Epigallocatechin-3-gallate-induced tolerance to cadmium stress involves increased flavonoid synthesis and nutrient homeostasis in tomato roots.

Cadmium (Cd) is a toxic heavy metal, increasingly accumulating in the environment and its presence in various environmental compartments represents a significant risk to human health via the food chain. Epigallocatechin-3-Gallate (EGCG) is a prominent secondary metabolite, which can safeguard plants from biotic and abiotic stress. However, the role of EGCG in flavonoid synthesis, nutrient acquisition and reactive oxygen species (ROS) metabolism under Cd stress remains unclear. Here, we examined the effects of EGCG and Cd treatment on leaf photochemical efficiency, cell ultrastructure, essential element acquisition, antioxidant system, and secondary metabolism in tomato (Solanum lycopersicum L.). The results showed that O2•-, H2O2, and malondialdehyde levels increased after Cd treatment, but Fv/Fm decreased significantly, suggesting that Cd induced oxidative stress and photoinhibition. However, EGCG mitigated the adverse effects of Cd-induced phytotoxicity in both the roots and leaves. A decrease in ROS accumulation under EGCG + Cd treatment was mainly attributed to the significant enhancement in antioxidant enzyme activity, flavonoid content, and PHENYLALANINE AMMONIA-LYASE expression in roots. Moreover, EGCG reduced Cd content but increased some essential nutrient contents in tomato plants. Transmission electron microscopy-based observations revealed that EGCG treatment safeguards leaf and root cell ultrastructure under Cd stress. This implies that tomato plants subjected to Cd stress experienced advantageous effects upon receiving EGCG treatment. The present work elucidated critical mechanisms by which EGCG induces tolerance to Cd, thereby providing a basis for future investigations into environmentally sustainable agricultural practices in areas contaminated with heavy metals, for utilizing naturally occurring substances found in plants.

Visfatin induces the apoptosis of endothelial progenitor cells via the induction of pro-inflammatory mediators through the NF-κB pathway.

Endothelial progenitor cells (EPCs) are an independent factor predicting cardiovascular events. Visfatin plays an important role in the pathogenesis of various metabolic disorders. In this study, we examined the effects of visfatin on the apoptosis of EPCs and the mechanisms underlying these effects. Cultured EPCs pre-treated with various concentrations of visfatin, FK866 (visfatin inhibitor) and BAY11-7085 [referred to as BAY11; nuclear factor-κB (NF-κB) inhibitor] were used to investigate the association between visfatin and EPC apoptosis. Following treatment with visfatin for 48 h, the EPCs exhibited a dose-dependent increase in apoptosis and an upregulated expression of Bax, caspase-3 and NF-κB at both the mRNA and protein level, and a decreased protein expression of Bcl-2. Compared with the untreated control group, the increase in EPC apoptosis, as well as in Bax and caspase-3 expression was significant following treatment with 150 ng/ml visfatin, which also induced a dose-dependent and significant increase in the protein expression of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1). All the visfatin-induced effects were suppressed by pre-treatment with FK866. Pre-incubation of the EPCs with BAY11 for 1 h followed by treatment with visfatin (150 ng/ml) for 48 h also abolished visfatin-induced apoptosis; it also abolished the promoting effects of visfatin on the expression of caspase-3, Bax, ICAM-1 and IL-6, and its suppressive effects on the protein expression of Bcl-2. On the whole, our data indicate that visfatin induces EPC apoptosis by increasing the expression of pro-inflammatory mediators partly through the regulation of NF-κB.

Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, ß-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

Leucine and histidine independently regulate milk protein synthesis in bovine mammary epithelial cells via mTOR signaling pathway.

The aim of this study is to investigate the effects of leucine (Leu) and histidine (His) on the expression of both the mammalian target of rapamycin (mTOR) signaling pathway-related proteins and caseins in immortalized bovine mammary epithelial cells (CMEC-H), using a single supplement through Western blotting. The Earle's balanced salt solution (EBSS) was set as the control group and other treatment groups, based on the EBSS, were added with different concentrations of Leu or His, respectively. The results showed that, compared with the control group, the expression of caseins and the phosphorylation of mTOR (Ser(2481)), Raptor (Ser(792)), eIF4E (Ser(209)), and eEF2 (Thr(56)) increased with the Leu concentrations ranging from 0.45 to 10.80 mmol/L (P<0.01). The P-4EBP1 (Thr(37)) at 10.80 mmol/L Leu, and P-RPS6 (Ser(235/236)) at 5.40 to 10.80 mmol/L Leu all decreased. Similarly, the His supplementation from 0.15 to 9.60 mmol/L increased the expression of αs2-casein, ß-casein, κ-casein, P-mTOR (Ser(2481)), P-Raptor (Ser(792)), P-S6K1 (Thr(389)), P-4EBP1 (Thr(37)), P-eIF4E (Ser(209)), and P-eEF2 (Thr(56)) (P<0.01) in CMEC-H, whereas the αs1-casein expression was only reduced at 9.60 mmol/L His, G protein ß subunit-like protein (GßL) at 0.15 and 9.60 mmol/L His, and P-RPS6 at 4.80 to 9.60 mmol/L His. Our linear regression model assay suggested that the αs1-casein expression was positively correlated with P-mTOR (P<0.01), P-S6K1 (P<0.01), and P-eEF2 (P<0.01) for the addition of Leu, while the expressions of ß-casein (P<0.01) and κ-casein (P<0.01) were positively correlated with P-eEF2 for the addition of His. In conclusion, the milk protein synthesis was up-regulated through activation of the mTOR pathway with the addition of Leu and His in CMEC-H.