Lipopolysaccharide (LPS) is the major of outer membrane of gram-negative bacteria and one of the most potent microbial initiators of inflammation. From the previous study showed that exposure to a low dose of LPS renders animals tolerant to a lethal dose of LPS, and protects against the toxicity of various chemicals. However, the effects of LPS treatment in thioacetamide (TA) - induced liver injury remain largely unknown. Liver injury caused by various toxic chemicals such as carbon tetrachloride, alcohol, dimethylnitrosamine. Here, we induced rat liver injury by intraperitoneal injection of TA, a representative hepatic fibrosis inducer. In this study, we investigated the effects of LPS in TA group, LPS group, LPS/TA group and vehicle control group on Sprague-Dawely rats (five rats for each group). All rats at the end of the experiment were sacrificed, and liver and serum were obtained. Serological analysis and hematoxylin and eosin staining showed that LPS/TA co-treatment was associated with decrease of aspartate aminotransferase (AST), alanine transaminase (ALT) and totalbilirubin and fibrosis than in TA-treated rats. RT-PCR showed that the levels of IL-6 and Cox2 mRNA were lower in the liver of LPS/TA-cotreated rats than in TA-treated rats. There were no significant differences ALT, ALP, AST, total-bilirubin, IL-6 and Cox2 between vehicle control and LPS-treated rats. These results imply that LPS/TA cotreatment partially alleviates the TA-induced liver injury of rats by reducing inflammatory response.

Potassium channels, the largest group of pore proteins, selectively regulate the flow of potassium (K+) ions across cell membranes. The activity and expression of K+ channels are critical for the maintenance of normal functions in vessels and neurons, and for the regulation of cell differentiation and maturation. However, their role and expression in stem cells have been poorly understood. In this study, we isolated perivascular stem cells (PVCs) from human umbilical cords and investigated the expression patterns of big-conductance Ca2+-activated K+ (BKCa) and voltage-dependent K+ (Kv) channels using the reverse transcription polymerase chain reaction. We also examined the effect of high glucose (HG, 25 mM) on expression levels of BKCa and Kv channels in PVCs. KCa1.1, KCaβ3, Kv1.3, Kv3.2, and Kv6.1 were detected in undifferentiated PVCs. In addition, HG treatment increased the amounts of BKCaβ3a, BKCaβ4, Kv1.3, Kv1.6, and Kv6.1 transcripts. These results suggested that ion channels may have important functions in the growth and differentiation of PVCs, which could be influenced by HG exposure.

Harmonized actions of ovarian estrogen (E2) and progesterone (P4) regulate cell proliferation and differentiation in the uterus with a spatiotemporal manner. Imbalance between the actions and levels of two major regulators often lead to infertility and gynecological diseases, such as endometriosis and endometrial cancer. While numerous works have shown that reduced expression and/or deletion of uterine factors associated with P4 signaling could disturb uterine physiology, local factor(s) to mediate E2 actions has not been extensively studied yet. Here we demonstrate that early growth response 1 (Egr1), a transcription factor which is rapidly induced in the uterus by E2, is required to maintain coordinated actions of E2 and P4 for uterine receptivity for embryo implantation. Given exogenous gonadotrophins to overcome LHβ deficiency in the pituitary of Egr1(－/－) mice, ovulation, fertilization and embryo development normally occurred in these mice. However, they showed complete failure of embryo implantation with reduced uterine responses to artificial decidualization stimuli. While serum levels of E2 and P4 in Egr1(－/－) mice were comparable, genes regulated by E2 and/or P4 in uterine epithelial cells (ECs) were aberrantly expressed on day 4 of pregnancy. Impaired P4 signaling along with absence of PR in ECs caused hypersensitive E2 responses shown as enhanced expression of E2-responsive genes such Muc1 and Ltf as well as reduced levels of P4-dependent genes, such as Ihh and Areg, in ECs of Egr1(－/－) mice. This is consistent with persistent proliferation in ECs and severely impaired proliferation in stromal cells (SCs) in Egr1(－/－) mice treated with E2+P4. Furthermore, primary co-culture of Egr1(－/－) ECs with Egr1(+/+) SCs and vice versa supported a notion that Egr1 itself is required for proper responses to two major regulators, E2 and P4, in both uterine cell compartments. Collectively, our results show that E2-induced Egr1 participates in P4-dependent modulation on E2 activities in the uterus by regulating a spectrum of genes essential for uterine receptivity and embryo implantation.

DGCR8 is a RNA-binding protein working with DROSHA to produce pre-microRNA in the nucleus, while DICER does not only mature microRNA but also endogenous siRNAs in the cytoplasm. Here, we have produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8flox/flox; PRcre/+ mice, Dgcr8d/d) and demonstrated that canonical microRNAs dependent of DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8d/d females did not undergo regular reproductive cycle and produce any pups when housed with fertile males, whereas administration of exogenous gonadotropins induced normal ovulation with corpus luteal formation in these mice. Ovulated oocytes from Dgcr8d/d mice had comparable fertilization potentials and were normally developed to the blastocyst after fertilization as compared to those in control Dgcr8f/f mice. Interestingly, PR-Cre-dependent Dgcr8 deletion showed aberrant infiltration of acute inflammatory immune cells to female reproductive organs only when Dgcr8d/d mice were mated with male mice. With respect to uterine development, gross morphology, histology, and weight of Dgcr8d/d uterus were similar to those of control at 3-week-old age. However, multiple uterine abnormalities were noticeable at 4-week-old age when PR expression is significantly increased, and these deformities became severe onwards. Gland formation and myometrial layers were significantly reduced, and stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced cell proliferation in stromal cell compartments of Dgcr8d/d mice. Collectively, our results suggest that DGCR8 dependent-canonical microRNAs are essential for development and physiology of the uterus with respect to morphogenesis, proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.

Multipotent perivascular stem cells (PVCs) have gained much attention as an alternative source for cell based regenerative medicine in recent years. Due to their rarity in human tissues, developing methods to efficiently isolate and expand PVCs from various fetal and adult tissues is necessary to obtain a clinically relevant number of cells that maintain progenitor potency. Here, we report on a non-enzymatic isolation (NE) method of PVCs from human umbilical cords (HUCs) and compare its efficiency with the conventional collagenase treatment method (CT) in terms of proliferation and immunophenotypes. The cells isolated by NE displyed acceptable surface marker profile of PVCs and showed multilineage (osteogenic, chondrogenic, and adipogenic) differentiation potential. While both methods provided similar levels or patterns of proliferation and immunophenotypes, PVCs by NE retained a higher level of CD146(+) frequency compared to that of CT over passage. Furthermore, we have investigated potentials of various exogenous factors to promote proliferation of HUCPVCs in vitro. Among these factors, supplementation of basic fibroblast growth factor (bFGF) provided the optimal condition to significantly enhance the proliferation rate of HUCPVC and increased a proportion of stage-specific antigen-4 (SSEA-4) positive subset. Collectively, our study suggests that NE method with bFGF supplementation offers an alternative way to obtain sufficient numbers of HUCPVCs with higher number of primitive SSEA-4(+) subpopulation that are applicable in therapeutic doses for regenerative medicine.

Early growth response 1 (Egr1) is a zinc-finger transcription factor to direct second-wave gene expression leading to cell growth, differentiation and/or apoptosis. While it is well-known that Egr1 controls transcription of an array of targets in various cell types, downstream target gene(s) whose transcription is regulated by Egr1 in the uterus has not been identified yet. Thus, we have tried to identify a list of potential target genes of Egr1 in the uterus by performing multi-step in silico promoter analyses. Analyses of mRNA microarray data provided a cohort of genes (102 genes) which were differentially expressed (DEGs) in the uterus between Egr1(+/+) and Egr1(–/–) mice. In mice, the frequency of putative EGR1 binding sites (EBS) in the promoter of DEGs is significantly higher than that of randomly selected non-DEGs, although it is not correlated with expression levels of DEGs. Furthermore, EBS are considerably enriched within –500 bp of DEG’s promoters. Comparative analyses for EBS of DEGs with the promoters of other species provided power to distinguish DEGs with higher probability as EGR1 direct target genes. Eleven EBS in the promoters of 9 genes among analyzed DEGs are conserved between various species including human. In conclusion, this study provides evidence that analyses of mRNA expression profiles followed by two-step in silico analyses could provide a list of putative Egr1 direct target genes in the uterus where any known direct target genes are yet reported for further functional studies.

Recent genomic evidences from unfractionated embryonic stem cell (ESC) cultures have demonstrated high levels of concomitant activating (H3K4me3) and repressive (H3K27me3) histone methylations, termed “bivalent marks”, at lineage specific gene loci, demonstrating that all cells residing within the cultures are developmentally equipotent. However, this dogma has been challenged, indicating that ESC cultures are heterogeneous, with individual cells displaying dynamic metastability and failed to make a connection with the variations between cell lines, a broad spectrum of differentiation, continuous phenotypic oscillation, and the expression of lineage specific genes in undifferentiated state. Recently, functional in vitro assays via fractionation of ESC cultures based on comparable expression of some phenotypes (c‐KIT, A2B5, SSEA3, Nanog, Rex‐1, IGFR1, and Stella) revealed a plastic gradient of clonogenicity and lineage specification within ESC cultures reflected by the presence of bivalent marks, which are resolved down to activating “monovalent marks”. More interestingly, dynamic heterogeneity represents a conserved feature on both mouse ESCs and human ESCs as being essentially required for self‐renewal and, more importantly, differentiation. However, it is the most substantive obstacle to control and specify ESCs into desirable cell types. Mostly, differentiation from ESCs has been evaluated by measuring the responses of whole EB populations under the specific inducible conditions, making it difficult to identify, which cell populations are dominantly contributing to differentiated progeny from ESCs. Therefore, further identification of novel transcriptional and phenotypic markers may allow for the isolation and enrichment of more promising target cells for stem cell‐based clinical therapy.