Xuanjun Wang

Mechanism of action of (–)-epigallocatechin-3-gallate: auto-oxidation-dependent activation of extracellular signal-regulated kinase 1/2 in Jurkat cells

AIM (-)-Epigallocatechin-3-gallate (EGCG), a major compound of tea polyphenols, exhibited antitumor activity in previous studies. In these studies, EGCG usually inhibits EGFR, and impairs the ERK1/2 phosphorylation in tumor cells. The aim was to clarify the mechanism of ERK1/2 activation induced by EGCG. METHOD Jurkat and 293T cells were treated with EGCG in different culture conditions. Western Blotting (WB) was employed to analyze ERK1/2 and MEK phosphorylation. Cetuximab and FR180204 were used to inhibit cell signaling. The stability of EGCG was assessed by HPLC. The concentration of hydrogen peroxide generated by the auto-oxidation of EGCG was determined by photocolorimetric analysis. RESULTS Activation of ERK1/2 was observed to be both time-and dose-dependent. Stimulation of cell signaling was dependent on MEK activity, but independent of EGFR activity. Unexpectedly, EGCG was depleted within one hour of incubation under traditional culture conditions. Auto-oxidation of EGCG generated a high level of hydrogen peroxide in the medium. Addition of catalase and SOD to the acidic medium inhibited the oxidation of EGCG. However, this particular condition also prevented the phosphorylation of ERK1/2. The generation of ROS by hydrogen peroxide may also induce ERK1/2 activation in Jurkat cells. CONCLUSION ERK1/2 phosphorylation was caused by auto-oxidation of EGCG. Traditional culture conditions were determined to be inappropriate for EGCG research.

High quality reference genome of drumstick tree ( Moringa oleifera Lam.), a potential perennial crop

The drumstick tree (Moringa oleifera Lam.) is a perennial crop that has gained popularity in certain developing countries for its high-nutrition content and adaptability to arid and semi-arid environments. Here we report a high-quality draft genome sequence of M. oleifera. This assembly represents 91.78% of the estimated genome size and contains 19,465 protein-coding genes. Comparative genomic analysis between M. oleifera and related woody plant genomes helps clarify the general evolution of this species, while the identification of several species-specific gene families and positively selected genes in M. oleifera may help identify genes related to M. oleifera’s high protein content, fast-growth, heat and stress tolerance. This reference genome greatly extends the basic research on M. oleifera, and may further promote applying genomics to enhanced breeding and improvement of M. oleifera.

Caffeine is responsible for the bloodglucose-lowering effects of green tea and Puer tea extractsin BALB/c mice.

The present study was designed to determine the effects of Puer tea and green tea on blood glucose level. Male BALB/c mice were administered green tea extract (GTE) or Puer tea extract (PTE), either intragastrically or in their drinking water. The major components of these teas are epigallocatechin gallate (EGCG) and caffeine, respectively. Blood glucose measurement results showed that mice fed intragastrically or mice that drank GTE, PTE or caffeine showed significantly lower blood glucose levels compared to the control group. However, EGCG exhibited no influence on the blood glucose levels. When caffeine was eliminated from the GTE and PTE, the effect on the blood glucose levels was abolished, but the effect was recovered when caffeine was re-introduced into the extracts. Evaluation of hematological and biochemical indices at the time of the greatest caffeine-induced decrease in blood glucose levels showed that the effect of caffeine was specific. Microarray analyses were performed in 3T3-L1 preadipocytes and mature adipocytes treated with 0.1 mg · mL(-1) caffeine to identify factors that might be involved in the mechanisms underlying these effects. The results showed that few genes were changed after caffeine treatment in adipocytes, and of them only phospholipid transfer protein (PLTP) may be ralated to blood glucose. In conclusion, this study indicates that caffeine may be the key constituent of tea that decreases blood glucose levels, and it may be used to treat type 2 diabetes.

Pu-erh Tea Water Extract Mediates Cell Cycle Arrest and Apoptosis in MDA-MB-231 Human Breast Cancer Cells

Pu-erh tea is believed to have health benefits, the growth inhibition activity of Pu-erh tea on breast cancer cell has not been investigated. In this study, we examined the activity of Pu-erh tea water extract on apoptosis and cell cycle arrest in the human breast adenocarcinoma cell line MDA-MB-231 and clarified its underlying mechanism of action. We found that Pu-erh tea extract inhibited cell proliferation and induced apoptosis in a dose-dependent manner. We also found that Pu-erh tea extract inhibited tumor cell growth within 24 h via accumulation of cells in S phase. Further experiments showed that at 24 h, Pu-erh tea extract up-regulated the expressions of P-p53 (Ser15), p21 and P-JNK and down-regulated the expressions of PCNA, CyclinD1 and CyclinE at the protein level in MDA-MB-231 cells. In particular, the JNK-specific inhibitor SP600125 restored the induction of P-JNK, P-p53 (Ser15), p21, CyclinD1 and CyclinE by Pu-erh tea extract. Our results indicate that Pu-erh tea water extract inhibits cell proliferation of MDA-MB-231 cells through the induction of apoptosis and the stimulation of cell cycle arrest, which is mediated via activation of the JNK-related pathway.

Pu-erh Tea Extract Ameliorates Ovariectomy-Induced Osteoporosis in Rats and Suppresses Osteoclastogenesis In Vitro

Background and Objective: Tea drinking is associated with positive effects on bone health and may protect against osteoporosis, especially in elderly women. Pu-erh tea has many beneficial effects on human health; however, whether Pu-erh tea has anti-osteoporotic potential remains unclear. Thus, we investigated the effects of Pu-erh tea extract (PTE) on ovariectomy-induced osteoporosis in rats and on osteoclastogenesis in vitro. Methods: Female Wistar rats were divided into six groups: the sham, model, and Xian-Ling-Gu-Bao capsule (XLGB) groups, and the low-, medium-, and high-dose PTE groups. Ovariectomized (OVX) rats were used as an animal model of osteoporosis. The animals were intragastrically administered distilled water, XLGB, or different concentrations of PTE for 13 weeks. Body weight, blood biochemical indicators, relative organ coefficients, femoral bone mineral density (BMD), bone biomechanical properties, and bone microarchitecture were examined and analyzed. Additionally, the in vitro effects of PTE on osteoclastic activities were investigated using the RAW 264.7 cell line as an osteoclast differentiation model. The effects of PTE on osteoclast differentiation and the expression of osteoclast-specific genes and proteins were determined. Results: PTE reduced OVX-induced body weight gain after 6 weeks of treatment, and the high-dose exerted a significant effect. High-dose PTE significantly ameliorated OVX-induced estradiol (E2) deficiency. PTE treatment maintained calcium and phosphorus homeostasis and improved other blood biochemical parameters to various degrees. In addition, PTE treatment improved organ coefficients of the femur, uterus, and vagina and improved femoral BMD and bone biomechanical properties. PTE treatment strikingly ameliorated bone microarchitecture. Moreover, in the in vitro studies, osteoclast differentiation using the differentiation cell model was significantly inhibited by PTE without cytotoxic effects. Additionally, PTE efficaciously suppressed the expression of key osteoclast-specific genes and proteins. Conclusion: PTE can ameliorate ovariectomy-induced osteoporosis in rats and suppress osteoclastogenesis in vitro.

Anti-Skin-Aging Effect of Epigallocatechin Gallate by Regulating Epidermal Growth Factor Receptor Pathway on Aging Mouse Model Induced by D-Galactose.

Epigallocatechin gallate(EGCG) is a monomer separated from tea catechins, as an well-known antioxidant, which helps fight wrinkles and rejuvenate skin cells. In this study, we investigated the anti-aging effect of EGCG, and to clarify underlying mechanism of skin aging in a d-galactose-induced aging mouse model. Forty-five male mice were divided into 5 groups and treated with different dose of EGCG, Vitamin C (VitC) to mice as a positive control. All groups except vehicle were established aging model induced by d-galactose (200mg/kg/day) that was subcutaneously injected to mice for 8 weeks. Two weeks after injection of d-galactose, EGCG and Vit C groups were simultaneously administered once a day by subcutaneously inject after 5h for injecting d-galactose. The results show that EGCG can be absorbed by the skin. Overall, the conditions of the skin of EGCG-treatment groups were improved, the whole structure of skin were better than control groups, and the levels of oxidative stress and the expression of relate with EGFR proteins were significantly higher than control group after EGCG treatment. All these findings suggest that EGCG can resist skin senility effectively. And the EGFR with relate of downstream proteins are implicated in the skin aging.

Tea polysaccharide inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells and ameliorates ovariectomy-induced osteoporosis in rats

BACKGROUND AND PURPOSE Tea drinking has positive effects on bone health and may prevent and treat osteoporosis, especially in older and postmenopausal women. Tea polysaccharide (TPS) is a major bioactive constituent in tea. Despite its profound effects on human health, whether TPS has anti-osteoporotic effects remains largely unknown. As such, we investigated the anti-osteoporotic effects of TPS. METHODS In vitro, TPS effects on osteoclastogenesis were examined using osteoclast precursor RAW264.7 cells. TPS effects on osteoclastogenesis-related expression of marker genes and proteins were determined by gene expression and immunoblotting analyses, respectively. For in vivo studies, 12-week-old female Wistar rats were divided randomly into a sham-operated group (sham) and four ovariectomized (OVX) subgroups: OVX with vehicle (model) and OVX with low-, medium-, and high-dose TPS (0.32, 0.64 and 1.28 g/kg body weight/day, respectively). TPS was administered intragastrically to rats for 13 weeks. Body weight, blood biochemical parameters, organ weight, organ coefficients, femoral length, bone mineral density (BMD), biomechanical properties, and bone microarchitecture were documented. RESULTS TPS inhibited osteoclast differentiation significantly and dose-dependently, and its inhibitory effect was not due to toxicity to RAW264.7 cells. TPS suppressed expression of osteoclastogenesis-related marker genes and proteins significantly. In in vivo studies, medium-dose TPS treatment ameliorated OVX-induced calcium loss significantly. Low-dose TPS treatment decreased the activity of acid phosphatase (ACP) in OVX rats significantly. In addition, TPS treatment improved other blood biochemical parameters and femoral biomechanical properties to a certain extent. More importantly, TPS treatment ameliorated bone microarchitecture in OVX rats strikingly because of increased cortical bone thickness and trabecular bone area in the femur. CONCLUSION TPS can inhibit receptor activator nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells and ameliorate ovariectomy-induced osteoporosis in rats.

Epigallocatechin Gallate Targets Notch to Attenuate the Inflammatory Response in the Immediate Early Stage in Human Macrophages

Inflammation plays important roles at different stages of diabetes mellitus, tumorigenesis, and cardiovascular diseases. (−)-Epigallocatechin gallate (EGCG) can attenuate inflammatory responses effectively. However, the immediate early mechanism of EGCG in inflammation remains unclear. Here, we showed that EGCG attenuated the inflammatory response in the immediate early stage of EGCG treatment by shutting off Notch signaling and that the effect did not involve the 67-kDa laminin receptor, the common receptor for EGCG. EGCG eliminated mature Notch from the cell membrane and the nuclear Notch intercellular domain, the active form of Notch, within 2 min by rapid degradation via the proteasome pathway. Transcription of the Notch target gene was downregulated simultaneously. Knockdown of Notch 1/2 expression by RNA interference impaired the downregulation of the inflammatory response elicited by EGCG. Further study showed that EGCG inhibited lipopolysaccharide-induced inflammation and turned off Notch signaling in human primary macrophages. Taken together, our results show that EGCG targets Notch to regulate the inflammatory response in the immediate early stage.

Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb) against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilized for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level.

Aflatoxin B1 can be complexed with oxidised tea polyphenols and the absorption of the complexed aflatoxin B1 is inhibited in rats.

BACKGROUND Aflatoxin B1 (AFB1) is the most prevalent and carcinogenic form of the aflatoxins. In this report, we explored the interaction between AFB1 and oxidised tea polyphenols (OTP). Then, the influence of OTP on the absorption and toxicity of AFB1 in rats was investigated. RESULTS We found that AFB1 can be complexed with OTP, and a transmembrane bidirectional transport experiment verified the absorption of complexed AFB1 (C-AFB1) was inhibited by OTP dramatically (P < 0.001). Animal experiments results showed that the AFB1 plus OTP group had significantly (P < 0.05) decreased AFB1-albumin (AFB1-alb) compared to the AFB1 group at 4 h after ingestion. OTP could significantly (P < 0.01) promote the elimination of AFB1 in faeces. Moreover, the liver injury induced by AFB1 was significantly inhibited by OTP. CONCLUSION Our results demonstrated AFB1 can be complexed with OTP and the absorption of the C-AFB1 is inhibited in rats. Consequently, the liver injury induced by AFB1 can be inhibited by OTP. These results provide insight that consuming OTP-containing products, like fermented Pu-er tea, can protect damage from AFB1, and OTP may be used as a kind of food additive.