Anti-Inflammatory Effects of the LK5 Herbal Complex on LPS- and IL-4/IL-13-Stimulated HaCaT Cells and a DNCB-Induced Animal Model of Atopic Dermatitis in BALB/c Mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease influenced by a complex interplay of genetic and environmental factors. The activation of the JAK-STAT pathway increases the expression of inflammatory cytokines such as IL-4 and IL-13, further deteriorating AD. Therefore, for the treatment of AD, the JAK-STAT pathway is emerging as a significant target, alongside inflammatory cytokines. This study investigates the potential therapeutic effects of a novel herbal complex, LK5, composed of Scutellaria baicalensis, Liriope platyphylla, Sophora flavescens, Dictammus dasycarpus, and Phellodendron schneider, known for their anti-inflammatory and immune-modulating properties. We examined the anti-inflammatory and anti-AD effects of the LK5 herbal complex in HaCaT cells stimulated by LPS and IL-4/IL-13, as well as in a mouse model of AD induced by DNCB. In HaCaT cells stimulated with LPS or IL-4/IL-13, the LK5 herbal complex demonstrated anti-inflammatory effects by inhibiting the expression of inflammatory cytokines including TNF-α, IL-6, and IL-1ß, and downregulating the phosphorylation of STAT proteins. In a murine AD-like model induced by DNCB, administration of the LK5 herbal complex significantly ameliorated clinical symptoms, including dermatitis, ear thickness, and TEWL. Histological analysis revealed a reduction in epidermal thickness and mast cell infiltration. The LK5 herbal complex also inhibited pruritus induced by compound 48/80. Furthermore, the LK5 herbal complex treatment significantly decreased the levels of inflammatory cytokines such as TSLP, IL-6, and IgE in plasma and ear tissue of AD-induced mice. These findings suggest that the LK5 herbal complex may modulate the immune response and alleviate AD symptoms by inhibiting STAT pathways.

A phase-II clinical trial of laparoscopy-assisted distal gastrectomy with D2 lymph node dissection for gastric cancer patients.

OBJECTIVES: This study was conducted to determine whether laparoscopy-assisted distal gastrectomy (LADG) with complete D2 lymph node dissection for gastric cancer is a safe and effective surgical option. METHODS: During an 8-month period, 64 patients, who were diagnosed preoperatively as having T1-2, N0-1 or M0 gastric cancer, were prospectively enrolled to undergo LADG with D2 lymph node dissection; two surgeons with experience of over 50 cases of laparoscopic gastrectomy performed the procedures. The compliance rate, defined as cases with no more than one missing lymph node station according to the Japanese Research Society of Gastric Cancer (JRSGC) lymph node grouping, for the open gastrectomy with D2 lymph node dissection was 66.0% in a pilot study and was used for calculations of sample size. Compliance rate and other surgical outcomes, including the number of retrieved lymph nodes from each lymph node station, morbidities, mortalities and conversion rate, were analyzed. RESULTS: The compliance rate was 67.2% and was similar to that of open distal gastrectomy reported in the pilot study. The mean number of retrieved lymph nodes was 50.1 (range 20-100). The most frequently missed lymph node station was no. 5 (31.2%) followed by no. 3 (25.0%). There were no missed lymph nodes at stations no. 6 and 9. The complication rate was 3.1% (2/66); there were two conversions (3.0%) and no mortalities. CONCLUSIONS: The current study suggests that LADG with D2 lymph node dissection is oncologically feasible, and phase-III clinical trials will be needed.

Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

Polyacetylenes from a marine sponge Petrosia sp. inhibit DNA replication at the level of initiation.

In the course of our search for bioactive metabolites from the marine sponges collected from Korean water, we found that the polyacetylenes of marine sponge, genus Petrosia, deliver significant selective cytotoxicity against several human tumor cell lines. The effects of polyacetylene on DNA replication were examined using simian virus 40 DNA replication system in vitro. We found that polyacetylenes inhibited DNA replication, and predominantly inhibited the initiation stage of DNA replication. Polyacetylenes inhibited the DNA cleavage by topoisomerase I, and also significantly reduced polymerase alpha-primase activity. The ssDNA binding activity of replication protein A was little affected by polyacetylenes. We suggest that polyacetylenes might inhibit proteins required to establish replication forks during the initiation reaction, and their cytotoxicities might be related to the inhibitory effect they have on this fundamental cellular process.

Effects of adenine-nucleotides on the sequence-specificity of origin recognition complex-binding to DNA.

We examined effects of adenine-nucleotides on the sequence-specificity of origin recognition complex (ORC)-binding to DNA, using a gel electrophoretic mobility shift assay. The sequence-specific DNA binding of ORC was observed in the presence of ATP or ATP-gamma-S but not in the presence of adenosine 5'-diphosphate (ADP) or in the absence of any adenine-nucleotides. In contrast, the sequence-independent DNA binding of ORC was observed under any one of these conditions. These results suggest that ATP increases the sequence-specificity of ORC-binding to DNA. In relation to the requirement for incubation at high temperature and inhibition by cardiolipin, there was no significant difference between the sequence-specific and the sequence-independent DNA binding activities of ORC.

Acidic phospholipids with unsaturated fatty acids inhibit the binding of origin recognition complex to origin DNA.

Origin Recognition Complex (ORC) is a candidate initiator of chromosomal DNA replication in eukaryotes. We recently reported that cardiolipin inhibits the interaction of Origin Recognition Complex ORC with origin DNA, as is the case of DnaA, the initiator of chromosomal DNA replication in prokaryotes. We report here that another acidic phospholipid, phosphatidylglycerol (PG), also inhibits the interaction. Synthetic PG with only unsaturated fatty acids inhibits ORC-binding to origin DNA more strongly than PG with only saturated fatty acids. On the other hand, phosphatidylcholine (neutral phospholipid) does not affect the ORC-origin interaction, regardless of the presence of saturated or unsaturated fatty acids. These results suggest that an acidic moiety and unsaturated fatty acids are important factors for the inhibitory effect of phospholipids on ORC binding to origin DNA, as is the case for DnaA. The inhibitory effect of cardiolipin on ORC binding to origin DNA was more apparent at 30 degrees C than at 4 degrees C. Furthermore, chlorpromazine restored the ORC-origin interaction in the presence of cardiolipin. Since the presence of unsaturated fatty acids, low incubation temperatures, and the addition of chlorpromazine all decrease membrane fluidity, these results suggest that membrane fluidity is important for the inhibitory effect of acidic phospholipids on ORC-binding to origin DNA, as is the case for DnaA.

Inhibitory effects of acidic phospholipids on the binding of origin-recognition complex to origin DNA.

Origin-recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, shares certain biochemical characteristics with DnaA, the initiator of chromosomal DNA replication in prokaryotes. These similarities include origin-specific DNA binding, ATP binding and ATPase activity. DnaA interacts with acidic phospholipids, such as cardiolipin, and its activity is regulated by these phospholipids. In this study, we examined whether Saccharomyces cerevisiae ORC also interacts with phospholipids. Among the various phospholipids tested, ORC was found to bind specifically to cardiolipin. This binding was inhibited by excess concentrations of salts but unaffected by ATP, adenosine 5'-[gamma-thio]triphosphate or the origin DNA. Cardiolipin weakly inhibited the ATP-binding activity of ORC, whereas it strongly inhibited ORC binding to origin DNA. Acidic phospholipids other than cardiolipin (phosphatidylglycerol and phosphatidylinositol) weakly inhibited ORC binding to origin DNA. Furthermore, total phospholipids extracted from yeast nuclear membranes inhibited ORC binding to origin DNA. We consider that phospholipids may modulate initiation of DNA replication in eukaryotes in a similar manner to that found in prokaryotes.