Discovery of anti-neuroinflammatory agents from 1,4,5,6-tetrahydrobenzo[2,3]oxepino[4,5-d]pyrimidin-2-amine derivatives by regulating microglia polarization.

Neuroinflammation mediated by microglia activation leads to various neurodegenerative and neurological disorders. In order to develop more and better options for this disorders, a series of 3,4-dihydrobenzo[b]oxepin-5(2H)-one derivatives (BZPs, 6-19) and novel 1,4,5,6-tetrahydrobenzo[2,3]oxepino[4,5-d]pyrimidin-2-amine derivatives (BPMs, 20-33) were synthesized and screened the anti-neuroinflamamtion effects. 3,5-bis-trifluoromethylphenyl-substituted BPM 29 showed more potent anti-neuroinflammatory activity and no toxicity to BV2 microglia cells in vitro. 29 significantly reduced the number of M1 phenotype of microglia cells, but significantly increased the number of M2 phenotype of microglia cells in lipopolysaccharide (LPS)-induced BV2 microglia cells. 29 significantly reduced the secretion of inflammatory cytokines (IL-18, IL-1ß, TNF-α), but increased the secretion of anti-inflammatory cytokines (IL-10) from LPS-induced BV2 microglia cells. Also, 29 inhibited the NOD-like receptor NLRP3 inflammasome formation, and down-regulated the expression of M2 isoform of pyruvate kinase in LPS-induced BV2 microglia cells. In vivo, 29 reduced the neuroinflammation in cuprizone-induced inflammatory and demyelinating mice by reducing the expression of inducible nitric-oxide synthase, but increased the expression of CD206. Taken together, 29 might be a prospective anti-neuroinflammatory compound for neuroinflammatory and demyelinating disease by alleviating microglia activation.

A nanofiber/sponge double-layered composite membrane capable of inhibiting infection and promoting blood coagulation during wound healing.

Uncontrolled bleeding and bacterial infections cause severe damage to the wounds and remain a clinical challenge. Here, we developed a nanofiber/sponge bilayered composite membrane (QCP) containing quaternized silicone (QP12) and quaternized chitosan (QCS12) by joint approaches of electrospinning and freeze-drying and investigated their potential for wound dressing. The QCP was composed of a sponge (QCC) containing collagen (COL) and QCS12 and a nanofibrous membrane (MQP) containing poly-ε-caprolactone (PCL) and QP12. The QCP composite membrane possessed feasible permeability (0.22 ± 0.01 g/(cm2·24 h)), available thermal stability, suitable mechanical properties with natural skin, and in vivo hemostatic efficiency. The bonds of the N-quaternary and Schiff base endow composite membranes with significant anti-microbial invasion, potentially enhancing the wound healing process with an eligible microenvironment. Meanwhile, QCP evinced fine hemocompatibility, low cytotoxicity, negligible skin irritation, and other desirable biosafety as an excellent wound dressing. QCP promoted collagen deposition and re-epithelization to accelerate healing and suppress scars in the full-thickness acute wound models. Furthermore, the evaluation in the chronic skin incision model of diabetes mellitus manifested high healing efficiency with a certain resistance to bacterial infection of the composite membrane. Taken together, the QCP composite membrane may be a potential antibacterial and hemostatic wound dressing.

Functionalized Electrospun Double-Layer Nanofibrous Scaffold for Wound Healing and Scar Inhibition.

Considerable advances have been made in developing materials that promote wound healing and inhibit scar formation in clinical settings. However, some challenges, such as cumbersome treatment processes and determination of optimal treatment time, remain unresolved. Thus, developing a multifunctional wound dressing with both wound healing and scar inhibition properties is crucial. Here, we present an integrated electrospun fibrous composite membrane (MPC12) for wound healing and scar inhibition, consisting of a quaternized chitosan-loaded inner membrane (PCQC5) and quaternized silicone-loaded outer membrane (MQP12). The inner membrane effectively coagulates blood and promotes wound healing, and the outer membrane moisturizes, resists bacteria, and inhibits scar formation. In vivo evaluation in a rabbit ear model revealed that MPC12 treatment results in faster wound healing and better alleviation of scar hypertrophy than treatment with commercial products (KELO-COTE and MSSG). Our strategy offers an excellent solution for the potential integration of wound healing and scar inhibition.

Discovery of anti-hepatoma agents from 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine by inhibiting PI3K/AKT/NF-κB pathway activation.

In order to obtain new anti-hepatoma drugs with low toxicity, some 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidines (PPMs, 4a-t) were synthesized in this study. Many of them showed significant anti-hepatoma effects against HCC cells and low toxicity toward HHL-5 cells. Combined with their anti-hepatoma activity and toxicity, 4-CF3-substituted 4k was selected as an effective lead compound. Preliminary mechanistic studies revealed that 4k could up-regulate the expression levels of Bax and caspase-3 proteins, down-regulate the expression levels of Bcl-2 protein, promote significant apoptosis of HepG2, and block cells in G2-M phase to prevent cells from completing mitosis. Also, 4k could significantly inhibit the activation of PI3K/AKT/NF-κB pathway by blocking the phosphorylation of PI3K, AKT, NF-κB/p65 and IFN-γ-induced nuclear transport. Docking analysis showed that 4k could reasonably bind to the active sites of Bcl-2, NF-κB/p65, PI3K and AKT. This result suggested that 4k could be used as a new type of NF-κB inhibitor, which provides a scientific basis for further research into the treatment of hepatoma.

Trifluoromethyl-substituted 3,5-bis(arylidene)-4-piperidones as potential anti-hepatoma and anti-inflammation agents by inhibiting NF-кB activation.

Some methoxy-, hydroxyl-, pyridyl-, or fluoro-substituted 3,5-bis(arylidene)-4-piperidones (BAPs) could reduce inflammation and promote hepatoma cell apoptosis by inhibiting activation of NF-κB, especially after introduction of trifluoromethyl. Herein, a series of trifluoromethyl-substituted BAPs (4-30) were synthesised and the biological activities were evaluated. We successfully found the most potential 16, which contains three trifluoromethyl substituents and exhibits the best anti-tumour and anti-inflammatory activities. Preliminary mechanism research revealed that 16 could promote HepG2 cell apoptosis in a dose-dependent manner by down-regulating the expression of Bcl-2 and up-regulating the expression of Bax, C-caspase-3. Meanwhile, 16 inhibited activation of NF-κB by directly inhibiting the phosphorylation of p65 and IκBα induced by LPS, together with indirectly inhibiting MAPK pathway, thereby exhibiting both anti-hepatoma and anti-inflammatory activities. Molecular docking confirmed that 16 could bind to the active sites of Bcl-2, p65, and p38 reasonably. The above results suggested that 16 has enormous potential to be developed as a multifunctional agent for the clinical treatment of liver cancers and inflammatory diseases.

Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis.

The peritoneum, especially the omentum, is a common site for gastric cancer (GC) metastasis. Our aim was to expound the role and mechanisms of Piezo1 on GC omentum metastasis. A series of functional assays were performed to examine cell proliferation, clone formation, apoptosis, Ca2+ influx, mitochondrial membrane potential (MMP) and migration after overexpression or knockdown of Piezo1. A GC peritoneal implantation and metastasis model was conducted. After infection by si-Piezo1, the number and growth of tumours were observed in abdominal cavity. Fibre and angiogenesis were tested in metastatic tumour tissues. Piezo1 had higher expression in GC tissues with omentum metastasis and metastatic lymph node tissues than in GC tissues among 110 patients. High Piezo1 expression was associated with lymph metastasis, TNM and distant metastasis. Overexpressed Piezo1 facilitated cell proliferation and suppressed cell apoptosis in GC cells. Moreover, Ca2+ influx was elevated after up-regulation of Piezo1. Piezo1 promoted cell migration and Calpain1/2 expression via up-regulation of HIF-1α in GC cells. In vivo, Piezo1 knockdown significantly inhibited peritoneal metastasis of GC cells and blocked EMT process and angiogenesis. Our findings suggested that Piezo1 is a key component during GC omentum metastasis, which could be related to up-regulation of HIF-1α.

Discovery of novel NF-кB inhibitor based on scaffold hopping: 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine.

NF-κB is a key signaling pathway molecule linking hepatoma and chronic inflammation. Inhibition of NF-κB activation can alleviate inflammation, and promote hepatoma cell apoptosis. In this study, a series of fluoro-substituted 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidines (PPMs, 31-57) were synthesized from 3,5-bis(arylidene)-4-piperidones (BAPs, 4-30) based on scaffold hopping. We successfully discovered the most potent 43 substituted by electron-withdrawing substitutes (3-F and 4-CF3) exhibited less toxicity and higher anti-inflammatory activity. Preliminary mechanistic studies revealed that 43 induced dose-dependent cell apoptosis at cell and protein level, while inhibited NF-κB activation by suppressing LPS-induced phosphorylation levels of p65, IκBα and Akt, and by indirectly suppressing MAPK signaling, and by inhibiting the nuclear translocation of NF-κB induced by TNF-α or LPS. Docking analysis verified simulated 43 could reasonably bind to the active site of Bcl-2, p65 and p38 proteins. This compound, as a novel NF-κB inhibitor, also demonstrated both anti-inflammatory and anti-hepatoma activities, warranting its further development as a potential multifunctional agent for the clinical treatment of liver cancers and inflammatory diseases.

Synthesis, crystal structures and anti-inflammatory activity of fluorine-substituted 1,4,5,6-tetrahydrobenzo[h]quinazolin-2-amine derivatives.

Two fluorine-substituted 1,4,5,6-tetrahydrobenzo[h]quinazolin-2-amine (BQA) derivatives, namely 2-amino-4-(2-fluorophenyl)-9-methoxy-1,4,5,6-tetrahydrobenzo[h]quinazolin-3-ium chloride, (8), and 2-amino-4-(4-fluorophenyl)-9-methoxy-1,4,5,6-tetrahydrobenzo[h]quinazolin-3-ium chloride, (9), both C19H19FN3O+·Cl-, were generated by Michael addition reactions between guanidine hydrochloride and the α,ß-unsaturated ketones (E)-2-(2-fluorobenzylidene)-7-methoxy-3,4-dihydronaphthalen-1(2H)-one, C18H15FO2, (6), and (E)-2-(4-fluorobenzylidene)-7-methoxy-3,4-dihydronaphthalen-1(2H)-one, (7). Because both sides of α,ß-unsaturated ketones (6) or (7) can be attacked by guanidine, we obtained a pair of isomers in (8) and (9). Single-crystal X-ray diffraction indicates that each isomer has a chiral C atom and both (8) and (9) crystallize in the achiral space group P21/c. The chloride ion, as a hydrogen-bond acceptor, plays an important role in the formation of multiple hydrogen bonds. Thus, adjacent molecules are connected through intermolecular hydrogen bonds to generate a banded structure. Furthermore, these bands are linked into an interesting 3D network via hydrogen bonds and π-π interactions. Fortunately, the solubilities of (8) and (9) were distinctly improved and can exceed 50 mg ml-1 in water or PBS buffer system (pH 7.4) at room temperature. In addition, the results of an investigation of anti-inflammatory activity show that (8) and (9), with o- and p-fluoro substituents, respectively, display more potential for inhibitory effects on LPS-induced NO secretion than starting ketones (6) and (7).

Corona-directed nucleic acid delivery into hepatic stellate cells for liver fibrosis therapy.

Strategies to modify nanoparticles with biological ligands for targeted drug delivery in vivo have been widely studied but met with limited clinical success. A possible reason is that, in the blood circulation, serum proteins could rapidly form a layer of protein "corona" on the vehicle surface, which might block the modified ligands and hamper their targeting functions. We speculate that strategies for drug delivery can be designed based upon elegant control of the corona formation on the vehicle surfaces. In this study, we demonstrate a retinol-conjugated polyetherimine (RcP) nanoparticle system that selectively recruited the retinol binding protein 4 (RBP) in its corona components. RBP was found to bind retinol, and direct the antisense oligonucleotide (ASO)-laden RcP carrier to hepatic stellate cells (HSC), which play essential roles in the progression of hepatic fibrosis. In both mouse fibrosis models, induced by carbon tetrachloride (CCl4) and bile duct ligation (BDL), respectively, the ASO-laden RcP particles effectively suppressed the expression of type I collagen (collagen I), and consequently ameliorated hepatic fibrosis. Such findings suggest that this delivery system, designed to exploit the power of corona proteins, can serve as a promising tool for targeted delivery of therapeutic agents for the treatment of hepatic fibrosis.

A tumor environment responsive doxorubicin-loaded nanoparticle for targeted cancer therapy.

Doxorubicin (DOX) is a potent cancer chemotherapeutic agent, but its clinical use is severely limited by potentially lethal cardiotoxicity. Delivery of DOX by particulate carriers can be an effective way to reduce its distribution in cardiac tissue. In the present study, we developed a self-assembled, tumor-microenvironment-responsive delivery system for DOX. The core of the carrier was built upon the DOX/DNA intercalation, which was further combined with cationic gelatin (C-gel) to form the complex GDD. GDD was then packaged into a complex, namely, HDD, based on the electrostatic interactions between the positively charged C-gel and negatively charged human serum albumin (HSA). The HSA molecules on the surface of the complex HDD effectively helped the particle evade the filtration of the body when injected into the circulation and passively accumulate into the tumor sites. After entering the tumor tissue, where albumin is rapidly consumed, GDD was release from HDD and the C-gel was then digested by the tumor-specific matrix metalloproteinase (MMPs) to free the DOX/DNA intercalation. Deoxyribonucleases (DNases) in the tissue could completely destroy the DNA molecules to release DOX into the microenvironments. After a series of in vitro optimization tests, we evaluated the anticancer capacity and cardiac toxicity of HDD in two animal models with cancer. The results suggested that HDD had a higher anticancer efficacy and a significantly lower cardiotoxicity than free DOX. Additionally, the main components of the carrier are all clinically approved materials. Taken together, our present delivery system is safe and efficient and has high potential for further clinical trials.

The autoregulatory feedback loop of microRNA-21/programmed cell death protein 4/activation protein-1 (MiR-21/PDCD4/AP-1) as a driving force for hepatic fibrosis development.

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-ß (TGF-ß) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.

3,3'-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR-21 expression.

BACKGROUND AND PURPOSE: Hepatic fibrosis is a type of liver disease characterized by excessive collagen deposition produced by activated hepatic stellate cells (HSCs), and no appropriate drug treatment is available clinically. The microRNA, miR-21 exhibits an important role in the pathogenesis and progression of hepatic fibrosis. 3,3'-Diindolylmethane (DIM) is a natural autolytic product in plants and can down-regulate miR-21 expression. Here we have assessed the therapeutic effects of DIM against hepatic fibrosis and investigated the underlying mechanisms. EXPERIMENTAL APPROACH: The effects of DIM on HSC activation were measured by analysing the expression of α-smooth muscle actin and collagen I in both HSC-T6 cell line and primary HSCs. Expression of miR-21 was also measured after DIM treatment and the therapeutic effect of DIM was further studied in vivo, using the model of hepatic fibrosis induced by thioacetamide in mice. The antagonist oligonucleotide, antagomir-21, was also used to suppress the effects of miR-21. KEY RESULTS: DIM suppressed the central TGF-ß signalling pathway underlying HSC activation by down-regulating the expression of miR-21. The decreased miR-21 expression was achieved by inhibiting the activity of the transcription factor, AP-1. Moreover, DIM blunted the activation phenotype of primary HSCs. Administration of DIM in vivo attenuated liver fibrosis induced by thioacetamide, as assessed by collagen deposition and profiles of profibrogenic markers. CONCLUSIONS AND IMPLICATIONS: DIM shows potential as a therapeutic agent for the treatment of hepatic fibrosis.