Nicotine exposure of male mice protects offspring against carbon tetrachloride-induced acute liver injury.

Paternal nicotine exposure can cause a phenotypic change in offspring. To study whether paternal nicotine exposure influences acute liver injury and repair of the offspring, we established a paternal nicotine exposure model in mice and treated the offspring mice with carbon tetrachloride (CCl4 ) to induce acute liver injury. After the treatment of CCl4 , the levels of alanine aminotransferase and aspartate aminotransferase in offspring serum of paternal nicotine exposed mice are about 37.44%, and 30.21% lower than the control mice, respectively. Transcription profiling screen and bioinformatics analysis of differently expressed genes in F1 mice liver revealed that the Wnt pathway was altered. The results demonstrate that nicotine exposure in male mice could enhance the activation of the Wnt pathway in F1 mice liver. The Wnt pathway facilitates cell proliferation and tissue repair. In conclusion, our findings showed that nicotine exposure of male mice protects hepatic against CCl4 -induced acute injury in offspring by activating the Wnt pathway in the F1 liver.

MiR-3571 modulates the proliferation and migration of vascular smooth muscle cells by targeting claudin 1.

Background and aims: The miRNA-based post-transcription modification has been extensively studied in hypertension. It however remains elusive how miRNA expression is regulated in this pathological process. We hypothesize that hydroxymethylation in the promoter regions tightly controls the levels of key miRNAs, which in turn affects the development of hypertension. Methods: The levels of hydroxymethylation in the promoter regions from thoracic aortic tissues were compared between spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs), using hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. The altered hydroxymethylation level of miR-3571 was confirmed by glucosylation-coupled hydroxymethylation-sensitive qPCR. We further identified claudin 1(CLDN1) as a key target of miR-3571 via bioinformatic prediction (targetscan) and dual-luciferase activity assays. Finally, we analyzed the contribution of miR-3571/CLDN1 axis in the proliferation and migration of vascular smooth muscle cells (VSMCs). Results: The hydroxymethylation level of miR-3571 promoter region in thoracic aortic tissue from spontaneously hypertensive rats was lower than that from normotensive Wistar-Kyoto rats. Accordingly, the expression of miR-3571 was lower during hypertension, with up-regulated CLDN1 protein levels. More importantly, we found that miR3571 overexpression led to phenotypic changes of VSMCs, and inhibited the proliferation and migration of muscle cells via suppressing CLDN1 as well. Our findings further suggested that CLDN1 up-regulation increase the activity of ERK1/2 in VSMCs. Conclusions: Our study suggested that hydroxymethylation in the promoter regions controlled the level of miR-3571 and revealed the important roles of miR-3571 and CLDN1 in VSMCs during the development of hypertension. In addition, our results also indicated that miR-3571/CLDN1 axis regulated the functions of VSMCs via the ERK1/2 pathway. Taken together, our findings support miR-3571 as a novel biomarker for the diagnosis and prevention of hypertension.

Nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway.

Nicotine, the main toxic substance in cigarette smoke, significantly reduced the differentiation and maturation ratio of Leydig cell in murine testes. To investigate the underlying mechanism, C57BL/6J mice were divided into control (CT) and nicotine treated (NT) groups. Next generation RNA sequencing and bio-informatics analysis were carried out to analysis the effects of nicotine on the RNA profile of Leydig cells. Expression level of 7 pathways remarkably changed after nicotine treatment. As the positive regulating pathway of Leydig cell differentiation, Hedgehog signaling pathway was found among these pathways. PTCH1 and ß-TrCP were down-regulated in nicotine treated mice Leydig cells, while GSK3ß, Gli2 and Gli2 fragments increased significantly. Nicotine stimulated the destabilization of Gli2 via ß-TrCP induced ubiquitination and degradation. Gli2 was phosphorylated by up-expressed GSK3ß during this process. Destabilization of Gli2 reduced the activation rate of target genes of Hedgehog signaling pathway such as Ptch1. The differentiation of Leydig cell positively regulated by Hh pathway was thus inhibited by nicotine exposure. Consequently, the male reproduction process powered by Leydic cell-mediated androgen secretion was thus influenced. In conclusion, we find that nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway.

Gx-50 reduces ß-amyloid-induced TNF-α, IL-1ß, NO, and PGE2 expression and inhibits NF-κB signaling in a mouse model of Alzheimer's disease.

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx-50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx-50 possesses anti-inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF-α, IL-1ß, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx-50 in amyloid ß (Aß) treated rat microglia; additionally, microglial activation and the expression of IL-1ß, iNOS, and COX2 were also significantly suppressed by gx-50 in APP(+) transgenic mice. Furthermore, gx-50 inhibited the activation of NF-κB and MAPK cascades in vitro and in vivo in APP-Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx-50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aß-induced upregulation of IL-1ß and TRAF6 to levels similar to gx-50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx-50. These findings provide strong evidence that gx-50 has anti-inflammatory effects against Aß-triggered microglial overactivation via a mechanism that involves the TLR4-mediated NF-κBB/MAPK signaling cascade.

Epidermal growth factor receptor signaling mediates aldosterone-induced profibrotic responses in kidney.

Aldosterone has been recognized as a risk factor for the development of chronic kidney disease (CKD). Studies have indicated that enhanced activation of epidermal growth factor receptor (EGFR) is associated with the development and progression of renal fibrosis. But if EGFR is involved in aldosterone-induced renal fibrosis is less investigated. In the present study, we examined the effect of erlotinib, an inhibitor of EGFR tyrosine kinase activity, on the progression of aldosterone-induced renal profibrotic responses in a murine model underwent uninephrectomy. Erlotinib-treated rats exhibited relieved structural lesion comparing with rats treated with aldosterone alone, as characterized by glomerular hypertrophy, mesangial cell proliferation and expansion. Also, erlotinib inhibited the expression of TGF-ß, α-SMA and mesangial matrix proteins such as collagen Ⅳ and fibronectin. In cultured mesangial cells, inhibition of EGFR also abrogated aldosterone-induced expression of extracellular matrix proteins, cell proliferation and migration. We also demonstrated that aldosterone induced the phosphorylation of EGFR through generation of ROS. And the activation of EGFR resulted in the phosphorylation of ERK1/2, leading to the activation of profibrotic pathways. Taken together, we concluded that aldosterone-mediated tissue fibrosis relies on ROS induced EGFR/ERK activation, highlighting EGFR as a potential therapeutic target for modulating renal fibrosis.

Nicotine induces Nme2-mediated apoptosis in mouse testes.

In mouse testes, germ cell apoptosis can be caused by cigarette smoke and lead to declining quality of semen, but the exact molecular mechanisms remain unclear. To evaluate the effects of nicotine exposure on apoptosis during spermatogenesis, we first constructed a nicotine-treated mouse model and detected germ cell apoptosis activity in the testes using the TUNEL method. Then we analyzed the variation of telomere length and telomerase activity by real-time PCR and TRAP-real-time PCR, respectively. Further, we investigated a highly expressed gene, Nme2, in mouse testes after nicotine treatment from our previous results, which has close correlation with the apoptosis activity predicted by bioinformatics. We performed NME2 overexpression in Hela cells to confirm whether telomere length and telomerase activity were regulated by the Nme2 gene. Finally, we examined methylation of CpG islands in the Nme2 promoter with the Bisulfite Sequencing (BSP) method. The results showed that apoptosis had increased significantly, and then telomerase activity became weak. Further, telomere length was shortened in the germ cells among the nicotine-treated group. In Hela cells, both overexpression of the Nme2 gene and nicotine exposure can suppress the activity of telomerase activity and shorten telomere length. BSP results revealed that the Nme2 promoter appeared with low methylation in mouse testes after nicotine treatment. We assume that nicotine-induced apoptosis may be caused by telomerase activity decline, which is inhibited by the up expression of Nme2 because of its hypomethylation in mouse germ cells.

Nicotine Induced Murine Spermatozoa Apoptosis via Up-Regulation of Deubiquitinated RIP1 by Trim27 Promoter Hypomethylation.

Nicotine significantly promoted apoptosis in stages I, VII, VIII, and XI spermatogonia, stages I, VII, VIII, X, and XI spermatocytes, and stages I-V, VII, and VIII elongating spermatids. To explore the underlying molecular mechanisms, sperm mRNA next-generation sequencing of nicotine-treated mice was conducted. Out of the 86 genes related to apoptosis, Tnf (tumor necrosis factor alpha) was screened to be the most significant varied transcript, and the Onto-pathway analysis indicated that the TNF apoptotic pathway was especially activated by nicotine exposure. The TNF pathway was further studied at the gene and protein levels. The results showed that RIP1, the key component in the TNF apoptotic pathway, was up-expressed in its deubiquitinated form in nicotine-treated mice testis. TRIM27, an E3 ubiquitin ligase that activated TNF apoptotic pathway through up-regulating deubiquitinated RIP1, was also overexpressed in nicotine-treated spermatocytes; moreover, four consecutive CpG sites near the Trim27 transcription start site were less frequently methylated. Finally, in vitro experiments of Trim27 overexpression and RNA interference in GC-1 spermatogonial cells confirmed that the RIP1 deubiquitination and TRIM27 hyopmethylation were both positively correlated with spermatocyte apoptosis. In summary, our study suggests that nicotine may induce murine spermatozoal apoptosis via the TNF apoptotic pathway through up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation.

Protein profile screening: reduced expression of Sord in the mouse epididymis induced by nicotine inhibits tyrosine phosphorylation level in capacitated spermatozoa.

Many studies have revealed the hazardous effects of cigarette smoking and nicotine exposure on male fertility, but the actual, underlying molecular mechanism remains relatively unclear. To evaluate the detrimental effects of nicotine exposure on the sperm maturation process, two-dimensional gel electrophoresis and mass spectrometry analyses were performed to screen and identify differentially expressed proteins from the epididymal tissue of mice exposed to nicotine. Data mining analysis indicated that 15 identified proteins were mainly involved in the molecular transportation process and the polyol pathway, indicating impaired epididymal secretory functions. Experiments in vitro confirmed that nicotine inhibited tyrosine phosphorylation levels in capacitated spermatozoa via the downregulated seminal fructose concentration. Sord, a key gene encoding sorbitol dehydrogenase, was further investigated to reveal that nicotine induced hyper-methylation of the promoter region of this gene. Nicotine-induced reduced expression of Sord could be involved in impaired secretory functions of the epididymis and thus prevent the sperm from undergoing proper maturation and capacitation, although further experiments are needed to confirm this hypothesis.

Alteration of sperm protein profile induced by cigarette smoking.

Cigarette smoking is associated with lower semen quality, but how cigarette smoking changes the semen quality remains unclear. The aim of this study was to screen the differentially expressed proteins in the sperm of mice with daily exposure to cigarette smoke. The 2D gel electrophoresis (2DE) and mass spectrometry (MS) analyses results showed that the mouse sperm protein profile was altered by cigarette smoking. And 22 of the most abundant proteins that correspond to differentially expressed spots in 2DE gels of the sperm samples were identified. These proteins were classified into different groups based on their functions, such as energy metabolism, reproduction, and structural molecules. Furthermore, the 2DE and MS results of five proteins (Aldoa, ATP5a1, Gpx4, Cs, and Spatc1) were validated by western blot analysis and reverse transcriptase-polymerase chain reaction. Results showed that except Spatc1 the other four proteins showed statistically significant different protein levels between the smoking group and the control group (P < 0.05). The expressions of three genes (Aldoa, Gpx4, and Spatc1) were significantly different (P < 0.05) at transcription level between the smoking group and the control group. In addition, five proteins (Aldoa, ATP5a1, Spatc1, Cs, and Gpx4) in human sperm samples from 30 male smokers and 30 non-smokers were detected by western blot analysis. Two proteins (Aldoa and Cs) that are associated with energy production were found to be significantly altered, suggesting that these proteins may be potential diagnostic markers for evaluation of smoking risk in sperm. Further study of these proteins may provide insight into the pathogenic mechanisms underlying infertility in smoking persons.

Estimated diversity of messenger RNAs in each murine spermatozoa and their potential function during early zygotic development.

To study the diversity of mRNAs in murine spermatozoa and their potential function during zygotic development, total RNAs in murine spermatozoa were sequenced via RNA-Seq and analyzed through bioinformatics techniques. The delivery and translation of sperm-borne mRNA in fertilized oocyte were detected using RT-PCR (reverse transcription-polymerase chain reaction), Western blot, and immunofluorescence. A total of 35 288 825 reads matching 33 039 transcripts, including 27 310 coding transcripts, were obtained. Based on our analyses, we hypothesized that the transcripts with RPKM (reads per kilobase of exon model per million mapped reads) higher than six may exist in each sperm cell as consistently retained transcripts. There were 4885 consistent transcripts in each sperm, and the remainder were randomly retained. If the baseline RPKM increased, the remaining coding transcripts were more likely related to reproduction and development. The sperm-borne transcripts Wnt4 and Foxg1 were delivered into fertilized oocytes on fertilization. Furthermore, Wnt4 was translated into protein in zygotes, whereas Foxg1 was not translated. In conclusion, approximately 4885 mRNAs were present in each murine spermatozoon, and the spermatozoal mRNAs related to reproduction and development were more likely retained. The sperm-borne mRNA Wnt4 was delivered into the fertilized oocyte and translated, evidence of a paternal effect on zygotic development.

Cigarette smoking exposure alters pebp1 DNA methylation and protein profile involved in MAPK signaling pathway in mice testis.

Many studies have addressed the role of cigarette smoking on semen quality, but the exact mechanisms remain inconclusive. To evaluate the detrimental effects of smoking on the spermatogenesis process, we initially screened and investigated 31 differentially expressed proteins extracted from the testes of mice exposed daily to cigarette smoke using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. Data mining analysis showed that these 31 proteins were categorized into five functional clustering groups: metabolic process, cell growth and/or maintenance, RNA and protein processing, stress response, and spermatogenesis. Additionally, 23 of 31 proteins were involved in a main pathway network, including Pkc (s), ERK1/2, Akt, and NF-kappaB, which are known to be involved in cell communication, proliferation, and differentiation. Interestingly, among the 31 proteins, a spermatogenesis-associated protein, phosphatidylethanolamine-binding protein 1 (PEBP1), was especially expressed in serial sections of spermatids of spermiogenesis and interacted with ERKs. The bisulfite sequencing result showed four CpGs near the Pebp1 transcriptional start site were largely methylated in the treated group. A 5-aza-2'-deoxycytidine treatment on GC-1 spg cells reversed the hypermethylation status and elevated both Pebp1 mRNA and protein expression levels. ERK1/2 phosphorylation levels were also increased with upregulation of Pebp1 expression in GC-1 spg cells. In conclusion, protein profile in testes could be altered by cigarette smoking. Moreover, we also suggest that epigenetic Pebp1 inactivation may affect activation of ERK, and it could impair spermatogenesis of mice. Our data could provide further insight into the mechanisms of spermatogenesis.

Heparanase induced by advanced glycation end products (AGEs) promotes macrophage migration involving RAGE and PI3K/AKT pathway.

BACKGROUND: Advanced glycation end products (AGEs), inflammatory-associated macrophage migration and accumulation are crucial for initiation and progression of diabetic vascular complication. Enzymatic activity of heparanase (HPA) is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, HPA enhances the phosphorylation of selected signaling molecules including AKT pathway independent of enzymatic activity. However, virtually nothing is presently known the role of HPA during macrophage migration exposed to AGEs involving signal pathway. METHODS: These studies were carried out in Ana-1 macrophages. Macrophage viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. HPA and AKT protein expression in macrophages are analysed by Western blotting and HPA mRNA expression by real time quantitative RT-PCR. Release of HPA was determined by ELISA. Macrophage migration was assessed by Transwell assays. RESULTS: HPA protein and mRNA were found to be increased significantly in AGEs-treated macrophages. Pretreatment with anti-HPA antibody which recognizes the nonenzymatic terminal of HPA prevented AGEs-induced AKT phosphorylation and macrophage migration. LY294002 (PI3k/AKT inhibitor) inhibited AGEs-induced macrophage migration. Furthermore, pretreatment with anti-receptor for advanced glycation end products (RAGE) antibody attenuated AGEs-induced HPA expression, AKT phosphorylation and macrophage migration. CONCLUSIONS: These data indicate that AGEs-induced macrophage migration is dependent on HPA involving RAGE-HPA-PI3K/AKT pathway. The nonenzymatic activity of HPA may play a key role in AGEs-induced macrophage migration associated with inflammation in diabetic vascular complication.

Heat shock cognate 70 protein like-2 protein in camphor pollen is one of the major culprits of asthma.

AIMS: In recent decades, Cinnamomum camphora have gradually become the main street trees in Shanghai. This study aims to investigate the allergenicity of camphor pollen. MAIN METHODS: A total of 194 serum samples from patients with respiratory allergy were collected and analyzed. Through protein profile identification and bioinformatics analysis, we hypothesized that heat shock cognate protein 2-like protein (HSC70L2) is the major potential allergenic protein in camphor pollen. Recombinant HSC70L2 (rHSC70L2) was expressed and purified, and a mouse model of camphor pollen allergy was established by subcutaneous injection of total camphor pollen protein extract (CPPE) and rHSC70L2. KEY FINDINGS: Specific IgE was found in the serum of 5 patients in response to camphor pollen and three positive bands were identified by Western blotting. Enzyme-linked immunosorbent assay (ELISA), Immune dot blot and Western blot experiments confirmed that CPPE and rHSC70L2 can cause allergies in mice. Moreover, rHSC70L2 induces polarization of peripheral blood CD4+ T cells to Th2 cells in patients with respiratory allergies and mice with camphor pollen allergy. Finally, we predicted the T cell epitope of the HSC70L2 protein, and through the mouse spleen T cell stimulation experiment, we found that the 295EGIDFYSTITRARFE309 peptide induced T cells differentiation to Th2 and macrophages differentiation to the alternatively activated (M2) state. Moreover, 295EGIDFYSTITRARFE309 peptide increased the serum IgE levels in mice. SIGNIFICANCE: The identification of HSC70L2 protein can provide novel diagnostic and therapeutic targets for allergies caused by camphor pollen.

Effect of mifepristone on COX-2 both in eutopic and ectopic endometrium in mouse endometriotic model.

OBJECTIVE: To study the influence of mifepristone on the expression of cyclooxygenase 2 (COX-2) protein and COX-2 mRNA and then to evaluate the mechanism. METHODS: After the establishment of 30 mice endometriosis models, the mice were randomly divided into six groups with 5 mice each group and assigned to experimental and control groups of 1-, 4- and 6-week circle according to whether mifepristone (0.13 mg d(-1)) was taken or not. Small animal optical imaging system was used to detect the fluorescent intensity of the ectopic tissue. Reverse transcript-polymerase chain reaction and western blot was used to examine COX-2 protein and COX-2 mRNA expression. ELISA was used to examine concentration of PGE(2) in serum. RESULT(S): Mifepristone could not affect the fluorescent intensity of the ectopic endometrium after it was taken 1, 4, and 6 (P > 0.05). However, it could decrease the transcription of COX-2 mRNA in the 1 and 4 week groups (P < 0.05), while the difference in the 6 week group was not significant (P > 0.05). It could decrease the expression of COX-2 protein after it was taken 4 and 6 weeks (P < 0.05). The serous PGE(2) in the trial groups was lower than that in the control groups, but the difference was not significant (P > 0.05). CONCLUSION(S): This study showed that mifepristone could not affect the size of the ectopic endometrium, but it could decrease the transcription of COX-2 gene and then reduce the expression of COX-2 protein and its product PGE(2) which is an important factor which mediate pain. This maybe another mechanism that mifepristone takes effect through anti-inflammatory path.

Astragalus membranaceus inhibits inflammation via phospho-P38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways in advanced glycation end product-stimulated macrophages.

Advanced glycation end products (AGEs) and inflammation contribute to the development of diabetic complications. Astragalus membranaceus has properties of immunological regulation in many diseases. The aim of this study was to determine the function of A. membranaceus extract (AME) on the AGE-induced inflammatory response in Ana-1 macrophages. The viability of cells treated with AME or AGEs was evaluated with the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] method. The secretion and mRNA levels of IL-1ß and TNF-α were measured by ELISA and RT-PCR, respectively. The activity of NF-κB was assayed by EMSA. The phosphorylation of p38 MAPK was assessed by western blotting. The results showed that AME was not toxic to macrophages. The treatment of macrophages with AME effectively inhibited AGE-induced IL-1ß and TNF-α secretion and mRNA expression in macrophages. These effects may be mediated by p38 MAPK and the NF-κB pathway. The results suggest that AME can inhibit AGE-induced inflammatory cytokine production to down-regulate macrophage-mediated inflammation via p38 MAPK and NF-κB signaling pathways and indicate that AME could be an immunoregulatory agent against AGE-induced inflammation in diabetes.

Astragalus embranaceus extract activates immune response in macrophages via heparanase.

Astragalus membranaceus (AM), a traditional Chinese medicinal herb, has immunoregulatory properties in many diseases. We investigated the effects and mechanism of Astragalus membranaceus extract (AME) in the macrophage migration and immune response mediator release. The viability of Ana-1 macrophages treated with AME was evaluated by the MTT method. The secretion and mRNA levels of IL-1ß and TNF-a were measured by ELISA and RT-PCR, respectively. Macrophage migration was assayed by transwell assay. The activity of heparanase (HPA) was determined by a heparin-degrading enzyme assay. Our results didn't show any toxicity of AME in macrophages. AME increased the activity of HPA, cell migration, mRNA levels and secretion of IL-1ß and TNF-a in macrophages. Pretreatment with anti-HPA antibody reduced cell migration, secretion of IL-1ß and TNF-a did not change the mRNA levels of IL-1ß and TNF-a significantly in AME-treated macrophages. This suggests that AME may increase the release of immune response mediator and cell migration via HPA to activate immune response in macrophages.

Urine protein in patients with type I hypersensitivity is indicative of reversible renal tube injury.

AIMS: In our clinical work, some patients with type I hypersensitivity could be detected protein in their urine. This study focused on the early renal injury in patients with type I hypersensitivity. MAIN METHODS: From 43 type I hypersensitivity patients with proteinuria, 10 patients were randomly selected for mass spectrometry analysis of 24-h urine together with 5 healthy volunteers. Mice were vaccinated with Dermatophagoides farina (Der f) and ovalbumin (OVA) were used as antigen to establish the type I hypersensitivity animal models. KEY FINDINGS: The urine protein of hypersensitivity patients was significantly increased in the alpha-1-microglobulin/ bikunin precursor (Protein AMBP) (t = 3.140, P = 0.008), retinol binding protein 4 (RBP4) (t = 2.426, P = 0.031), kininogen-1 (t = 2.501, P = 0.027), and transferrin appeared only in patients' urine. After immunizing mice with antigens, significant increases of the total serum immunoglobulin E (IgE) were observed in both Der f (86.92 ± 36.01 U/mL, t = 5.231, P = 0.0004) and OVA group (34.65 ± 24.72 U/mL, t = 2.891, P = 0.0161) compared with the negative control group (2.68 ± 0.47 U/mL). Meanwhile, definite eosinophil infiltration around the impaired renal tubules as well as the bronchus in Der f mice were observed, and urine protein appeared. After stopping the allergen stimulation, proteinuria disappeared. Instead, when the mice were treated with the antigen again, proteinuria reappeared. SIGNIFICANCE: Our findings suggest that renal tubular damage in patients with type I hypersensitivity is reversible, and proteinuria disappears with allergy symptoms remission.

Paternal nicotine exposure promotes hepatic fibrosis in offspring.

Paternal nicotine exposure can alter phenotypes in future generations. The aim of this study is to explore whether paternal nicotine exposure affects the hepatic repair to chronic injury which leads to hepatic fibrosis in offspring. Our results demonstrate that nicotine down regulates mmu-miR-15b expression via the hyper-methylation on its CpG island shore region in the spermatozoa. This epigenetic modification imprinted in the liver of the offspring. The decreased mmu-miR-15b promotes the expression of Wnt4 and activates the Wnt pathway in the offspring mice liver. The activation of the Wnt pathway improves the activation and proliferation of hepatic stellate cells (HSCs) leading to liver fibrosis. Moreover, the Wnt pathway promotes the activation of the TGF-ß pathway and the two pathways cooperate to promote the transcription of extracellular matrix (ECM) genes. In conclusion, this study found that nicotine promotes hepatic fibrosis in the offspring via the activation of Wnt pathway by imprinting the hyper-methylation of mmu-miR-15b.

A study of Y chromosome gene mRNA in human ejaculated spermatozoa.

We asked if messenger ribonucleic acid (mRNA) of Y chromosome genes was selectively retained in human ejaculated spermatozoa. A panel of genes on the nonrecombining region of Y chromosome (NRY) and their X homologues were selected and screened in human testis and ejaculated spermatozoa by reverse transcription-polymerase chain reaction (RT-PCR). Then, the cellular localization of DBY, RPS4Y and SMCY mRNA in testis and matured spermatozoa were investigated by in situ hybridization. The expression pattern of these three genes in human ejaculated spermatozoa was also tested by Western blot and Immunofluorescence. All 11 Y chromosome gene mRNAs were found in the testis, but the transcripts of only three genes DBY, SRY, and RPS4Y were detected in ejaculated sperm by RT-PCR. Further investigation showed that DBY and RPS4Y mRNA were present in both human testis and the post-acrosome region comprising nearly 50% of spermatozoa, while the encoded proteins were not detected in spermatozoa. In contrast, although SMCY mRNA was not detected in mature spermatozoa, the H-Y antigen encoded by SMCY was expressed on the surface of spermatozoa. We therefore conclude that the spermatozoa mRNA could be selectively reserved in mature ejaculated spermatozoa. They might be useful in early zygotic and embryonic development.

Paternal nicotine exposure induces hyperactivity in next-generation via down-regulating the expression of DAT.

Many substances in cigarette smoke can induce changes in DNA methylation. Our previous studies have confirmed paternal nicotine exposure causes hyperactivity in the offspring via mmu-miR-15b. The main aim of the present study is to explore the molecular mechanism underlying the cross-generation effects of paternal nicotine exposure more comprehensively. The male C57BL/6 mice were exposed to 2 mg/kg/d nicotine for 5 weeks, and then mated with wild-type females. The offspring male mice were subjected to behavioral tests at 8 weeks after birth. The results suggested that, paternal nicotine exposure led to hyperactivity in the offspring. An analysis of the changes in DNA methylation revealed that nicotine exposure induced a rise in the total DNA methylation level of Dat in murine spermatozoa, and the hyper-methylation could imprint in the brains of the offspring mice. Then these epigenetic modifications reduced the expression of DAT in the brain of the offspring, resulting in a rise in the level of extracellular dopamine. The activation of D2 receptors caused the dephosphorylation of AKT, which led to increased activation of GSK3α/ß, and ultimately caused hyperactivity in the offspring mice. Further, in wild-type mice, injection of DAT inhibitors simulated this hyperactive phenotype, while the injection of D2s inhibitors reversed the hyperactivity of the offspring caused by paternal nicotine exposure. In conclusion, all results indicated that paternal nicotine exposure could induce hyperactivity in the offspring via the hyper-methylation of Dat. Consequently, Dat may be one of the genes that mediate the cross-generation effects of nicotine besides mmu-mmiR-15b.