Quercetin alleviates chronic unpredictable mild stress-induced depression-like behavior by inhibiting NMDAR1 with α2δ-1 in rats.

BACKGROUND: Depression is a serious mental disorder and the most prevalent cause of disability and suicide worldwide. Chronic unpredictable mild stress (CUMS) can lead to a significant acceleration of depression development. Quercetin (Que) is a flavonoid compound with a wide range of pharmacological effects. Recent studies have shown that quercetin can improve CUMS-induced depression-like behavior, but the mechanism of its improvement is still unclear. α2δ-1 is a regulatory subunit of voltage-gated calcium channel, which can interact with N-methyl-D-aspartate receptor (NMDAR) to form a complex. OBJECTIVE: In this study, we found that Que could inhibit the increase of α2δ-1 and NMDAR expression in rat hypothalamus induced by CUMS. In pain, chronic hypertension and other studies have shown that α2δ-1 interacts with the NMDAR to form a complex, which subsequently affects the expression level of NMDAR. Consequently, the present study aimed to investigate the antidepressant effect of Que in vivo and in vitro and to explore its mechanism of action in terms of the interaction between α2δ-1 and NMDAR. METHODS: Rats were randomly exposed to two stressors every day for 4 weeks to establish a CUMS rat model, then sucrose preference test (SPT), forced swimming test (FST), tail suspension test (TST), and open field test (OFT) were performed to detect the behavior of CUMS rats, so as to evaluate whether the CUMS rat model was successfully established and the improvement effect of Que on CUMS-induced depression-like behavior in rats. Experimental techniques such as serum enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blot, and co-immunoprecipitation, as well as in vitro experiments, were used to investigate the mechanisms by which Que exerts its antidepressant effects. RESULTS: Behavioral and ELISA test results showed that Que could produce a reduction in the excitability of the hypothalamic-pituitary-adrenal (HPA) axis in CUMS rats and lead to significant improvements in their depressive behavior. Western blot, immunofluorescence, and co-immunoprecipitation experiments showed that Que produced a decrease in NMDAR1 and α2δ-1 expression levels and interfered with α2δ-1 and NMDAR1 binding. In addition, the neural regulation mechanism of Que on antidepressant effect in PC12 cells knocked out α2δ-1 gene was further verified. Cellular experiments demonstrated that Que led to a reversal of up-regulation of NMDAR1 and α2δ-1 expression levels in corticosterone-injured PC12 cells, while Que had no effects on NMDAR1 expression in PC12 cells with the α2δ-1 gene knockout. CONCLUSIONS: Que has a good antidepressant effect and can significantly improve the depression-like behavior caused by CUMS. It exerts antidepressant effects by inhibiting the expression level of α2δ-1, interfering with the interaction between α2δ-1 and NMDAR, and then reducing the excitability of the HPA axis.

ß-Indole-3-acetic acid attenuated collagen-induced arthritis through reducing the ubiquitination of Foxp3 via the AhR-TAZ-Tip60 pathway.

Massive evidence shows that intestinal tryptophan metabolites affected by intestinal flora can modulate the progression of rheumatoid arthritis (RA). However, the effects and mechanisms of intestinal tryptophan metabolites on RA are not yet detailed. Herein, we investigated the protective effects of intestinal tryptophan metabolites on RA and its detailed mechanisms. In this study, the collagen-induced arthritis (CIA) rat model was established. Based on metabolomics analysis, the contents of ß-indole-3-acetic acid (IAA), indolylpropionic acid, and indole-3-ß-acrylic acid in the sera of CIA rats were significantly less compared with those of the normal rats. Under the condition of Treg or Th17 cell differentiation, IAA significantly promoted the differentiation and activation of Treg cells instead of Th17 cells. Intestinal tryptophan metabolites are well-known endogenic ligands of aryl hydrocarbon receptor (AhR). Not surprisingly, IAA increased the level of Foxp3 through activating the AhR pathway. Interestingly, IAA had little impact on the level of Foxp3 mRNA, but reducing the ubiquitination and degradation of Foxp3. Mechanically, IAA reduced the expression of the transcriptional coactivator TAZ, which was almost completely reversed by either AhR antagonist CH223191 or siRNA. In vitro, IAA decreased the combination of TAZ and the histone acetyltransferase Tip60, while it increased the combination of Tip60 and Foxp3. In CIA rats, oral administration of IAA increased the number of Treg cells and relieved the inflammation. A combined use with CH223191 almost abolished the effect of IAA. Taken together, IAA attenuated CIA by promoting the differentiation of Treg cells through reducing the ubiquitination of Foxp3 via the AhR-TAZ-Tip60 pathway.

Identification of evodiamine and rutecarpine as novel TMEM16A inhibitors and their inhibitory effects on peristalsis in isolated Guinea-pig ileum.

The transmembrane member 16A (TMEM16A)-encoded Ca2+-activated Cl- channel (CaCC) is expressed in interstitial cells of Cajal (ICCs) and involved in the generation of the slow-wave currents of gastrointestinal (GI) smooth muscles. TMEM16A modulators have been shown to positively or negatively regulate the contraction of gastrointestinal smooth muscle. Therefore, targeting the pharmacological modulation of TMEM16A may represent a novel treatment approach for gastrointestinal dysfunctions such as constipation and diarrhoea. In this study, evodiamine and rutecarpine were extracted from the traditional Chinese medicine Evodia rutaecarpa and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on intestinal peristalsis were examined. Whole-cell patch clamp results show that evodiamine and rutecarpine inhibited TMEM16A Cl- currents in CHO cells. The half-maximal inhibition values (IC50) of evodiamine and rutecarpine on TMEM16A Cl- currents were 11.8 ± 1.3 µΜ and 9.2 ± 0.4 µM, and the maximal effect values (Emax) were 95.8 ± 5.1% and 99.1 ± 1.6%, respectively. The Lys384, Thr385, and Met524 in TMEM16A are critical for evodiamine and rutecarpine's inhibitory effects. Further functional studies show that both evodiamine and rutecarpine can significantly suppress the peristalsis in isolated guinea-pig ileum. These findings demonstrate that evodiamine and rutecarpine are new TMEM16A inhibitors and support the regulation effect of TMEM16A modulators on gastrointestinal motility.

Tetrandrine enhances the ubiquitination and degradation of Syk through an AhR-c-src-c-Cbl pathway and consequently inhibits osteoclastogenesis and bone destruction in arthritis.

Recently, we reported that tetrandrine, a natural alkaloid, could inhibit the osteoclastogenesis and bone erosion through enhancing the ubiquitination and degradation of spleen tyrosine kinase (Syk). Herein, we addressed whether and how aryl hydrocarbon receptor (AhR) mediate the effect of tetrandrine. In vitro, tetrandrine was shown to repress RANKL-induced osteoclastogenesis and the expression of osteoclast-related marker genes, which was almost completely reversed by either AhR antagonist CH223191 or siRNA. In pre-osteoclasts, tetrandrine enhanced the ubiquitination and degradation of Syk through the AhR/c-src/c-Cbl signaling pathway, downregulated the expression of phospho-Syk and phospho-PLCγ2, and inhibited the nuclear translocation of NFATc1, a master transcription factor for osteoclastogenesis. Notably, tetrandrine acted through the non-genomic pathway of the ligand-activated AhR, as evidenced by the fact that the effect of tetrandrine did not change in the absence of AhR nuclear translocator. In collagen-induced arthritis rats, oral administration of tetrandrine decreased the number of phospho-Syk-positive cells and osteoclasts, and reduced the bone erosion in the areas of the proximal tibial epiphysis excluding the cortical bone. A combined use with CH223191 almost abolished the effect of tetrandrine. These findings revealed that tetrandrine enhanced the ubiquitination and degradation of Syk and consequently repressed the osteoclastogenesis and bone destruction through the AhR-c-src-c-Cbl pathway.

Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Tetrandrine, a bisbenzylisoquinoline alkaloid, was previously demonstrated to attenuate inflammation and cartilage destruction in the ankles of mice with collagen-induced arthritis (CIA). Here, we explored the underlying mechanism by which tetrandrine prevented arthritis-induced bone erosion by focusing on the differentiation and function of osteoclasts. We found that daily administration of tetrandrine (30 mg/kg) markedly reduced the bone damage and decreased the number of osteoclasts in CIA rats. In vitro, tetrandrine inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis at the early stage and reduced the expressions of osteoclast-related marker genes. In bone marrow-derived macrophages and RAW264.7 cells, tetrandrine inhibited RANKL-induced translocation of NF-κB-p65 and nuclear factor of activated T cell 1 (NFATc1) through suppressing spleen tyrosine kinase (Syk)-Bruton's tyrosine kinase-PLCγ2-Ca2+ signaling. Of interest, tetrandrine did not affect the phosphorylation of immunoreceptor tyrosine-based activation motifs, the conventional upstream of Syk, but it inhibited the activity of Syk by enhancing its ubiquitination and degradation. The anti-osteoclastogenesis effect of tetrandrine nearly disappeared when it was used in combination with the Syk inhibitor piceatannol or in constitutively activated Syk-overexpressing cells. Taken together, tetrandrine attenuated CIA-induced bone destruction by inhibiting osteoclastogenesis through hindering the translocation of NF-κB-p65 and NFATc1 via reducing the activation of Syk.-Jia, Y., Miao, Y., Yue, M., Shu, M., Wei, Z., Dai, Y. Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation.

BACKGROUND: Natural products from plants have been proven to be important resources of antitumor agents. In this study, we exploited the antitumor activity of (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of scopoletin, by in vitro and in vivo experiments. METHODS: Human hepatocellular carcinoma cell line HepG2 cells and xenograft of HepG2 cells in BALB/c nude mice were used to investigate the effects of SC-III3 on hepatocellular cancers. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Cell cycle arrest, apoptosis and ATM-Chk pathway-related proteins were characterized by western blot. RESULTS: SC-III3 selectively inhibited the viability of HepG2 cells without significant cytotoxicity against human normal liver cells LO2. In mouse xenograft model of HepG2 cells, SC-III3 showed a marked inhibition of tumor growth in a dose-dependent manner. Cell cycle analysis revealed that SC-III3 induced cells to accumulate in S phase, which was accompanied by a marked decrease of the expressions of cyclin A, cyclin B, cyclin E and Cdk2 proteins, the crucial regulators of S phase cell cycle. SC-III3 treatment resulted in DNA breaks in HepG2 cells, which might contribute to its S phase arrest. The S arrest and the activation of ATM-Chk1/Chk2-Cdc25A-Cdk2 pathways induced by SC-III3 in HepG2 cells could be efficiently abrogated by pretreatments of either Ku55933 (an inhibitor of ATM) or UCN-01 (an inhibitor of Chk1/Chk2). The activation of p53-p21 pathway by SC-III3 was also reversed by Ku55933 treatment. SC-III3 led to significant accumulation of intracellular reactive oxygen species (ROS), a breaker of DNA strand, in HepG2 cells but not LO2 cells. Pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, DNA damage, activation of signal pathways relevant to DNA damage, S phase arrest and cell viability decrease in HepG2 cells. CONCLUSION: SC-III3 is able to efficiently inhibit the growth of hepatocellular carcinoma through inducing the generation of intracellular ROS, DNA damage and consequent S phase arrest, but lack of significant cytotoxicity against normal liver cells. This compound deserves further studies as a candidate of anticancer drugs.