[Baicalin improves inflammatory response of human microglia by regulating cAMP-PKA-NF-κB/CREB pathway].

This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 µmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 µmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 µmol·L~(-1) BAI significantly increased the SOD activity and 5 µmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1ß, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.

Synthesis of a New Phenyl Chlormethine-Quinazoline Derivative, a Potential Anti-Cancer Agent, Induced Apoptosis in Hepatocellular Carcinoma Through Mediating Sirt1/Caspase 3 Signaling Pathway.

Quinazoline derivatives display multiple pharmacological activities and target various biological receptors. Based on the skeleton of quinazoline core, we designed and synthesized three new quinazoline-phenyl chlormethine conjugates (I-III) bearing a Schiff base (C = N) linker, and investigated their anti-tumor effects on HepG2-xenografted tumor and human cancer cell line HepG2. Among these compounds, compound II showed better inhibitory effect against HepG2 cells. In the present study, TUNEL staining, western blot, molecular docking, and siRNA were used to investigate the inhibitory mechanism of compound II towards hepatoma. Compound II inhibited HepG2-xenografted tumor growth in nude mice. Moreover, Compound II not only up-regulated Bax/Bcl-2 ratio and active-caspase 3 level, but also down-regulated Sirt1 expression and its activity, as well as PGC-1α expression. Furthermore, compound II also significantly suppressed the promotion of HepG2 cell proliferation, as evidenced by MTT assay and lactate dehydrogenase (LDH) release assay. Of note, the cytotoxicity of Compound II on HepG2 cells mainly via regulating Sirt1/caspase 3 signaling pathway, consisting with the results in vivo. Intriguingly, z-DEVD-FMK, a caspase 3 inhibitor, almost abolished the inhibitory effects of compound II. Of note, knockdown of caspase 3 by siRNA significantly reversed the inhibitory effect of compound II on HepG2. Interestingly, compound II directly bonded to Sirt1, indicating that Sirt1 might be a promising therapeutic target of compound II. In summary, our findings reveal that compound II, a new synthetical phenyl chlormethine-quinazoline derivative, contributes to the apoptosis of HepG2 cells both in vivo and in vitro through mediating Sirt1/caspase 3 singling pathway. These findings demonstrate that compound II may be a new potent agent against hepatocellular carcinoma.

LncRNA ADAMTS9-AS2 inhibits cell proliferation and decreases chemoresistance in clear cell renal cell carcinoma via the miR-27a-3p/FOXO1 axis.

Accumulating evidence reveals the principal role of long noncoding RNAs in the progression of clear cell renal cell carcinoma (ccRCC). However, little is known about the underlying mechanism of ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2 (ADAMTS9-AS2) in ccRCC. Here, bioinformatics analyses verified ADAMTS9-AS2 is a long noncoding RNA and its high expression was associated with better prognosis of ccRCC. ADAMTS9-AS2 was clearly downregulated in ccRCC clinical samples and cell lines. Clinical data showed low-expressed ADAMTS9-AS2 was correlated with worse overall survival in ccRCC patients. Next, miR-27a-3p was identified as an inhibitory target of ADAMTS9-AS2 by dual-luciferase reporter and RNA immunoprecipitation assays. Both overexpressed ADAMTS9-AS2 and underexpressed miR-27a-3p in ccRCC cell lines led to the inhibition of cell proliferation and the reduction of chemoresistance. Additionally, Forkhead Box Protein O1 (FOXO1) was confirmed as the inhibitory target of miR-27a-3p. Induced by ADAMTS9-AS2 overexpression, cell proliferation and chemoresistance exhibited an obvious reduction, FOXO1 expression showed an evident increase, but all were reversed after miR-27a-3p was simultaneously overexpressed. Collectively, these results suggest ADAMTS9-AS2 inhibits the progression and impairs the chemoresistance of ccRCC via miR-27a-3p-mediated regulation of FOXO1 and may serve as a prognostic biomarker and therapeutic target for ccRCC.

Effect of survivin downregulation by simvastatin on the growth and invasion of salivary adenoid cystic carcinoma.

Simvastatin, an inhibitor of 3­hydroxy­3-methylglutaryl­coenzyme A reductase, is been used in the clinic due to its pleiotropic effects, such as breast cancer, prostate cancer, pancreatic cancer. Simvastatin has recently been demonstrated to serve a potential role in the prophylaxis and therapeutics of a number of human cancers. The majority of reports concerning simvastatin treatment in the majority of human cancers have demonstrated that survivin is significantly decreased as a result and has been implicated in tumorigenesis. However, only a limited number of studies have investigated the use of simvastatin for the treatment of salivary gland adenoid cystic carcinoma (SACC). Therefore, this agent is a candidate for further investigation. The aim of the present study was to investigate the effects of simvastatin on the proliferation, invasion and apoptosis of the human salivary adenoid cystic carcinoma cell line, SACC­83, as well as survivin expression in the cells. The Cell Counting kit­8 assay results revealed that simvastatin inhibited the proliferation of SACC­83 cells in a dose­dependent (10 to 50 µM) and time­dependent (24 to 48 h) manner when compared with the untreated cells. Flow cytometry analysis indicated that simvastatin increased the percentage of cells in early and late apoptosis. Invasion assays revealed that simvastatin treatment inhibited the invasiveness of SACC­83 cells in a dose­dependent manner. In addition, simvastatin downregulated survivin expression in SACC­83 cells. In conclusion, simvastatin significantly inhibited the proliferation and invasion of SACC­83 cells, induced apoptosis, and reduced the expression of survivin, which suggests that simvastatin may be a novel target for SACC therapy.

[Characterization and pathophysiological changes of cerebral infarction rat model with qi-deficiency and blood-stasis Syndrome].

This study aimed to observe the general state and changes in pathophysiological indexes of multiple cerebral infarction rat model with Qi-deficienty and Blood-stasis syndrome. Rats were randomly divided into 4 groups(with 30 in each group): the normal group, the sham group, the model group and the Yiqi Huoxue recipe group. Rats in the model group and Yiqi Huoxue group were provided with interruptable sleep deprivation for 7 days before the multiple cerebral infarction operation, and followed by another 4 weeks of sleep deprivation; rats in the Yiqi Huoxue group were intragastrically administrated with drug at a dose of 26 g·kg⁻¹, once a day for 4 weeks. The general state was observed, and the pathophysiological indexes were measured at 48 h, 2 weeks and 4 weeks after administration. The results showed that rats in the normal group and the sham group represented a good general state and behaviors, with a normal morphological structure of brain tissues; rats in the model group featured yellow fur, depression, accidie, loose stools and movement disorder, with obvious brain histomorphological damage, which became aggravated with the increase of modeling time; rats in the Yiqi Huoxue group showed release in the general state and above indexes. Compared with the sham group at three time points, rats in the model group showed decrease in body weight, exhaustive swimming time and RGB value of tongue surface image, and increase in whole blood viscosity of the shear rate under 5, 60 and 150 S⁻¹, reduction in cerebral cortex Na⁺-K⁺-ATPase, Ca²âº-ATPase activity and contents of 5-HT, rise in TXB2 levels and decline in 6-keto-PGF1a in serum(P<0.05, P<0.01). Compared with the model group, rats in the Yiqi Huoxue group showed alleviations in the above indexes at 2 w and 4 w(P<0.05, P<0.01). The results showed that the characterization and pathophysiological indexes in the multiple cerebral infarction rat model with Qi-deficiency and blood-stasis syndrome were deteriorated; Yiqi Huoxue recipe could significantly alliviate the abnormal conditions, which suggested of the model was stable and reliable and the pathophysiologic evolutionary mechanism might be related to energy metabolism dysfunction, vasoactive substance abnormality and changes in neurotransmitters.

Occurrence and Biological Effects of Endocrine Disrupting Chemicals in the Yellow River (Zhengzhou Section).

Estrogenic activities of river water from four representative cross-sections of the Yellow River (Zhengzhou section) and their effects on reproduction and development of fish were assessed. MVLN assay showed estradiol equivalents of river water from Yiluohe, Xinmanghe, Qinhe and Huayuankou cross-sections were 1.09 ± 0.11, 0.72 ± 0.01, 1.19 ± 0.19 and 0.80 ± 0.04 ng/L, respectively. Significant vitellogenin (VTG) inductions were observed in adult male Japanese madaka (Oryzias latipes) after 30 days of exposure to river water from Yiluohe and Qinhe cross-sections (p < 0.05). Hepatic-somatic index was significantly elevated in fish exposed to water from Qinhe cross-section (p < 0.05). A significant delay in time to hatching was observed in embryos treated by water from Xinmanghe cross-section (p < 0.05). Significant lower survivals were observed in fish treated by water from Yiluohe and Xinmanghe cross-sections after a full life cycle exposure (p < 0.05). Exposure of water from Yiluohe and Qinhe cross-sections induced significantly elevated VTG levels in the first sexually mature male fish (p < 0.05). Both the in vitro and in vivo bioassay demonstrate endocrine disrupting chemicals exist in the Yellow River (Zhengzhou section) and fish in Yiluohe and Qinhe cross-sections can be at a risk of reproductive and developmental impairment.

Effects of individual and binary mixtures of estrogens on male goldfish (Carassius auratus).

Adverse effects of five typical environmental estrogens, namely estrone (E1), 17ß-estradiol (E2), 4-n-octylphenol (4-n-OP), 4-n-nonylphenol (4-n-NP) and bisphenol A (BPA) on adult male goldfish (Carassius auratus) were investigated both individually and in binary mixtures, using serum vitellogenin (VTG) induction and gonadosomatic index (GSI) as the endpoints. Doses of individual and binary mixtures of estrogens were chosen at broad ranges. Five individual estrogens induced common dose-dependent increases of serum VTG in the experimental fish when injection doses of the estrogen series were comparatively low. The levels of VTG induction in fish descended after peaked at a certain dose of the individual estrogen. Significant GSI decreases were observed in fish treated by all dose series of E1 and E2, and comparatively high doses of 4-n-OP, 4-n-NP and BPA when compared with that of solvent control (SC). Effects of binary mixtures of the five typical estrogens on VTG induction in male goldfish were in additive manner at low-effect doses, but divergences occurred at high dose levels, with the predicted effects by additive manner exceeding those were observed. All of GSI of fish treated by the binary mixtures were about or lower than 10(-3)%. Serious atrophy of gonads was observed in all the mixture treatment groups when compared with that of SC. These findings highlight the potential reproductive risk of fish resulted from existing mixtures of hormones in the aquatic environment, and they have important implications for environmental estrogen hazard assessment.

Improved expression of secretory and trimeric proteins in mammalian cells via the introduction of a new trimer motif and a mutant of the tPA signal sequence.

Ideal immunogenicity in antigens is a prerequisite to eliciting a sufficiently strong immune and memory response via either DNA or protein vaccines. To improve immunogenicity, efforts have focused on high-level expression of target proteins and on maintaining their natural conformations. In the present work, two trimer motifs (MTQ and MTI) were designed and introduced into a plasmid vector with the tissue plasminogen activator signal peptide (tPA-SP). Next, we examined the efficacy and the efficiency of the two motifs as well as the introduction of tPA-SP and its mutant forms, 22P/A and 22P/G, in facilitating the secretory expression of trimeric proteins in mammalian cells. We found that both trimer motifs could produce the target protein in a trimeric form at a high level. Introduction of tPA-SP 22P/A markedly increased the secretory expression level. The combination of the trimer motif, MTQ, and the signal peptide, 22P/A, may serve as a universal mammalian vector for producing trimeric proteins in vaccine development.