Insulin-like growth factor 1 in heat stress-induced neuroinflammation: novel perspective about the neuroprotective role of chromium.

Heat stress (HS) can cause a series of stress responses, resulting in numerous negative effects on the body, such as the diminished food intake, carcass quality and reproductive capacity. In addition to the negative effects on the peripheral system, HS leads to central nervous system (CNS) disorders given its toll on neuroinflammation. This neuroinflammatory process is mainly mediated by microglia and astrocytes, which are involved in the activation of glial cells and the secretion of cytokines. While the regulation of inflammatory signaling has a close relationship with the expression of heat shock protein 70 (Hsp70), HS-induced neuroinflammation is closely related to the activation of the TLR4/NF-κB pathway. Moreover, oxidative stress and endoplasmic reticulum (ER) stress are key players in the development of neuroinflammation. Chromium (Cr) has been widely shown to have neuroprotective effects in both humans and animals, despite the lack of mechanistic evidence. Evidence has shown that Cr supplementation can increase the levels of insulin-like growth factor 1 (IGF-1), a major neurotrophic factor with anti-inflammatory and antioxidant effects. This review highlights recent advances in the attenuating effects and potential mechanisms of Cr-mediated IGF-1 actions on HS-induced neuroinflammation, providing presently existing evidence supporting the neuroprotective role of Cr.

Dietary nitrate supplementation prevents radiotherapy-induced xerostomia.

Management of salivary gland hypofunction caused by irradiation (IR) therapy for head and neck cancer remains lack of effective treatments. Salivary glands, especially the parotid gland, actively uptake dietary nitrate and secrete it into saliva. Here, we investigated the effect of dietary nitrate on the prevention and treatment of IR-induced parotid gland hypofunction in miniature pigs, and elucidated the underlying mechanism in human parotid gland cells. We found that nitrate administration prevented IR-induced parotid gland damage in a dose-dependent manner, by maintaining the function of irradiated parotid gland tissue. Nitrate could increase sialin expression, a nitrate transporter expressed in the parotid gland, making the nitrate-sialin feedback loop that facilitates nitrate influx into cells for maintaining cell proliferation and inhibiting apoptosis. Furthermore, nitrate enhanced cell proliferation via the epidermal growth factor receptor (EGFR)-protein kinase B (AKT)-mitogen-activated protein kinase (MAPK) signaling pathway in irradiated parotid gland tissue. Collectively, nitrate effectively prevented IR-induced xerostomia via the EGFR-AKT-MAPK signaling pathway. Dietary nitrate supplementation may provide a novel, safe, and effective way to resolve IR-induced xerostomia.

Head and neck cancers are commonly treated using radiotherapy, where a beam of high-energy radiation is targeted at the tumour. This often severely damages the surrounding salivary glands, leading to chronic dry mouth and impairing a patient's sense of taste, nutrient intake, speech and immune system. Despite this significant impact on quality of life, there is no effective treatment yet for this side effect. In the body, salivary glands are one of the primary users of a compound known as nitrate, which is commonly found in the diet. In the glands, it is ushered into cells thanks to a protein known as sialin. The nutrient supports the activity and maintenance of the glands, before it is released in the saliva. Feng, Wu et al. therefore decided to test whether nitrate could offer protection during neck and head radiotherapy. The experiments used miniature pigs, which have similar salivary glands to humans. The animals that received sodium nitrate before and after exposure to radiation preserved up to 85% of their saliva production. By comparison, without any additional nitrate, saliva production fell to 20% of pre-radiation levels. To understand how this protective effect emerged, Feng, Wu et al. added nitrate to cells from a human salivary gland known as the parotid. This led to the cells producing more sialin, creating a feedback loop which increases the amount of nitrate in the salivary glands. Further examination then showed that the compound promotes growth of cells and reduce their death. These findings therefore suggest that clinical studies may be worthwhile to test if nitrate could be used to prevent dry mouth in head and neck cancer patients who undergo radiotherapy.

Repressed Gene Expression of Photosynthetic Antenna Proteins Associated with Yellow Leaf Variation as Revealed by Bulked Segregant RNA-seq in Tea Plant Camellia sinensis.

The young leaves and shoots of albino tea cultivars are usually characterized as having a yellow or pale color, high amino acid, and low catechin. Increasing attention has been paid to albino tea cultivars in recent years because their tea generally shows high umami and reduced astringency. However, the genetic mechanism of yellow-leaf variation in albino tea cultivar has not been elucidated clearly. In this study, bulked segregant RNA-seq (BSR-seq) was performed on bulked yellow- and green-leaf hybrid progenies from a leaf color variation population. A total of 359 and 1134 differentially expressed genes (DEGs) were identified in the yellow and green hybrid bulked groups (Yf vs Gf) and parent plants (Yp vs Gp), respectively. The significantly smaller number of DEGs in Yf versus Gf than in Yp versus Gp indicated that individual differences could be reduced within the same hybrid progeny. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed that the photosynthetic antenna protein was most significantly enriched in either the bulked groups or their parents. Interaction was found among light-harvesting chlorophyll a/b -binding proteins (LHC), heat shock proteins (HSPs), and enzymes involved in cuticle formation. Combined with the transcriptomic expression profile, results showed that the repressed genes encoding LHC were closely linked to aberrant chloroplast development in yellow-leaf tea plants. Furthermore, the photoprotection and light stress response possessed by genes involved in HSP protein interaction and cuticle formation were discussed. The expression profile of DEGs was verified via quantitative real-time PCR analysis of the bulked samples and other F1 individuals. In summary, using BSR-seq on a hybrid population eliminated certain disturbing effects of genetic background and individual discrepancy, thereby helping this study to intensively focus on the key genes controlling leaf color variation in yellow-leaf tea plants.

Novel insight into theacrine metabolism revealed by transcriptome analysis in bitter tea (Kucha, Camellia sinensis).

Kucha (Camellia sinensis) is a kind of unique wild tea resources in southwest China, containing sizeable amounts of theacrine (1,3,7,9-tetramethyluric acid) and having a special bitter taste both in fresh leaves and made tea. Theacrine has good healthy function locally. But the molecular mechanism of theacrine metabolism in Kucha was still unclear. In order to illuminate the biosynthesis and catabolism of theacrine in Kucha plants, three tea cultivars, C. sinensis 'Shangyou Zhongye' (SY) with low-theacrine, 'Niedu Kucha 2' (ND2) with middle-theacrine and, 'Niedu Kucha 3' (ND3) with high-theacrine, were used for our research. Purine alkaloid analysis and transcriptome of those samples were performed by High Performance Liquid Chromatography (HPLC) and RNA-Seq, respectively. The related gene expression levels of purine alkaloid were correlated with the content of purine alkaloid, and the results of quantitative real-time (qRT) PCR were also confirmed the reliability of transcriptome. Based on the data, we found that theacrine biosynthesis is a relatively complex process, N-methyltransferase (NMT) encoded by TEA024443 may catalyze the methylation at 9-N position in Kucha plant. Our finding will assist to reveal the molecular mechanism of theacrine biosynthesis, and be applied to selection and breeding of Kucha tea cultivars in the future.

Neuroprotective Effects and Hepatorenal Toxicity of Angong Niuhuang Wan Against Ischemia-Reperfusion Brain Injury in Rats.

Angong Niuhuang Wan (AGNHW) is a classic prescription in traditional Chinese medicine (TCM) used for stroke treatment, but its efficacies remain to be confirmed. With its arsenic- and mercury-containing materials, the application of AGNHW raises great safety concerns. Herein, we aim to explore the neuropharmacological effects against cerebral ischemia-reperfusion injury and evaluate the toxicological effects of AGNHW for better use. Male SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) and following 22 h of reperfusion. AGNHW (257 mg/kg, 1× AGNHW) were orally administered for pharmacological effects and 257, 514, and 1,028 mg/kg (equivalent to 1×, 2×, 4× AGNHW) were used for the toxicological study. The results revealed that AGNHW treatment reduced the infarct size and protected the blood-brain barrier (BBB) integrity in the MCAO rat ischemic stroke model. AGNHW treatment up-regulated bcl-2 expression and down-regulated the expressions of Bax, p47phox, inducible nitric oxide synthase (iNOS), and 3-nitrotyrosine (3-NT), and inhibited the expressions and activities of matrix metalloproteinase-2 (MMP-2), MMP-9, and reserved tight junction protein zonula occludens-1 (ZO-1) and claudin-5 in the ischemic brains. These results indicated that the neuroprotective mechanisms of AGNHW could be associated with its antioxidant properties by inhibiting oxidative/nitrative stress-mediated MMP activation and protecting tight junction proteins in the ischemic brains. Administration of 1× AGNHW for 7 days would not induce the accumulation of mercury in blood, liver, and kidney at day 14. Administration of 2× AGNHW and 4× AGNHW for 7 days increased the level of mercury in the kidney. For arsenic level, administration of 1× AGNHW for 7 days would increase the level of arsenic in the liver and blood without increase of arsenic in the kidney at day 14. Administration of 2× AGNHW and 4× AGNHW for 7 days would further increase the level of arsenic in the liver and blood. There is no influence on body weight, organ index, histological structures, and renal and liver functions. These results suggest that short-term treatment of AGNHW within 1 week should be safe and has neuroprotective effects against cerebral ischemia-reperfusion injury.

Realgar and cinnabar are essential components contributing to neuroprotection of Angong Niuhuang Wan with no hepatorenal toxicity in transient ischemic brain injury.

Realgar and cinnabar are commonly used mineral medicine containing arsenic and mercury in Traditional Chinese Medicine (TCM). Angong Niuhuang Wan (AGNHW) is a representative realgar- and cinnabar-containing TCM formula for treating acute ischemic stroke, but its toxicology and neuropharmacological effects are not well addressed. In this study, we compared the neuropharmacological effects of AGNHW and modified AGNHW in an experimental ischemic stroke rat model. Male SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus 22 h of reperfusion. Although oral administration of AGNHW for 7 days in the rats increased arsenic level in the blood and liver tissue, there were no significant changes in the arsenic level in kidney, mercury level in the blood, liver and kidney as well as hepatic and renal functions in MCAO rats. AGNHW revealed neuroprotective properties by reducing infarction volume, preserving blood-brain barrier integrity and improving neurological functions against cerebral ischemia-reperfusion injury. Interestingly, removing realgar and/or cinnabar from AGNHW abolished the neuroprotective effects. Meanwhile, AGNHW could scavenge peroxynitrite, down-regulate the expression of p47phox, 3-NT and MMP-9 and up-regulate the expression of ZO-1 and claudin-5 in the ischemic brains, which were abolished by removing realgar and/or cinnabar from AGNHW. Notably, realgar or cinnabar had no neuroprotection when used alone. Taken together, oral administration of AGNHW for one week should be safe for treating ischemic stroke with neuroprotective effects. Realgar and cinnabar are necessary elements with synergetic actions with other herbal materials for the neuroprotective effects of AGNHW against cerebral ischemia-reperfusion injury.

Vitamin D reduces the serum levels of inflammatory cytokines in rat models of periodontitis and chronic obstructive pulmonary disease.

The purpose of this study was to investigate the effects of vitamin D in rat models of chronic obstructive pulmonary disease (COPD) and periodontitis. Animals with both periodontitis and COPD, or with periodontitis only, were established. Once the animal model was established, experimental groups received intraperitoneal injections of 25-hydroxyvitamin D3 (25-OHD3) for 8 weeks, while control groups received refined peanut oil. After sacrifice, inflammatory status was examined in terms of the serum levels of receptor activator of the nuclear factor κB ligand (RANKL), tumor necrosis factor alpha (TNF-α) and interleukins (IL-1 and IL-10), as well as alveolar bone loss, forced expiratory volume (0.20) (FEV 0.20), and the ratio of FEV0.2 to forced vital capacity. The results showed that 25-OHD3 treatment significantly alleviated inflammation by decreasing the serum levels of RANKL, TNF-α and IL-1 and increasing that of IL-10, while reducing alveolar bone loss and slightly improving lung function. These findings suggest that vitamin D supplementation could be a new clinical approach for the treatment of COPD and periodontitis.

Nitrate and Nitrite in Health and Disease.

The source of dietary nitrate (NO3) is mainly green, leafy vegetables, partially absorbed into blood through intestinal mucosa. The recycled nitrate is reabsorbed and concentrated by the salivary glands and then secreted into saliva. In 2012, sialin was first discovered as the mammalian membrane nitrate transporter in salivary glands and plays a key role in circulation of inorganic nitrate, providing a scientific basis for further investigation into the circulation and functions of nitrate. Dietary nitrate can be converted to nitrite (NO2) by oral commensal bacteria under the tongue or in the stomach, following which nitrite is converted to nitric oxide (NO) through non-enzymatic synthesis. Previously, nitrate and nitrite were thought to be carcinogenic due to the potential formation of nitrogen compounds, whereas the beneficial functions of NO3 --NO2 --NO pathway were ignored. Under conditions of hypoxia and ischemia, the production of endogenous NO from L-arginine is inhibited, while the activity of exogenous NO3 --NO2 --NO is enhanced. Recently, a greater amount of evidence has shown that nitrate and nitrite serve as a reservoir and perform positive biological NO-like functions. Therefore, exogenous dietary nitrate plays an important role in various physiological activities as an effective supplement of nitrite and NO in human body. Here we generally review the source, circulation and bio-functions of dietary nitrate.

Dietary nitrate protects submandibular gland from hyposalivation in ovariectomized rats via suppressing cell apoptosis.

Xerostomia, a major oral symptom of menopause, is a subjective feeling of dry mouth associated with oral pain and difficulties in deglutition and speech, which significantly reduces patient's quality of life. Dietary nitrate, which can be converted to nitric oxide, has multiple physiological functions in the body, including antioxidant activity and vasodilatation; however, its protective effect against xerostomia remains poorly understood. The present study aimed to evaluate the effects of dietary nitrate on estrogen deficiency-induced xerostomia. We established an ovariectomized (OVX) rat model, which included five groups: sham-operated, OVX, OVX + 0.4 mM nitrate, OVX + 2 mM nitrate, and OVX + 4 mM nitrate (n = 6). After ovariectomy, animals in the nitrate treatment groups received appropriate amounts of sodium nitrate dissolved in distilled water for 3 months. The results showed that nitrate treatment reduced body weight and water intake, and increased serum nitrate and nitrite levels. Furthermore, nitrate uptake increased saliva secretion as evidenced by saliva flow rates and aquaporin 5 expression, and alleviated histological lesions as evidenced by reduction of the fibrotic area and cell atrophy in the salivary glands. Although protective effects of nitrate against estrogen deficiency-induced xerostomia were observed at all doses, treatment with 2 mM nitrate was more effective than that with 0.4 mM and 4 mM nitrate. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 expression analyses showed that nitrate also protected cells from apoptosis, possibly through upregulation of Cu-Zn superoxide dismutase (Cu-Zn SOD) known to inhibit oxidative stress-related apoptosis. Our findings indicate that nitrate could improve functional activity of the salivary glands in OVX rats by suppressing apoptosis and upregulating Cu-Zn SOD expression, suggesting that dietary nitrate may potentially prevent hyposalivation in menopausal women.

Inorganic nitrate alleviates the senescence-related decline in liver function.

Senescence-related decline in liver function is a common complication in the elderly that can lead to impaired health in older individuals. Dietary nitrate supplements have physiological and therapeutic effects on organ function by nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway. However, the role of dietary nitrate on the senescence-related decline in liver function is unclear. The findings of the present study showed that nitrate levels in the serum and liver decreased, whereas the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum increased in ageing mice. Consistently, cell senescence, decreased glycogen levels and increased lipid deposition were found in the liver of aged mice, both glucokinase (GCK) and phosphoenolpyruvate carboxykinase (PCK) were down-regulated (n=10). Daily nitrate intake (0.5 mmol L-1) restored nitrate levels, decreased ALT and AST levels, and prevented cell senescence and structural and glucose and lipid metabolism degeneration in liver tissue both in D-galactose (D-gal)-induced ageing mice (n=10) and in natural aged mice (n=10). In conclusion, the present study demonstrated that the reduction of nitrate levels was correlated with liver degeneration in ageing individuals and that dietary supplement of nitrate could restore the nitrate levels in serum and the liver and prevent ageing-related liver degeneration.

Aspirin promotes bone marrow mesenchymal stem cell-based calvarial bone regeneration in mini swine.

INTRODUCTION: Stem cells have great therapeutic potential due to their capacity for self-renewal and their potential for differentiating into multiple cell lineages. It has been recently shown that the host immune system has fundamental effects on the fate of transplanted mesenchymal stem cells during bone repair, where the topical administration of aspirin is capable of improving calvarial bone repair in rodents by inhibiting tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production. This study investigates whether aspirin is capable of accelerating the regenerative potential of bone marrow mesenchymal stem cells (BMSC) in a mini swine calvarial bone defect model. METHODS: Calvarial bone defects (3 cm × 1.8 cm oval defect) in mini swine were treated with BMSC pretreated with 75 µg/ml aspirin for 24 h seeded onto hydroxyaptite/tricalcium phosphatel (HA/TCP), or with BMSC with HA/TCP, or with HA/TCP only, or remained untreated. Animals were scanned with micro-computed tomography (microCT) at 2 days and 6 months postsurgery and were sacrificed at 6 months postsurgery with decalcified tissues being processed for histomorphometric examination. The cytokine levels, including TNF-α and IFN-γ, were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Aspirin at 75 µg/ml promoted the osteogenesis of BMSC in vitro and in vivo, shown by Alizarin Red staining and new bone volume in the nude mice transplantation model (p < 0.01), respectively. Defects treated with aspirin-BMSC showed significantly greater new bone fill compared with other three groups at 6 months postsurgery (p < 0.01). Aspirin-BMSC treatment has significantly decreased the concentration of TNF-α and IFN-γ (p < 0.05). CONCLUSIONS: The present study shows that BMSC pretreated with aspirin have a greater capacity to repair calvarial bone defects in a mini swine model. The results suggest that the administration of aspirin is capable of improving BMSC-mediated calvarial bone regeneration in a big animal model.

Histological characteristics following a long-term nitrate-rich diet in miniature pigs with parotid atrophy.

The aim of this study was to investigate the histological characteristics following a 2-year nitrate-rich diet in miniature pigs with parotid atrophy. Using averages collected data from three time points at 6, 12, and 24 months following the induction of parotid gland atrophy, salivary nitrate levels of the nitrate-diet parotid-atrophied group (17.3 ± 3.9 ng/µl) were close to those of the control group (19.6 ± 5.1 ng/µl). Compared to the control group, the nitrate-diet group had significantly higher nitrate levels in blood (P < 0.05) and urine (P < 0.001). Histological and electron microscopy analyses showed no abnormalities in the organs of experimental or control animals. No significant differences on apoptosis rate were found in liver and kidney tissues between the standard- and nitrate-diet groups. Therefore, dietary nitrate supplementation could restore salivary nitrate levels. High-dose nitrate loading for 2 years had no observed systemic toxicity in miniature pigs with parotid atrophy.

[Chromatographic Fingerprinting Study of Zhenyuan Granules Dry Extract by HPLC-DAD and HPLC-MS/MS].

OBJECTIVE: To establish a novel, accurate and valid fingerprint method of Zhenyuan granules dry extract by using HPLC-DAD method, to study herbs belonging of fingerprint peaks and to identify some of the chromatographic peaks by HPLC-MS/MS analysis, for providing the basis for scientific evaluation of the quality. METHODS: The sample solutions were analyzed by an Agilent SB C18 (250 mm x 4.6 mm, 5 µm) column, and gradiently eluted with acetonitrile (containing 0.1% formic acid) and aqueous phase (containing 0.1% formic acid) as the mobile phase. The flow rates were 1.2 mL/min (0~70 min) and 0.8 mL/min (70~150 min); the column temperature was 30 °C; and the detection wavelength was 254 nm. RESULTS: 40 peaks were selected as fingerprint peaks under the optimal chromatographic condition, and the similarity coefficients of 10 batches of Zhenyuan granules dry extract were all greater than 0.98. 27 peaks were tentatively identified with reference to literature data based on their mass spectrometry. CONCLUSION: The chromatographic fingerprint of Zhenyuan granules is proved to be a reliable method for comprehensive quality control and assessment.

Structural and functional characteristics of irradiation damage to parotid glands in the miniature pig.

PURPOSE: To evaluate the effects of a solitary megadose protocol of ionizing radiation (IR) on the structure and function of the miniature pig (minipig) parotid gland. METHODS AND MATERIALS: Fourteen minipigs were subjected to either 15 or 20 Gy to one parotid gland with a linear accelerator, whereas another four minipigs served as non-IR controls. Salivary flow rates and salivary chemistries were measured pre-IR and 4 and 16 weeks post-IR. A quantitative assessment of gland weight and acinar area and detailed serum chemistry and hematologic analyses were also performed. RESULTS: Parotid flow rates decreased by approximately 50% either with 20 Gy at 4 weeks, or 15 Gy at 16 weeks post-IR. In the 20 Gy group, salivary flow rates were reduced by approximately 80% at 16 weeks post-IR. A significant decrease in salivary calcium and amylase and an increase of salivary potassium levels were found in both IR groups. There were also transient alterations in serum chemistry and hematology parameters post-IR. Parotid gland weights were significantly decreased (-50%) in the 15 and 20 Gy groups at 4 and 16 weeks post-IR. Additionally, the acinar cell area in glands of both IR groups was significantly reduced from that in control glands at both the 4 and 16 weeks time points. CONCLUSION: Structural changes in salivary gland parenchyma occurred relatively early after IR, whereas the alterations in salivary output were relatively delayed. Further, reductions in salivary flow were not proportional to acinar cell area loss. Together, these findings suggest that nonparenchymal IR damage likely contributes to IR-induced salivary hypofunction.