Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Synthesis and anti-inflammatory activities of glycyrrhetinic acid derivatives containing disulfide bond.

A series of glycyrrhetinic acid (GA, aglycone of glycyrrhizic acid) derivatives containing disulfide bond were synthesized and their anti-inflammatory and anti-fibrosis activities were evaluated in vivo and in vitro. Among them, compound 7 displayed the highest toxicity to all the tested cell lines including macrophages. Compounds 3 and 4 showed higher activities than GA in the cell and animal model. In the anti-inflammatory tests, compounds 3 and 4 down-regulated the expressions of several inflammatory factors, such as HMGB1, TLR4, IL-1ß, TNF-α and TGF-ß1 in LPS-treated RAW264.7 cells in a dose-dependent manner. Compounds 3 and 4 at 30 µM respectively reduced the levels of HMGB1 in the LPS group to 42.7% and 38.2%. In addition, the level of TLR4 decreased to close to that of control group when treated by compound 4 at the concentration of 30 µM. In the process of anti-fibrosis tests using TGF-ß1-induced A549 cell line as the model, compounds 3 and 4 also decreased the expression levels of Col1 and α-SMA in a dose-dependent manner. Compound 3 and 4 at 30 µM respectively reduced the expression of α-SMA level by 2.2-fold and 2.6-fold compared to the TGF-ß1-treated control group. Moreover, they influenced the ROS level and mitochondrial membrane potential (MMP) in A549 cells. In the paraquat-induced pulmonary fibrosis mice model, the symptoms of inflammation and fibrosis of mice were alleviated after administration of compound 3 or 4. The above results suggest that compounds 3 and 4 may be promising candidates for inflammation and lung fibrosis treatment.

Protective effect of glycyrrhizin on osteoarthritis cartilage degeneration and inflammation response in a rat model.

This study was conducted to investigate the protective effects of glycyrrhizin on a rat model of osteoarthritis and elucidate the underlying mechanism. Rat osteoarthritis was established by using medial meniscectomy (MMx) and an anterior cruciate ligament transaction (ACLT). Glycyrrhizin (2, 4, and 10 mg/kg) was administered by intra-articular knee injection for 12 weeks. Incapacitance test was performed to determine mechanical hyperalgesia. Enzyme-linked immunosorbent assay (ELISA) was performed to measure cartilage degradation and inflammation-related markers. Quantitative reverse transcription PCR (RT-qPCR) and Western blot were performed to determine the mRNA and protein levels of genes, respectively. The results demonstrated that treatment with glycyrrhizin ameliorated mechanical hyperalgesia and bilateral joints oedema in a rat model of osteoarthritis. Treatment with 10 mg/kg glycyrrhizin also suppressed serum enzymes including matrix metalloproteinase (MMP)-1, MMP-3, prostaglandin E2, and C-telopeptide of type II collagen (CTX-II). In addition to inhibition of cartilage matrix catabolic related markers, treatment with glycyrrhizin also decreased the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and iNOS in serum and cartilage. The underlying mechanism study demonstrated that treatment with glycyrrhizin inhibited HMGB1 and the TLR4/NF-κB signaling pathway. In summary, treatment with glycyrrhizin ameliorated cartilage degeneration and inflammation in osteoarthritis rats by the regulation of HMGB1 and the TLR4/NF-κB signaling pathway.