Longitudinal characterization of serum metabolome and lipidome reveals that the ceramide profile is associated with metabolic health in early postpartum cows experiencing different lipolysis.

Reduced feed intake in early lactation prompts increased fat mobilization to meet dairy cow energy needs for milk production. The increased lipolysis in cows presents significant health risks with unclear mechanisms. The objectives of our study were to compare the longitudinal profiles of metabolites and lipids of serum from high- and low-lipolysis cows. Forty multiparous Holstein dairy cows were enrolled in the retrospective study. Serum samples were collected on d 7 before expected calving, as well as on d 5, 7, 14, and 21 postpartum. Dairy cows were grouped according to mean serum nonesterified fatty acids on d 5 and 7 after parturition as low (<0.600 mmol/L; n = 8; LFM) and high (>0.750 mmol/L; n = 8; HFM), indicating fat mobilization during early lactation. Lactational performance and serum metabolic parameters related to glucose and lipid metabolism, liver functions, oxidative status, and inflammatory responses were determined. Serum samples were subjected to liquid chromatography-MS-based metabolomics and lipidomics. Despite differences in postpartum BW change, there were no observed variations in milk yield and composition between the 2 groups. Serum ß-hydroxybutyric acid, glucose, leptin, aspartate aminotransferase, IL-6, and tumor necrosis factor alpha were greater in cows with HFM than in cows with LFM. Serum adiponectin, revised quantitative insulin sensitivity check index, and albumin were lower in cows with HFM than in cows with LFM. Intensified fat mobilization in the HFM cows came along with reduced estimated insulin sensitivity, impaired liver functions, and increased oxidative stress and inflammatory responses. Differences in metabolic patterns were observed across the transition period when comparing serum blood matrixes (e.g., in different amino acids, acylcarnitines, and sphingolipids). The serum metabolome of the HFM cows was characterized by higher concentrations of glycine, acylcarnitines, carnosine, Cer(d20:0/18:0), Cer(d18:1/16:0), and Cer(t18:0/24:0) compared with LFM cows. The differential serum metabolites and lipids at different sampling times during the peripartum period were enriched in the sphingolipid metabolism. Differences in serum metabolic status parameters suggest that cows adopt varied metabolic adaptation strategies to cope with energy deficits postpartum. Our investigation found a comprehensive remodeling of the serum metabolic profiles in transition dairy cattle, highlighting the significance of alterations in sphingolipid species, as they play a crucial role in insulin resistance and metabolic disorders.

[Efficacy of video-assisted thoracoscopic surgical decortication for stage â ¢ tuberculous empyema].

Objective: To investigate the clinical efficacy, safety and feasibility of "double-portal" video-assisted thoracoscopic surgical(VATS) decortication among patients with stage Ⅲ tuberculous empyema, and then to evaluate the recovery of chest deformity. Method: This study was a single center retrospective study. A total of 49 patients with stage Ⅲ tuberculous empyema who underwent VATS pleural decortication at the Department of Thoracic Surgery, Public Health Clinical Center of Chengdu between June 2017 and April 2021 were enrolled, including 38 males, and 11 females, aged 13-60 (27.5±10.4) years. The safety and feasibility of VATS were further evaluated. The inner circumference of the chest on sternal and xiphoid planes on chest CT scans before and 1, 3, 6, 12months after decortication were collected through the measuring software of the CT. The samples in-pair test was used to compare the changes in the chest to reflect the recovery of the chest deformity. Results: In the 49 patients, The surgical time was (186±61) min, and the volume of blood loss was (366±267) ml. There were 8 cases (16.33%) with postoperative complications during the perioperative period. Constant air leak and pneumonia were the main postoperative complications. No relapse of empyema or dissemination of tuberculosis occured during the period of follow-up. Before surgery, the inner thoracic circumference of the thorax at the level of the carina plane was (655±54) mm, and the inner thoracic circumference of the thorax at the level of the xiphoid plane was (720±69) mm. Patients were followed for 12-36 months. The inner thoracic circumference of the thoracic cavity at the level of carina was (666±51), (667±47) and (671±47) mm at the 3rd, 6th and 12th months after operation, which were significantly larger than that at the level of carina before operation (all P<0.05). The inner thoracic circumference diameter of the thoracic cavity measured at the xiphoid level at the 3rd, 6th and 12th months after the operation was (730±65), (733±63) and (735±63) mm respectively(all P<0.05).The inner thoracic circumference of the thoracic cavity increased significantly than that before surgery (P<0.05). At 6 months after operation, there was significant difference in the improvement of the inner thoracic circumference of the carina plane in patients with age less than 20 years and FEV1% less than 80% (P=0.015, P=0.003). The improvement in the inner thoracic circumference of the carina plane in patients with pleural thickening≥8 mm compared with those with less than 8 mm was not statistically different(P=0.070). Conclusions: For some patients with stage Ⅲ tuberculous empyema, pleural decortication under thoracoscopy is safe and feasible, and can significantly restore the inner thoracic circumference of the patient's chest, improve the collapse of the patient's chest, and have significant clinical effect. The "double-portal VATS" surgical technology has the advantage of less trauma, wide operation field, large operation space and is easy to master, which is worth further exploring for clinical application.

Development of ruminating behavior in Holstein calves between birth and 30 days of age.

Ruminating behavior accompanies the development of the rumen and the intake of solid feed in calves. However, few studies have reported on the emergence and development of rumination. In this study, we observed ruminating behavior changes of 56 Holstein calves (body weight at birth = 40.1 ± 3.96 kg; mean ± standard deviation) from birth to 30 d of age under the feeding management of suckling calves that were only fed pelleted concentrate feed and milk. All calves were housed in individual pens equipped with infrared cameras. We explored feed intake within 30 d of age, body weight on 61 d of age, and other apparent indicators, including the age of first eating the bedding, duration of non-nutritive oral behavior at 25 and 30 d of age, total starter feed intake within 30 d of age, average daily starter feed intake within 30 d of age, and duration of ruminating behavior at 25 and 30 d of age for all calves, to further explore the effects of the age of first ruminating behavior (AFR). The AFR fitted the normal distribution and ranged from 15 to 20 d of age for 50% of the experimental population. The AFR was positively correlated with the age of first eating the bedding and duration of non-nutritive oral behavior at 30 d of age. Total starter feed intake within 30 d of age, average daily starter feed intake within 30 d of age, duration of ruminating behavior at 25 and 30 d of age, and duration of eating the bedding at 25 and 30 d of age were negatively correlated with AFR. Overall, to the best of our knowledge, this is the first study that has analyzed the correlation between AFR and other indicators. We found that earlier AFR was associated with shorter duration of non-nutritive oral behavior, longer durations of rumination and eating the bedding, and higher feed intake by 30 d of age.

Effects of propylene glycol on in vitro ruminal fermentation, methanogenesis, and microbial community structure.

We evaluated the effects of propylene glycol (PG) on in vitro ruminal fermentation, methanogenesis, and microbial community structure. A completely randomized design was conducted in the in vitro incubation, and 4 culture PG dose levels (0, 7.5, 15, and 22.5 µL/g of dry matter) were used in the trial. Based on the fermentation results, the control group (0 µL/g of dry matter, CON) and the second treatment group (15.0 µL/g of dry matter, TRT) were chosen for further analysis to explore the effects of PG on the bacterial and archaeal community structure. The concentrations of propanol, propanal, and succinate increased linearly, whereas the concentration of l-lactate decreased linearly as PG doses increased. The molar proportion of propionate demonstrated a linear increase with increasing PG doses. In contrast with propionate, the molar proportion of acetate and butyrate, and acetate-to-propionate ratio decreased linearly with increasing PG doses. The addition of PG markedly decreased methane production without negative effects on nutrient degradability. In the archaeal level, the relative abundance of Methanobrevibacter tended to decrease, but that of Methanomassiliicoccus significantly increased in TRT group. At the bacterial level, the relative abundance of Bacteroidetes and Prevotella in TRT group was numerically higher than that in CON group. The analysis of the Negativicutes class showed that the relative abundance of Succiniclasticum tended to increase, whereas that of Selenomonas tended to decrease in TRT group. These results demonstrated that PG might be used as an inhibitor to mitigate methane emission. However, the small decrease in methane production will limit the application of PG as a methane inhibitor in production practices. Further research is needed to determine whether use together with other inhibitors may improve the effects of PG on the utilization of reducing equivalents ([H]) and methane production.

[HBx promotes ubiquitination and degradation of ZO1 and increases the migration and invasion of liver cancer cells].

Objective: To study the effect of hepatitis B virus X protein (HBx) expression level on migration and invasion of zonula occludens protein-1 (ZO-1) in HepG2 liver cancer cells. Methods: Liver cancer cells were transfected with HBV full gene plasmid (pcDNA3.1-HBV1. 1 or pcDNA3.1-HBV1.3), empty plasmid (pcDNA3.1) and HBV-encoded protein plasmids (pHBc, pHBs, pHBp and pHBx), respectively. Western blot and RT-PCR were used to detect ZO1 protein and mRNA levels. Immunoprecipitation was used to detect transfected pHBx. Western blot was used to detect ZO1 ubiquitination levels. Transwell chambers were used to assess cell migration and invasion. Cell proliferation and lactate dehydrogenase assay was used to detect siRNA transfecting targeting ZO1. Flow cytometry was used to detect cell apoptosis and cycle. The data was compared between two and multiple groups by using an independent sample t-test and one-way analysis of variance. Results: Compared with the empty plasmid, ZO1 protein level in HepG2 cells after transiently transfected with pHBV1.1 and pHBV1.3 was decreased by 42.99% ± 6.8% and 55.0% 5 ± 4.56%, respectively, and their mRNA levels did not change significantly. ZO1 protein level in Huh7 cells was decreased by 17.46% ± 4.94% and 47.53% ± 3.38%, respectively. ZO1 protein level after transfection with pHBx was decreased by 47.02% ± 3.4%, while the ZO1 protein level after transfection with pHBc, pHBs and pHBp did not change significantly. ZO1 mRNA level was unaffected with pHBx transfection. ZO1 ubiquitin level and cell migration and invasion ability in HepG2 cells was significantly increased with transfected pHBx. HepG2 cells proliferation, apoptosis and cycle after transfection with ZO1-targeted siRNA did not change significantly, but the migration and invasion ability were significantly increased. Conclusion: HBx can increase the migration and invasion of liver cancer cells by promoting the ubiquitination and degradation of ZO1 protein level.

Amino acid profiles of rumen undegradable protein: a comparison between forages including cereal straws and alfalfa and their respective total mixed rations.

Optimizing the amino acid (AA) profile of rumen undegradable protein (RUP) can positively affect the amount of milk protein. This study was conducted to improve knowledge regarding the AA profile of rumen undegradable protein from corn stover, rice straw and alfalfa hay as well as the total mixed ratio diets (TMR) based on one of them as forage source [forage-to-concentrate ratio of 45:55 (30% of corn stover (CS), 30% of rice straw (RS), 23% of alfalfa hay (AH) and dry matter basis)]. The other ingredients in the three TMR diets were similar. The RUP of all the forages and diets was estimated by incubation for 16 hr in the rumen of three ruminally cannulated lactating cows. All residues were corrected for microbial colonization, which was necessary in determining the AA composition of RUP from feed samples using in situ method. Compared with their original AA composition, the AA pattern of forages and forage-based diets changed drastically after rumen exposure. In addition, the extent of ruminal degradation of analysed AA was not constant among the forages. The greatest individual AA degradability of alfalfa hay and corn stover was Pro, but was His of rice straw. A remarkable difference was observed between microbial attachment corrected and uncorrected AA profiles of RUP, except for alfalfa hay and His in the three forages and TMR diets. The ruminal AA degradability of cereal straws was altered compared with alfalfa hay but not for the TMR diets. In summary, the AA composition of forages and TMR-based diets changed significantly after ruminal exposure, indicating that the original AA profiles of the feed cannot represent its AA composition of RUP. The AA profile of RUP and ruminal AA degradability for corn stover and rice straw contributed to missing information in the field.

Effects of dietary threonine and immune stress on growth performance, carcass trait, serum immune parameters, and intestinal muc2 and NF-κb gene expression in Pekin ducks from hatch to 21 days.

An experiment was conducted to investigate the effects of different dietary threonine (Thr) levels and immune stress on Pekin ducklings' growth performance, carcass traits, serum immune parameters, and intestinal mucin 2 (MUC2) and nuclear factor kB (NF-κB) gene expressions. A total of 320 Pekin ducklings was randomly assigned to a 5 × 2 factorial arrangement of treatments. Each treatment group consisted of 4 replicate pens with 8 ducks per pen. Ducklings were fed 5 graded levels of Thr: 0.49, 0.56, 0.60, 0.65, and 0.76% from hatch to 21 d of age. At 11 d of age, ducks in the stressed groups were challenged with bovine serum albumin (BSA), and ducks in the unstressed groups were injected with normal saline water. The results showed that increasing Thr supplementation from 0.49 to 0.56% in the diet can improve BWG; feed consumption; weight and relative weight of breast and leg; weight of liver, bursa of Fabricius, spleen, and thymus; serum natural immune globulin A (IgA) concentration; and MUC2 gene expression in the ileum of 21-day-old Pekin ducks, significantly (P < 0.05). Immune stress with BSA had a significant effect on 21-day-old Pekin ducklings' BWG, feed consumption, and weight and relative weight of breast and thymus (P < 0.05), but no interaction between BSA and dietary Thr content was noticed in our experiment in 21-day-old Pekin ducks (P < 0.05). Dietary Thr requirements of the unstressed groups and stressed groups based on broken-line model analyses for ducks' BWG were 0.705 and 0.603%, respectively, and for ducks' feed consumption were 0.724 and 0.705%, respectively.

Effect of cereal straw and alfalfa hay diet on amino acid profile of gastrointestinal digesta in lactating dairy cows.

This study was conducted to evaluate the effects of replacing alfalfa hay with rice straw (RS) or corn stover (CS) on amino acid (AA) profiles of gastrointestinal digesta in lactating cows. Eighteen lactating dairy cows were randomly assigned to one of three groups (n = 6) and fed identical concentrate and corn silage with different forages on dry matter basis: (i) 23% alfalfa hay and 7% Chinese wild rye hay (AH); (ii) 30% CS; and (iii) 30% RS. After the 14-week feeding, a total of 18 cows were slaughtered to collect digesta from four representative organs, including rumen, omasum, duodenum and jejunum. The AA profiles of ruminal microbial fraction were similar among the treatments, except for greater Arg in cows fed RS than in cows fed AH or CS. Most of the analysed AA changed under different diets. Significant differences were found among the microbial fraction, rumen fluid and rumen digesta, with greater essential AA in digesta than in microbial fraction or rumen fluid and greater essential AA in microbial fraction than rumen fluid. Significant differences in individual AA profiles of digesta and relevant fluid were found across the four representative digestive tract parts, including rumen, omasum, duodenum and jejunum, showing much lower Leu proportion in CS and RS than in AH in duodenal fluid. In summary, ruminal microbes may prefer using essential AA, rather than non-essential AA. The AA profile of ruminal microbes was constant except for Arg. The AA composition of digesta across the four digestive tracts changed dramatically, which indicated differences in the ability and efficiency of AA absorption. The lower duodenum absorbable Leu proportion in cows fed CS or RS indicated the shortage of Leu in CS or RS diets, which might also restrict the balanced AA absorption.

Long non-coding RNA Loc344887 is a potential prognostic biomarker in non-small cell lung cancer.

OBJECTIVE: Long non-coding RNA NmrA-like family domain containing 1 pseudogene (Loc344887) has been shown to be aberrantly expressed in non-small cell lung cancer (NSCLC). However, the biological function of Loc344887 in NSCLC remains unknown. The present study aimed to explore the prognosis value of Loc344887 in NSCLC patients. PATIENTS AND METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression level of Loc344887 in NSCLC tissues and adjacent non-tumor lung tissues. Correlations between Loc344887 expression and the clinicopathological features of NSCLC patients were then evaluated. Overall survival was examined using Kaplan-Meier curves. Cox regression analyses were performed to judge whether plasma Loc344887 was an independent predictor of survival. RESULTS: The expression levels of Loc344887 were increased in NSCLC tissues compared with those in normal lung tissues (p < 0.05). High Loc344887 expression level was significantly associated with lymph node metastasis (p = 0.005), advance stage (p = 0.001), and poorer differentiation (p = 0.014). Furthermore, patients with high expression of Loc344887 had a significantly shorter overall survival time compared with those with low Loc344887 expression in NSCLC (p < 0.001). Further analysis by Cox regression showed that high expression of Loc344887 in NSCLC was an independent predictor of poor prognosis. CONCLUSIONS: These findings, for the first time, suggested the potential roles of Loc344887as a prognostic factor and as a therapeutic target for NSCLC.

Effect of tea saponins on milk performance, milk fatty acids, and immune function in dairy cow.

This study investigated the effects of tea saponins (TSP) on milk performance, milk fatty acids, and blood immune function in dairy cows. A total of 20 early-lactation Holstein cows (days in milk = 66.4 ± 16.8 d; parity = 1.75 ± 0.91; and milk yield = 36.3 ± 7.32 kg/d; mean ± standard deviation) were randomly divided into 4 homogeneous treatment groups, with TSP added at 0, 20, 30, and 40 g/d per head, respectively. All cows had 2 wk of adaptation and 6 wk of treatments. Feed, milk, and blood were sampled and analyzed weekly. At the end of the experimental period (wk 6), the dry matter intake and yields of energy-corrected milk, milk, and milk protein, fat, and lactose in the cows fed TSP showed a quadratic response, with the lowest values in cows fed TSP at 40 g/d. The milk fat content of cows fed TSP increased linearly. Significant interactions for treatment by week were found in milk C16:1 cis-9 and C18:1 cis-9, with the highest values at wk 2, 3, and 4 in the cows fed TSP at 40 g/d. The levels declined quickly after 4 wk of feeding to values similar to those for other TSP treatments and the control at wk 5 and 6. Plasma malondialdehyde concentration decreased as the supplement level of TSP increased. The concentration of superoxide dismutase increased as the supplement level of TSP increased. The plasma concentration of tumor necrosis factor-α increased as the supplement level of TSP increased. In summary, this study showed that an intermediate dose of TSP (20 and 30 g/d) had no significant effect on feed intake, but the supplementation of 40 g/d TSP decreased feed intake, resulting in a lower milk yield. The energy-corrected milk of cows fed 40 g/d TSP declined at first but increased after 3 wk of feeding, indicating the potential adaptation to high doses of TSP supplements in dairy cows. The supplementation of TSP could reduce oxidative stress in cows and improve the immunity of dairy cows during 6 wk of feeding.

Thiamine supplementation facilitates thiamine transporter expression in the rumen epithelium and attenuates high-grain-induced inflammation in low-yielding dairy cows.

An experiment was conducted to uncover the effects of increasing dietary grain levels on expression of thiamine transporters in ruminal epithelium, and to assess the protective effects of thiamine against high-grain-induced inflammation in dairy cows. Six rumen-fistulated, lactating Holstein dairy cows (627 ± 16.9 kg of body weight, 180 ± 6 d in milk; mean ± standard deviation) were randomly assigned to a replicated 3 × 3 Latin square design trial. Three treatments were control (20% dietary starch, dry matter basis), high-grain diet (HG, 33.2% dietary starch, DM basis), and HG diet supplemented with 180 mg of thiamine/kg of dry matter intake. On d 19 and 20 of each period, milk performance was measured. On d 21, ruminal pH, endotoxic lipopolysaccharide (LPS), and thiamine contents in rumen and blood, and plasma inflammatory cytokines were detected; a rumen papillae biopsy was taken on d 21 to determine the gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. The HG diet decreased ruminal pH (5.93 vs. 6.49), increased milk yield from 17.9 to 20.2 kg/d, and lowered milk fat and protein from 4.28 to 3.83%, and from 3.38 to 3.11%, respectively. The HG feeding reduced thiamine content in rumen (2.89 vs. 8.97 µg/L) and blood (11.66 vs. 17.63 µg/L), and the relative expression value of thiamine transporter-2 (0.37-fold) and mitochondrial thiamine pyrophosphate transporter (0.33-fold) was downregulated by HG feeding. The HG-fed cows exhibited higher endotoxin LPS in rumen fluid (134,380 vs. 11,815 endotoxin units/mL), and higher plasma concentrations of lipopolysaccharide binding protein and pro-inflammatory cytokines when compared with the control group. The gene and protein expression of tumor necrosis factor α (TNFα), IL1B, and IL6 in rumen epithelium increased when cows were fed the HG diet, indicating that local inflammation occurred. The depressions in ruminal pH, milk fat, and protein of HG-fed cows were reversed by thiamine supplementation. Thiamine supplementation increased thiamine contents in rumen and blood, and also upregulated the relative expression of thiamine transporters compared with the HG group. Thiamine supplementation decreased ruminal LPS (49,361 vs. 134,380 endotoxin units/mL) and attenuated the HG-induced inflammation response as indicated by a reduction in plasma IL6, and decreasing gene and protein expression of pro-inflammatory cytokines in rumen epithelium. Western bottling analysis showed that thiamine suppressed the protein expression of TLR4 and the phosphorylation of nuclear factor kappa B (NFκB) unit p65. In conclusion, HG feeding inhibits thiamine transporter expression in ruminal epithelium. Thiamine could attenuate the epithelial inflammation during high-grain feeding, and the protective effects may be due to its ability to suppress TLR4-mediated NFκB signaling pathways.

Relationship between thiamine and subacute ruminal acidosis induced by a high-grain diet in dairy cows.

Two experiments were conducted to reveal the effects of grain-induced subacute rumen acidosis (SARA) on thiamine status in blood and rumen fluid in dairy cows. In both experiments, 6 multiparous, rumen-fistulated Holstein dairy cows were used in a 2-treatment, 2-period crossover design. Each experimental period consisted of 21d (total of 42d). Experiment 1 was to investigate the effects of SARA on thiamine status in blood and rumen fluid. Treatments were either control (20% starch, dry matter basis) or SARA-inducing diet (SAID, 33.2% starch, dry matter basis). In experiment 2, the effects of dietary thiamine supplementation on attenuating SARA and ruminal fermentation characteristics in dairy cows were studied. All cows received the same SAID diet during the whole experimental period; treatments were with or without thiamine (180mg of thiamine/kg of dry matter intake). In both experiments, rumen fluid samples were collected at 0, 3, 6, 9, and 12h after morning feeding on d 21 and 42 of the experiments for measurement of pH, thiamine, volatile fatty acid, and lactate contents. Peripheral blood was also collected at 3h after morning feeding on d 21 and 42 to measure thiamine, carbohydrate metabolites, and enzyme activities. In experiment 1, cows fed the SAID diet had lower ruminal and plasma thiamine concentrations and higher lactate than cows fed the control diet. The ruminal thiamine contents were positively related to pH and the concentrations of acetate in the rumen, and negatively correlated with the lactate contents. Experiment 2 demonstrated that ruminal pH and the concentrations of thiamine, acetate, and total volatile fatty acids in the rumen were increased, whereas ruminal lactate contents were reduced by thiamine supplementation. The concentrations of lactate and the activity of lactate dehydrogenase in blood were reduced in the thiamine supplemented group, and the opposite was true for the nonesterified fatty acids and α-ketoneglutarate dehydrogenase contents. In conclusion, the thiamine status was affected by SARA in dairy cows and ruminal infusion of thiamine could help attenuate SARA by improving theproportions of ruminal volatile fatty acids and reducing lactate contents in rumen fluid and blood.

[Hydrogen peroxide induces oxidative stress and the mitochondrial pathway of apoptosis in RAT intestinal epithelial cells (IEC-6)].

UNLABELLED: In order to investigate the mechanism of apoptosis in rat intestinal epithelial cells (IEC-6) induced by hydrogen peroxide (H(2)O(2)), IEC-6 cells were subjected to 20 µmol/L H(2)O(2) and cell proliferation activity was determined using 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide. Cell morphology was observed by microscopy and cell apoptosis was detected by acridine orange and ethidium bromide staining and the portion of apoptotic cells was measured by flow cytometry. Genes and proteins related to cell apoptosis were detected by RT-PCR and Western blotting, and the mitochondrial membrane potential was evaluated by fluorescence probes. RESULTS: Significant morphology damage was caused by exposure to H(2)O(2), and results showed that ROS generation significantly increased (P < 0.01). The activity of superoxide dismutase decreased significantly (P < 0.05), malondialdehyde content increased (P < 0.05), and expression of both catalase and glutathione peroxidase decreased significantly (P < 0.05) in the H(2)O(2) treatment group. Mitochondrion membrane potential was reduced, cytochrome released into the cytoplasm and caspase-9 and caspase-3 were significantly increased (P < 0.01) after treatment with H(2)O(2). Moreover, the ratio of Bax/Bcl-2 and apoptosis were significantly increased (P < 0.01) in the H(2)O(2) group. In conclusion, the present study indicated that the mitochondrial pathway plays a vital role in H(2)O(2) induced IEC-6 cell apoptosis.

Spinal ephrinB/EphB signalling contributed to remifentanil-induced hyperalgesia via NMDA receptor.

BACKGROUND: One of the major unresolved issues in treating pain is the paradoxical hyperalgesia produced by opiates, and accumulating evidence implicate that EphBs receptors and ephrinBs ligands are involved in mediation of spinal nociceptive information and central sensitization, but the manner in which ephrinB/EphB signalling acts on spinal nociceptive information networks to produce hyperalgesia remains enigmatic. The objective of this research was to investigate the role of ephrinB/EphB signalling in remifentanil-induced hyperalgesia (RIH) and its downstream effector. METHODS: We characterized the remifentanil-induced pain behaviours by evaluating thermal hyperalgesia and mechanical allodynia in a rat hind paw incisional model. Protein expression of EphB1 receptor and ephrinB1 ligand in spinal dorsal horn cord was determined by Western blotting, and Fos was determined by immunohistochemistry assay, respectively. To figure out the manner in which ephrinB/EphB signalling acts with N-methyl-d-aspartic acid (NMDA) receptor, we used MK-801, an antagonist of NMDA receptor, trying to suppressed the hyperalgesia induced by ephrinB1-Fc, an agonist of ephrinB/EphB. RESULTS: Continuing infusion of remifentanil produced a thermal hyperalgesia and mechanical allodynia, which was accompanied with increased protein expression of spinal-level EphB1 receptor, ephrinB1 ligand and Fos; what appeared above was suppressed by pretreatment with EphB1-Fc, an antagonist of ephrinB/EphB or MK-801, and increased pain behaviours induced by intrathecal injection of ephrinB1-Fc, an agonist of ephrinB/EphB, were suppressed by MK-801. CONCLUSIONS: Our findings indicated that ephrinB/EphB signalling is involved in RIH. EphrinB/EphB signalling might be the upstream of NMDA receptor.

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Reduced expression of insulin-like growth factor 1 receptor leads to accelerated intervertebral disc degeneration in mice.

Insulin-like growth factor 1 (IGF-1) and its receptor (insulin-like growth factor 1 receptor, IGF1R) can regulate the extracellular matrix synthesis and play a crucial role in maintaining the normal functions of the intervertebral disc (IVD). The objective of this study was to investigate whether there would be accelerated IVD degeneration (IVDD) in IGF1R+/- mice. Three IGF1R+/- male mice and three wild-type male mice were sacrificed respectively at 6, 12, and 18 weeks after birth. Six lumbar disc samples were harvested from each mouse, with a total of 54 disc samples taken from each genotype. Histomorphological analysis for the IVD was performed to assess the degenerative extent according to the classification system proposed by Boos et al. Quantitative real-time PCR and semi-quantitative histologic scoring (HScore) for immunohistochemical staining were used to evaluate the expression level of type-II collagen, aggrecan and matrix metallopeptidase 13 (MMP-13). Histomorphological analysis for the discs revealed significantly less amounts of proteoglycan and type-II collagen, and significantly higher total degenerative score in IGF1R+/- mice than in wild-type mice. Real-time PCR showed that the mRNA expressions of type-II collagen and aggrecan in the discs were significantly lower, while MMP-13 was significantly higher in IGF1R+/- mice than in wild-type mice. The results of HScore analysis were similar to those obtained from the quantitative real-time PCR. Taken together, our study indicates that reduced expression of IGF1R would lead to accelerated degeneration of IVD. IGF1R+/- mice could be regarded as a good animal model to study IVD degeneration (IVDD), and studies on the IVD of IGF1R+/- mice could provide further insight into the pathogenesis of IVDD.

Differential responsiveness to 17ß-estradiol of mesenchymal stem cells from postmenopausal women between osteoporosis and osteoarthritis.

UNLABELLED: Differential osteogenic potential and responsiveness to 17ß-estradiol (E2) of mesenchymal stem cells (MSCs) were found between postmenopausal women with osteoporosis (OP) and osteoarthritis (OA). These results suggest differential biological mechanisms of estrogen deficiency in regulation of bone remodeling between OP and OA. INTRODUCTION: OP and OA are two common disorders in postmenopausal women. The inverse relationship has been suggested between OP and OA, but their mechanisms that relate to estrogen deficiency are not fully understood. The aim of this study was to compare the differential responsiveness to E2 of MSCs from osteoporotic versus osteoarthritic donors. METHODS: Twenty postmenopausal patients, ten with osteoporotic hip fractures and ten with hip osteoarthritis, were included into this study. MSCs were derived from cancellous bones of femoral heads from OA and OP donors and cultured in osteogenic and adipogenic medium with or without E2 added. The alkaline phosphatase (ALP) activity, calcium content, calcified nodules, lipid droplets, messenger RNA (mRNA) expression of ALP, osteocalcin (OC), collagen 1α (COL1α), peroxisome proliferators-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) were measured and compared between two groups with OP and OA. RESULTS: In osteogenic medium, ALP activity, calcium content and mRNA expression of OC and COL1α in MSCs from OA were significantly higher than those from OP group. In adipogenic condition, there was no significant difference in lipid droplets formation and mRNA expression of PPARγ2 and LPL between OP and OA groups. With E2 added in osteogenic medium, ALP activity, calcium content and OC mRNA were significantly higher in OP group than in OA group, whereas E2 had no significant effect on lipid droplet formation and mRNA expression of PPARγ2 and LPL. CONCLUSION: Differential osteogenic potential and responsiveness to E2 of MSCs were found between postmenopausal women with OP and OA. These results may provide information for clinical application of MSCs in the differential setting of estrogen deficiency.

Bone loss and impaired fracture healing in spinal cord injured mice.

UNLABELLED: Spinal cord injury (SCI) results in impaired fracture healing in mice while leading to significant bone loss. Poor fracture healing following SCI is consistent with significant bone loss. INTRODUCTION: SCI leads to significant bone loss in sublesional limbs, but there is few data concerning the relationship between fracture healing and bone loss following SCI. This study was undertaken to investigate the effect of SCI on fracture healing using a mouse femur fracture model. METHODS: One hundred twenty male C57BL/6J mice were randomly divided into SCI and control groups (n=60, respectively). A femoral shaft fracture was generated and fixed with intramedullary pins 3 weeks after SCI. Fracture healing was evaluated by micro-computed tomography (micro-CT) for callus formation and mineralization and neovascularization, and bone mineral density (BMD) was measured by DXA at 1, 2, and 4 weeks after fracture. Serum vascular endothelial growth factor (VEGF), osteocalcin, and alkaline phosphatase (ALP) were assessed using ELISA at each time point. Biomechanical testing was performed at 2 and 4 weeks. RESULTS: BMD in SCI mice was significantly lower compared to control mice at each time point, with callus volume and all vessel parameters reduced as measured by micro-CT. Ultimate stress of the femora was significantly lower in SCI mice than in control mice at 2 and 4 weeks after fracture, whereas Young's modulus between the SCI and control mice turned to be significantly different at 4 weeks. Serum VEGF was lower in SCI mice than in the control group at 2 and 4 weeks, whereas serum osteocalcin and ALP were lower in SCI mice than in control ones at each time point. CONCLUSION: Significant bone loss and fracture healing impairment was noted in SCI mice. Decreased angiogenesis is consistent with the changes of microarchitecture and biomechanical properties during fracture healing.