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Objective To investigate the effect of caffeine consumption on skin wound healing and scar formation in mice. Methods The mouse model was established according to our previous method of chronic intervention with caffeine combined with the model of full-thickness skin wound on the back. Wound closure was traced every 2 days in each group using transparent film until the wound closed, and the scar areas were calculated at 30 d after healing. The sections were stained with hematoxylin-eosin (HE) to observe the changes in epithelial cells crawling and thickness of granulation tissue at 3, 6 and 9 d after wounding, and scar width and the thinness-to-thickness ratio of the epithelium in the scar tissue at 30 d after healing with the aid of Labwork 4.0 software. Masson staining was used to analyze the collagen changes of the wound at 6 d after wounding and of the scar tissue at 30 d after healing. Immunohistochemical assay was used to observe the changes of CD31 and CD11b protein levels in wound tissue at 6 d after wounding. Results Results The healing time of caffeine group was delayed by 28.3%, and the scar area was reduced by 56.8% when compared with that of the control group. In the process of wound healing, the crawling length of epithelium and the thickness of granulation tissue were significantly decreased in the caffeine group than the control group (P < 0.01), the collagen content and the expression level of CD31 in the wound were also decreased (P < 0.01), and the expression level of CD11b was obviously enhanced (P < 0.01). Meanwhile, compared with the control group, the caffeine group had significantly decreased scar width (P < 0.01), higher thickness of scar tissue epithelium (P < 0.01), but similar collagen content in the scar and the surrounding undamaged subcutaneous collagen. Conclusion Caffeine consumption can delay the wound healing in mice, but attenuate scar formation.

Hypoxia‐induced vascular smooth muscle cells (VSMCs) migration plays an important role in vascular remodeling and is implicated in vascular diseases, such as atherosclerosis and pulmonary hypertension. We previously observed the increased expression of krüppel‐like factor 4 (KLF4) in VSMCs under hypoxia. However, whether the upregulation of KLF4 participates in hypoxia‐induced VSMCs migration is still unknown. In this study, we demonstrated that KLF4 was an important player in the process of VSMCs migration under hypoxia since interference of KLF4 by small interfering RNA mostly dampened hypoxia‐induced migration of VSMCs. In addition, using luciferase reporter and ChIP assays, we confirmed two hypoxia‐inducible factor 1α (HIF1α) binding elements (located at ‐150 to ‐163 and ‐3922 to ‐3932) in the upstream regulatory region of klf4 locus and identified KLF4 as a novel direct target gene of HIF1α. Our findings unveil a novel regulatory mechanism that involves HIF1α‐induced upregulation of KLF4, which plays a vital role in VSMCs migration under hypoxia. [ABSTRACT FROM AUTHOR]

Abstract: The inactivation of the A2A receptor (A2AR) has been shown to neuroprotect against brain injury in several animal models of neurological disorders including stroke and Parkinson''s disease. However, despite marked elevation of adenosine level, the role of the A2A in traumatic brain injury (TBI) remains unclear. In the present study, we investigated the effects of genetic inactivation of A2ARs in the acute stage. The A2AR knock-out (KO) mice and their wild-type (WT) littermates were subjected to cortical impact injury by a dropping weight. The control group was only craniotomized without TBI. At 24 h post-TBI, the neurological deficit scores of the KO mice were significantly lower than that of WT littermates. Consistent with the behavioral changes, the brain water contents as well as histological changes and the TUNEL-positive cells of the injured cortex of the KO mice were significantly lower than that of WT littermates. Furthermore, the glutamate level in the cerebral spinal fluid (CSF) of the KO mice was also significantly lower than that of WT littermates. In addition, we found that at 12 h post-TBI the mRNA and protein levels of TNF-α and IL-1β were higher in the KO mice than that in the WT littermates. However, at 24 h post-TBI, the level of TNF-α and IL-1β continually increased in the WT mice but largely declined in the KO mice. These results suggest that the genetic inactivation of A2AR protects against TBI, which is mainly associated with the suppression of glutamate level. [Copyright &y& Elsevier]

Exercise-induced fatigue is a complex physiological phenomenon and is greatly influenced by central mechanisms in brain. As one of the most abundant circulating carbon metabolites, l-lactate in brain has been considered to be an important supplementary fuel during exercise; however, whether it plays a signaling role in fatigue remains largely obscure. In this study, our results initially revealed that brain l-lactate levels were increased after an exhaustive swimming session in several brain regions including motor cortex, hippocampus, and cerebellum. Then, we examined the specific role of brain lactate receptor, also known as hydroxycarboxylic acid receptor 1 (GPR81), in exercise-induced fatigue. We found that intracerebroventricular injection of either d-lactate (an enantiomer that could mediate activation of GPR81 as l-lactate) or a potent GPR81 agonist 3-chloro-5-hydroxybenzoic acid (CHBA), significantly decreased the swimming time to fatigue. After being subjected to the same weight-loaded swimming for 30 min, no obvious changes of blood lactate levels, gastrocnemius pAMPK/AMPK ratio, and glycogen contents were observed between intracerebroventricular CHBA-injected mice and vehicle-treated ones, which suggested a comparable degree of peripheral fatigue. Meanwhile, there were higher extracellular γ-aminobutyric acid (GABA) levels and lower extracellular glutamate levels and glutamate/GABA ratio in motor cortex of the intracerebroventricular CHBA-injected mice than that of vehicle-treated ones, indicating a greater extent of central fatigue in CHBA-injected mice than that in vehicle animals. Collectively, our results suggested that an increased level of brain l-lactate acts as a signaling molecule via activating GPR81, which in turn exacerbates central fatigue during exercise.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O 2 ) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

Objective: To observe the protein expression related to cognitive and learning memory function, and to investigate the effect of aquaporin 4 (AQP4) silence on learning and memory function in traumatic brain injury (TBI) rats., Methods: Ninety-six healthy adult male Wistar rats were divided into groups according to the random number table. (1) Forty-eight rats were divided into sham operation (sham) group, TBI group (by using modified Feeney method), AQP4 RNA interference (RNAi) negative group [TBI+meaningless small interfering RNA (siRNA)-AQP4 liposome solution 10 μL], and AQP4 RNAi group (TBI+siRNA-AQP4 liposome solution 10 μL). In each group, brain tissues of 4 rats were harvested at 1, 6 and 12 hours respectively. The protein expressions of hippocampus AQP4, general control nonderepressible 2 kinase (GCN2), cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (p-CREB) were detected by Western Blot. (2) In addition, 48 rats were divided into normal control group (control group), sham group, TBI group and AQP4 RNAi group, brain water content were measured in 6 of them after 12 hours of injury, and 6 were used in Morris water maze test., Results: (1) The protein expressions of hippocampus AQP4 and GCN2 in TBI group were significantly higher than those in sham group, and increased gradually with time with statistical difference at 12 hours (AQP4 protein: 5.03±0.09 vs. 1, GCN2 protein: 4.01±0.13 vs. 1, both P < 0.01); the protein expressions of hippocampus CREB and p-CREB were significantly lower than those in sham group, and decreased gradually with time with statistical difference at 12 hours (CREB protein: 0.38±0.03 vs. 1, p-CREB protein: 0.38±0.03 vs. 1, both P < 0.01). Compared with TBI group, the protein expressions of AQP4 in AQP4 RNAi group was significantly decreased (1 hour: 1.02±0.04 vs. 2.23±0.05, 6 hours: 1.23±0.03 vs. 2.59±0.04, 12 hours: 2.20±0.08 vs. 5.03±0.09, all P < 0.01), but there were no significant difference in the expressions of GCN2, CREB or p-CREB. There was no significant difference in the expression of protein between AQP4 RNAi negative group and TBI group. (2) The brain water content in TBI group was significantly higher than that in control group and sham group [(83.7±0.4)% vs. (76.2±0.2)%, (76.2±0.3)%, both P < 0.01]. The brain water content in AQP4 RNAi group [(78.8±0.3)%] was significantly decreased as compared with that in TBI group (P < 0.01). The latency of Morris water maze test was significantly prolonged in the day 11, 13 and 15 after the injury of the TBI group and AQP4 RNAi group, and the exploration time was significantly shortened. Compared with TBI group, the incubation period of AQP4 RNAi group was significantly shortened at 15 days (s: 60.2±11.1 vs. 62.0±11.5, P < 0.05), and the exploration time was significantly prolonged (s: 37.0±8.5 vs. 32.7±9.2, P < 0.05)., Conclusions: The impairment of cognitive and learning memory function in rats after TBI was significantly related to the changes in CREB and GCN2 in cognitive and learning memory function. After RNAi treatment, the cognitive and learning and memory function of rats was not improved obviously, but the brain edema could be alleviated.

The bi-directional regulation of TGF-beta1 (transforming growth factor-beta1) on fibroblast proliferation with stimulation at low concentration, but inhibition at high concentration, has important significance during tissue repair. The mechanism has not been defined. c-Ski is a major co-repressor of TGF-beta1/Smad3 signalling; however, the exact role of c-Ski in the bi-directional regulation of fibroblast proliferation remains to be determined. In the present study, we established a dose-effect relationship of bi-directional regulation of TGF-beta1-mediated proliferation in rat skin fibroblasts, and found that c-Ski overexpression promoted fibroblast proliferation by inhibiting Smad3 activity. Importantly, c-Ski expression was decreased at the high concentration of TGF-beta1, but increased at the low concentration of TGF-beta1. This dose-dependent change in TGF-beta1 action did not affect Smad3 phosphorylation or nuclear translocation, but altered Smad3 DNA-binding activity, transcriptional activity and expression of the downstream gene p21 that both increased at the high concentration and decreased at the low concentration. Furthermore, c-Ski overexpression exerted synergistic stimulation with TGF-beta1 at the low concentration, but reversed the inhibitory effect of TGF-beta1 at high concentrations, while knockdown of c-Ski by RNA interference abrogated bi-directional role of TGF-beta1 on fibroblast proliferation. Thus our data reveal a new mechanism for this bi-directional regulation, i.e. c-Ski expression change induced by low or high TGF-beta1 concentration in turn determines the promoting or inhibiting effects of TGF-beta1 on fibroblast proliferation, and suggests an important role of c-Ski that modulates the local availability of TGF-beta1 within the wound repair microenvironment.

Objective: To investigate the expression of placental vascular endothelial growth factor (VEGF) and its relationship with construction of placental vascular network, placental weight and the newborns' weight in patients with pregnancy induced hypertension (PIH)., Methods: Twenty-five patients with PIH and twenty-five normal late pregnant (NLP) women in the third trimester were observed in this study. The expression of VEGF protein in placental, the placental vascular density were determined by Western blot and immunohistochemical F8 staining respectively., Results: The VEGF values in patients with PIH and NLP were 24 793 +/- 6 579 and 41 903 +/- 11 009, and the vascular densities were (61 +/- 11) and (78 +/- 11) counts/400 x vision respectively. They were both significantly different in both groups and were closely related to the severity of PIH (P < 0.01). Placental weight and the newborns' weight in patients with PIH [(460 +/- 59) g, (3 176 +/- 503) g respectively] were also significantly decreased compared with NLP [(573 +/- 99) g, (3 468 +/- 493) g respectively], but the new newborns' length had no significant difference between the two groups (P > 0.05). In NLP group, placental vascular density, placental weight and the newborns' weight all had a significantly positive correlation with VEGF (P < 0.01, r = 0.823, 0.671, 0.888 respectively) and in PIH group, the situation was the same (P < 0.01, r = 0.905, 0.859, 0.732 respectively), but the new newborns' length had no correlation with VEGF (P > 0.05)., Conclusion: It shows that the reduced expression of VEGF in placenta of patients with PIH may be one of the important factors responsible for decreased placental vascular density and fetal intrauterine growth restriction.