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.

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.

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.

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.

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.

RNA profile of control and nicotine treated Murine Leydig single cell represented in TPM data.

Data provided in this article is RNA profile represented in RPKM and RPKM based TPM value for the research article titled Nicotine inhibits Murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway Jiajie et al., 2019. Nicotine treatment changes the RNA profile of Murine Leydig cells. RNA of 12 control group Leydig cells and 12 nicotine treated Leydig cells are sequenced and the data of 29943 genes are achieved. The information of the gene symbol, gene description, gene type, position and transcript length are provided.

Estrogen reverses nicotine-induced inflammation in chondrocytes via reducing the degradation of ECM.

PROBLEM: Osteoarthritis (OA) is a chronic disease with a very high incidence and the pathology of which is quite complex. Epidemiological investigation showed that OA may be related to smoking and estrogen levels, but there are few studies focused on the cross-effect of these two factors. This research aims to investigate the molecular mechanism of nicotine and estrogen effects on chondrocytes to study the effect of smoking on the incidence of osteoarthritis in women. METHOD OF THE STUDY: Nicotine was added to obtain inflammatory supernatants of macrophages, which were used to induce chondrocyte inflammation. Toluidine staining and immunohistochemistry were used to detect the extracellular matrix (ECM) of chondrocytes, while the important proteins in the metabolism of chondrocytes were detected by Western blot. RESULTS: Nicotine-induced inflammatory supernatant promoted the degradation of ECM, such as type II collagen, aggrecan and proteoglycan 4. While in the presence of physiological concentrations of estrogen, this destructive effect is reversed. On the molecular level, estrogen (17ß-estradiol, 1 nmol/L) can inhibit the matrix degrading enzymes and promote the transforming growth factor (TGF)-ß1 pathway which is involved in matrix synthesis. However, in the presence of inflammatory induction, although estrogen could still inhibit the expression of matrix degrading enzymes, it inhibited the TGF-ß1 pathway. Moreover, the different inflammatory factors in the inflammatory supernatant, mainly tumor necrosis factor-α, interleukin-1ß, had different effects on the metabolic processes of chondrocytes. CONCLUSION: Estrogen reverses nicotine-induced inflammation mainly via reducing the degradation of ECM. The cross-effect of estrogen and inflammatory factor inhibitors can be a potential clinical reference for OA patients.

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.

Emission of sulfur dioxide from polyurethane foam and respiratory health effects.

Recently, health damage to children exposed to synthetic polyurethane (PU) running tracks has aroused social panic in China. Some possible toxic volatiles may be responsible for these damages. However, the exact cause remains unclear. We have detected a low concentration of sulfur dioxide (SO2; 1.80-3.30 mg/m3) on the surface of the PU running track. Surprisingly, we found that SO2 was generated from the PU running track, and even such a low concentration of SO2 could induce severe lung inflammation with hemorrhage, inflammatory cell infiltration, and inflammatory factor secretion in mice after 2-week exposure. Prolonged exposure (5 weeks) to the SO2 caused chronic pulmonary inflammation and pulmonary fibrosis in the mice. Peripheral hemogram results showed that platelet concentration increased significantly in the SO2 group compared to that in the control group, and the proportion of blood neutrophils and monocytes among total leukocytes was more imbalanced in the SO2 group (16.6%) than in the control group (8.0%). Further histopathology results of sternal marrow demonstrated that hematopoietic hyperplasia was severely suppressed with increased reticular stroma and adipocytes under SO2 exposure. These data indicate that a low concentration of SO2 generated spontaneously from PU running track outdoors as a secondary product is still harmful to health, as it impairs the respiratory system, hematopoiesis, and immunologic function. This indicates that the low-concentration SO2 could be a major cause of diseases induced by air pollution, such as chronic obstructive pulmonary disease.

Nicotine induced autophagy of Leydig cells rather than apoptosis is the major reason of the decrease of serum testosterone.

A new report has shown that nicotine exposure can decrease serum testosterone by apoptosis in Leydig cells; however, in our previous studies, we have almost never observed apoptosis there. The purpose of this study is to ensure whether apoptosis or autophagy in Leydig cells occurred. Our results confirmed again that the concentration of testosterone in the sera of nicotine-treated mice statistically decreased (P < 0.05). Furthermore, the data of single cell transcriptome indicated that the expression of autophagy-related genes was increased after nicotine exposure. Likewise, chemical and immune-histological staining demonstrated that autophagy of the Leydig cells increased after nicotine treatment rather than apoptosis. Apoptosis mainly exists in spermatids. Further, the expression of autophagy-related genes, such as Beclin1 and LC3, were up-regulated after nicotine exposure (P < 0.05). Additionally, the data of transmission electron microscopy showed more autophagosomes in the Leydig cells of the nicotine-exposed groups than the cells of the control groups. Moreover, immunofluorescent staining of LC3 in the TM3 Leydig cell line indicated that rapamycin and nicotine exposure up-regulates the autophagy phenotype/process and down-regulates their testosterone synthesis. In addition, the methylation level of the promoter region of TCL1 is increased in the nicotine-treated group compared to the control group, consequently decreasing the expression of TCL1. In conclusion, the autophagy in Leydig cells induced by nicotine, which is set by the hyper-methylation of the TCL1 promoter region via the TCL1-mTOR-autophagy signaling pathway.

Maternal nicotine exposure has severe cross-generational effects on offspring behavior.

Our previous studies showed that paternal nicotine exposure can lead to hyperactivity in the offspring. Nevertheless, the cross-generational effects of maternal and biparental nicotine exposure remain unclear. In this study, female and male mice were exposed respectively by nicotine before pregnancy. The maternal pre-pregnancy nicotine exposure led to depression-like behaviors in the F1 offspring. However, after biparental pre-pregnancy nicotine exposure, seventy percentage of the offspring exhibited a depressive phenotype while 20% were hyperactive, and the remaining exhibited no obvious abnormal behavior. The cross-generational effects appeared to be mediated via disruption of the balance between GSK3 and p-GKS3 by nicotine. These results suggested that pre-pregnancy nicotine exposure can induce alterations in the behavior of the offspring, and the cross-generational effects of maternal nicotine exposure were particularly serious.

Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response.

Diabetic vasculopathy is intensified by macrophage inflammation caused by advanced glycation end products (AGEs). Heparanase (HPA) is a unique endoglycosidase, which cleaves heparan sulfate proteoglycans (HSPGs) including syndecan-1 (Syn-1) to further stimulate macrophage cell migration and inflammation. The present study was planned to evaluated the role of C-domain (if any) of HPA in AGE inflammatory response in macrophages. Cell viability was assessed using MTT assay, migration assay, ELISA for tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) levels, mRNA expression by RT-PCR and heparan degrading enzyme assay for HPA activity. In the present study, we found that pretreatment with anti-HPA antibody, which recognizes the C-domain of HPA inhibited macrophage migration, secretion of IL-1ß and TNF-α as well as decreased HPA enzymatic activity and increased Syn-1 protein expression in AGE-induced macrophages. Compared with anti-HPA antibody pretreatment, co-pretreatment with anti-HPA plus Syn-1 antibodies promoted macrophage migration, and secretion of IL-1ß and TNF-α significantly in AGE-induced macrophages. In addition, pretreatment with anti-HPA or anti-HPA plus Syn-1 antibodies did not markedly change the mRNA levels of IL-1ß and TNF-α concentration AGE-treated macrophages. The results showed that C-domain of HPA mediates AGE-induced macrophage migration and inflammatory cytokine release via Syn-1 protein expression. Furthermore, C-domain of HPA may have a key role in diabetic vascular complication-associated inflammatory response.

Paternal nicotine exposure defines different behavior in subsequent generation via hyper-methylation of mmu-miR-15b.

The neurobehavioral effects of paternal smoking and nicotine use have not been widely reported. In the present study, nicotine exposure induced depression in the paternal generation, but reduced depression and promoted hyperactivity in F1 offspring. While this intergenerational effect was not passed down to the F2 generation. Further studies revealed that nicotine induced the down-regulation of mmu-miR-15b expression due to hyper-methylation in the CpG island shore region of mmu-miR-15b in both the spermatozoa of F0 mice and the brains of F1 mice. As the target gene of mmu-miR-15b, Wnt4 expression was elevated in the thalamus of F1 mice due to the inheritance of DNA methylation patterns from the paternal generation. Furthermore, the increased expression of Wnt4 elevated the phosphorylation level of its downstream protein GSK-3 through the canonical WNT4 pathway which involved in the behavioral alterations observed in F1 mice. Moreover, in vivo stereotaxic brain injections were used to induce the overexpression of mmu-miR-15b and WNT4 and confirm the neurobehavioral effects in vitro. The behavioral phenotype of the F1 mice resulting from paternal nicotine exposure could be attenuated by viral manipulation of mmu-miR-15b in the thalamus.

Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

Nicotine can induce the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs). We have previously shown that cytoskeletal proteins and RhoGDIA, a negative regulator of the Rho GTPase pathway, are involved in the nicotine-induced dysfunction of VSMCs. Here, we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways, including targets such as MYPT1, PAK1 and PI3K/AKT. Upon nicotine treatment, the mRNA level of RhoGDIA is increased but protein level is decreased both in vitro and in vivo, which suggested a mechanism of post-translational regulation. By the dual luciferase reporter assay, we identified the microRNA-200b (miR-200b) as a modulator of the behavioural changes of VSMCs in response to nicotine through targeting RhoGDIA directly. Introducing miR-200b inhibitors into cultured VSMCs significantly attenuated cell proliferation and migration. Additionally, we found that hypomethylation in the CpG island shore region of miR-200b was responsible for the nicotine-induced miR-200b up-regulation in VSMCs. The study demonstrates that nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module through the hypomethylation of miR-200b promoter and suggests that epigenetic modifications may play an important role in the pathological progression.

Heparanase-driven inflammation from the AGEs-stimulated macrophages changes the functions of glomerular endothelial cells.

AIMS: Amounts of macrophages were infiltrated in glomeruli in diabetic nephropathy. Heparanase has been thought to be closely related to proteinuria. Our aims were to determine the effect of heparanase on the inflammation in AGEs-stimulated macrophages and its role on the functions of glomerular endothelial cells (GEnCs). METHODS: The expression of inflammation cytokines in macrophages were assayed by q-RT PCR, western, and ELISA. Then western was used to measure the expression of RAGE and key proteins in NF-κB pathway in macrophages. The expression of the adherence molecules and tight junction proteins in GEnCs were assessed by western. The adherence of mononuclear cells to GEnCs were observed by HE staining and transendothelial FITC-BSA were tested for the permeability of GEnCs. RESULTS: HPA siRNA and heparanase inhibitor sulodexide could attenuate the increasing inflammatory factors (TNF-α and IL-1ß) in AGEs-stimulated macrophages. NF-κB inhibitor PDTC could also decrease the augmented inflammation cytokines through inhibiting the activation of the NF-κB pathway induced by AGEs. The phosphorylation of NF-κB signaling pathway could be also attenuated by HPA siRNA and sulodexide, the same to the receptor of AGEs RAGE. When the macrophage-conditioned culture medium were added to the glomerular endothelial cells, we found HPA siRNA and sulodexide groups could decrease the increasing adherence and permeability of GEnCs induced by AGEs. CONCLUSIONS: Heparanase increases the inflammation in AGEs-stimulated macrophages through activating the RAGE-NF-κB pathway. Heparanase driven inflammation from AGEs-stimulated macrophages increases the adherence of GEnCs and augments the permeability of GEnCs.

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.

Gx-50 Inhibits Neuroinflammation via α7 nAChR Activation of the JAK2/STAT3 and PI3K/AKT Pathways.

Recent studies have revealed that the α7 nicotinic acetylcholine receptor (α7 nAChR) is a critical link between inflammation and neurodegeneration, which is closely associated with Alzheimer's disease (AD). The JAK2/STAT3 and PI3K/AKT signaling pathways contribute to the neuroprotective and anti-inflammatory effects of α7nAChR. Our previous studies have shown that treatment with gx-50 improves cognitive function and is neuroprotective. Here, we investigated the effect of gx-50 on α7 nAChR and Aß-induced inflammation in microglia. First, the binding affinity of gx-50 to α7 nAChR was examined using the fluorescence-based Octet RED system, and the expression of α7 nAChR was detected using real-time PCR and western blotting. We also investigated downstream events of α7 nAChR activity, including the translocation of p-STAT3 and the phosphorylation of JAK2, STAT3, PI3K, and AKT. Finally, the effect of gx-50 on Aß-induced inflammation via α7 nAChR-mediated signaling pathways was investigated using cytokine assays. The results showed that gx-50 is able to act as a specific ligand to activate α7 nAChR, which then upregulates the JAK2/STAT3 and PI3K/AKT signaling pathways to inhibit the secretions of pro-inflammatory cytokines, such as IL-1ß. In conclusion, the results suggest that gx-50 could inhibit the Aß-induced inflammatory response in microglia via α7 nAChR activity, which might be a successful therapeutic target against AD.

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.