Protection of Icariin Against Hydrogen Peroxide‐Induced MC3T3‐E1 Cell Oxidative Damage

ObjectiveThe aim of the present study was to evaluate the potential protective mechanism of icariin against oxidative damage caused by hydrogen peroxide in MC3T3‐E1 cells.MethodsMC3T3‐E1 cells were treated with different concentrations of icariin to explore the optimal dose of icariin. MC3T3‐E1 cells were divided into groups treated with various concentrations of hydrogen peroxide (H₂O₂; 0, 0.1, 0.2, 0.5, 1, and 2 mM) for 24 h to induce oxidative damage and cell viability was assessed by Cell Counting Kit‐8 (CCK‐8) assay. Then, cells were divided into five groups: control, H₂O₂ (0.2 mM), icariin (0.1 μM) and H₂O₂ (0.2 mM), + icariin (0.1 μM). Cell viability was detected by CCK‐8 assay. In addition, the content of glutathione and superoxide dismutase and the activity level of malondialdehyde in these treatment groups were determined. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were also performed to measure the early and late osteogenesis, respectively. Protein expression of β‐catenin and cyclin D1 was measured by western blot assay. Then, we used an antagonist of Wnt/β‐catenin signaling pathway (DKK‐1) and western blot analysis to further explore potential mechanism.ResultsAfter 24 h of exposure to 0.2 mM H₂O₂, the viability of MC3T3‐E1 cells was significantly decreased compared to that of the control cells. We first found that icariin can promote cell proliferation of MC3T3‐E1 cells in a dose‐dependent manner, with the dosage 0.1 μM showing the best pro‐proliferative effect. Furthermore, icariin could promote the protein expression of OSX and RUNX2. The results showed that icariin can reverse the inhibitory osteogenic effects of MC3T3‐E1 caused by H₂O₂. In addition, icariin could increase the Wnt‐signaling related proteins. The results showed that MC3T3‐E1 cells in the H₂O₂ (0.2 mM) + icariin (0.1 μM) + Wnt‐signaling antagonist (DKK‐1) group had weaker ALP and ARS staining compared with that observed in the control and H₂O₂ (0.2 mM) + icariin (0.1 μM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H₂O₂ + 0.1 μM icariin + DKK‐1 group compared to that observed in the 0.2 mM H₂O₂ + 0.1 μM icariin group.ConclusionThe results showed that icariin can increase the viability of MC3T3‐E1 cells, reverse the oxidative stress induced by H₂O₂ and protect MC3T3‐E1 cells against H₂O₂‐induced inhibition of osteogenic differentiation, which may occur through the Wnt/β‐catenin signaling pathway.     

Mild Recessive Mutations in Six Fraser Syndrome–Related Genes Cause Isolated Congenital Anomalies of the Kidney and Urinary Tract

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately 40% of children with ESRD in the United States. Hitherto, mutations in 23 genes have been described as causing autosomal dominant isolated CAKUT in humans. However, >90% of cases of isolated CAKUT still remain without a molecular diagnosis. Here, we hypothesized that genes mutated in recessive mouse models with the specific CAKUT phenotype of unilateral renal agenesis may also be mutated in humans with isolated CAKUT. We applied next-generation sequencing technology for targeted exon sequencing of 12 recessive murine candidate genes in 574 individuals with isolated CAKUT from 590 families. In 15 of 590 families, we identified recessive mutations in the genes FRAS1, FREM2, GRIP1, FREM1, ITGA8, and GREM1, all of which function in the interaction of the ureteric bud and the metanephric mesenchyme. We show that isolated CAKUT may be caused partially by mutations in recessive genes. Our results also indicate that biallelic missense mutations in the Fraser/MOTA/BNAR spectrum genes cause isolated CAKUT, whereas truncating mutations are found in the multiorgan form of Fraser syndrome. The newly identified recessive biallelic mutations in these six genes represent the molecular cause of isolated CAKUT in 2.5% of the 590 affected families in this study.     

Genomic insights into abdominal aortic aneurysms

Introduction

An individual’s genetic background plays a significant role in his or her chances of developing an abdominal aortic aneurysm (AAA). This risk is likely to be due to a combination of multiple small effect genetic factors acting together, resulting in considerable difficulty in the identification of these factors.

Methods

Methods for the identification of genetic factors associated with disease are usually based on the analysis of genetic variants in case-control studies. Over the last decade, owing to advances in bioinformatics and laboratory technology, these studies have progressed from focusing on the examination of a single genetic variant in each study to the examination of many millions of variants in a single experiment. We have conducted a series of such experiments using these methods.

Results

Our original methods using candidate gene approaches led to the initial identification of a genetic variant in the interleukin-10 gene associated with AAA. However, further studies failed to confirm this association and highlighted the necessity for adequately powered studies to be conducted, as well as the need for confirmatory studies to be performed, prior to the acceptance of a variant as a risk for disease. The subsequent application of genomic techniques to our sample set, in a global collaboration, has led to the identification of three robustly verified risk loci for AAA in the LRP1, LDLR and SORT1 genes.

Conclusions

Genomic studies of AAA have led to the identification of new pathways involved in the pathogenesis of AAA. The exploration of these pathways has the potential to unlock new avenues for therapeutic intervention to prevent the development and progression of AAA.     

Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca²⁺ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP₂ in two ways: first, PIP₂ acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP₂ and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.     

Effect of prostaglandin E1 on preservation injury of canine liver grafts preserved in UW solution

This study investigated whether prostaglandin E₁ (PGE₁) could reduce hepatic injury to the liver graft caused by harvesting and 24-h preservation in University of Wisconsin (UW) solution in a canine model. The PGE₁-treated group was intravenously administered 0.5 g/kg per minute of PGE₁ for 30 min before harvesting, as well as a concentration of 1 mg/l PGE₁ in the washout and UW solutions. In both the PGE₁-treated and the control group, all recipients survived for 1 week or more after transplantation. Arterial ketone body ratio (AKBR) remained over 1.0 in the early postoperative period. The PGE₁ group showed significant reductions in guanase, GOT, and LDH during the early postoperative period compared to the untreated control group. Histological examination disclosed partial mitochondrial swelling, hepatocyte vacuolation, and necrosis in the control group, while such abnormalities were rarely seen in the PGE₁ group. These results suggest that PGE₁ can effectively reduce hepatic injury to liver grafts preserved in UW solution prior to transplantation.