Enteric glial cells contribute to chronic stress-induced alterations in the intestinal microbiota and barrier in rats.

Background: Emerging evidence has demonstrated the impact of psychological stress on intestinal microbiota, however, the precise mechanisms are not fully understood. Enteric glia, a unique type of peripheral glia found within the enteric nervous system (ENS), play an active role in enteric neural circuits and have profound effects on gut functions. In the present study, we tested the hypothesis that enteric glia are involved in the alterations in the intestinal microflora and barrier induced by chronic water-avoidance stress (WAS) in the gut. Methods and results: Western blotting and immunohistochemical (IHC) staining were used to examine the expression of glial fibrillary acidic protein (GFAP), nitric oxide synthetase (NOS) and choline acety1transferase (ChAT) in colon tissues. 16S rDNA sequencing was performed to analyse the composition of the intestinal microbiota in rats. Changes in the tight junction proteins Occludin, Claudin1 and proliferating cell nuclear antigen (PCNA) in the colon tissues were detected after WAS. The abundance of Firmicutes, Proteobacteria, Lactobacillus and Lachnospiraceae_NK4A136 decreased significantly, whereas the abundance of Actinobacteria, Ruminococcaceae_UCG-005 and Christensenellaceae-R-7 increased significantly in stressed rats. Meanwhile, the expression of Occludin, Claudin1 and PCNA significantly decreased after WAS. Treatment with L-A-aminohexanedioic acid (L-AA), a gliotoxin that blunts astrocytic function, obviously decreased the abundance of Actinobacteria, Ruminococcaceae_UCG-005 and Christensenel-laceae_R-7 in stressed rats and significantly increased the abundance of Proteobacteria, Lactobacillus and Lachnospiraceae_NK4A136. In addition, the protein expression of colon Occludin, Claudin1, and PCNA increased after intraperitoneal injection of L-AA. Furthermore, the expression level of NOS in colon tissues was significantly decreased, whereas that of ChAT was significantly increased following L-AA treatment. Conclusions: Our results showed that enteric glial cells may contribute to WAS-induced changes in the intestinal microbiota and barrier function by modulating the activity of NOS and cholinergic neurones in the ENS.

PLEK2 mediates metastasis and invasion via α5-nAChR activation in nicotine-induced lung adenocarcinoma.

Evidence has shown a strong relationship between smoking and epithelial mesenchymal transition (EMT). α5-nicotinic acetylcholine receptor (α5-nAChR) contributes to nicotine-induced lung cancer cell EMT. The cytoskeleton-associated protein PLEK2 is mainly involved in cytoskeletal protein recombination and cell stretch migration regulation, which is closely related to EMT. However, little is known about the link between nicotine/α5-nAChR and PLEK2 in lung adenocarcinoma (LUAD). Here, we identified a link between α5-nAChR and PLEK2 in LUAD. α5-nAChR expression was correlated with PLEK2 expression, smoking status and lower survival in vivo. α5-nAChR mediated nicotine-induced PLEK2 expression via STAT3. α5-nAChR/PLEK2 signaling is involved in LUAD cell migration, invasion and stemness. Moreover, PLEK2 was found to interact with CFL1 in nicotine-induced EMT in LUAD cells. Furthermore, the functional link among α5-nAChR, PLEK2 and CFL1 was confirmed in mouse xenograft tissues and human LUAD tissues. These findings reveal a novel α5-nAChR/PLEK2/CFL1 pathway involved in nicotine-induced LUAD progression.

α5-nAChR/STAT3/CD47 axis contributed to nicotine-related lung adenocarcinoma progression and immune escape.

OBJECTIVES: The CHRNΑ5 gene, which encodes the α5-nicotinic acetylcholine receptor (α5-nAChR), is related to lung cancer and nicotine addiction. Smoking is closely related to the immunosuppressive effect of macrophages. CD47, a phagocytosis checkpoint in macrophages, is a therapeutic target in various cancer types. Nevertheless, the relationship between α5-nAChR and CD47 in lung cancer is still unclear. METHODS AND RESULTS: The present study showed that α5-nAChR-mediated CD47 expression via STAT3 signaling, consequently leading to tumor progression and immune suppression in lung adenocarcinoma (LUAD). α5-nAChR expression was correlated with STAT3 expression, CD47 expression, smoking status and poor prognosis of LUAD in vivo. In vitro, α5-nAChR expression mediated the phosphorylation of STAT3, and phosphorylated STAT3 bound to the CD47 promoter and mediated CD47 expression. Downregulation of α5-nAChR and/or CD47 significantly reduced cell proliferation, migration, invasion, stemness and IL-10 expression, but increased TNF-α expression and phagocytosis of macrophages in LUAD. Furthermore, α5-nAChR/CD47 signaling contributed to the growth of subcutaneous xenograft tumors and liver metastasis of tumors in mice. CONCLUSION: The α5-nAChR/STAT3/CD47 axis contributed to the progression and immune escape of lung cancer and may be a potential target for LUAD immunotherapy.

Melatonin reduced colon inflammation but had no effect on energy metabolism in ageing Mongolian gerbils (Meriones unguiculatus).

In photoperiod-sensitive wild animals, the secretion of melatonin (MT) is modulated by external photoperiod, and MT affects inflammation and the ageing process. The beneficial effects of MT in delaying the progress of ageing have been reported in laboratory mice and rats. However, little is known about MT in wild mammals. In the current study, we investigated energy metabolism, microbial community structure and colon homeostasis in ageing Mongolian gerbils (Meriones unguiculatus) through exogenous supplementation of MT to test the hypothesis that MT has beneficial effects on gut homeostasis in ageing gerbils. Exogenous MT supplementation had no effect on energy metabolism in Mongolian gerbils but reduced the levels of circulating tumor necrosis factor-α (TNF-α), immune globulin G (IgG) and corticosterone (CORT). The increase in the level of inflammation in ageing animals was related to changes in the structure and diversity of the gut microbiota. At the genus level, the relative abundance of Prevotella, Treponema, Corynebacterium, and Sphingomonas was increased in ageing animals and decreased significantly by the treatment of MT. Christensenella and Lactobacillus were attenuated in ageing animals, and tended to be enhanced by MT treatment. Functions related to glycosphingolipid biosynthesis-ganglio series and lipopolysaccharide biosynthesis (metabolisms of cofactors, vitamins and glycan) were increased in ageing animals and decreased significantly by the treatment of MT. Our data suggest that a supplement of MT could improve colon homeostasis through changing the composition of gut microbiota and reducing inflammation in ageing gerbils.

Astrocytes in the spinal cord contributed to acute stress-induced gastric damage via the gap junction protein CX43.

Rat restraint water-immersion stress (RWIS) is a compound stress of high intensity and is widely used to study the pathological mechanisms of stress gastric ulcers. The spinal cord, as a part of the central nervous system, plays a dominant role in the gastrointestinal tract, but whether the spinal cord is involved in rat restraint water-immersion stress (RWIS)-induced gastric mucosal damage has not been reported. In this study, we examined the expression of spinal astrocytic glial fibrillary acidic protein (GFAP), neuronal c-Fos, connexin 43 (Cx43), and p-ERK1/2 during RWIS by immunohistochemistry and Western blotting. In addition, we intrathecally injected the astrocytic toxin L-a-aminoadipate (L-AA), gap junction blocker carbenoxolone (CBX), and ERK1/2 signaling pathway inhibitor PD98059 to explore the role of astrocytes in the spinal cord in RWIS-induced gastric mucosal damage and its possible mechanism in rats. The results showed that the expression of GFAP, c-Fos, Cx43, and p-ERK1/2 was significantly elevated in the spinal cord after RWIS. Intrathecal injection of both the astrocyte toxin L-AA and the gap junction blocker CBX significantly attenuated RWIS-induced gastric mucosal damage and decreased the activation of astrocytes and neurons induced in the spinal cord. Meanwhile, the ERK1/2 signaling pathway inhibitor PD98059 significantly inhibited gastric mucosal damage, gastric motility and RWIS-induced activation of spinal cord neurons and astrocytes. These results suggest that spinal astrocytes may regulate the RWIS-induced activation of neurons via CX43 gap junctions and play a critical role in RWIS-induced gastric mucosa damage through the ERK1/2 signaling pathway.

Acetylcholine promotes chronic stress-induced lung adenocarcinoma progression via α5-nAChR/FHIT pathway.

Chronic stress significantly elevates the expression levels of various neurotransmitters in the tumour microenvironment, thereby promoting the cell growth and metastasis of lung adenocarcinoma (LUAD). However, the role of chronic stress in the progression of LUAD remains unclear. In this study, we found that chronic restraint stress increases the levels of the neurotransmitter acetylcholine (ACh), and the α5-nicotinic acetylcholine receptor (α5-nAChR) and decreased fragile histidine triad (FHIT) expression in vivo. Crucially, the increased ACh levels promoted LUAD cell migration and invasion via modulation of the α5-nAChR/DNA methyltransferase 1 (DNMT1)/FHIT axis. In a chronic unpredictable stress (CUMS) mouse model, chronic stress promotes tumour development, accompanied by changes in α5-nAChR, DNMT1, FHIT, and vimentin. Together, these findings reveal a novel chronic stress-mediated LUAD signalling pathway: chronic stress enforces lung adenocarcinoma cell invasion and migration via the ACh/α5-nAChR/FHIT axis, which could be a potential therapeutic target for chronic stress-related LUAD.

Acute toxicity and genotoxicity studies on new melatonergic antidepressant GW117.

GW117, a novel derivate compound of agomelatine that acts as both a 5-HT2C receptor antagonist and a MT1/MT2 receptor agonist, likely underlines the potent antidepressant action with less hepatotoxicity than agomelatine. We evaluated the acute toxicity of GW117, and the genotoxicity of GW117 using bacterial reverse mutation test, mammalian chromosomal aberration test in Chinese hamster lung cells (CHL) and mouse bone marrow micronucleus test. The acute toxicity test results showed that maximum tolerated dose (MTD) of GW117 was 2000 mg/kg, under which mean Cmax and AUC0→t was 10,782 ng/mL and 81,046 ng/mL × h, respectively. The result of bacterial reverse mutation test showed that the number of bacterial colonies in each dose group of GW117 did not increase significantly compared with that in the solvent control group with or without S9 metabolic activation system. In vitro chromosome aberration test of CHL cells, the chromosome aberration rate of each dose group of GW117 did not increase with or without S9 metabolic activation system. In mouse micronucleus test, the highest dose was 2000 mg/kg, the micronucleus rate did not increase significantly. Under the conditions of this study, the MTD of a single GW117 administration was 2000 mg/kg, there was no genotoxicity effect of GW117.

Synthesis and Biological Evaluation of Substituted Pyrazole-Fused Oleanolic Acid Derivatives as Novel Selective α-Glucosidase Inhibitors.

A series of novel substituted pyrazole-fused oleanolic acid derivative were synthesized and evaluated as selective α-glucosidase inhibitors. Among these analogs, compounds 4a-4f exhibited more potent inhibitory activities compared with their methyl ester derivatives, and standard drugs acarbose and miglitol as well. Besides, all these analogs exhibited good selectivity towards α-glucosidase over α-amylase. Analog 4d showed potent inhibitory activity against α-glucosidase (IC50 =2.64±0.13 µM), and greater selectivity towards α-glucosidase than α-amylase by ∼33-fold. Inhibition kinetics showed that compound 4d was a non-competitive α-glucosidase inhibitor, which was consistent with the result of its simulation molecular docking. Moreover, the in vitro cytotoxicity of compounds 4a-4f towards hepatic LO2 and HepG2 cells was tested.

Alpha5 nicotinic acetylcholine receptor mediated immune escape of lung adenocarcinoma via STAT3/Jab1-PD-L1 signalling.

BACKGROUND: Immunotherapy has proven to be an emerging treatment for non-small-cell lung cancer in recent years. Notably, smokers show higher programmed cell death ligand-1 (PD-L1) expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Genome-wide association studies show that the CHRNΑ5 encoding α5-nicotinic acetylcholine receptor (α5-nAChR) is especially relevant to lung cancer and nicotine dependence. Jab1 is a key regulatory factor and promotes the stabilization of PD-L1. Our previous study reported that α5-nAChR mediates lung adenocarcinoma (LUAD) epithelial-mesenchymal transition (EMT) and metastasis via STAT3/Jab1. However, the link between α5-nAChR and PD-L1 is unclear in LUAD. METHODS: We used various bioinformatics databases to analyze the expression of related genes and their correlations. Expression and clinicopathologic significance of α5-nAChR and PD-L1 were detected by immunohistochemistry in a tissue microarray. α5-nAChR regulated LUAD cell immune escape by targeting the STAT3/Jab1-PD-L1 signalling by Western-blotting and ChIP in vitro. We used T cell coculture, flow cytometry, ELISA, CCK8 assay and crystal violet staining to detect the expression of regulatory T cell (Tregs), IFN-γ, IL-2 and the ability of T cell-mediated tumour cell killing respectively. IF assays were performed in both cancer cells and tumour xenograft paraffin sections to analyze the protein expression. The in vivo experiments in mouse model were performed to show the α5-nAChR-mediated immune escape via PD-L1 pathway. RESULTS: The expression of α5-nAChR was correlated with PD-L1 expression, smoking status and lower survival of LUAD in vivo. In vitro, the expression of α5-nAChR mediated phosphorylated STAT3 (pSTAT3), Jab1 and PD-L1 expression. STAT3 bound to the Jab1 or PD-L1 promoter and mediated PD-L1 expression. Jab1 stabilized PD-L1 expression in LUAD cells. Furthermore, in primary T cell cocultured system, downregulation of α5-nAChR suppressed the function of CD4+CD25+FOXP3+ Tregs, enhanced IFN-γ secretion, and increased T cell-mediated killing of LUAD cells. In the Jurkat T cells and LUAD cells coculture assay, inhibition of α5-nAChR increased IL-2 secretion. In tumour xenograft tissues, α5-nAChR expression was related to PD-L1, Jab1, pSTAT3, CD4 and granzyme B expression (GB). CONCLUSIONS: Our results suggest that the novel α5-nAChR/STAT3-Jab1-PD-L1 axis is involved in LUAD immune escape, which could lead to potential therapeutic strategies for cancer immunotherapy. Video abstract.

Functional Interactions Between the Parafascicular Thalamic Nucleus and Motor Cortex Are Altered in Hemiparkinsonian Rat.

Parkinson's disease (PD) is characterized by aberrant discharge patterns and exaggerated oscillatory activity within basal ganglia-thalamocortical circuits. We have previously observed substantial alterations in spike and local field potential (LFP) activities recorded in the thalamic parafascicular nucleus (PF) and motor cortex (M1), respectively, of hemiparkinsonian rats during rest or catching movements. This study explored whether the mutual effects of the PF and M1 depended on the amplitude and phase relationship in their identified neuron spikes or group rhythmic activities. Microwire electrode arrays were paired and implanted in the PF and M1 of rats with unilateral dopaminergic cell lesions. The results showed that the identified PF neurons exhibited aberrant cell type-selective firing rates and preferential and excessive phase-locked firing to cortical LFP oscillations mainly at 12-35 Hz (beta frequencies), consistent with the observation of identified M1 neurons with ongoing PF LFP oscillations. Experimental evidence also showed a decrease in phase-locking at 0.7-12 Hz and 35-70 Hz in the PF and M1 circuits in the hemiparkinsonian rats. Furthermore, anatomical evidence was provided for the existence of afferent and efferent bidirectional reciprocal connectivity pathways between the PF and M1 using an anterograde and retrograde neuroanatomical tracing virus. Collectively, our results suggested that multiple alterations may be present in regional anatomical and functional modes with which the PF and M1 interact, and that parkinsonism-associated changes in PF integrate M1 activity in a manner that varies with frequency, behavioral state, and integrity of the dopaminergic system.

Moderate Treadmill Exercise Modulates Gut Microbiota and Improves Intestinal Barrier in High-Fat-Diet-Induced Obese Mice via the AMPK/CDX2 Signaling Pathway.

OBJECTIVE: The aim of this study was to investigate the effects of moderate treadmill exercise on gut microbiota, expression of proteins associated with gut barrier and to elucidate the mechanisms underlying their role in high-fat-diet-induced obese mice. METHODS: Six-week-old male C57BL/6 mice were randomly divided into standard chow diet control group (SD + Sed, n=6), chow diet exercise group (SD + Exe, n=6), high-fat diet control group (HFD + Sed, n=6) and high-fat diet exercise group (HFD + Exe, n=6). Exercise groups were trained on a motorized treadmill for 45 min/d at running speeds of 12 m/min, 5 days/week, for 12 consecutive weeks. The body weight and fasting blood glucose of the mice were recorded before euthanasia. Thereafter, the mice were sacrificed and the alteration of adipose mass, colonic histopathology, gut microbiome and gut barrier-related molecules were tested. RESULTS: It was found that the moderate treadmill exercise prevented the development of adiposity and hyperglycemia and effectively improved the loss of diversity and the relative abundance of intestinal microflora induced by high-fat diet. Moreover, regular exercise reversed the intestinal pathology and elevated the number of goblet cells in obesity. Besides, compared with the sedentary obese mice, the protein expression levels of colonic ZO-1 and occludin were enhanced and AMPK/CDX2 signaling pathway was significantly upregulated in obese mice that underwent exercise. CONCLUSION: Long-term moderate treadmill exercise can markedly reduce the degree of obesity, modulate the colonic gut microbiota, and effectively activating AMPK/CDX2 signaling pathway to improve intestinal barrier in obese mice induced by high-fat diet.

Neurons and Astrocytes in Ventrolateral Periaqueductal Gray Contribute to Restraint Water Immersion Stress-Induced Gastric Mucosal Damage via the ERK1/2 Signaling Pathway.

BACKGROUND: The restraint water immersion stress (RWIS) model includes both psychological and physical stimulation, which may lead to gastrointestinal disorders and cause gastric mucosal damage. The ventrolateral periaqueductal gray (VLPAG) contributes to gastrointestinal function, but whether it is involved in RWIS-induced gastric mucosal damage has not yet been reported. METHODS: The expression of glial fibrillary acidic protein, neuronal c-Fos, and phosphorylated extracellular signal regulated kinase 1/2 in the VLPAG after RWIS was assessed using western blotting and immunocytochemical staining methods. Lateral ventricle injection of astrocytic toxin L-a-aminoadipate and treatment with extracellular signal-regulated kinase (ERK)1/2 signaling pathway inhibitor PD98059 were further used to study protein expression and distribution in the VLPAG after RWIS. RESULTS: The expression of c-Fos, glial fibrillary acidic protein, and phosphorylated extracellular signal regulated kinase 1/2 in the VLPAG significantly increased following RWIS and peaked at 1 hour after RWIS. Lateral ventricle injection of the astrocytic toxin L-a-aminoadipate significantly alleviated gastric mucosal injury and decreased the activation of neurons and astrocytes. Treatment with the ERK1/2 signaling pathway inhibitor PD98059 obviously suppressed gastric mucosal damage as well as the RWIS-induced activation of neurons and astrocytes in the VLPAG. CONCLUSIONS: These results suggested that activation of VLPAG neurons and astrocytes induced by RWIS through the ERK1/2 signaling pathway may play a critical role in RWIS-induced gastric mucosa damage.

Effects of Exogenous Hydrogen Sulfide in the Hypothalamic Paraventricular Nucleus on Gastric Function in Rats.

Background: Hydrogen sulfide (H2S) is a new type of gas neurotransmitter discovered in recent years. It plays an important role in various physiological activities. The hypothalamus paraventricular nucleus (PVN) is an important nucleus that regulates gastric function. This study aimed to clarify the role of H2S in the paraventricular nucleus of the hypothalamus on the gastric function of rats. Methods: An immunofluorescence histochemistry double-labelling technique was used to determine whether cystathionine-beta-synthase (CBS) and c-Fos neurons are involved in PVN stress. Through microinjection of different concentrations of NaHS, physiological saline (PS), D-2-Amino-5-phosphonovaleric acid (D-AP5), and pyrrolidine dithiocarbamate (PDTC), we observed gastric motility and gastric acid secretion. Results: c-Fos and CBS co-expressed the most positive neurons after 1 h of restraint and immersion, followed by 3 h, and the least was at 0 h. After injection of different concentrations of NaHS into the PVN, gastric motility and gastric acid secretion in rats were significantly inhibited and promoted, respectively (p < 0.01); however, injection of normal saline, D-AP5, and PDTC did not cause any significant change (p > 0.05). The suppressive effect of NaHS on gastrointestinal motility and the promotional effect of NaHS on gastric acid secretion could be prevented by D-AP5, a specific N-methyl-D-aspartic acid (NMDA) receptor antagonist, and PDTC, an NF-κB inhibitor. Conclusion: There are neurons co-expressing CBS and c-Fos in the PVN, and the injection of NaHS into the PVN can inhibit gastric motility and promote gastric acid secretion in rats. This effect may be mediated by NMDA receptors and the NF-κB signalling pathway.

Exogenous Hydrogen Sulfide Within the Nucleus Ambiguus Inhibits Gastrointestinal Motility in Rats.

Hydrogen sulfide (H2S) is a neuromodulator in the central nervous system. However, the physiological role of H2S in the nucleus ambiguus (NA) has rarely been reported. This research aimed to elucidate the role of H2S in the regulation of gastrointestinal motility in rats. Male Wistar rats were randomly assigned to sodium hydrosulfide (NaHS; 4 and 8 nmol) groups, physiological saline (PS) group, capsazepine (10 pmol) + NaHS (4 nmol) group, L703606 (4 nmol) + NaHS (4 nmol) group, and pyrrolidine dithiocarbamate (PDTC, 4 nmol) + NaHS (4 nmol) group. Gastrointestinal motility curves before and after the injection were recorded using a latex balloon attached with a pressure transducer, which was introduced into the pylorus through gastric fundus. The results demonstrated that NaHS (4 and 8 nmol), an exogenous H2S donor, remarkably suppressed gastrointestinal motility in the NA of rats (P < 0.01). The suppressive effect of NaHS on gastrointestinal motility could be prevented by capsazepine, a transient receptor potential vanilloid 1 (TRPV1) antagonist, and PDTC, a NF-κB inhibitor. However, the same amount of PS did not induce significant changes in gastrointestinal motility (P > 0.05). Our findings indicate that NaHS within the NA can remarkably suppress gastrointestinal motility in rats, possibly through TRPV1 channels and NF-κB-dependent mechanism.

Nervous mechanisms of restraint water-immersion stress-induced gastric mucosal lesion.

Stress-induced gastric mucosal lesion (SGML) is one of the most common visceral complications after trauma. Exploring the nervous mechanisms of SGML has become a research hotspot. Restraint water-immersion stress (RWIS) can induce GML and has been widely used to elucidate the nervous mechanisms of SGML. It is believed that RWIS-induced GML is mainly caused by the enhanced activity of vagal parasympathetic nerves. Many central nuclei, such as the dorsal motor nucleus of the vagus, nucleus of the solitary tract, supraoptic nucleus and paraventricular nucleus of the hypothalamus, mediodorsal nucleus of the thalamus, central nucleus of the amygdala and medial prefrontal cortex, are involved in the formation of SGML in varying degrees. Neurotransmitters/neuromodulators, such as nitric oxide, hydrogen sulfide, vasoactive intestinal peptide, calcitonin gene-related peptide, substance P, enkephalin, 5-hydroxytryptamine, acetylcholine, catecholamine, glutamate, γ-aminobutyric acid, oxytocin and arginine vasopressin, can participate in the regulation of stress. However, inconsistent and even contradictory results have been obtained regarding the actual roles of each nucleus in the nervous mechanism of RWIS-induced GML, such as the involvement of different nuclei with the time of RWIS, the different levels of involvement of the sub-regions of the same nucleus, and the diverse signalling molecules, remain to be further elucidated.

Comparative study of the distribution of NOS-positive neurons in pigeon intestine.

Nitric oxide (NO) plays an important role in regulating gut motility, mucosal barrier function and secretions in the enteric nervous system. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining has been used to identify nitrergic neurons of the enteric nervous system in different species. However, NADPH-d staining lacks specificity because it also reflects the presence of enzymes other than nitric oxide synthase (NOS). Therefore, NOS immunohistochemistry techniques are needed to test for nitrergic neurons in the avian gut. In the present work, the morphology, density and size of NOS-positive neurons in the duodenum, jejunum, ileum, caecum and rectum myenteric plexus of adult pigeons were investigated using NOS immunohistochemistry and whole-mount preparations techniques. The density of NOS-positive ganglion was highest in the ileum, similar to the caecum and rectum, and the lowest staining levels were observed in the duodenum. The staining intensity of NOS-positive neurons in the duodenum, jejunum and ileum was dark, followed by the rectal regions, with weak staining in the caecum. These results suggested that NOS immunohistochemistry and whole-mount preparation techniques provide an effective assessment method of the ganglia in the pigeon intestinal myenteric nerve plexus and are more accurate for cell counting compared with conventional sections.

Advances in salt tolerance molecular mechanism in tobacco plants.

Tobacco, an economic crop and important model plant, has received more progress in salt tolerance with the aid of transgenic technique. Salt stress has become a key research field in abiotic stress. The study of tobacco promotes the understanding about the important adjustment for survival in high salinity environments, including cellular ion transport, osmotic regulation, antioxidation, signal transduction and expression regulation, and protection of cells from stress damage. Genes, which response to salt, have been studied using targeted transgenic technologies in tobacco plants to investigate the molecular mechanisms. The transgenic tobacco plants exhibited higher seed germination and survival rates, better root and shoot growth under salt stress treatments. Transgenic approach could be the promising option for enhancing tobacco production under saline condition. This review highlighted the salt tolerance molecular mechanisms of tobacco.

α5-nAChR contributes to epithelial-mesenchymal transition and metastasis by regulating Jab1/Csn5 signalling in lung cancer.

Recent studies have showed that α5 nicotinic acetylcholine receptor (α5-nAChR) is closely associated with nicotine-related lung cancer. Our previous studies also demonstrated that α5-nAChR mediates nicotine-induced lung carcinogenesis. However, the mechanism by which α5-nAChR functions in lung carcinogenesis remains to be elucidated. Jab1/Csn5 is a key regulatory factor in smoking-induced lung cancer. In this study, we explored the underlying mechanisms linking the α5-nAChR-Jab1/Csn5 axis with lung cancer epithelial-mesenchymal transition (EMT) and metastasis, which may provide potential therapeutic targets for future lung cancer treatments. Our results demonstrated that the expression of α5-nAChR was correlated with the expression of Jab1/Csn5 in lung cancer tissues and lung cancer cells. α5-nAChR expression is associated with Jab1/Csn5 expression in lung tumour xenografts in mice. In vitro, the expression of α5-nAChR mediated Stat3 and Jab1/Csn5 expression, significantly regulating the expression of the EMT markers, N-cadherin and Vimentin. In addition, the down-regulation of α5-nAChR or/and Stat3 reduced Jab1/Csn5 expression, while the silencing of α5-nAChR or Jab1/Csn5 inhibited the migration and invasion of NSCLC cells. Mechanistically, α5-nAChR contributes to EMT and metastasis by regulating Stat3-Jab1/Csn5 signalling in NSCLC, suggesting that α5-nAChR may be a potential target in NSCLC diagnosis and immunotherapy.

Effects of Restraint Water-Immersion Stress-Induced Gastric Mucosal Damage on Astrocytes and Neurons in the Nucleus Raphe Magnus of Rats via the ERK1/2 Signaling Pathway.

Restraint water-immersion stress (RWIS) consists of psychological and physical stimulation, and it has been utilized in the research of gastric mucosal damage. It has been shown by previous studies that the nucleus raphe magnus (NRM) is closely involved in the gastrointestinal function, but its functions on the stress-induced gastric mucosal injury (SGMI) have not been thoroughly elucidated to date. Consequently, in this research, we aim to measure the expression of astrocytic glial fibrillary acidic protein (GFAP), neuronal c-Fos, and phosphorylation extracellular signal regulated kinase 1/2 (p-ERK1/2) in the process of RWIS with immunohistochemistry and western blot methods. What is more, we detect the relation between astrocytes and neurons throughout the stress procedure and explore the regulation of the ERK1/2 signaling pathway on the activity of astrocytes and neurons after RWIS. The results indicated that all three proteins expression multiplied following peaked 3 h substantially. The SMGI, astrocyte and neuron activity were affected after the astrocytotoxin L-A-aminohexanedioic acid (L-AA) and c-fos antisense oligonucleotide (ASO) injections. After the injection of PD98059, the gastric mucosal injury, astrocyte and neuron activity significantly fell off. These results suggested that RWIS-induced activity of astrocytes and neurons in the NRM may play a significant part in gastric mucosa damage via the ERK1/2 signaling pathway.

Two RING-Finger Ubiquitin E3 Ligases Regulate the Degradation of SPX4, An Internal Phosphate Sensor, for Phosphate Homeostasis and Signaling in Rice.

SPX-domain-containing proteins (SPXs) play an important role in inorganic phosphate (Pi) sensing, signaling, and transport in eukaryotes. In plants, SPXs are known to integrate cellular Pi status and negatively regulate the activity of Pi central regulators, the PHOSPATE STARVATION RESPONSE proteins (PHRs). The stability of SPXs, such as SPX4, is reduced under Pi-deficient conditions. However, the mechanisms by which SPXs are degraded remain unclear. In this study, using a yeast-two-hybrid screen we identified two RING-finger ubiquitin E3 ligases regulating SPX4 degradation, designated SDEL1 and SDEL2, which were post-transcriptionally induced by Pi starvation. We found that both SDELs were located in the nucleus and cytoplasm, had ubiquitin E3 ligase activity, and directly ubiquitinated the K213 and K299 lysine residues in SPX4 to regulate its stability. Furthermore, we found that PHR2, a Pi central regulator in rice, could compete with SDELs by interacting with SPX4 under Pi-sufficient conditions, which protected SPX4 from ubiquitination and degradation. Consistent with the biochemical function of SDEL1 and SDEL2, overexpression of SDEL1 or SDEL2 resulted in Pi overaccumulation and induced Pi-starvation signaling even under Pi-sufficient conditions. Conversely, their loss-of-function mutants displayed decreased Pi accumulation and reduced Pi-starvation signaling. Collectively, our study revealed that SDEL1 and SDEL2 facilitate the degradation of SPX4 to modulate PHR2 activity and regulate Pi homeostasis and Pi signaling in response to external Pi availability in rice.