Design,synthesis and preliminary structure–activity relationship investigation of nitrogen-containing chalcone derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors: a further study based on Flavokawain B Mannich base derivatives

In order to study the structure–activity relationship of Flavokawain B Mannich-based derivatives as acetylcholinesterase (AChE) inhibitors in our recent investigation, 20 new nitrogen-containing chalcone derivatives (4?a–8d) were designed, synthesized, and evaluated for AChE inhibitory activity in vitro. The results suggested that amino alkyl side chain of chalcone dramatically influenced the inhibitory activity against AChE. Among them, compound 6c revealed the strongest AChE inhibitory activity (IC₅₀ value: 0.85?μmol/L) and the highest selectivity against AChE over BuChE (ratio: 35.79). Enzyme kinetic study showed that the inhibition mechanism of compound 6c against AChE was a mixed-type inhibition. The molecular docking assay showed that this compound can both bind with the catalytic site and the peripheral site of AChE.     

哺乳动物雷帕霉素靶蛋白（mTOR）在不同发育阶段SD大鼠睾丸中的表达及作用

精子发生是男性生殖中的主要过程,精原细胞的不断分裂增殖又保证了精子发生的顺利进行。随着年龄的不断增长,男性精子的数量、质量出现下降趋势。mTOR信号转导通路在细胞增殖分化中发挥着中心调控作用,因此,mTOR信号通路可能在精子发生过程中有着重要的地位。为了探明mTOR信号通路与精子发生的关系,首先,通过SD大鼠睾丸组织切片的免疫组化,发现mTOR是在生精小管的精原细胞胞浆中表达;其次,采用FQ-PCR检测mTOR mRNA在SD大鼠睾丸中的表达。结果显示,80周龄组mTOR的转录与8周龄组相比差异显著。最后利用Western blot检测出mTOR蛋白的表达及其对下游靶蛋白P70S6K的磷酸化效率均随年龄的增长逐渐下降。同时,在用雷帕霉素处理8周龄SD大鼠中,发现精子数量减少,P70S6K磷酸化效率降低并伴随生精小管萎缩和空泡化。通过这些结果,可以看出mTOR信号转导通路可能在精子发生中发挥着重要作用。     

A crosstalk between the Smad and JNK signaling in the TGF-β-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells

Transforming growth factor β (TGF-β) induces the process of epithelial-mesenchymal transition (EMT) through the Smad and JNK signaling. However, it is unclear how these pathways interact in the TGF-β1-induced EMT in rat peritoneal mesothelial cells (RPMCs). Here, we show that inhibition of JNK activation by introducing the dominant-negative JNK1 gene attenuates the TGF-β1-down-regulated E-cadherin expression, and TGF-β1-up-regulated α-SMA, Collagen I, and PAI-1 expression, leading to the inhibition of EMT in primarily cultured RPMCs. Furthermore, TGF-β1 induces a bimodal JNK activation with peaks at 10 minutes and 12 hours post treatment in RPMCs. In addition, the inhibition of Smad3 activation by introducing a Smad3 mutant mitigates the TGF-β1-induced second wave, but not the first wave, of JNK1 activation in RPMCs. Moreover, the inhibition of JNK1 activation prevents the TGF-β1-induced Smad3 activation and nuclear translocation, and inhibition of the TGF-β1-induced second wave of JNK activation greatly reduced TGF-β1-induced EMT in RPMCs. These data indicate a crosstalk between the JNK1 and Samd3 pathways during the TGF-β1-induced EMT and fibrotic process in RPMCs. Therefore, our findings may provide new insights into understanding the regulation of the TGF-β1-related JNK and Smad signaling in the development of fibrosis.     

柠檬苦素类似物的生物活性和 构效关系研究进展

柠檬苦素类似物;生物活性;构效关系;结构修饰     

Over-expression of l-galactono-γ-lactone dehydrogenase increases vitamin C, total phenolics and antioxidant activity in lettuce through bio-fortification

Epidemiological studies have shown that regular consumption of fruits and vegetables is associated with reduced risk of chronic diseases. Vegetables can provide vitamins, phenolics, flavonoids, minerals and dietary fibers for optimal health benefits. However, some nutrients contained in many fruits and vegetables cannot meet of the complete nutrition need in the human body. Biotechnology has the potential to improve the nutritional value of crops. Considering the high consumption of romaine lettuce in human diet worldwide, the objective of study is to enhance the contents of vitamin C, phenolics and antioxidant activity in lettuce leaves by genetic engineering techniques. The gene expression level, vitamin C content, total phenolics, as well as total and cellular antioxidant activities were analyzed by real-time PCR, HPLC, Folin–Ciocalteu, Hydro-PSC and CAA methods, respectively. The bio-fortification of genetically engineered lettuce increased vitamin C up to 48.94 ± 1.34 mg/100 g FW following the increased over-expression of _At GLDH. This is almost a 3.2-fold increase as the content when compared with wild type lettuce (p < 0.05). In addition, phenolic compounds in transgenic lettuce contained 120.4 ± 1.62 mg GA equiv./100 g FW, almost double the phenolic content of the wild type. Total antioxidant activities were 735.4 ± 47.7 μmol vitamin C equiv./100 g FW, cellular antioxidant activities were 7.33 ± 0.86 μmol quercetin equiv./100 g FW (PBS wash) and 18.14 ± 0.68 μmol quercetin equiv./100 g FW (No PBS wash) in transgenic lettuce, respectively, 1.5, 4 and twofold increases when compared with the wild type. This study suggests that bio-fortification by genetic engineering has great potential to improve vitamin C, phenolic contents and antioxidant activity in lettuce.     

The alteration of 5-HT(2A) and 5-HT(2C) receptors is involved in neuronal apoptosis of goldfish cerebellum following traumatic experience

5-HT receptor changes remain controversial in posttraumatic stress disorder (PTSD) models. This study looks at the relationship between traumatic injuries and the alterations in 5-HT(2A) and 5-HT(2C) receptors in the goldfish brain. The effect of treatment with doxepin and fluoxetine, known to be selective serotonin reuptake inhibitor (SSRI) antidepressants, on 5-HT receptor expression in goldfish with fin ablation was also investigated. We demonstrated that fin ablation induced anxiety-like behavioural alterations and significant up-regulation of c-fos expression in goldfish cerebellum. The behavioural alterations correlated well with an increased expression of 5-HT(2A) receptors in the cerebellum of the fish with traumatic injury. An increase in the number of apoptotic cells and a higher caspase-8 protein level was present in the brains of goldfish with fin ablation compared to the control. Our findings suggest that neuronal apoptosis occured in the cerebellum as a result of fin ablation and may be related to the alterations of 5-HT(2A) and 5-HT(2C) levels and that the beneficial clinical effects of doxepin/fluoxetine treatment are due to the down-regulation of 5-HT(2A) and up-regulation of 5-HT(2C) receptors in the brain.     

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and ¹H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.     

Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg⁻¹·day ⁻¹ BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.     

Possible Roles of mmu-miR-141 in the Endometrium of Mice in Early Pregnancy Following Embryo Implantation

Objective

Embryo implantation is directly affected by genes related to uterine receptivity. Studies have demonstrated the important roles of miRNAs in the regulation of gene expression. Our early miRNA chip analyses revealed that the mmu-miR-141 expression in endometrial tissue is lower after embryo implantation than before it. However, the possible roles of miR-141 in embryo implantation have not yet been elucidated. Here, mmu-miR-141 was designed to detect the expression and role of miR-141 in the endometria of mice in early pregnancy following embryo implantation.

Methods

Real-time PCR and in-situ hybridization were used to study mmu-miR-141 expression in mouse uterus. Cell proliferation was detected by tetrazolium dye (MTT) assay and flow cytometry. Real-time PCR and Western blot analysis were used to confirm the mRNA and protein levels of phosphatase and tensin homolog (PTEN) to determine whether it was the target gene of mmu-miR-141. Enhanced green fluorescent protein (EGFP) fluorescence reporter vector analysis was also performed. A functional study was performed by injecting mice uteri with mmu-miR-141 inhibitor or mimic vectors.

Results

mmu-miR-141 expression was lower on day 6 (D6) than day 4 (D4) and could be increased by progesterone. Reduced mmu-miR-141 could decrease the proliferation activity of stromal cells and promote apoptosis. Upregulation of mmu-miR-141 inhibited PTEN protein expression but downregulation of mmu-miR-141 increased it, while the mRNA level remained unchanged. EGFP fluorescence reporter vector analysis showed that miR-141 targets the 3′-untranslated region of the PTEN mRNA. In addition, when the physiological mmu-miR-141 level was altered on D2 by injecting with inhibitor or mimic, the embryo implantation sites were significantly decreased on D7.

Conclusions

This study demonstrated that mmu-miR-141 might influence cell proliferation and apoptosis in the endometrium by negatively regulating PTEN expression, and could also influence the number of embryo implantation sites. mmu-miR-141 plays an essential role in embryo implantation.