Nrf2 Is Involved in Hyperglycemia-induced Abnormal Lung Development through both Antioxidation-Dependent and Antioxidation-Independent Activation.

Accumulating evidence has shown that hyperglycemia during pregnancy negatively affects lung development. However, the pathological mechanism of lung dysplasia caused by hyperglycemia remains unclear. In this study, we demonstrated the phenotypes of the impaired lung epithelial cell differentiation of mouse lungs in pregestational diabetes mellitus (PGDM) and gestational diabetes mellitus (GDM), and increased levels of oxidative stress and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways occurred. Nrf2 deficiency during pregnancy led to the aforementioned similar and aggravated phenotypes of the poor saccular process as in diabetes, implying the Nrf2 signaling pathway played a very important role in both physiological and pathological conditions. Based on RNA sequencing and luciferase reporter gene analysis, we revealed that Nrf2 could regulate Wnt signaling by targeting Ctnnd2. In summary, we revealed the pathological mechanism of how diabetes affected late lung development during embryogenesis, especially elucidating the bilateral roles of Nrf2-mediated oxidative stress responses and Wnt signaling. This finding also indicated that Nrf2 could potentially be used in preventing or treating pulmonary anomalies induced by hyperglycemia during pregnancy.

Alpha-2-macroglobulin is involved in the occurrence of early-onset pre-eclampsia via its negative impact on uterine spiral artery remodeling and placental angiogenesis.

BACKGROUND: Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood. METHODS: Human placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague-Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors. RESULTS: In this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFß1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats. CONCLUSIONS: Our data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.

Gut-Lung Dysbiosis Accompanied by Diabetes Mellitus Leads to Pulmonary Fibrotic Change through the NF-κB Signaling Pathway.

Growing evidence shows that the lungs are an unavoidable target organ of diabetic complications. However, the pathologic mechanisms of diabetic lung injury are still controversial. This study demonstrated the dysbiosis of the gut and lung microbiome, pulmonary alveolar wall thickening, and fibrotic change in streptozotocin-induced diabetic mice and antibiotic-induced gut dysbiosis mice compared with controls. In both animal models, the NF-κB signaling pathway was activated in the lungs. Enhanced pulmonary alveolar well thickening and fibrotic change appeared in the lungs of transgenic mice expressing a constitutively active NF-κB mutant compared with wild type. When lincomycin hydrochloride-induced gut dysbiosis was ameliorated by fecal microbiota transplant, enhanced inflammatory response in the intestine and pulmonary fibrotic change in the lungs were significantly decreased compared with lincomycin hydrochloride-treated mice. Furthermore, the application of fecal microbiota transplant and baicalin could also redress the microbial dysbiosis of the gut and lungs in streptozotocin-induced diabetic mice. Taken together, these data suggest that multiple as yet undefined factors related to microbial dysbiosis of gut and lungs cause pulmonary fibrogenesis associated with diabetes mellitus through an NF-κB signaling pathway.

Maternal and infant outcomes during the COVID-19 pandemic: a retrospective study in Guangzhou, China.

In late December 2019, the COVID-19 pandemic caused a great threat to people's lives worldwide. As a special category of the population, pregnant women are vulnerable during emergencies. This study was designed to explore whether or not the COVID-19 pandemic has influenced maternal and infant outcomes. We collected maternal characteristics, laboratory results, condition in the third trimester, maternal outcome, fetal or neonatal outcomes, and characteristics of amniotic fluid, umbilical cord and placenta from pregnant women and fetals or newborns in the first affiliated hospital of Jinan university from 24 January to 31 March 2020 (peak period), chose the same types of data at the hospital during the same period in 2019 and 1 January-23 January 2020 (prior to the outbreak of COVID-19 in 2020) as a control. Our study focused on uncomplicated singleton pregnancies among women not infected by COVID-19. The results demonstrated that there was not an increase in adverse outcomes of pregnant women and newborns during the COVID-19 pandemic; This might be associated with the updated design of major epidemic prevention and control systems in Guangzhou, and the extension of pregnant women's rest time during the third trimester of pregnancy. Nevertheless, the survey showed an increased incidence rate of 25-hydroxyvitamin D and zinc deficiency in newborns during the epidemic, implying that pregnant women should participate in appropriate physical exercise, increase their exposure to outdoor sunlight and improve nutrition intake to ensure healthy newborns during the quarantine period. Our study has provided some guidance for maternal management during the COVID-19 pandemic.

Effects of oxidative stress on hyperglycaemia-induced brain malformations in a diabetes mouse model.

Pregestational diabetes mellitus (PGDM) enhances the risk of fetal neurodevelopmental defects. However, the mechanism of hyperglycaemia-induced neurodevelopmental defects is not fully understood. In this study, several typical neurodevelopmental defects were identified in the streptozotocin-induced diabetes mouse model. The neuron-specific class III beta-tubulin/forkhead box P1-labelled neuronal differentiation was suppressed and glial fibrillary acidic protein-labelled glial cell lineage differentiation was slightly promoted in pregestational diabetes mellitus (PGDM) mice. Various concentrations of glucose did not change the U87 cell viability, but glial cell line-derived neurotrophic factor expression was altered with varying glucose concentrations. Mouse maternal hyperglycaemia significantly increased Tunel(+) apoptosis but did not dramatically affect PCNA(+) cell proliferation in the process. To determine the cause of increased apoptosis, we determined the SOD activity, the expression of Nrf2 as well as its downstream anti-oxidative factors NQO1 and HO1, and found that all of them significantly increased in PGDM fetal brains compared with controls. However, Nrf2 expression in U87 cells was not significantly changed by different glucose concentrations. In mouse telencephalon, we observed the co-localization of Tuj-1 and Nrf2 expression in neurons, and down-regulating of Nrf2 in SH-SY5Y cells altered the viability of SH-SY5Y cells exposed to high glucose concentrations. Taken together, the data suggest that Nrf2-modulated antioxidant stress plays a crucial role in maternal hyperglycaemia-induced neurodevelopmental defects.

Hyperactive neuronal autophagy depletes BDNF and impairs adult hippocampal neurogenesis in a corticosterone-induced mouse model of depression.

Background: Depression is a mental disorder that poses a serious threat to human health. Adult hippocampal neurogenesis (AHN) is closely associated with the efficacy of antidepressants. Chronic treatment with corticosterone (CORT), a well-validated pharmacological stressor, induces depressive-like behaviors and suppresses AHN in experimental animals. However, the possible mechanisms of chronic CORT action remain elusive. Methods: A chronic CORT treatment (0.1 mg/mL, drinking water for 4 weeks) was applied to prepare a mouse model of depression. Immunofluorescence was performed to analyze the hippocampal neurogenesis lineage, and immunoblotting, immunofluorescence, electron microscopy, and adeno-associated virus (AAV) expressing a pH-sensitive tandemly tagged light chain 3 (LC3) protein were used to analyze neuronal autophagy. AAV-hSyn-miR30-shRNA was used to knock down autophagy-related gene 5 (Atg5) expression in the neurons. Results: Chronic CORT induces depressive-like behaviors and decreases the expression of neuronal brain-derived neurotrophic factor (BDNF) in the dentate gyrus (DG) of the hippocampus in mice. Moreover, it markedly diminishes the proliferation of neural stem cells (NSCs), neural progenitor cells, and neuroblasts and impairs the survival and migration of newborn immature and mature neurons in the DG, which may be attributed to changes in the cell cycle kinetics and induction of NSCs apoptosis. Furthermore, chronic CORT induces hyperactive neuronal autophagy in the DG, possibly by increasing the expression of ATG5 and causing excess lysosomal degradation of BDNF in neurons. Notably, inhibiting hyperactive neuronal autophagy in the DG of mice by knocking down Atg5 in neurons using RNA interference reverses the decrease of neuronal BDNF expression, rescues AHN, and exerts antidepressant effects. Conclusion: Our findings reveal a neuronal autophagy-dependent mechanism that links chronic CORT to reduced neuronal BDNF levels, AHN suppression and depressive-like behavior in mice. In addition, our results provide insights for treating depression by targeting neuronal autophagy in the DG of the hippocampus.

NF-κB activation impedes the transdifferentiation of hypertrophic chondrocytes at the growth plate of mouse embryos in diabetic pregnancy.

BACKGROUND: Diabetes mellitus could cause numerous complications and health problems including abnormality of endochondral bone formation during embryogenesis. However, the underlying mechanisms still remain obscure. METHODS: Streptozotoci (STZ) was injected to induce pregestational diabetes mellitus (PGDM) mouse model. The femurs of E18.5 mouse embryos from control and PGDM groups were harvested. Morphological staining was implemented to determine the abnormality of the bone development. The expressions of the key genes participating in osteogenesis (e.g., Sox9, Runx2, and Osterix), the NF-κB signaling molecules (e.g., P50, P65, IκBα), and the corresponding regulatory factors (e.g., Bmp2, phospho-p38) were evaluated by immunofluorescence, quantitative PCR and western blot. Finally, in vitro chondrocyte differentiation model was employed to verify the role of NF-κB on the expressions of chondro-osteogenic markers. RESULTS: Alcian blue/alizarin red double staining and H&E staining demonstrated the restriction of skeletal development and relatively extended hypertrophic zone at growth plate in E18.5 STZ-induced diabetic mouse embryos compared to the control. Immunofluorescent staining and qPCR showed that Sox9 expression increased, while Runx2 and Osterix expressions decreased in the growth plate of the offspring of PGDM mice. Immunofluorescence of P65 manifested the activation of NF-κB signaling in growth plate in PGDM mouse embryos. Furthermore, the relatively extended hypertrophic zone was also observed in the growth plate of the NF-κB-activated transgenic mice, as well as the activated p65 up-regulated the expression of Bmp2 and p-p38. In ATDC5 cells, we could observe the high glucose up-regulated the P50 and P65 expressions and down-regulated IκBα expression, but the high glucose-activated NF-κB signaling could be reversed by addition of Bay (inhibitor of NF-κB signaling). The expression changes of Bmp2, Sox9 and Runx2 in presence of high glucose were resumed too. CONCLUSION: Our data revealed that NF-κB signaling was involved in mediation effects of dysfunctional trans-differentiation of hypertrophic chondrocytes in the embryonic growth plate induced by maternal diabetic mellitus.

The regulative effects of levetiracetam on adult hippocampal neurogenesis in mice via Wnt/ß-catenin signaling.

Adult hippocampal neurogenesis plays the pivotal roles in central nervous system diseases. Recently, it has been reported that levetiracetam (LEV), a new antiepileptic drug with novel chemical construction and unique pharmacological properties, suppressed aberrant adult subventricular zone (SGZ) neurogenesis in kainite-induced epileptic mice, while promoted adult SGZ neuroblast differentiation in normal mice. These studies indicate LEV can modulate adult hippocampal neurogenesis, but the exact mechanism remained unknown. Thus, the present study aimed to investigate the effects of subchronic and chronic LEV treatments on neural stem cell by lineage tracing in adult hippocampal dentate gyrus of mice, as well as the potential mechanism related to Wnt/ß-catenin signaling pathway. The data showed that both subchronic and chronic LEV treatments had no effects on body weight, locomotor activity and anxiety-like behavior in mice. Notably, subchronic LEV treatment significantly suppressed the proliferation of intermediate progenitor cell and neuroblast, decreased the number of intermediate progenitor cell and neuroblast, but increased the number of quiescent neural stem cell. On the contrary, chronic LEV treatment promoted the proliferation of neural stem cell, intermediate progenitor cell and neuroblast, increased the number of neural stem cell, intermediate progenitor cell and neuroblast, and promoted differentiation of newborn immature neuron and mature neuron. Furthermore, subchronic LEV treatment decreased the level of Wnt 3a and nuclear ß-Catenin expression, which led to the inhibition on Wnt/ß-catenin signaling pathway. Chronic LEV treatment increased the level of Wnt 3a, cytosolic ß-catenin and nuclear ß-Catenin, decreased the expression of GSK-3ß, p-Tyr216-GSK-3ß and Axin2, resulting in the enhancement of Wnt/ß-catenin signaling pathway. These results demonstrated that LEV significantly suppressed or promoted adult hippocampal neurogenesis in mice by subchronic or chronic treatment possibly through the regulation of Wnt/ß-catenin signaling pathway. Our findings provided the new perspectives of LEV on adult hippocampal neurogenesis underlying its clinical application.

Revealing Antidepressant Mechanisms of Baicalin in Hypothalamus Through Systems Approaches in Corticosterone- Induced Depressed Mice.

Baicalin, the main active flavonoid constituent of Scutellaria baicalensis Georgi, has been reported to exert antidepressant effects. Hypothalamic-pituitary-adrenal (HPA) axis plays important roles in depression. However, antidepressant effect and mechanism of baicalin on HPA axis in hypothalamus are still unknown. In present study, we find baicalin significantly attenuates the increase of immobility time in tail suspension and forced swimming, improves the decrease of spending time in open arms, and restores the aberrant negative feedback of HPA axis in chronic corticosterone (CORT)-induced depressed mice. Moreover, proteomics finds 370 differentially expressed proteins after baicalin treatment, including 114 up-regulation and 256 down-regulation in hypothalamus. Systems biology analysis indicates the functions of differentially expressed proteins focus on phosphoserine binding and phosphorylation, especially participate in GR signaling pathway. Finally, our findings demonstrate that baicalin normalizes hypothalamic GR nuclear translocation via reducing GR phosphorylation to remodel negative feedback of HPA axis in CORT-induced mice.

Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation. AIM OF THE STUDY: To discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis. MATERIALS AND METHODS: The antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network. RESULTS: Our data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT's antidepressant network. CONCLUSION: Our results indicate that XCHT plays the important roles in antidepressant action by restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression.

CNTF and Nrf2 Are Coordinately Involved in Regulating Self-Renewal and Differentiation of Neural Stem Cell during Embryonic Development.

There is high risk of fetal neurodevelopmental defects in pregestational diabetes mellitus (PGDM). However, the effective mechanism of hyperglycemia-induced neurodevelopmental negative effects, including neural stem cell self-renewal and differentiation, still remains obscure. Neuropoietic cytokines have been shown to play a vital part during nervous system development and in the coordination of neurons and gliocytes. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) dysfunction might be related to a reduction of self-protective response in brain malformation induced by hyperglycemia. We therefore evaluated the role of Nrf2 and neuropoietic cytokines in fetal neurodevelopmental defects induced by PGDM and determined the mechanisms involved. Our data reveal that PGDM dramatically impairs the developmental switch of neural stem cells from neurogenesis to gliogenesis, principally under the cooperative mediation of neuropoietic cytokine CNTF and Nrf2 antioxidative signaling. This indicates that CNTF and Nrf2 could be potentially used in the prevention or therapy of neurodevelopmental defects of PGDM offspring.

Negative impact of hyperglycaemia on mouse alveolar development.

Diabetes mellitus in pregnancy has been known to affect the embryonic development of various systems, including cardiovascular and nervous systems. However, whether this disease could have a negative impact on embryonic respiratory system remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in lung development in mice are mainly characterized by the changes in the morphological structure of the lung. Immunostaining and Western blotting showed that proliferation increased and apoptosis decreased in PGDM. Hyperglycaemia caused pulmonary tissue fibrationas manifested by an increase in Masson staining and decorin expression in PGDM lungs, and the immunofluorescent pro-SPC+ type II pulmonary epithelial cell number was decreased. The alteration of pulmonary epithelial cell differentiation might be due to hyperglycaemia-activated Wnt signalling and suppressed GATA6 expression in PGDM mouse lung tissues and MLE-12 cells. The treatment of MLE-12 cells with high glucose in the presence/absence of XAV939 or su5402 further proved that hyperglycaemia suppressed the expression of GATA6 and pro-SPC by activating Wnt signalling and induced the expression of decorin, α-SMA and TGF-ß by activating Fgf signalling. Therefore, in this study, we revealed that hyperglycemia induced dysfunctional pulmonary cell apoptosis and proliferation, as well as pulmonary myofibroblast hyperplasia, which contributed to the formation of aberrant structure of alveolar walls. Furthermore, the hyperglycaemia also inhibited the differentiation of pulmonary epithelial cells through the canonical Wnt and Fgf signalling, and the alteration of Fgf and Wnt signalling activated TGF-ß, which would promote the AECII EMT process.

Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta.

It is widely accepted that diabetes mellitus impairs placental development, but the mechanism by which the disease operates to impair development remains controversial. In this study, we demonstrated that pregestational diabetes mellitus (PGDM)-induced defects in placental development in mice are mainly characterized by the changes of morphological structure of placenta. The alteration of differentiation-related gene expressions in trophoblast cells rather than cell proliferation/apoptosis is responsible for the phenotypes found in mouse placenta. Meanwhile, excess reactive oxygen species (ROS) production and activated nuclear factor erythroid2-related factor 2 (Nrf2) signalling were observed in the placenta of mice suffering from PGDM. Using BeWo cells, we also demonstrated that excess ROS was produced and Nrf2 signalling molecules were activated in settings characterized by a high concentration of glucose. More interestingly, differentiation-related gene expressions in trophoblast cells were altered when endogenous Nrf2 expression is manipulated by transfecting Nrf2-wt or Nrf2-shRNA. In addition, PGDM interferes with autophagy in both mouse placenta and BeWo cells, implying that autophagy is also involved, directly or indirectly, in PGDM-induced placental phenotypes. Therefore, we revealed that dysfunctional oxidative stress-activated Nrf2 signalling and autophagy are probably responsible for PGDM-induced defects in the placental development of mice. The mechanism was through the interference with differentiation-related gene expression in trophoblast cells.

Investigating the Mechanism of Hyperglycemia-Induced Fetal Cardiac Hypertrophy.

Hyperglycemia in diabetic mothers enhances the risk of fetal cardiac hypertrophy during gestation. However, the mechanism of high-glucose-induced cardiac hypertrophy is not largely understood. In this study, we first demonstrated that the incidence rate of cardiac hypertrophy dramatically increased in fetuses of diabetic mothers using color ultrasound examination. In addition, human fetal cardiac hypertrophy was successfully mimicked in a streptozotocin (STZ)-induced diabetes mouse model, in which mouse cardiac hypertrophy was diagnosed using type-M ultrasound and a histological assay. PH3 immunofluorescent staining of mouse fetal hearts and in vitro-cultured H9c2 cells indicated that cell proliferation decreased in E18.5, E15.5 and E13.5 mice, and cell apoptosis in H9c2 cells increased in the presence of high glucose in a dose-dependent manner. Next, we found that the individual cardiomyocyte size increased in pre-gestational diabetes mellitus mice and in response to high glucose exposure. Meanwhile, the expression of ß-MHC and BMP-10 was up-regulated. Nkx2.5 immunofluorescent staining showed that the expression of Nkx2.5, a crucial cardiac transcription factor, was suppressed in the ventricular septum, left ventricular wall and right ventricular wall of E18.5, E15.5 and E13.5 mouse hearts. However, cardiac hypertrophy did not morphologically occur in E13.5 mouse hearts. In cultured H9c2 cells exposed to high glucose, Nkx2.5 expression decreased, as detected by both immunostaining and western blotting, and the expression of KCNE1 and Cx43 was also restricted. Taken together, alterations in cell size rather than cell proliferation or apoptosis are responsible for hyperglycemia-induced fetal cardiac hypertrophy. The aberrant expression of Nkx2.5 and its regulatory target genes in the presence of high glucose could be a principal component of pathogenesis in the development of fetal cardiac hypertrophy.