Immunoregulatory Effect and Mechanism of Epigallocatechin-3-Gallate in A Mouse Oral Cancer Model

Objective:This investigation delineates the anti-cancer potency of epigallocatechin-3-gallate(EGCG)in an oral cancer mouse model,with a focus on its effect on T-cell activation.Methods:An oral cancer model was established in male Balb/c mice using 4-nitroquinoline 1-oxide(4-NQO).The mice were systematically grouped and administered graded concentrations of EGCG.Key parameters such as body weight,hydration levels,tumor volume,and mass were meticulously tracked.T-cell activity and cytokine expression profiles,focusing on interleukin-2(IL-2),interferon-gamma(IFN-γ),and tumor necrosis factor-alpha(TNF-α),were quantified using ELISA.A comprehensive statistical evaluation included one-way ANOVA,Tukey’s HSD multiple comparison test,and the Kruskal-Wallis non-parametric assessment.Results:EGCG-administered cohorts exhibited a pronounced reduction in tumor size and mass,with the high-dose group showing the greatest efficacy.ELISA findings corroborated a significant increase in T-cell activity and concomitant upregulation of key cytokines,including IL-2,IFN-γ,and TNF-α(P<0.05).Conclusion:This investigation confirms the tumor-suppressive efficacy of EGCG in a murine oral squamous cell carcinoma model.The therapeutic effects of EGCG are mediated through T-cell activation and the upregulation of pivotal cytokine expression,highlighting its potential immunomodulatory role in oral cancer treatment.

Epigallocatechin-3-gallate exerts antihypertensive effects and improves endothelial function in spontaneously hypertensive rats

Objective:To investigate the effect of epigallocatechin-3-gallate(EGCG)on endothelial dysfunction in spontaneously hypertensive rats(SHR).Methods:Wistar-Kyoto(WKY)rats and SHR were divided into four groups;WKY control,SHR control and SHR treated with EGCG(50 mg/kg/day)or losartan(10 mg/kg/day).The treatment was given daily for 4 weeks by oral gavage and the blood pressure was monitored by tail-cuff method every 3 days.Acetylcholineinduced endothelium-dependent relaxations were assessed in isolated phenylephrine-precontracted aortic rings at the end of treatment.The vascular levels of reactive oxygen species,nitric oxide,tetrahydrobiopterin,and cyclic guanosine monophosphate were also measured.Moreover,the expression of angiotensinⅡtype 1(AT_(1))receptor protein was determined.Results:The systolic blood pressure was significantly decreased in SHR treated with EGCG.The impaired endothelium-dependent relaxation was significantly improved in aortic ring isolated from the EGCG-treated SHR group.EGCG also significantly increased the levels of nitric oxide,tetrahydrobiopterin,and cyclic guanosine monophosphate,while decreasing the level of reactive oxygen species and the protein expression of AT_(1)receptor in SHR.Conclusions:EGCG attenuates endothelial dysfunction in SHR by decreasing oxidative stress and increasing vascular nitric oxide bioavailability,which may be modulated partly by inhibition of vascular AT_(1)receptors.An increase in endothelium-dependent relaxation may contribute to a decrease in blood pressure in hypertensive animals.

(-)-Epigallocatechin-3-gallate inhibits growth of gastric cancer by reducing VEGF production and angiogenesis

AIM： To investigate the effect of （-）-epigallocatechin-3-gallate （EGCG） on growth of gastric cancer and its possible mechanism. METHODS： Heterotopic tumors were induced by subcutaneously injection of SGC-7901 cells in nude mice. Tumor growth was measured by calipers in two dimensions. Tumor angiogenesis was determined with tumor microvessel density （MVD） by immunohistology. Vascular endothelial growth factor （VEGF） protein level and activation of signal transducer and activator of transcription 3 （Star3） were examined by Western blotting. VEGF mRNA expression was determined by RT-PCR and VEGF release in tumor culture medium by ELISA. VEGF-induced cell proliferation was studied by MTT assay, cell migration by gelatin modified Boyden chamber （Transwell） and in vitro angiogenesis by endothelial tube formation in Matrigel. RESULTS： Intraperitoneal injection of EGCG inhibited the growth of gastric cancer by 60.4%. MVD in tumor tissues treated with EGCG was markedly reduced. EGCG treatment reduced VEGF protein level in vitro and in vivo. Secretion and mRNA expression of VEGF in tumor cells were also suppressed by EGCG in a dose-dependent manner. This inhibitory effect was associated with reduced activation of Star3, but EGCG treatment did not change the total Star3 expression. EGCG also inhibited VEGF-induced endothelial cell proliferation, migration and tube formation. CONCLUSION： EGCG inhibits the growth of gastric cancer by reducing VEGF production and angiogenesis, and is a promising candidate for anti-angiogenic treatment of gastric cancer.

(-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 via Stat3 in gastric cancer

AIM: To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer. METHODS: Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditionedmedium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RTPCR). Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay. RESULTS: There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490, VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited Stat3 nucleus translocation, and Stat3-DNA binding activity was also markedly decreased by EGCG. Furthermore, EGCG inhibited IL-6 induced vascular endothelial cell proliferation and tube formation in vitro and angiogenesis in vitro . CONCLUSION: EGCG inhibits IL-6-induced VEGF expression and angiogenesis via suppressing Stat3 activity in gastric cancer, which has provided a novel mechanistic insight into the anti-angiogenic activity of EGCG.

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells

AIM: To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21waf1, and p27kip1 expression and phosphorylation of PDGF β-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. RESULTS: EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin Dl and increased p27kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF p-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/ Akt pathways. CONCLUSION: EGCG inhibited PDGFBB-induced proliferation and migration of PSCs through the inhibition of PDGFmediated signaling pathways.

Apoptotic effect of Epigallocatechin-3-gallate on the human gastric cancer cell line MKN45 via activation of the mitochondrial pathway

To investigate whether Epigallocatechin-3-gallate （EGCG） can induce apoptosis of the gastric cancer cell line MKN45 and its apoptotic pathway.METHODS： To determine this, apoptotic rates of MKN45 cells after EGCG treatment with or without caspase-3 inhibitor were evaluated by Annexin V-FITC ＋ PI staining The influence of EGCG on the activity of caspase-3 in the MKN45 cells was determined by ELISA. By Rhodamine123 staining, the membrane potential change of the mitochondrion was also investigated, and mRNAs and protein expression of the bcl-2 family were analyzed by RT-PCR and Western blot.RESULTS： EGCG can induce apoptosis of MKN45 cells in time- and dose-dependent manner. Eight hours after EGCG treatment, the activity of caspase-3 in the MKN45 increased, especially 12 h after treatment. The mitochondrial membrane potential was significantly weakened 4 h after EGCG insult. The mRNA and protein expression levels of pro-apoptotic members, such as Bax, Bid and Bad, were upregulated gradually as treated time increased. Moreover, the mRNA and protein expression levels of anti-apoptotic members, such as Bcl-xL and Bcl-2, were inhibited. CONCLUSION： These data support that EGCG can induce apoptosis of the human gastric cancer cell line MKN45, and the effect is in a time- and dose-dependent manner. The apoptotic pathway triggered by EGCG in MKN45 is mitochondrial-dependent.

Cytotoxicity of epigallocatechin-3-gallate to LNCaP cellsin the presence of Cu^(2+)

Epigallocatechin-3-gallate (EGCG) has shown remarkably anti-cancer activity, with its bioactivity being related to reactive conditions, such as pH and metal ions. The present study investigated the degradation of EGCG and its effect on prostate cancer cell in the presence of Cu2+. EGCG was incubated with prostate cancer cells, LNCaP, pretreated with or without Cu2+.EGCG in F-12 medium was quantified using HPLC and the viability of cells was assessed by gel electrophoresis, flow cytometry,and electron microscope. The results of HPLC showed that EGCG degraded completely within 12 h in F-12 medium with or without Cu2+. Gel electrophoresis and flow cytometry did not detect apoptosis of LNCaP cells when they were incubated with EGCG. Electron microscopy examination revealed that EGCG-Cu2+ complex led to damage of cytoplasm membrane in LNCaP cells. It was speculated that not EGCG, but its oxide and complex with Cu2+, are the bioactive components responsible for its cytotoxicity to LNCaP prostate cancer cells.

Epigallocatechin-3-gallate Alleviates Cognitive Deficits in APP/PS1 Mice

Alzheimer's disease(AD)shows cognitive impairments in clinic,which is multifactorial with different etiopathogenic mechanisms such as A|3 deposition,neuroinflammation and neuronal dystrophy involved.Therefore,multi-targets drugs with neuroprotective,anti-amyloidogenic and anti-inflammatory properties will be effective in AD treatment.Epigallocatechin-3-gallate(EGCG)possesses a broad spectrum of pharmacological activities in the prevention and treatment of multiple neurodegenerative diseases.In the present study,we showed that oral administration of EGCG(50 mg/kg)for 4 months significantly attenuated the cognitive deficits in APP/PS1 transgenic mice,which served as AD model.Moreover,EGCG induced an improvement in dendritic integrity and expression levels of synaptic proteins in the brain of APP/PS 1 mice.And EGCG exerted obvious anti-inflammatory effects,which was manifested by alleviating microglia activation,decreasing pro-inflammatory cytokine(IL-β)and increasing anti-inflammatory cytokines(IL-10,IL-β).Furthermore,p-amyloid(AP)plaques were markedly reduced in the hippocampus of 6-month old APP/PS 1 mice after EGCG treatment.In conclusion,these findings indicate that EGCG improves AD-like cognitive impairments through neuroprotective,anti-amyloidogenic and anti-inflammatory effects,thus is a promising therapeutic can didate for AD.

Suppression of esophageal cancer cell growth using curcumin,(-)-epigallocatechin-3-gallate and lovastatin

AIM:To determine the effects of curcumin,(-)-epigallocatechin-3-gallate (EGCG),lovastatin,and their combinations on inhibition of esophageal cancer.METHODS:Esophageal cancer TE-8 and SKGT-4 cell lines were subjected to cell viability methyl thiazolyl tetrazolium and tumor cell invasion assays in vitro and tumor formation and growth in nude mouse xenografts with or without curcumin,EGCG and lovastatin treatment.Gene expression was detected using immunohistochemistry and Western blotting in tumor cell lines,tumor xenografts and human esophageal cancer tissues,respectively.RESULTS:These drugs individually or in combinations significantly reduced the viability and invasion capacity of esophageal cancer cells in vitro.Molecularly,these three agents reduced the expression of phosphorylated extracellular-signal-regulated kinases (Erk1/2),c-Jun and cyclooxygenase-2 (COX-2),but activated caspase 3 in esophageal cancer cells.The nude mouse xenograft assay showed that EGCG and the combinations of curcumin,EGCG and lovastatin suppressed esophageal cancer cell growth and reduced the expression of Ki67,phosphorylated Erk1/2 and COX-2.The expression of phosphorylated Erk1/2 and COX-2 in esophageal cancer tissue specimens was also analyzed using immunohistochemistry.The data demonstrated that 77 of 156 (49.4%) tumors expressed phosphorylated Erk1/2 and that 121 of 156 (77.6%) esophageal cancers expressed COX-2 protein.In particular,phosphorylated Erk1/2 was expressed in 23 of 50 (46%) cases of esophageal squamous cell carcinoma (SCC) and in 54 of 106 (50.9%) cases of adenocarcinoma,while COX-2 was expressed in 39 of 50 (78%) esophageal SCC and in 82 of 106 (77.4%) esophageal adenocarcinoma.CONCLUSION:The combinations of curcumin,EGCG and lovastatin were able to suppress esophageal cancer cell growth in vitro and in nude mouse xenografts,these drugs also inhibited phosphorylated Erk1/2,c-Jun and COX-2 expression.

Neuroprotective effect of epigallocatechin-3-gallate on hemisection-induced spinal cord injury in rats

Epigallocatechin-3-gallate （EGCG）, a naturally occurring compound in green tea, has been widely used as an antioxidant agent. In the present study, model rats with acute spinal cord injury were intraperitoneally injected with 25, 50, and 100 mg/kg EGCG, and spinal cord ultrastructure, oxidative stress reaction, inflammatory factors, and apoptosis-associated gene expression were observed. Results showed that EGCG attenuated neuronal and axonal injury 24 hours post injury. It also decreased serum intedeukin-113, tumor necrosis factor-a, and intercellular adhesion molecule-1 release, and decreased apoptosis-associated gene expression. Furthermore, it increased the level of the superoxide anion （O2-）, superoxide dismutase, and B-cell lymphoma/leukemia-2, and reduced malondialdehyde levels. Furthermore, it reduced the expression of the pro-apoptotic protein Bax. Noticeably, EGCG at the 100 mg/kg dosage exhibited similar effects as methylprednisolone sodium succinate, which has been frequently used for clinical acute spinal cord injury. The results demonstrated that EGCG can significantly inhibit inflammation, suppress oxidation, and reduce apoptosis in acute spinal cord injury.

Protective Effects of Epigallocatechin-3-gallate on Intestinal Ischemia Reperfusion Injury through Enhanced Activation of PI3K/Akt Pathway in Rats

Inflammation plays a critical role in intestinal ischemia reperfusion injury（IRI）. Epigallocatechin-3-gallate（EGCG） has been demonstrated to possess anti-inflammatory effect. This study examined the effect of EGCG on intestinal IRI and explored the possible mechanisms. Male Wistar rats were randomly divided into three groups： sham-operated group（Sham）, IRI control group（IRI） and IRI-EGCG group（EGCG）. Rats in IRI-EGCG group were administered dissolved EGCG in drinking water（0.4 mg/m L） for 14 days prior to IRI induction. A rat model of intestinal IRI was established by ligating the superior mesenteric artery（SMA） for 30 min, followed by reperfusion for 1 h. Intestinal histology, pro-inflammatory cytokines and mediators were examined and the effect of EGCG on PI3K/Akt signalling was assessed. EGCG significantly alleviated the pathological changes of the intestine and suppressed the IRI-induced up-regulation of TNF-α, IL-1 and IL-6 m RNA and protein expression in the serum and intestine. The mechanism might be that EGCG enhanced the activation of PI3K/Akt signalling pathway. In conclusion, the administration of EGCG can significantly mitigate the acute intestinal IRI in rats by enhancing the activation of PI3K/Akt signalling pathway to suppress inflammatory response and might be a promising alternative for the prevention or treatment of intestinal IRI in the clinical practice.

Inhibition of Invasion and Up-regulation of E-cadherin Expression in Human Malignant Melanoma Cell Line A375 by(-)-Epigallocatechin-3-gallate

The inhibitory effects of （-）-epigallocatechin-3-gallate （EGCG） on the invasion of human malignant melanoma cell line A375 and the possible molecular mechanisms of this effect were investiaged. A375 cells were pretreated with 20 μg/mL EGCG for 24, 48 and 72 h respectively and the E-cadherin expression was detected by Western blot analysis. A375 cells were also pretreated with different concentrations of EGCG （1, 5, 10 and 20 μg/mL） for 72 h and the expression of E-cadherin was measured by RT-PCR. The adhesion and invasion of A375 cells were tested by cell-matrigel adhesion assay and matrigel invasion assay respectively. The results showed that EGCG could significantly up-regulate the expression of E-cadherin time-and concentration-dependently （both P〈0.05）. Statistical analysis showed that A375 cells invasion was inhibited by EGCG and correlated with the up-regulation of E-cadherin expression. It was suggested that EGCG strongly inhibited invasion of A375 cells, and the inhibition mechanism was possibly associated with the up-regulation of E-cadherin expression.

Epigallocatechin-3-gallate treatment to promote neuroprotection and functional recovery after nervous system injury

Traumatic spinal cord injury （SCI） causes motor paralysis, sensory anesthesia and autonomic dysfunction below the le- sion site and additionally some SCI patients refer neuropathic pain together with these signs and symptoms. Clinical and experimental studies have revealed the main pathological changes of injured spinal cord implicated in all these signs and symptoms, including neuropathic pain. After few hours of traumatic SCI, it is usual to observe broken blood brain barrier with plasma and blood cells extravasation, cell necrosis, disruption of ascending and descending spinal cord pathways and increased potassium and glutamate. Glutamate contributes to excitotoxicity of neurons whereas potassium facilitates ectopic depolarization of survival neurons and activation of resident microglia.

Epigallocatechin-3-gallate attenuates lipopolysaccharideinduced inflammation in human retinal endothelial cells

AIM:To investigate the mechanism underlying the anti-inflammatory effects of epigallocatechin-3-gallate(EGCG)in lipopolysaccharide(LPS)-stimulated human retinal endothelial cells(HRECs).METHODS:HRECspre-treatedwithEGCG(0-100μmol/L)were stimulated with LPS(250 ng/mL).Levels of tumor necrosis factor alpha(TNF-α),vascular endothelial growth factor(VEGF),monocyte chemotactic protein-1(MCP-1)and nitric oxide(NO)in the supernatants were determined by enzyme-linked immunosorbent assay(ELISA)and Griess assay.The protein expression of phosphorylated extracellular signal-regulated kinase(ERK)1/2 and p38 mitogen-activated protein kinases(p38)were determined by Western blot analysis.RESULTS:EGCG pre-treatment significantly inhibited the secretion of TNF-α,VEGF,MCP-1 and NO in LPSstimulated HRECs.Moreover,EGCG effectively attenuated LPS-induced activation and phosphorylation of ERK1/2 and p38 in HRECs in a dose-dependent manner.CONCLUSION:EGCG exhibited inhibitory effects on LPS-induced pro-inflammatory cytokines production by modulating ERK1/2 and p38 pathways in HRECs,suggesting EGCG as a potential candidate for antiinflammatory intervention.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes

AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Epigallocatechin-3-gallate suppresses transforming growth factor-beta signaling by interacting with the transforming growth factor-beta typeⅡreceptor

AIM: To investigate the(-)-epigallocatechin-3-gallate(EGCG) binding to transforming growth factor-β(TGF-β) type Ⅱ receptor(TGFRⅡ).METHODS: The expression of α-smooth muscle actin(α-SMA) was used as a marker for fibrotic change inhuman lung fibroblast MRC-5 cells. The α-SMA expression level was determined by western blotting and immunohistological analysis. We examined whether the anti-fibrotic effects of EGCG on MRC-5 cells was dependent on antioxidant mechanism by using edaravone and N-acetylcysteine(NAC). The suppression effects of EGCG on Smad2/3 activation were studied by confocal fluorescence microscopy. The binding of EGCG to recombinant TGFRⅡ protein was analyzed by immunoprecipitation and affinity chromatography.RESULTS: When MRC-5 cells were treated with TGF-β, EGCG decreased the expression of α-SMA in a dose dependent manner, whereas catechin did not influence the α-SMA expression in the cells. Except for EGCG, antioxidant compounds(e.g., edaravone and NAC) had no effects on the TGF-β-induced α-SMA expression. Nuclear localization of phosphorylated Smad2/3 was observed after TGF-β treatment; however, EGCG treatment attenuated the nuclear transportation of Smad2/3 in the presence or absence of TGF-β. After a TGFRⅡ expression vector was introduced into COS-7 cells, cell lysates were untreated or treated with EGCG or catechin. The immunoprecipitation experiments using the lysates showed that EGCG dose-dependently bound to TGFRⅡ and that catechin did not at all. Affinity chromatography study indicated that EGCG would bind to TGFRⅡ.CONCLUSION: Our results demonstrate that EGCG interacts with TGFRⅡ and inhibits the expression of α-SMA via the TGF-β-Smad2/3 pathway in human lung fibroblast MRC-5 cells.

Protective effects of (-)-epigallocatechin-3-gallate on D-galactose-induced neuronal apoptosis in mice

BACKGROUND： The neuroprotective effects of （-）-epigallocatechin-3-gallate （EGCG）, the main polyphenolic constituent of green tea, have been widely reported. However, the action mechanisms, in particular in D-galactose-induced aging mice, remain poorly understood. OBJECTIVE： The present study investigated the protective effects of EGCG on D-galactose-induced hippocampus neuronal apoptosis in aging mice, as well as the relationship with expression of p751CD, JNK2, and p53 proteins. DESIGN, TIME AND SETTING： A randomized, controlled, molecular biological, animal experiment was performed at the Laboratory of Pharmacology, Pharmaceutical College of China Medical University, China, from September 2006 to July 2008. MATERIALS： D-galactose and EGCG （Sigma, USA）, as well as terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling （TUNEL） In Situ Cell Apoptosis Detection Kit （Promega, USA）, were used in this study. METHODS： A total of 64 mice were equally and randomly divided into D-galactose model, low-dose EGCG, high-dose EGCG, and control groups. Mice in the D-galactose model, low-dose EGCG, and high-dose EGCG groups were subcutaneously injected with 3% D-galactose （150 mg/kg）, daily for 6 weeks, to establish a mouse model of aging. Mice in the control group were treated with saline （5 mL/kg）. At 3 weeks following injection, mice in the low-dose EGCG and high-dose EGCG groups were orally administered EGCG at a dose of 2 mg/kg and 6 mg/kg, respectively, once a day, for 4 consecutive days. Mice in the control and D-galactose model groups received distilled water （5 mL/kg）. MAIN OUTCOME MEASURES： Memory function was evaluated using a step-through passive avoidance test. Neuronal apoptosis in the mouse hippocampus was detected using TUNEL staining. Expression levels of the intracellular domain of the p75 neurotrophin receptor （p75NTR）-p751CD, JNK2, and p53 proteins in the hippocampus were determined using Western blot analysis. RESULTS： The aging mouse model was induced by subcutaneous injection of D-galactose, which resulted in obvious memory impairment, increased apoptotic index, and increased protein expression levels of p751CD, JNK2, and p53 in the hippocampus, compared with control mice （P 〈 0.01）. Oral EGCG administration （2 or 6 mg/kg） for 4 weeks significantly improved levels of memory deficit in the aging mice and reduced apoptotic indices and protein expression levels of p751CD, JNK2, and p53 in the mouse hippocampus （P 〈 0.01）. CONCLUSION： Results from this study demonstrated increased protein expression levels of p751CD, JNK2, and p53, as well as increased hippocampal neuronal apoptosis in a D-galactose-induced mouse model of aging. EGCG provided protective effects against D-galactose-induced neuronal apoptosis in the hippocampus by reducing protein expression levels of p751CD, JNK2, and p53 proteins in the hippocampus of aging mice.

Integrating network pharmacology and pharmacological evaluation for deciphering the mechanism of(-)-epigallocatechin-3-gallate alleviating ethanol-induced endothelial cells injury

Objective To investigate the potential therapeutic targets and pharmacological mechanism of(-)-epigallocatechin-3-gallate(EGCG)based on network pharmacology and experimental verification.METHODS The druggability of EGCG was measured by the traditional Chinese medicine systems pharmacology(TCMSP)server,and potential targets of EGCG were identified by Pharm Mapper and Drug Repositioning and Adverse drug Reaction via Chemical-Protein Interactome(DRAR-CPI).The potential targets were imported into GeneMANIA database to obtain the protein-protein direct interaction network,and target physical interaction,co-expression,prediction,genetic interaction,and shared protein domains.The biological process,molecular functions,cellular components and KEGG signaling pathways of potential targets were analyzed using DAVID database.For further study,ethanol was used to establish a model of endothelial injury in vitro.The cell viability was assayed by MTT method,the cellular apoptosis was stained by Annexin V/PI,and the expression levels of Bcl-2,Bax and cleved-caspase-3 were tested by Western blotting.Then,JC-1 and nuclear translocation of NF-κB experiments were used to study the mitochondrial membrane potential and nuclear translocation.RESULTS The oral availability of EGCG was 55.09%(≥30%)and drug-like index was 0.77(≥0.18),which were considered pharmacokinetically active.17 potential targetable proteins of EGCG were predicted by Pharm Mapper and DRAR-CPI.Further research showed that 68.13%displayed similar co-expression characteristics,26.11%physical interactions,and 2.74%shared the same protein domain.The depth network analysis results showed that the biofunctions of EGCG were mainly by regulating glutathione derivative biosynthetic process,glutathione metabolic process,nitrogen compound metabolic process etc..via drug binding,catalytic activity,glutathione transferase activity,anion binding etc..in sarcoplasmic reticulum,spindle pole,microtubule cytoskeleton and cytoplasm.KEGG enrichment analysis showed that Glutathione metabolism,IL^(-1)7 signaling pathway,EGFR tyrosine kinase inhibitor resistance,PI3K-Akt signaling pathway and other pathways were involves in the biofunction of EGCG.The above analyses indicated that EGCG exerts its biofunction through antioxidant and anti-inflammatory mechanisms.The experimental results showed that ethanol 20.0 mmol·L^(-1) decreased cell viability,Bcl-2 expression,and increased cell apoptosis,the intracellular ROS,as well as the expression of Bax and cleaved-caspase-3 of human endothelial cells.However,treatment of the cells with EGCG can significantly alleviate ethanol induced endothelial cells injury.Further study showed that EGCG significantly alleviates ethanol induced mitochondrial depolarization and nuclear translocation of NF-κB.CONCLUSIONS EGCG exerts pharmacological efficacies on ethanol induced endothelial cell injury through multi-target,multi-function and multi-pathway mode.Protective effect of EGCG on ethanol induced cell injury was mainly through alteration of mitochondrial function and NF-κB translocation.Therefore,EGCG have great potential in protecting against endothelial dysfunction of the persons who are chronically abuse of ethanol.This study also provides a new understanding of EGCG in clinical application on cardiovascular and cerebrovascular diseases.

Influence of (-)-epigallocatechin-3-gallate on the expression of connexin43 and gap junction intercellular communication of the bladder cancer cell lines BIU-87 in vitro

Objective： The aim of our study was to investigate the effects of （-）-epigallocatechin-3-gallate （EGCG, the major phytochemistry component in green tea） on the expression of connexin43 （Cx43） gene and detect the intercellular communication of the human bladder cancer cell lines BIU-87, and explore its possible mechanisms of prevention and cure for the bladder tumor. Methods： The methyl thiazolyl tetrazolium and Annexin-V/PI double-labeled flow cytometry methods were used to observe the growth inhibitory rate （IR） and apoptosis rate （AR） of BlU-87 cells treated by EGCG at different concentrations （0, 5, 10 and 20 mg/L）, respectively. The reverse transcription-polymerase chain reaction （RT-PCR） and Western Blotting analysis were employed to detect the relative expression levels of the Cx43 mRNA and its protein. The scrape-loading fluorescence dye transfer method was used to assess the gap junction intercellular communication （GJIC） under fluorescence microscope. Results： EGCG at concentrations （10 and 20 mg/L） both could significantly inhibit the proliferation and induce the apoptosis of BIU-87 cells. The IR and AR were （15.67 ± 1.15）%, （18.33 ± 1.53）% and （42.00 ± 4.34）%, （27.33 ± 3.21）%, respectively. And compared with the control groups of 0 mg/L and 5 mg/L （P 〈 0.05）, EGCG could significantly up-regulate the expression of Cx43 mRNA and its protein and enhance the function of BIU-87 cells. The effects had the significant correlation with the dose-dependent of EGCG. Conclusion： EGCG （10, 20 mg/L） could effectively up-regulate Cx43 expression and enhance the GJIC of BlU-87 cells. The results may indicate the effects of EGCG inducing bladder tumor cells apoptosis and inhibiting its growth which provides the experimental evidence for further demonstrating the mechanism of chemical prevention and cure for the bladder tumor by EGCG.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.