L-Theanine Down-Regulates the JAK/STAT3 Pathway to Attenuate the Proliferation and Migration of Vascular Smooth Muscle Cells Induced by Angiotensin II.

L-Theanine, a green tea amino acid derivative, has cardiovascular qualities. The focus of the current evaluation was to examine the suppression of L-theanine on cultured vascular smooth muscle cell (VSMC) proliferation and migration that is prompted by angiotensin II (Ang II). The VSMCs were treated with non-cytotoxic concentrations of L-theanine and then stimulated with Ang II. The CCK-8 and Transwell chamber assays were monitored on the proliferation and migration rate, respectively. We discovered that L-theanine (50 and 100 µM) significantly halted Ang II-induced VSMC proliferation and migration. This was joined by a decline in the amount of cyclin D1. An additional discovery was that L-theanine lowered the proportion of S-phase cells, whereas the number of G1/G0-phase cells in Ang II-stimulated VSMCs was elevated, based on flow cytometry. Western blotting analyses indicated that L-theanine had no impact on extracellular-signal-regulated kinase 1/2 (ERK1/2) activation prompted by Ang II. Nevertheless, L-theanine significantly lowered Ang II-prompted phosphorylation of Janus kinase 2 (JAK2), c-Src tyrosine kinase, and signal transducer and activators of transcription 3 (STAT3). The outcomes revealed that L-theanine subdued the Ang II-prompted proliferation and migration of VSMC, partly via the obstruction of the JAK/STAT3 pathway instead of via just the ERK pathway.

Protective Effect of L-Theanine on Cadmium-Induced Apoptosis in PC12 Cells by Inhibiting the Mitochondria-Mediated Pathway.

L-Theanine is an amino acid derivative from green tea. The present work was aimed at the effect of L-theanine on neuron-like rat pheochromocytoma (PC12) cells stimulated with cadmium chloride. Treatment with L-theanine before cadmium exposure increased cell viability; the experiments of Annexin V/PI staining indicated that L-theanine inhibited cadmium-induced cell apoptosis. Meanwhile, L-theanine decreased ROS production and protected from cadmium-induced disruption of mitochondrial transmembrane potential. Compared with cadmium-treated cells, L-theanine could also decrease the ratio of Bax/Bcl-2, as well as the level of cleaved caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Furthermore, L-theanine depresses cadmium-induced up regulation of phosphorylations of PI3K/Akt, MAPK ERK1/2, and JNK signaling. These data suggest that L-theanine pretreatment reduces severity of cadmium toxicity probably via antioxidant action. Therefore, it may be concluded that L-theanine could be exploited for prevention of cadmium-induced diseases.

Theanine, an antitumor promoter, induces apoptosis of tumor cells via the mitochondrial pathway.

Theanine, an active component of green tea (Camellia sinensis), is considered a modulator of chemotherapy. To further investigate the anticancer activity of theanine, the present study investigated the cytotoxic effect of theanine at the concentration of 600 µg/ml, in the human HepG2 hepatoblastoma and HeLa adenocarcinoma cell lines, in comparison with the normal L02, H9c2 and HEK293 cell lines using a MTT assay. It was found that theanine induced cell death in the tumor cells, but not in the normal cells. Notably, when glutamine was restricted or reduced in the cell culture medium, the cell death induced by theanine was significantly enhanced. A terminal deoxynucleotidyl­transferase­mediated dUTP nick end labeling assay indicated that DNA damage was induced in theanine­treated HepG2 cells. Further experiments demonstrated that theanine caused HepG2 cell apoptosis through the mitochondrial pathway, with a loss of membrane potential and the release of apoptosis­inducing factor, endonuclease G and cytochrome c. Western blot analysis and caspase activity detection also revealed that caspase­9 and caspase­3 were activated, whereas caspase­8 remained inactive. These observations suggested that theanine exerted potent cytotoxicity on tumor cells when glutamine was restricted.

Glutathione S-Transferase Pi Prevents Sepsis-Related High Mobility Group Box-1 Protein Translocation and Release.

Glutathione S-transferase Pi (GSTP) was originally identified as one of cytosolic phase II detoxification enzymes and also was considered to function via its non-catalytic, ligand-binding activity. We have reported that GSTP played an anti-inflammatory role in macrophages, suggesting that GSTP may have a protective role in inflammation. In this study, we deleted the murine Gstp gene cluster and found that GSTP significantly decreased the mortality of experimental sepsis and reduced related serum level of high mobility group box-1 protein (HMGB1). As HMGB1 is the key cytokine involved in septic death, we further studied the effect of GSTP on HMGB1 release. The results demonstrated that a classic protein kinase C (cPKC) dependent phosphorylation of cytoplasmic GSTP at Ser184 occurred in macrophages in response to lipopolysaccharide (LPS) stimulation. Phosphorylated GSTP was then translocated to the nucleus. In the nucleus, GSTP bound to HMGB1 and suppressed LPS-triggered and cPKC-mediated HMGB1 phosphorylation. Consequently, GSTP prevented the translocation of HMGB1 to cytoplasm and release. Our findings provide the new evidence that GSTP inhibited HMGB1 release via binding to HMGB1 in the nucleus independent of its transferase activity. cPKC-mediated GSTP phosphorylation was essential for GSTP to translocate from cytoplasm to nucleus. To our knowledge, we are the first to report that nuclear GSTP functions as a negative regulator to control HMGB1 release from macrophages and decreases the mortality of sepsis.

l-Theanine attenuates cadmium-induced neurotoxicity through the inhibition of oxidative damage and tau hyperphosphorylation.

Cadmium (Cd) has long been known to induce neurological degenerative disorders. We studied effects of l-theanine, one of the major amino acid components in green tea, on Cd-induced brain injury in mice. Male ICR mice were intraperitoneally injected with l-theanine (100 or 200mg/kg/day) or saline and after one hour these mice were orally administrated with CdCl2 (3.75-6mg/kg). The treatment was conducted for 8 weeks. l-Theanine significantly reduced Cd level in the mouse brain and plasma. Cd-induced neuronal cell death in the mouse cortex and hippocampus were apparently inhibited by l-theanine treatment. l-Theanine also decreased the levels of malondialdehyde (MDA) and ROS, and obviously elevated the levels of glutathione (GSH) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the mouse brain. Hyperphosphorylation of tau protein is proposed to be an early event for the evolution of tau pathology, and may play an important role in Cd-induced neurodegeneration. Our results showed that l-theanine significantly suppressed Cd-induced tau protein hyperphosphorylation at Ser199, Ser202, and Ser396. Mechanism study showed that l-theanine inhibited the activation of glycogen synthase kinase-3ß (GSK-3ß) which contributed to the hyperphosphorylation of tau and Cd-induced cytotoxicity. Furthermore, l-theanine reduced Cd-induced cytotoxicity possibly by interfering with the Akt/mTOR signaling pathway. In conclusion, our study indicated that l-theanine protected mice against Cd-induced neurotoxicity through reducing brain Cd level and relieved oxidative damage and tau hyperphosphorylation. Our foundings provide a novel insight into the potential use of l-theanine as prophylactic and therapeutic agents for Cd-induced neurodegenerative diseases.

Phosphorylated heat shock protein 27 promotes lipid clearance in hepatic cells through interacting with STAT3 and activating autophagy.

Nonalcoholic fatty liver disease (NAFLD) has become the major liver disease worldwide. Recently, several studies have identified that the activation of autophagy attenuates hepatic steatosis. Heat shock protein 27 (Hsp27) is involved in autophagy in response to various stimuli. In this study, we demonstrate that phosphorylated Hsp27 stimulates autophagy and lipid droplet clearance and interacts with STAT3. In vivo study showed that high fat diet (HFD) feeding increased Hsp25 (mouse orthology of Hsp27) phosphorylation and autophagy in mouse livers. Inhibition of Hsp25 phosphorylation exacerbated HFD-induced hepatic steatosis in mice. In vitro study showed that palmitate-induced lipid overload in hepatic cells was enhanced by Hsp27 knockdown, KRIBB3 treatment and Hsp27-3A (non-phosphorylatable) overexpression but was prevented by Hsp27-WT (wild type) and Hsp27-3D (phosphomimetic) overexpression. Mechanism analysis demonstrated that palmitate could induce Hsp27 phosphorylation which promoted palmitate-induced autophagy. Phosphorylated Hsp27 interacted with STAT3 in response to palmitate treatment, and disrupted the STAT3/PKR complexes, facilitated PKR-dependent eIF2α phosphorylation, and thus stimulated autophagy. To conclude, our study provides a novel mechanism by which the phosphorylated Hsp27 promotes hepatic lipid clearance and suggests a new insight for therapy of steatotic diseases such as nonalcoholic fatty liver disease (NAFLD).

Curcumin promotes degradation of inducible nitric oxide synthase and suppresses its enzyme activity in RAW 264.7 cells.

Curcumin, a natural polyphenolic compound, has been reported to possess anti-inflammatory properties. Previous works showed that curcumin decreased lipopolysaccharide (LPS)-induced iNOS up-regulation at transcription level. However, whether curcumin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that curcumin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Mechanically, such degradation of iNOS protein is due to ubiquitination and proteasome-dependency since it was almost completely blocked by N-benzoyloxycarbonyl-Leu-Leu-leucinal (MG132), a specific inhibitor of proteasome. Furthermore, curcumin decreased iNOS tyrosine phosphorylation through inhibiting ERK 1/2 activation and subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that curcumin can promote the ubiqitination and degradation of iNOS after LPS stimulation.

beta-Arrestins facilitate ubiquitin-dependent degradation of apoptosis signal-regulating kinase 1 (ASK1) and attenuate H2O2-induced apoptosis.

beta-Arrestins are ubiquitously expressed proteins that play important roles in receptor desensitization, endocytosis, proteosomal degradation, apoptosis and signaling. It has been reported that beta-Arrestin2 acts as a scaffold by directly interacting with the JNK3 isoform and recruiting MKK4 and the apoptosis-signaling kinase-1 (ASK1). Here, we report a novel function of beta-Arrestins in regulating H(2)O(2)-induced apoptosis. Our study demonstrates that beta-Arrestins physically associate with C-terminal domain of ASK1, and moreover, both over-expression and RNA interference (RNAi) experiments indicate that beta-Arrestins down-regulate ASK1 protein. In detail, beta-Arrestin-induced reduction of ASK1 protein is due to ubiquitination and proteasome-dependent degradation of ASK1 in response to association of beta-Arrestins and ASK1. Upon H(2)O(2) stimulation, the protein binding between beta-Arrestins and ASK1 increases and ASK1 degradation is expedited. In consequence, beta-Arrestins prevent ASK1-JNK signaling and as a result attenuate H(2)O(2)-induced apoptosis. Structurally, C-terminal domain of ASK1 is essential for beta-Arrestins and ASK1 association. We also found that CHIP is required for beta-Arrestins-induced ASK1 degradation, which suggested that beta-Arrestins function as a scaffold of ASK1 and CHIP, leading to CHIP-mediated ASK1 degradation. All these findings indicate that beta-Arrestins play a negative regulatory role in H(2)O(2)-induced apoptosis signaling through associating with ASK1 and CHIP and facilitating ASK1 degradation, which provides a new insight for analyzing the effects of beta-Arrestins on protecting cells from oxidative stress-induced apoptosis.