Ketogenic diet attenuates cognitive dysfunctions induced by hypoglycemia via inhibiting endoplasmic reticulum stress-dependent pathways.

Hypoglycemia can potentially cause severe damage to the central nervous system. The ketogenic diet (KD), characterized by high-fat and extremely low-carbohydrate content, can modulate homeostasis and nutrient metabolism, thereby influencing body health. However, the effects and underlying mechanisms of KD on hypoglycemia-induced brain injury have not been thoroughly investigated. We aimed to explore the modulating effects of KD on cognitive functions and elucidate the underlying mechanisms. In this study, one-month-old mice were fed with KD for 2 weeks, and the changes in the gut microbiota were detected using the 16S rRNA gene amplicon sequencing method. The hypoglycemic model of mice was established using insulin, and the potential protective effect of KD on hypoglycemia-induced brain injury in mice was evaluated through immunofluorescence staining, western blotting, transmission electron microscopy, and Golgi staining. Our results showed that the intestinal flora of Dorea increased and Rikenella decreased in KD-fed mice. KD can not only alleviate anxiety-like behavior induced by hypoglycemia, but also increase the proportion of mushroom dendritic spines in the hippocampus by modulating changes in the gut microbiota. KD regulated synaptic plasticity by increasing the levels of SPN, PSD95, and SYP, which relieve cognitive impairment caused by hypoglycemia. Moreover, KD can promote the proliferation and survival of adult neural stem cells in the hippocampus, while reducing apoptosis by suppressing the activation of the IRE1-XBP1 and ATF6 endoplasmic reticulum stress pathways in mice with hypoglycemia. This study provides new evidence for demonstrating that KD may alleviate cognitive dysfunctions caused by hypoglycemia by modulating the gut microbiota.

Ketogenic Diet Alleviates Hypoglycemia-Induced Neuroinflammation via Modulation the Gut Microbiota in Mice.

SCOPE: This study aims to investigate the role of gut microbiota regulation with ketogenic diet (KD) in hypoglycemia-induced neuroinflammation. METHODS AND RESULTS: Immunofluorescence staining and western blotting show that KD alleviates blood-brain barrier injury induced by hypoglycemia by increasing Podxl and zonula occludens-1 (ZO-1) levels. KD-fed mice show reduced brain edema by decreasing aquaporin-4 (AQP4) content and maintaining its polarized expression. 16S rRNA gene amplicon sequencing results show that KD reduces the Chao 1 index of gut microbiota α-diversity, and significant separation is detected in the ß-diversity analysis between the control and KD-fed mice. KD increases the relative abundance of Firmicutes and Proteobacteria and decreases that of Bacteroidetes. Hypoglycemia can reduce SOD and GSH-PX levels while increasing TNF-α, IL-1ß, and IL-6 mRNA levels in the brain tissues of mice. KD alleviates hypoglycemia-induced neuroinflammation by inhibiting microglia activation and TLR4/p38MAPK/NF-κB signaling pathway. Importantly, antibiotic cocktail depletion of the gut microbiota weakens anti-inflammatory and antioxidation responses in KD-fed mice. CONCLUSION: Collectively, these findings suggest that KD alleviates hypoglycemia-induced brain injury via gut microbiota modulation, which may provide novel insights into the therapy for hypoglycemia.

Network Pharmacology Combined with an Experimental Validation Study to Reveal the Effect and Mechanism of Eucommia ulmoides Leaf Polysaccharide against Immunomodulation.

In the present study, the immuno-enhancing effect of Eucommia ulmoides leaf polysaccharide (ELP) was investigated in immunosuppressed mice induced by cyclophosphamide (CTX). To evaluate the immune enhancement mechanism of ELP, the immunoregulation effect of ELP was evaluated in vitro and in vivo. ELP is primarily composed of arabinose (26.61%), galacturonic acid (25.1%), galactose (19.35%), rhamnose (16.13%), and a small amount of glucose (12.9%). At 1000~5000 µg·mL-1, ELP could significantly enhance the proliferation and the phagocytosis of macrophages in vitro. Additionally, ELP could protect immune organs, reduce pathological damage, and reverse the decrease in the hematological indices. Moreover, ELP significantly increased the phagocytic index, enhanced the ear swelling response, augmented the production of inflammatory cytokines, and markedly up-regulated the expression of IL-1ß, IL-6, and TNF-α mRNA levels. Furthermore, ELP improved phosphorylated p38, ERK1/2, and JNK levels, suggesting that MAPKs might be involved in immunomodulatory effects. The results provide a theoretical foundation for exploring the immune modulation function of ELP as a functional food.

Dietary Supplementation With Acer truncatum Oil Promotes Remyelination in a Mouse Model of Multiple Sclerosis.

Background: Multiple sclerosis is a chronic demyelinating disease of uncertain etiology. Traditional treatment methods produce more adverse effects. Epidemiological and clinical treatment findings showed that unknown environmental factors contribute to the etiology of MS and that diet is a commonly assumed factor. Despite the huge interest in diet expressed by people with MS and the potential role diet plays in MS, very little data is available on the role of diet in MS pathogenesis and MS course, in particular, studies on fats and MS. The oil of Acer truncatum is potential as a resource to be exploited in the treatment of some neurodegenerative diseases. Objective: Here, we investigated the underlying influences of Acer truncatum oil on the stimulation of remyelination in a cuprizone mouse model of demyelination. Methods: Cuprizone (0.2% in chow) was used to establish a mouse model of demyelination. Acer truncatum oil was administrated to mice during remyelination. Following techniques were used: behavioral test, histochemistry, fluorescent immunohistochemistry, transmission electron microscope. Results: Mice exposed to cuprizone for 6 weeks showed schizophrenia-like behavioral changes, the increased exploration of the center in the open field test (OFT), increased entries into the open arms of the elevated plus-maze, as well as demyelination in the corpus callosum. After cuprizone withdrawal, the diet therapy was initiated with supplementation of Acer truncatum oil for 2 weeks. As expected, myelin repair was greatly enhanced in the demyelinated regions with increased mature oligodendrocytes (CC1) and myelin basic protein (MBP). More importantly, the supplementation with Acer truncatum oil in the diet reduced the schizophrenia-like behavior in the open field test (OFT) and the elevated plus-maze compared to the cuprizone recovery group. The results revealed that the diet supplementation with Acer truncatum oil improved behavioral abnormalities, oligodendrocyte maturation, and remyelination in the cuprizone model during recovery. Conclusion: Diet supplementation with Acer truncatum oil attenuates demyelination induced by cuprizone, indicating that Acer truncatum oil is a novel therapeutic diet in demyelinating diseases.

ß-Hydroxybutyric acid attenuates heat stress-induced neuroinflammation via inhibiting TLR4/p38 MAPK and NF-κB pathways in the hippocampus.

Heat stress causes many pathophysiological responses in the brain, including neuroinflammation and cognitive deficits. ß-Hydroxybutyric acid (BHBA) has been shown to have neuroprotective effects against inflammation induced by lipopolysaccharide. The aim of the present study was to evaluate the effects of BHBA on neuroinflammation induced by heat stress, as well as the underlying mechanisms. Mice were pretreated with vehicle, BHBA or minocycline (positive control group) and followed by heat exposure (43°C) for 15 min for 14 days. In mice subjected to heat stress, we found that treatment with BHBA or minocycline significantly decreased the level of serum cortisol, the expressions of heat shock protein 70 (HSP70), and the density of c-Fos+ cells in the hippocampus. Surprisingly, the ethological tests revealed that heat stress led to cognitive dysfunctions and could be alleviated by BHBA and minocycline administration. Further investigation showed that BHBA and minocycline significantly attenuated the activation of microglia and astrocyte induced by heat stress. Pro-inflammatory cytokines were attenuated in the hippocampus by BHBA and minocycline treatment. Importantly, compared with the heat stress group, mice in the BHBA treatment group and positive control group experienced a decrease in the expressions of toll-like receptor 4 (TLR4), phospho-p38 (p-p38), and nuclear factor kappa B (NF-κB). Our results elucidated that BHBA inhibits neuroinflammation induced by heat stress by suppressing the activation of microglia and astrocyte, and modulating TLR4/p38 MAPK and NF-κB pathways. This study provides new evidence that BHBA is a potential strategy for protecting animals from heat stress.

ß-Hydroxybutyrate Alleviates Low Glucose-Induced Apoptosis via Modulation of ROS-Mediated p38 MAPK Signaling.

Hypoglycemia has emerged as a prominent complication in anti-diabetic drug therapy or negative energy balance of animals, which causes brain damage, cognitive impairment, and even death. Brain injury induced by hypoglycemia is closely related to oxidative stress and the production of reactive oxygen species (ROS). The intracellular accumulation of ROS leads to neuronal damage, even death. Ketone body ß-hydroxybutyrate (BHBA) not only serves as alternative energy source for glucose in extrahepatic tissues, but is also involved in cellular signaling transduction. Previous studies showed that BHBA reduces apoptosis by inhibiting the excessive production of ROS and activation of caspase-3. However, the effects of BHBA on apoptosis induced by glucose deprivation and its related molecular mechanisms have been seldom reported. In the present study, PC12 cells and primary cortical neurons were used to establish a low glucose injury model. The effects of BHBA on the survival and apoptosis in a glucose deficient condition and related molecular mechanisms were investigated by using flow cytometry, immunofluorescence, and western blotting. PC12 cells were incubated with 1 mM glucose for 24 h as a low glucose cell model, in which ROS accumulation and cell mortality were significantly increased. After 24 h and 48 h treatment with different concentrations of BHBA (0 mM, 0.05 mM, 0.5 mM, 1 mM, 2 mM), ROS production was significantly inhibited. Moreover, cell apoptosis rate was decreased and survival rate was significantly increased in 1 mM and 2 mM BHBA groups. In primary cortical neurons, at 24 h after treatment with 2 mM BHBA, the injured length and branch of neurites were significantly improved. Meanwhile, the intracellular ROS level, the proportion of c-Fos+ cells, apoptosis rate, and nuclear translocation of NF-κB protein after treatment with BHBA were significantly decreased when compared with that in low glucose cells. Importantly, the expression of p38, p-p38, NF-κB, and caspase-3 were significantly decreased, while the expression of p-ERK was significantly increased in both PC12 cells and primary cortical neurons. Our results demonstrate that BHBA decreased the accumulation of intracellular ROS, and further inhibited cell apoptosis by mediating the p38 MAPK signaling pathway and caspase-3 apoptosis cascade during glucose deprivation. In addition, BHBA inhibited apoptosis by activating ERK phosphorylation and alleviated the damage of low glucose to PC12 cells and primary cortical neurons. These results provide new insight into the anti-apoptotic effect of BHBA in a glucose deficient condition and the related signaling cascade.

Neuroinflammation Induction and Alteration of Hippocampal Neurogenesis in Mice Following Developmental Exposure to Gossypol.

BACKGROUND: Neurogenesis in the neonatal period involves the proliferation and differentiation of neuronal stem/progenitor cells and the establishment of synaptic connections. This process plays a critical role in determining the normal development and maturation of the brain throughout life. Exposure to certain physical or chemical factors during the perinatal period can lead to many neuropathological defects that cause high cognitive dysfunction and are accompanied by abnormal hippocampal neurogenesis and plasticity. As an endocrine disruptor, gossypol is generally known to exert detrimental effects in animals exposed under experimental conditions. However, it is unclear whether gossypol affects neurogenesis in the hippocampal dentate gyrus during early developmental stages. METHODS: Pregnant Institute of Cancer Research mice were treated with gossypol at a daily dose of 0, 20, and 50 mg/kg body weight from embryonic day 6.5 to postnatal day (P) 21. The changes of hippocampal neurogenesis as well as potential mechanisms were investigated by 5-bromo-2-deoxyuridine labeling, behavioral tests, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western-blot analyses. RESULTS: At P8, maternal gossypol exposure impaired neural stem cell proliferation in the dentate gyrus and decreased the number of newborn cells as a result of reduced proliferation of BLBP+ radial glial cells and Tbr2+ intermediate progenitor cells. At P21, the numbers of NeuN+ neurons and parvalbumin+ γ-aminobutyric acid-ergic interneurons were increased following 50 mg/kg gossypol exposure. In addition, gossypol induced hippocampal neuroinflammation, which may contribute to behavioral abnormalities and cognitive deficits and decrease synaptic plasticity. CONCLUSIONS: Our findings suggest that developmental gossypol exposure affects hippocampal neurogenesis by targeting the proliferation and differentiation of neuronal stem/progenitor cells, cognitive functions, and neuroinflammation. The present data provide novel insights into the neurotoxic effects of gossypol on offspring.

Prenatal Exposure to Gossypol Impairs Corticogenesis of Mouse.

Gossypol is a yellow polyphenolic compounds extracted from roots, stems and seeds of cotton plants. Excessive intake of gossypol induces severe pathological signs of toxicity in livestock and wildlife. Currently, gossypol has received widespread attention for its toxic effects on the reproductive system. However, reports of the effects of gossypol during corticogenesis and the development of the mouse cerebral cortex are unavailable. In the present study, gossypol was orally administrated at a dose of 0, 20, and 50 mg/kg body weight/day to pregnant mice from embryonic day 6.5 to the time of sample collection. We used in utero electroporation and immunofluorescence to demonstrate that gossypol impaired cortical neuronal migration. Furthermore, labeling with 5-bromo-2-deoxyuridine and western blot analysis revealed that gossypol disturbed the balance between proliferation and differentiation of neural progenitors, inhibited neural progenitor cell proliferation, neuronal differentiation, and maturation. Additionally, cortical progenitor apoptotic cell death increased in the developing gossypol-treated cortex, which was associated with NF-κB and MAPK pathways. In conclusion, our findings indicate that gossypol exposure disrupted neurogenesis in the developing neocortex, suggesting the potentially harmful impact of gossypol on the cerebral cortex development of humans and livestock.

A new structure from cardiac cells cultured in vitro: Cardiomyocyte-annulation of neonatal rats.

To explore the formation, morphological characteristics, cell composition, and differentiation potential of cardiomyocyte annulation (cardio-annulation) during in vitro culture of cardiac cells. Cardiac cells were isolated and cultured. A live-cell imaging system was used to observe cardio-annulation. Cardiac troponin-T (cTnT) and vimentin were labeled with double immunofluorescence staining, and coexpressions of cTnT and connexin43 (Cx43), cTnT and nanog, c-kit and nanog, and c-kit and stem cell antigen (sca-1) were detected. The location of various types of cells within the cardio-annulation structure was observed. Adipogenic- and osteogenic-inducing fluids were used separately for in situ induction to detect the multidirectional differentiation potential of cells during the annulation process. After 3 to 6 days, cardiac cells migrated and formed an open or closed annulus with a diameter of 800 to 3500 µm. The annulus wall comprised the medial, middle, and lateral regions. The cells in the medial region were small, abundant, and laminated, while those in the middle region were larger with fewer layers, and those in the lateral region were less abundant, and loosely arranged in a single layer. Cardiomyocytes were distributed mainly on the surface of the medial region; nanog+ , c-kit+ , and sca-1+ cells were located mainly at the bottom of the annulus wall and fibroblasts were located mainly between these layers. The annulus cavity contained a large number of small, round cells, and telocytes. Cx43 was expressed in all cell types, and nanog, c-kit, and sca-1 were coexpressed in the cardio-annulation cells, which possess adipogenic and osteogenic differentiation potential. Cardio-annulation was discovered during an in vitro culture of cardiac cells. The structure contains cardiomyocytes, fibroblasts, telocytes, and abundant stem cells. These results provide insight into the relationship among cardiac cells in vitro.

CD90+ cardiac fibroblasts reduce fibrosis of acute myocardial injury in rats.

OBJECTIVE: To explore the differentiation tendency of CD90+ cardiac fibroblast (CFs) into cardiomyogenic cells in vitro and repair functions in acute myocardial infarction rats. METHODS: CD90+ subpopulation was sorted from rat CFs by flow cytometry. 10 µmoL/L of 5-Azacytosine (5-aza) was used to induce differentiation of CFs into cardiomyogenic cells. An acute myocardial infarction model was prepared by ligation of the rat left anterior descending coronary artery. After nuclei were labeled by DAPI, induced CD90+ CFs were injected into the infarction marginal zone. Before coronary ligation, 40 min after ligation, and at 7 and 14 days after cell transplantation, cardiac function changes were detected by ultrasound imaging system respectively. cTnT and endothelial cell marker VIII factor were detected by immunofluorescence staining. Infarct size was examined by TTC staining. Fibrosis was evaluated with masson's trichrome staining, vimentin, type I and type III collagen staining. RESULTS: CD90+ CFs sorted by flow cytometry was 34.9%. On day 28 after induction, the cTnT positive rate was 61.17 ±â€¯9.75%. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) at days 7 and 14 after transplantation were significantly increased compared with those before transplantation (P < 0.05). LVEF and LVFS at the 14th day after transplantation were also significantly increased compared with those at the 7th day (LVEF: 61.40 ±â€¯2.45% vs. 56.25 ±â€¯2.9%, LVFS: 33.21 ±â€¯0.68% vs. 30.26 ±â€¯2.06%, P < 0.01). Additionally, small numbers of CD90+ CFs differentiated into cardiomyocytes and became involved in neovascularization. CD90+ CFs and CFs reduced myocardial infarct size at days 14. It was significantly smaller in rats with CFs transplantation group than those in MI group(24.78 ±â€¯2.28% vs. 31.28 ±â€¯2.83%, P < 0.05), and it was also significantly smaller in rats with CD90+CFs transplantation group than those in CFs transplantation group (17.47 ±â€¯4.15% vs. 24.78 ±â€¯2.28%, P < 0.05). Meanwhile, The percentage of fibrotic area and vimentin, type I and type III collagen in the infarct border zone and infarct area were both significantly reduced in CD90 + CFs. CONCLUSION: CD90+ CFs is the preponderant subpopulation of cardiomyogenic differentiation, with potential use as seed cells in basic and clinical research of heart regeneration and repair.

Multiple Directional Differentiation Difference of Neonatal Rat Fibroblasts from Six Organs.

BACKGROUND/AIMS: Fibroblasts are abundantly distributed throughout connective tissues in the body and are very important in maintaining the structural and functional integrity. Recent reports have proved that fibroblasts and mesenchymal stem cells share much more in common than previously recognized. The aim of this study was to investigate comparative studies in fibroblasts on the differences in the expression of molecular markers and differentiation capacity from different organs. METHODS: Combined trypsin/collagenase enzymes digestion method was used to isolate and culture the fibroblasts derived from heart, liver, spleen, lung, kidney and skin. Cell activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Common molecular markers for fibroblasts such as vimentin, DDR2 and FSP1, stem cell markers nanog, c-kit and sca-1 were detected by RT-PCR, immunofluorescence and western blotting. The osteogenic, adipogenic and cardiogenic differentiations of fibroblasts were performed by inductive culture in special mediums, and analyzed by Alizarin red, Oil red O and immunofluorescence staining of cTnT respectively. RESULTS: The proliferation rate of fibroblasts in lung was faster than in other five organs. Common molecular markers for fibroblasts were expressed differently in different organs. DDR2 was strongly expressed in fibroblasts in the heart, partly expressed in the heart, skin, liver and spleen. Interestingly, no expression of DDR2 was detected in liver and kidney. However, vimentin and FSP1 were consistently expressed in fibroblasts from skin, liver, kidney, spleen and lung. nanog expression in fibroblasts from lung was less than that from heart, skin, liver and spleen (P < 0.01). c-kit expression in fibroblasts from heart, skin and kidney was higher than that from spleen (P < 0.05), while the c-kit positive fibroblasts from liver was obviously higher than that from spleen (P < 0.01). But sca-1 expression in fibroblasts from lung was the lowest among six organs (P < 0.01). Directed differentiation in vitro had demonstrated that skin fibroblasts had the strongest multiple differentiation potential, and the next was cardiac fibroblasts. And fibroblasts in liver and kidney had the advantage in myocardial differentiation, while fibroblasts in spleen only had the advantage in osteogenic differentiation. CONCLUSIONS: There are obvious heterogeneity in molecular markers and muti-directional differentiation in fibroblasts from six organs.

Telocytes in the Spleen.

Telocytes, a novel type of interstitial cells with very long and thin prolongations, have been identified in many organs in mammals. At present, the ultrastructural, immunocytochemical and electrophysiological properties of telocytes in multiple organs have been understood. However, telocytes in spleen, especially their roles in spleen have not been reported. The aim of this study was to investigate the ultrastructure, distribution and immunophenotypes of splenic telocytes. Rat spleen was harvested for the ultrastructure analysis by transmission electron microscopy (TEM). The primary culture of telocytes was performed after combined enzymatic digestion. The characteristic morphology was analyzed by a scanning electron microscopy (SEM). It was shown that telocytes displayed a piriform/spindle/triangular shape with long and slender telopods and extremely long prolongation contracting with surrounding cells in the spleen. Their dynamic profiles of cytoplasmic separation were recorded by the Live Cell Imaging System. The length of telopods was mostly distributing in 20-30 µm, in accordance with normal distribution. Most telocytes had three or two telopods (28.71% and 22.58% respectively). Immunostaining indicated that these cells were positive for vimentin, CD34, nanog and sca-1, but negative for c-kit. These data prove the existence of telocytes in the spleen, which may serve as the experimental base for exploring their roles in the spleen.

Multiple immunophenotypes of cardiac telocytes.

AIMS: Telocytes (TCs) form a 3-dimensional network in the myocardial interstitium, which most probably play important role(s) in heart development. However, the dynamics of their prolongations, continuous cell shape changes and adherence properties have not been well documented till recently. The aim of this study was to investigate dynamics of extension of prolongations (Telopods) and multiple phenotypes of cardiac TCs cultured in vitro. METHODS: Cardiac TCs were isolated from neonatal rats by a combined enzyme digestion process and identified by light microscopy, immunofluorescence analysis and scanning using electron microscopy (SEM). Their continuous changes in shape were analyzed by a Live Cell Imaging System and multiple phenotypes were identified by immunofluorescence analysis using various markers, like vimentin, c-kit, CD34, nanog and sca-1. RESULTS: Cardiac TCs displayed piriform/spindle/triangular shapes with long and slender telopodes showing extremely long prolongations. The morphology of cell body was continuously changing while their prolongations were extending gradually. After adhering to the surface, TCs' movement and extension of their prolongations lasted for approximately 1.5h. Cardiac TCs expressed mesenchymal cell marker vimentin, hematopoietic stem cell marker CD34, embryonic stem cell-associated gene of Nanog, and myocardial stem cell markers sca-1 and c-kit. CONCLUSION: These findings indicate that cultured TCs in vitro have multiple phenotypes, which are most likely important for evaluating their functional roles in heart development.

Gata4, Tbx5 and Baf60c induce differentiation of adipose tissue-derived mesenchymal stem cells into beating cardiomyocytes.

The adipose tissue-derived mesenchymal stem cells (ADMSCs) are extensively utilized in tissue engineering, regenerative medicine and cell therapy. ADMSCs can differentiate into cardiomyocytes, and it has been shown that over-expression of a cocktail of factors can induce ectopic heart formation and program cardiogenesis in ESCs. However, which genes are responsible for differentiation of ADMSCs into beating cardiomyocyte-like cells remains unknown. In this study we have shown that the combination of Gata4, Tbx5 and Baf60c is sufficient for inducing ADMSCs to form cardiomyocytes. It also appears that, while Gata4 and Baf60c are key inducers of myocardial differentiation, Tbx5 is essential for the ability of cardiac cells to contract. These findings provide additional experimental references for myocardial tissue engineering in the emerging field of cell-based therapy of heart diseases.