-specific vectors.

[Mechanism of hedysari polysaccharide in the apoptosis of human hepatocellular carcinoma HEP-G2 cells].

OBJECTIVE: To study the effects of Hedysari polysaccharide (HPS) on the proliferation and apoptosis of human hepatocellular carcinoma HEP-G2 cells and its mechanism. METHODS: MTT assay, flow cytometry and fluorescence microscopy were used to the effects of HPS in anti-proliferation, cell cycle arresting and apoptosis induction. Flow cytometry was also used to detect the effects detect of HPS on protein expressions of Bcl-2 and Bax. RESULTS: HPS could cause apparent inhibition on cell growth, induce G2/M phase arrest and apoptosis on HEP-G2 cells. HPS could down-regulate bcl-2 protein expression and up-regulate Bax protein expression. CONCLUSION: HPS has anti-tumor effects, maybe by the way of inhibiting the cell growth, arresting the G2/M phase, inducing apoptosis, down-regulating bcl-2 and up-regulating Bax protein expression.

Selection of optimal sites for TGFB1 gene silencing by chitosan-TPP nanoparticle-mediated delivery of shRNA.

Most human tumors produce high levels of TGF-beta1, whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Currently, many experimental therapies that target TGFB1 have utilized antisense DNA or RNA interference (RNAi). Despite the great potential of RNAi, the selection of effective target sites and proper delivery systems for short hairpin RNA (shRNA) remains a significant issue. Here, we used chitosan nanoparticle-mediated delivery of a shRNA-expressing vector to inhibit TGFB1 expression in the human rhabdomyosarcoma cell line RD. Knockdown of TGFB1 by shRNA resulted in a decrease in RD cell growth in vitro and tumorigenicity in nude mice. The efficiency of TGFB1 gene silencing varied with the selection of targeting sites. These data suggest that chitosan nanoparticle-mediated delivery of an shRNA produces efficient TGFB1 knockdown in rhabdomyosarcoma cells and may be a method of choice for shRNA delivery for gene therapy.

Two hour exposure to sodium butyrate sensitizes bladder cancer to anticancer drugs.

OBJECTIVES: To investigate the inhibitory effect of sodium butyrate (NaB) on the proliferation of human bladder cancer cell lines and its synergetic effect with anticancer drugs in treating bladder cancer in vitro and in vivo. METHODS: The inhibitory effects of NaB on human bladder cancer cell lines in vitro and the synergetic effect of NaB with mitomycin c, cisplatin (CDDP) and adriamycin were detected by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Hoechst staining and electron microscopy were used to observe morphology for apoptotic cells after NaB treatment. Fas, bcl-2 and caspase-3 were determined with flow cytometry. In vivo synergetic effects were detected in N-methyl-N-nitrosourea induced bladder cancer model rats. RESULTS: NaB significantly inhibited the growth of bladder cancer cell lines in a concentration and time dependent manner. Better results of tumor inhibition have been achieved when NaB was combined with CDDP, mitomycin c and adriamycin, rather than used alone. Furthermore, 2 h exposure to NaB can sensitize bladder cancer to chemotherapy agents. The Bcl-2 expression in bladder cancer cells is decreased and caspase-3 expression increased after NaB treatment. Intravesical application of NaB combined with CDDP can significantly inhibit tumor growth and progression. CONCLUSIONS: NaB has a direct anticancer effect and can markedly enhance the action of several chemotherapy agents. 2 h expose to NaB can also sensitize bladder cancer to anticancer drugs. NaB may be an excellent candidate agent for intravesical application in treating bladder cancer.

Eremophilane-type sesquiterpene derivatives from the roots of Ligularia lapathifolia.

Six new highly oxygenated eremophilane-type sesquiterpene derivatives (1-6), including a norbisesquiterpene, were isolated from an extract of the roots of Ligularia lapathifolia, and their structures were elucidated by spectroscopic methods. The structure of 1 was confirmed by single-crystal X-ray crystallography. In addition, the cytotoxicity of compounds 1, 2, 3, 5, and 6 was evaluated against selected cancer cell lines, including human stomach carcinoma (MGC-803), human hepatoma (HEP-G2), and murine sarcoma (S-180) cell lines.

Preparation and suppressive effect of astragalus polysaccharide in glomerulonephritis rats.

Astragalus membranaceus (AM) has been widely used for treating kidney diseases in traditional Chinese medicine. In this study, the Astragalus polysaccharide (APS), the main active ingredient was isolated and purified from the Rhizomes of AM, which consisted of d-glucopyranose and had the molecular weight of 3.6x10(4) Da. The effect of APS on glomerulonephritis rats induced by cationic Bovine Serum Albumin(C-BSA) was evaluated by flow cytometry using Nuclear Transcription Factor-kappaB (NF-kappaB) as marker. Interleukin-2 (IL-2), Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha) were determined by the ELISA method. The rats (model group and treatment group) were injected subcutaneously with C-BSA plus incomplete Freund's adjuvant on day 0, C-BSA was injected through the caudal vein from week 2 to week 7 to induce glomerulonephritis. The rats (treatment group) were given APS by intraperitoneal injection from week 2 to week 7. The expression of NF-kappaB and the concentration of IL-2, IL-6 and TNF-alpha were significantly decreased in the treatment group. This study clearly suggests that APS is effective in protecting against glomerulonephritis induced by C-BSA through the inhibition of NF-kappaB mediated-cytokine pathway.