Diisoprenyl Cyclohexene-Type Meroterpenoids with Cytotoxic Activity from a Mangrove Endophytic Fungus Aspergillus sp. GXNU-Y85.

Five new diisoprenyl cyclohexene-type meroterpenoids, aspergienynes J-N (1-5), along with three known analogues (6-8), were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85. The chemical structures, including their absolute configurations, were established via spectroscopic data and comparison of experimental and calculated ECD spectra. Cytotoxicity assay results indicated that compound 8 had strong cytotoxicity against HeLa cancer cells, and its IC50 value was 11.8 µM. In addition, flow cytometry analysis revealed that the cytotoxicity of 8 was due to the induction of G1 cell cycle arrest and apoptosis in HeLa cells.

Diisoprenyl-cyclohexene-type meroterpenoids from a mangrove endophytic fungus Aspergillus sp. GXNU-Y65 and their anti-nonalcoholic steatohepatitis activity in AML12 cells.

Nine previously undescribed diisoprenyl-cyclohexene-type meroterpenoids, aspergienynes A-I, together with five known analogues, were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y65. The diisoprenyl-cyclohexene-type meroterpenoids were elucidated based on multispectroscopic analysis, and the previously undescribed compounds' absolute configurations were established via electronic circular dichroism calculations. Biological activity results indicated that aspergienyne C (compound 3) had strong anti-nonalcoholic steatohepatitis activity against AML12 cells treated with PA (Palmitic acid) + OA (Oleic acid). At the same concentration of 20 µM, 3 significantly reduced triglyceride (TG) content compared with fenofibrate (positive control) in PA + OA treated AML12 cells, and obviously increased phosphorylation of acetyl-CoA carboxylase.

A Network Pharmacology Approach to Explore the Mechanisms of Shugan Jianpi Formula in Liver Fibrosis.

PURPOSE: We explored the mechanism of Shugan Jianpi Formula (SGJPF) and its effective components for the treatment of liver fibrosis (LF). MATERIALS AND METHODS: We collected the active ingredients in SGJPF through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and screened the effective components by absorption, distribution, metabolism, and excretion. Herb-associated target proteins were predicted and screened based on the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine and Search Tool for Interactions of Chemicals databases. LF-associated target proteins were predicted and screened based on the Online Mendelian Inheritance in Man® Database and Comparative Toxicogenomics Database. Common genes with LF and herbs were selected, and Cytoscape 3.5.1 software was used to construct an herb pathway and component-LF common target network. The Search Tool for the Retrieval of Interacting Genes/Proteins was used to build a protein-protein interaction, and quantitative PCR was used to verify the related target genes. Finally, clusterProfiler was applied for the analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. RESULTS: The pharmacological network contained 252 active compounds (e.g., Astragaloside A, saikosaponin, linoleic acid, and Poria acid A), 84 common target genes, and 94 significant signaling pathways. Among them, interleukin 6 (IL-6), tumor protein 53 p53 (TP53), prostaglandin-endoperoxide synthase 2 (PTGS2), AKT1, IL-1ß, and the nucleotide-binding and oligomerization domain-like receptor and Janus kinase-signal transducer and activator of transcription signaling pathways were selected as the critical target gene and critical signal pathway, respectively. CONCLUSION: The mechanisms of SGJPF in protecting against LF include the regulation of multiple targets such as IL-6, TP53, PTGS2, and AKT1. These target proteins affect LF through various signal transduction pathways.

Effect of astragalosides on long non-coding RNA expression profiles in rats with adjuvant-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, which occurs in ~1.0% of the general population. Increasing studies have suggested that long non­coding RNAs (lncRNAs) may serve important roles in various biological processes and may be associated with the pathogenesis of different types of disease, including RA. Astragalosides (AST) has been used as a traditional Chinese medicine for the treatment of RA. However, the mechanism underlying its therapeutic effect has remained unclear to date. Thus, there is an urgent need to elucidate the possible mechanism of AST in the treatment of RA from the perspective of lncRNAs. In the present study, the lncRNAs and mRNAs of a vehicle group, animal model group and AST treatment (control) group were determined by Arraystar Rat lncRNA/mRNA microarray. The differentially expressed genes with a fold change >1.5 and P<0.05 were selected and analyzed. Gene Ontology (GO) and pathway analysis was performed using the Database for Annotation, Visualization and Integration Discovery, and the coding­non­coding gene co­expression network was drawn based on the correlation analysis between the differentially expressed lncRNAs and mRNAs. Based on node degree and the correlation between bioinformatics analysis and RA, the critical differentially expressed lncRNAs were selected, analyzed and verified by reverse transcription­quantitative PCR (RT­qPCR) analysis. The results showed that, following AST treatment, up to 75 lncRNAs and 247 mRNAs were found to be differentially expressed among the three groups. GO and pathway analysis manifested that 135 GO terms and 17 pathways were enriched by differentially expressed genes. Four lncRNAs (MRAK012530, MRAK132628, MRAK003448 and XR_006457) were selected as the critical lncRNAs and their trend in expression showed consistency between the RT­qPCR and microarray data. In conclusion, AST had a regulatory effect on differentially expressed lncRNAs during the development of RA, and four lncRNAs could be selected as critical therapeutic targets of AST.

Effect of hesperidin on TGF-beta1/Smad signaling pathway in HSC.

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.

Tranilast attenuates TGF-ß1-induced epithelial-mesenchymal transition in the NRK-52E cells.

We previously reported that tranilast can halt the pathogenesis of chronic cyclosporine nephrotoxicity in rats via the transforming growth factor-ß (TGF-ß) /Smad pathway, an important signaling system involved in epithelial-mesenchymal transition (EMT), but the exact underlying cellular mechanisms are not yet clear. Thus, by selecting TGF-ß1-induced normal rat kidney proximal tubular epithelial cells (NRK-52E) as a model, we demonstrated potential modifying effect of tranilast on EMT-induced by TGF-ß1 in vitro. NRK-52E cells were incubated with the blank vehicle (Dulbecco's modified Eagle's medium and F-12 (DMEM/F12) added with 10% fetal bovine serum (FBS)), 10 ng/ml TGF-ß1 alone or together with 100, 200 or 400µM tranilast for 48 h after incubation in medium containing 1% FBS for 24 h. Cell morphological changes were observed to confirm occurrence of EMT. Protein expressions of two typical markers of EMT, E-cadherin and α-smooth muscle actin (α-SMA), were assessed by western blotting and flow cytometry, respectively. Our results showed that TGF-ß1 induced spindle-like morphological transition, the loss of E-cadherin protein and upregulation of expression of α-SMA. However, the TGF-ß1-produced changes in cellular morphology, E-cadherin and α-SMA were inversed by tranlilast in concentration-dependent manner. Our findings indicate that tranilast can directly inhibit EMT. Thus, it may be implied that regulation of EMT be the target to prevent renal tubulointerstitial fibrosis.

[Dynamic study on curative effect of Shuganjianpifang against hepatic fibrosis induced by CCl4 in rats].

OBJECTIVE: To observe the dynamic effects of Shuganjianpifang (SGJPF) in preventing rat hepatic fibrosis induced by CCl4. METHODS: The model rat was induced by subcutaneous injection of 50% CCl4 solution in olive oil (0.1 mL/100 g), twice a week for 12 weeks. The treatment groups were given by lavage with SGJPF and Colchicine respectively once a day for 16 weeks. After 4, 8, 12 and 16 weeks, six rats were randomly sacrificed in each group. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH) in serum as well as superoxide dismutase (SOD) and malondialdehyde (MDA) in hapatic tissues were examined by colorimetric methods dynamically. Meanwhile, the histopathologic changes of hepatic tissues were observed by the HE stained method. RESULTS: Compared with normal group, the levels of ALT and AST in serum and MDA in hepatic tissues were significantly elevated, the levels of SOD in hepatic tissues and GSH in serum were significantly decreased in model group after 4, 8, 12 and 16 weeks. Compared with model group, the levels of ALT and AST in serum and MDA in hepatic tissues were significantly reduced, the levels of SOD in hepatic tissues and GSH in serum were increased significantly after 8, 12 and 16 weeks in SGJPF group and Colchicine group. Histopathologic examination results also showed that SGJPF reduced the degree of liver fibrosis after 4, 8, 12 and 16 weeks. CONCLUSION: Shuganjianpifang can obviously inhibit the formation of liver fibrosis at different phases in rats.

CD133 silencing inhibits stemness properties and enhances chemoradiosensitivity in CD133-positive liver cancer stem cells.

Cancer stem cells (CSCs) are considered the source of the initial tumor formation and postoperative recurrence and metastasis. CD133(+) cells in hepatocellular carcinoma (HCC) display cancer stem-like properties and are thought to be responsible for chemoradioresistance. To explore the functional role of CD133 in liver cancer stem cells (LCSCs), we isolated CD133(+) cells from the HCC cell line HepG2, which were tested and confirmed to be CSC-like cells in HCC, downregulated CD133 expression in HepG2-CD133(+) cells by lentivirus-mediated short hairpin (shRNA) and analyzed the effects of CD133 on the modulation of stemness properties and chemoradiosensitivity in LCSCs. Our results showed that the in vitro cell proliferation, tumorsphere formation, colony formation and in vivo tumor growth in NOD/SCID mouse xenografts of LCSCs were significantly repressed after CD133 silencing. We also found that suppression of CD133 enhances the sensitivity of LCSCs to chemotherapy and radiotherapy. Knockdown of CD133 reduced G0/G1 phase cells and increased cellular apoptosis via modulation of Bcl-2 and Bax. Collectively, the stem-targeted therapy via CD133 could provide a novel strategy for the treatment of HCC.

[Study on the correlation between polymorphisms of genes with susceptibility to tuberculosis and drug-resistant tuberculosis in Chinese Han population].

OBJECTIVE: To investigate the distribution of polymorphisms of SLC11A1 gene, VDR gene, MBL gene and IFNG gene with susceptibility to tuberculosis (TB) in Chinese Han population suffering from drug-sensitive TB and drug-resistant TB so as to identify the correlation between gene polymorphisms and the development of drug-resistant TB. METHODS: Single nucleotide polymorphisms (SNP) of VDR gene, SLC11A1 gene, MBL gene, IFNG gene were typed and analyzed by pyrosequencing, Real-time Probe and SNaPshot among 229 patients with drug-sensitive TB and 230 patients with drug-resistant TB. RESULTS: The polymorphic foci of VDR gene from the drug-sensitive TB group and the drug-resistant TB group showed no significant difference (P > 0.05). The genotype of INT4 site and allelic frequency of SLC11A1 gene for drug-sensitive TB group were significantly different from those for drug-resistant TB group (P = 0.031, 0.046). If recessive inheritance was assumed, the genotypes of INT4 site from the two groups were significantly different (OR = 5.756, 95%CI: 1.261 - 26.269, P = 0.011). Considering the relationship between OR values under various combination, our findings confirmed that the genetic mode of INT4 site was in accordance with recessive inheritance. The genotypes of Q/P site and allelic frequencies of MBL gene from drug-sensitive and drug-resistant groups were significantly different (P = 0.029, 0.033). The difference still existed under the hypothesis of recessive inheritance (OR = 9.290, 95%CI: 1.167 - 73.949, P = 0.011). The polymorphic foci of IFNG gene from the two groups showed no significant difference. CONCLUSION: INT4 sites on SLC11A1 gene and Q/P site on MBL gene were probably associated with the development of drug-resistant TB in Chinese Han population. Further study on this issue would be helpful in locating the population at high risk of drug-resistant TB and exploring the effective intervention to decrease the incidence of this disease.

[The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in 459 Han patients].

OBJECTIVE: To study the correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of tuberculosis in Han population. METHODS: Four hundred and fifty-nine tuberculosis inpatients of Han population in Shanghai Pulmonary Hospital from Jan 2007 to Dec 2008 were recruited. The clinical characteristics of tuberculosis (gender, fever, extent of lesions, cavity formation, hemoptysis, initial treatment and retreatment) were observed. The polymorphisms of VDR gene (variants in FokI and TaqI), NRAMP1 gene (variants in INT4, D543N and 3 UTR), MBL gene (variants in HL, YX and QP) and IFNG gene (variants in 874AT) were genotyped by a variety of SNP genotyping techniques. The correlation between polymorphisms of genes with susceptibility to tuberculosis and the clinical characteristics of the disease was analyzed by ANOVAs. RESULTS: The frequency of CC, CT and TT variants of FokI in VDR gene in cases with fever were 54.7% (29/53), 13.2% (7/53) and 32.1% (17/53), respectively, compared to 40.6% (52/128), 30.5% (39/128) and 28.9% (37/128) in cases without fever, the difference being significant (χ² = 6.183, P < 0.05). In patients with CT variants, 15.2% (7/46) had fever, while in patients with non-CT variants, 34.1% (46/135) had fever (χ² = 5.891, P < 0.05), suggesting that patients with CT variants were less likely to have fever. The frequencies of TT + TC and CC variants of QP in the MBL gene in initial treatment cases were 28.3% (60/212) and 71.7% (152/212), respectively, compared to 19.1% (41/215) and 80.9% (174/215) in retreatment cases, the difference being significant (χ² = 5.038, P < 0.05). No significant correlation was observed between the other variants and the clinical characteristics of tuberculosis (χ² = 0.001 - 2.732, P > 0.05). CONCLUSIONS: The polymorphisms of FokI in VDR gene was associated with fever among the clinical characteristics of tuberculosis, and patients with CT variants might be protected from fever. The polymorphisms of QP in MBL gene might be associated with recurrence of tuberculosis.

[Study on revising the criteria of classification of hazard conditions of productive dust].

OBJECTIVE: To provide scientific basis data for revising the national hygiene criteria of "Classification of hazard conditions of productive dust" (GB5817-86). METHODS: The data of the retrospective study and the field survey data were analyzed with correlation and regression analysis. The product of total dust concentration of respiratory exposure (mg/m(3)), total ventilation during exposure (m(3)/d per psrson), and level of free SiO(2) in dust (%) was the respiratory exposure dose of free SiO(2) (mg per day per person) which was used as dose criteria value of classification of hazard degree of dust. RESULTS: Using free SiO(2) exposure dose and the dose-effect relationship, the hazard degrees of the dust were divided into 5 grades: 0, I, II, III, IV (0 - 8.0, 8.1 - 12.0, 12.1 - 16.0, > 24.1 mg per day per person). CONCLUSION: The exposure dose of free SiO(2) is closely related to the pathogenesis of silicosis. Using the exposure dose of free SiO(2) as the classification indicator of hazard degree of dust is reliable, simple and easy to execute.

[p33(ING1) gene expression and mutation in stomach cancer tissues and precarcinomatous tissues].

OBJECTIVE: To analyze the relationship of p33(ING1) gene expression and p33(ING1) exon-2 mutation to the pathogenesis, development and consequence of stomach cancer. METHODS: Envision immunohistochemical method was utilized to detect the p33(ING1) expression in 103 specimens of stomach cancer, 36 specimens of stomach mucosal atypical hyperplasia, and 32 specimens of normal stomach mucosa. PCR-SSCP was utilized to detect p33(ING1) exon-2 mutation in stomach cancer tissues. RESULTS: The p33(ING1) expression rate in stomach cancer was 54.4% (56/103), significantly lower than that in precarcinomatous tissues (94.4%, 34/36, P < 0.01) and that in normal tissues (100%, 32/32, P < 0.01). The p33(ING1) expression in stomach cancer was related to tumor growth, distant metastasis and tumor differentiation (all P < 0.05). p33(ING1) gene exon-2 mutation was detected in 3 cases of stomach cancer tissues (12%, 3/25), and not in other tissues by PCR-SSCP method. CONCLUSION: p33(ING1) low expression, and gene p33(ING1) exon-2 mutation may play an important role in the pathogenesis, development and consequence of stomach cancer.