The effects of ambient temperature and feeding regimens on cecum bacteria composition and circadian rhythm in growing rabbits.

Seasonal environmental shifts and improper eating habits are the important causes of diarrhea in children and growing animals. Whether adjusting feeding time at varying temperatures can modify cecal bacterial structure and improve diarrhea remains unknown. Three batches growing rabbits with two groups per batch were raised under different feeding regimens (fed at daytime vs. nighttime) in spring, summer and winter separately, and contents were collected at six time points in 1 day and used 16S rRNA sequencing to investigate the effects of feeding regimens and season on the composition and circadian rhythms of cecum bacteria. Randomized forest regression screened 12 genera that were significantly associated with seasonal ambient temperature changes. Nighttime feeding reduced the abundance of the conditionally pathogenic bacteria Desulfovibrio and Alistipes in summer and Campylobacter in winter. And also increases the circadian rhythmic Amplicon Sequence Variants in the cecum, enhancing the rhythm of bacterial metabolic activity. This rhythmic metabolic profile of cecum bacteria may be conducive to the digestion and absorption of nutrients in the host cecum. In addition, this study has identified 9 genera that were affected by the combination of seasons and feeding time. In general, we found that seasons and feeding time and their combinations affect cecum composition and circadian rhythms, and that daytime feeding during summer and winter disrupts the balance of cecum bacteria of growing rabbits, which may adversely affect cecum health and induce diarrhea risk.

Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-ß1 via the Hippo signaling pathway.

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-ß1 (TGF-ß1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-ß1 (Ad-sh-TGF-ß1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-ß1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-ß1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-ß1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD. Similar to Ad-sh-TGF-ß1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-ß1, Ad-ADM improved LPS-induced TGF-ß1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-ß1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-ß1 through Hippo signaling.

Local adrenomedullin gene delivery inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo.

Adrenomedullin (ADM) has beneficial effects on Leydig cells under pathological conditions, including lipopolysaccharide (LPS)-induced orchitis. Our previous studies demonstrated that ADM exerts a restorative effect on steroidogenesis in LPS-treated primary rat Leydig cells by attenuating oxidative stress, inflammation and apoptosis. In this study, we aim to investigate whether ADM inhibits Leydig cell dysfunction by rescuing steroidogenic enzymes in vivo. Rats were administered with LPS and injected with Ad-ADM, an adeno-associated virus vector that expressed ADM. Then, rat testes were collected for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunofluorescence staining. Steroidogenic enzymes or steroidogenic regulatory factors or protein, including steroidogenic factor-1 (SF-1), liver receptor homologue-1 (LRH1), Nur77, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17α-hydroxylase/17, 20 lyase (CYP17) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD), were detected via gene expression profiling and western blot analysis. Plasma testosterone concentrations were measured. Results showed that ADM may inhibit Leydig cell dysfunction by rescuing steroidogenic enzymes and steroidogenic regulatory factors in vivo. The reduction in the number of Leydig cells after LPS exposure was reversed by ADM. ADM rescued the gene or protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3ß-HSD, CYP17 and 17ß-HSD and plasma testosterone concentrations. To summarize ADM could rescue some important steroidogenic enzymes, steroidogenic regulatory factors and testosterone production in Leydig cells in vivo.

Greater hydraulic safety contributes to higher growth resilience to drought across seven pine species in a semi-arid environment.

Quantifying inter-specific variations of tree resilience to drought and revealing the underlying mechanisms are of great importance to the understanding of forest functionality, particularly in water-limited regions. So far, comprehensive studies incorporating investigations in inter-specific variations of long-term growth patterns of trees and the underlying physiological mechanisms are very limited. Here, in a semi-arid site of northern China, tree radial growth rate, inter-annual tree-ring growth responses to climate variability, as well as physiological characteristics pertinent to xylem hydraulics, carbon assimilation and drought tolerance were analyzed in seven pine species growing in a common environment. Considerable inter-specific variations in radial growth rate, growth response to drought and physiological characteristics were observed among the studied species. Differently, the studied species exhibited similar degrees of resistance to drought-induced branch xylem embolism, with water potential corresponding to 50% loss hydraulic conductivity ranging from -2.31 to -2.96 MPa. We found that higher branch hydraulic efficiency is related to greater leaf photosynthetic capacity, smaller hydraulic safety margin and lower woody density (P < 0.05, linear regressions), but not related to higher tree radial growth rate (P > 0.05). Rather, species with higher hydraulic conductivity and photosynthetic capacity were more sensitive to drought stress and tended to show weaker growth resistance to extreme drought events as quantified by tree-ring analyses, which is at least partially due to a trade-off between hydraulic efficiency and safety across species. This study thus demonstrates the importance of drought resilience rather than instantaneous water and carbon flux capacity in determining tree growth in water-limited environments.

Night-Restricted Feeding Improves Gut Health by Synchronizing Microbe-Driven Serotonin Rhythm and Eating Activity-Driven Body Temperature Oscillations in Growing Rabbits.

The circadian misalignment of the gut microbiota caused by unusual eating times in adult animals is related to disease development. However, whether the composition and diurnal rhythm of gut microbiota can be optimized by synchronizing the window period of eating with natural eating habits to reduce the risk of diarrhea remains unclear, especially in growing animals. In this study, 108 5-week-old weaned rabbits (nocturnal animals) were randomly subjected to daytime feeding (DF) and night-restricted feeding (NRF). At age 12 weeks, six rabbits were selected from each group, and caecum and cecal contents, as well as serum samples were collected at 4-h intervals during 24 h. Overall, NRF was found to reduce the risk of diarrhea in growing rabbits, improved the diurnal rhythm and abundance of beneficial microorganisms, along with the production of beneficial metabolites, whereas reduced the abundance of potential pathogens (Synergistes, Desulfovibrio, and Alistipes). Moreover, NRF improved diurnal rhythm of tryptophan hydroxylase isoform 1 and serotonin. Furthermore, NRF strengthened the diurnal amplitude of body core temperature, and promoted the diurnal expression of intestinal clock genes (BMAL1, CLOCK, REV-ERBα, and PER1), and genes related to the regulation of the intestinal barrier (CLAUDIN-1), and intestinal epithelial cell self-proliferation and renewal (BMI1). In vitro simulation experiments further revealed that synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, which are important zeitgebers, could promote the diurnal expression of clock genes and CLAUDIN-1 in rabbit intestinal epithelial cells (RIEC), and enhance RIEC proliferation. This is the first study to reveal that NRF reprograms the diurnal rhythm of the gut microbiome, promotes the diurnal expression of clock genes and tight junction genes via synchronization of microbial-driven serotonin rhythm and eating activity-driven body temperature oscillations, thereby improving intestinal health and reducing the risk of diarrhea in growing rabbits. Collectively, these results provide a new perspective for the healthy feeding and management of growing animals.

Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis.

Adrenomedullin (ADM) exerts anti-oxidant, anti-inflammatory and anti-apoptotic effects in Leydig cells. However, the role and mechanism of ADM in the pyroptosis of Leydig cells are poorly understood. This study first showed the protective effects of ADM on the pyroptosis and biological functions of Leydig cells exposed to lipopolysaccharide (LPS) by promoting autophagy. Primary rat Leydig cells were treated with various concentrations of LPS and ADM, together with or without N-acetyl-L-cysteine (NAC) or 3-methyladenine (3-MA). Cell proliferation was detected through CCK-8 and BrdU incorporation assays, and ROS level was measured with the DCFDA assay. Real-time PCR, western blot, immunofluorescence, transmission electron microscopy, TUNEL and flow cytometry were performed to examine ADM's effect on the pyroptosis, autophagy and steroidogenic enzymes of Leydig cells and AMPK/mTOR signalling. Like NAC, ADM dose-dependently reduced LPS-induced cytotoxicity and ROS overproduction. ADM also dose-dependently ameliorated LPS-induced pyroptosis by reversing the increased expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18, GSDMD, caspase-3, caspase-7, TUNEL-positive and PI and active caspase-1 double-stained positive rate, DNA fragmentation and LDH concentration, which could be rescued via co-incubation with 3-MA. ADM dose-dependently increased autophagy in LPS-induced Leydig cells, as confirmed by the increased expression of LC3-I/II, Beclin-1 and ATG-5; decreased expression of p62 and autophagosomes formation; and increased LC3-II/LC3-I ratio. However, co-treatment with 3-MA evidently decreased autophagy. Furthermore, ADM dose-dependently rescued the expression of steroidogenic enzymes, including StAR, P450scc, 3ß-HSD and CYP17, and testosterone production in LPS-induced Leydig cells. Like rapamycin, ADM dose-dependently enhanced AMPK phosphorylation but reduced mTOR phosphorylation in LPS-induced Leydig cells, which could be rescued via co-incubation with 3-MA. In addition, pyroptosis was further decreased, and autophagy was further promoted in LPS-induced Leydig cells upon co-treatment with ADM and rapamycin. ADM may protect the steroidogenic functions of Leydig cells against pyroptosis by activating autophagy via the ROS-AMPK-mTOR axis.

Human epididymis protein 4 in endometrial cancer: A meta-analysis.

BACKGROUND: Serum human epididymis protein 4 (HE4) is a potential marker for endometrial cancer (EC), however, the diagnostic value of HE4 for EC remains controversial. In this study, we performed a meta-analysis to estimate the diagnostic accuracy of serum HE4 for EC. METHODS: Literature reports of the diagnostic accuracy of serum HE4 for EC were systematically identified using online data-bases. The meta-analysis was performed using STATA 12.0, Meta-Disc 1.4, and Review Manager 5.2. RESULTS: A total of 4182 participants and 23 studies were included in our meta-analysis. The pooled sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) were 0.65 (95% CI: 0.56-0.73), 0.91 (95% CI: 0.84-0.95), (95% CI: 4.38-12.64), 0.38 (95% CI: 0.31-0.47), 19.46 (95% CI: 11.61-32.62) and 0.84 (95% CI: 0.81 to 0.87), respectively. Our overall analysis suggested that HE4 is a useful diagnostic marker for EC. Subgroup analysis indicated that studies with benign disease controls showed higher diagnostic accuracies than those with healthy controls. CONCLUSION: Serum HE4 may serve as a potential biomarker for EC diagnosis. Due to certain limitations, this conclusion should to be cautiously interpreted.

Adrenomedullin protects Leydig cells against lipopolysaccharide-induced oxidative stress and inflammatory reaction via MAPK/NF-κB signalling pathways.

This study aimed to explore the possible benefits of adrenomedullin (ADM) in preventing oxidative stress and inflammation by using an in vitro primary culture model of rat Leydig cells exposed to lipopolysaccharide (LPS). Cell proliferation was detected through CCK-8 and BrdU incorporation assays. ROS were determined with a DCFDA kit, and cytokine concentrations were measured with ELISA assay kits. Protein production was examined by immunohistochemical staining and Western blot, and gene expression was observed through RT-qPCR. Results revealed that ADM significantly reduced LPS-induced cytotoxicity, and pretreatment with ADM significantly suppressed ROS overproduction and decreased 4-HNE and 8-OHdG expression levels and concentrations. ADM pretreatment also significantly attenuated the overactivation of enzymatic antioxidants, namely, superoxide dismutase, catalase, thioredoxin reductase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase. ADM supplementation reversed the significantly increased gene expression levels and concentrations of TNF-α, IL-1ß, TGF-ß1, MCP-1 and MIF. ADM pretreatment significantly inhibited the gene expression and protein production of TLR-2 and 4. Furthermore, ADM pretreatment markedly reduced the phosphorylation of JNK, ERK 1/2 and p38, phosphorylation and degradation of IκBα and nuclear translocation of p65. Our findings demonstrated that ADM protects Leydig cells from LPS-induced oxidative stress and inflammation, which might be associated with MAPK/NF-κB signalling pathways.

Interference of fisetin with targets of the nuclear factor-κB signal transduction pathway activated by Epstein-Barr virus encoded latent membrane protein 1.

Fisetin is an effective compound extracted from lacquer which has been used in the treatment of various diseases. Preliminary data indicate that it also exerts specific anti-cancer effects. However, the manner in which fisetin regulates cancer growth remains unknown. In this study, we elucidated interference of fisetin with targets of the nuclear factorκB signal transduction pathway activated by Epstein-Barr virus encoding latent membrane protein 1 (LMP1)in nasopharyngeal carcinoma (NPC) cells, Results showed that fisetin inhibited the survival rate of CNE-LMP1 cells and NF-κB activation caused by LMP1. Fisetin also suppressed nuclear translocation of NF-κB (p65) and IκBα phosphorylation, while inhibiting CyclinD1, all key targets of the NF-κB signal transduction pathway. It was suggested that interference effects of fisetin with signal transduction activated by LMP1 encoded by the Epstein-Barr virus may play an important role in its anticancer potential.

[Effects of radix astragali on expression of transforming growth factor ß 1 and Smad 3 signal pathway in hypertrophic scar of rabbit].

OBJECTIVE: To observe the effects of radix astragali on expression of TGF-ß1 and Smad 3 signal pathway in hypertrophic scar of rabbits, and to analyze its therapeutic effect and mechanism on hypertrophic scar. METHODS: Twenty healthy adult Japanese big ear rabbits were inflicted with 4 full-thickness skin defects on ventral side of each ear, which formed scar later. Rabbits were divided into 5 groups: 1.00, 0.50, 0.25 g/mL radix astragali treatment groups [injected with radix astragali on post injury day (PID) 21, 25, 32, and 36 respectively], physiological saline group (PS, injected with 0.2 mL physiological saline in the same volume at the same time points as above groups), and blank control group (BC, without treatment) according to the random number table, with 32 scars in each group. Another 4 rabbits were enrolled as normal control group (NC). Structural changes of hypertrophic scar was observed with HE and Masson staining. Thickness and hardness of hypertrophic scar on PID 32 and 43 were respectively examined by chromoscope ultrasonic diagnostic equipment and hardness tester. Protein and mRNA expression of TGF-ß1 and Smad 3 in hypertrophic scar was respectively detected with RT-PCR and immunohistochemical analysis. Data were processed with t test and one-way analysis of variance. RESULTS: Compared with that in PS and BC groups, dermis of hypertrophic scar became thinner in radix astragali treatment groups on PID 32, 43, with fibroblasts and collagenous fibers arranged regularly on PID 43. Thickness and hardness of hypertrophic scar, levels of mRNA and protein of TGF-ß1 and Smad 3 decreased along with the increase in radix astragali concentration. Compared with those in PS group, levels of mRNA of TGF-ß1 and Smad 3 in 1.00 g/mL radix astragali treatment group on PID 32 decreased 26.1% and 28.2%. Protein levels of TGF-ß1 and Smad 3 in 1.00 g/mL radix astragali treatment group were 3.15 ± 0.80 and 4.72 ± 1.06, which were obviously lower than those in PS group (6.06 ± 0.85, 8.04 ± 0.63, with F value respectively 27.230 and 33.525, P < 0.05 or P < 0.01). There was significant statistical difference in all measurement indices except for mRNA of TGF-ß1 and Smad 3 among radix astragali treatment groups on PID 32 and 43 [with t values respectively 3.593-4.814 (thickness), 4.051-5.811 (hardness), 2.976-5.986 (TGF-ß1 protein), and 2.742-4.630 (Smad 3 protein), P < 0.05 or P < 0.01]. CONCLUSIONS: Radix astragali injection inhibits fibroblast proliferation in hypertrophic scars through down-regulating mRNA expression and protein synthesis of TGF-ß1 and Smad 3, thus inhibits hypertrophic scars formation. Its inhibition effect is drug concentration and duration dependent. The drug may be considered as a potential agent to prevent hypertrophic scar.

Effect of a combination of carnosine and aspirin eye drops on streptozotocin -- induced diabetic cataract in rats.

PURPOSE: To investigate the effect of a combination of carnosine and aspirin eye drops on the progression of diabetic cataract formation induced by streptozotocin (STZ). METHODS: Rats were made diabetic with STZ. Animals in the treated groups received carnosine, aspirin, or a combination of carnosine and aspirin as drops to the eyes. Cataract progression was monitored by slit lamp microscope and classified into four stages. At the end of 8 weeks, the animals were killed and biochemical changes were determined. Blood and urine glucose levels, body weights, food, and intake were also determined. RESULTS: About 84.4% of the rats responded to the STZ injection. There were statistically significant differences in the stage of cataract of lenses between the untreated and the treated diabetic animals and between the combination and the aspirin group at the 7th and 8th week. There was a significant decrease in the water-soluble protein in the diabetic groups compared with the control group. The three treatments improved the water-soluble protein levels, and the combination treatment had the greatest effect. The levels of thiol were remarkably decreased in the lenses of diabetic rats, except the combination group. The specific activity of glutathione peroxidase (GPx) was increased and the activities of glutathione reductase (GR) and catalase (CAT) were decreased in all the diabetic groups. CONCLUSIONS: The results indicated that carnosine, aspirin, and a combination eye drops are effective against the onset and development of diabetic cataract in rats. Most important, the effect of combination eye drops is better than aspirin only.

Hyperoxia-induced lens damage in rabbit: protective effects of N-acetylcysteine.

PURPOSE: To investigate the efficacies of different concentrations of N-acetylcysteine (NAC) in preventing hyperoxia-induced lens opacification and changes to biochemical parameters in organ-cultured rabbit lenses. METHODS: Thirty-six lenses from adult rabbits were divided into the control group (group A), the hyperoxia-exposed group (group B), and the hyperoxia-exposed, NAC-treated groups: 5 mM NAC (group C), 10 mM NAC (group D), 20 mM NAC (group E), and 40 mM NAC (group F). Groups B-F were incubated with hyperoxia (pO(2)>80%) for 4 h per day for 7 d. Lens transparency, histology, and enzymatic activities were measured after incubation. RESULTS: Gross examination of these lenses revealed some severe cortical opacification in group B, and moderate cortical opacification in the lenses of groups C and D. There was minimal cortical opacification in groups A, E, and F. The activities of Na, K-ATPase, and catalase were significantly (p<0.05) lower in group B (38.2%) than in group A (39.9%). It was also lower in group E and F lenses (p<0.05), which had higher levels of NAC-protected enzymes. The glutathione and water-soluble protein content were significantly lower in group B lenses than in group A, E, or F lenses (p<0.05). However, there was no difference between group E and F lenses (p>0.05). CONCLUSIONS: The present data suggests that NAC (20 mM-40 mM) significantly prevented experimental lenses' hyperoxia-induced cortical opacification, indicating NAC's potential role in protecting lenses against cataracts induced by high oxygen levels.