NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-ß and further enhanced by hypoxia. The effect of TGF-ß was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia.

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI. Methods: The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model. Results: TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb. Conclusions: TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application.

Polystyrene microplastics induce myocardial inflammation and cell death via the TLR4/NF-κB pathway in carp.

Microplastics (MPs) have attracted widespread attention as an emerging environmental pollutant. Especially in aquatic ecosystems, the harm of MPs to aquatic animals has increasingly become a severe environmental problem. In this study, we constructed a carp polystyrene microplastics (PS-MPs) exposure model to explore the damage and mechanism of PS-MPs exposure to carp myocardial tissue. The results of H&E, TUNEL, and AO/EB staining showed that PS-MPs exposure could induce inflammation, apoptosis, and necrosis in carp myocardial tissue and cardiomyocytes. In addition, our study explored the targeting relationship between PS-MPs and TLR4 and found that PS-MPs exposure could significantly increase the expression of TLR4 pathway-related factors. As the concentration of PS-MPs increased, the NF-κB pathway and inflammation-related factors increased dose-dependent. In addition, myocardial injury induced by exposure to PS-MPs was predominantly apoptotic, accompanied by necrosis. In short, our data suggest that PS-MPs cause damage to myocardial tissue via the TLR4\NF-κB pathway. The above findings enrich the theory of toxicological studies on PS-MPs and provide an essential reference for aquaculture.

Exploring metabolism in scleroderma reveals opportunities for pharmacological intervention for therapy in fibrosis.

Background: Recent evidence has indicated that alterations in energy metabolism play a critical role in the pathogenesis of fibrotic diseases. Studies have suggested that 'metabolic reprogramming' involving the glycolysis and oxidative phosphorylation (OXPHOS) in cells lead to an enhanced generation of energy and biosynthesis. The aim of this study was to assess the molecular basis of changes in fibrotic metabolism in systemic sclerosis (Scleroderma; SSc) and highlight the most appropriate targets for anti-fibrotic therapies. Materials and methods: Dermal fibroblasts were isolated from five SSc patients and five healthy donors. Cells were cultured in medium with/without TGF-ß1 and with/without ALK5, pan-PIM or ATM kinase inhibitors. Extracellular flux analyses were performed to evaluate glycolytic and mitochondrial respiratory function. The mitochondrial network in TMRM-stained cells was visualized by confocal laser-scanning microscopy, followed by semi-automatic analysis on the ImageJ platform. Protein expression of ECM and fibroblast components, glycolytic enzymes, subunits of the five OXPHOS complexes, and dynamin-related GTPases and receptors involved in mitochondrial fission/fusion were assessed by western blotting. Results: Enhanced mitochondrial respiration coupled to ATP production was observed in SSc fibroblasts at the expense of spare respiratory capacity. Although no difference was found in glycolysis when comparing SSc with healthy control fibroblasts, levels of phophofructokinase-1 isoform PFKM were significantly lower in SSc fibroblasts (P<0.05). Our results suggest that the number of respirasomes is decreased in the SSc mitochondria; however, the organelles formed a hyperfused network, which is thought to increase mitochondrial ATP production through complementation. The increased mitochondrial fusion correlated with a change in expression levels of regulators of mitochondrial morphology, including decreased levels of DRP1, increased levels of MIEF2 and changes in OPA1 isoform ratios. TGF-ß1 treatment strongly stimulated glycolysis and mitochondrial respiration and induced the expression of fibrotic markers. The pan-PIM kinase inhibitor had no effect, whereas both ALK5 and ATM kinase inhibition abrogated TGF-ß1-mediated fibroblast activation, and upregulation of glycolysis and respiration. Conclusions: Our data provide evidence for a novel mechanism(s) by which SSc fibroblasts exhibit altered metabolic programs and highlight changes in respiration and dysregulated mitochondrial morphology and function, which can be selectively targeted by small molecule kinase inhibitors.

Correlation between chronic periodontitis and pulmonary ventilation function / 中国基层医药

Objective:To investigate the correlation between chronic periodontitis and pulmonary ventilation function.Methods:A total of 135 patients with chronic periodontitis who received treatment in Yuyao People's Hospital of Zhejiang Province between June 2014 and December 2019 were included in this study. They were divided into group A (stage I, initial lesion, n = 45), group B (stage II, early lesion, n = 45), group C (stage III, confirmed lesion, n = 45) according to the severity of periodontal lesion. Lung ventilation function indexes and serum levels of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were compared among the three groups. The correlation between periodontal condition and lung ventilation function indexes was analyzed. Results:Probing depth (PD), clinical attachment loss (CAL), number of missing teeth, alveolar bone resorption level were (1.67 ± 0.65) mm, (2.48 ± 0.44) mm, 0 pieces, and (1.38 ± 0.23) mm in group A, (2.05 ± 0.30) mm, (4.04 ± 0.97) mm, 1 piece, (3.37± 0.73) mm in group B, and (2.23 ± 0.47) mm, (5.17 ± 0.75) mm, 3 pieces, (6.48 ± 0.62) mm in group C. With the worsening of the disease, PD, CAL, number of missing teeth, and alveolar bone resorption level were gradually increased. PD, CAL and alveolar bone resorption level in group C were significantly higher than those in group A ( t = 4.68, 20.75, 51.74, all P < 0.001) and group B ( t = 2.17, 6.18, 21.78, P = 0.033, < 0.001, < 0.001). PD, CAL and alveolar bone resorption level in group B were significantly higher than those in group A ( t = 3.56, 9.82, 17.44, all P < 0.001). There was no significant difference in the number of missing teeth ( P > 0.05). Serum IL-6, IL-8 and TNF-α levels were (11.28 ± 4.26) ng/L, (7.48 ± 1.97) ng/L, (14.59 ± 2.11) ng/L in group A, (17.09 ± 4.91) ng/L, (10.82 ± 2.10) ng/L, (19.95 ± 4.48) ng/L in group B, and (26.47 ± 5.86) ng/L, (15.06 ± 2.75) ng/L, (33.76 ± 6.30) ng/L] in group C. With the worsening of the disease, serum IL-6, IL-8 and TNF-α levels were gradually increased. Serum IL-6, IL-8 and TNF-α levels in group C were significantly higher than those in group A ( t = 14.06, 15.03, 19.36, P < 0.001) and group B ( t = 8.23, 8.22, 11.98, all P < 0.001). Serum IL-6, IL-8 and TNF-α levels in group B were significantly higher than those in group A ( t = 6.00, 7.78, 7.26, P < 0.001). The percentage of the maximum expiratory volume in the first second to the predicted value (FEV 1%pre) and the ratio of the maximum expiratory volume in the first second to the forced vital capacity (FEV 1/FVC) were (81.53 ± 6.30)% and (68.73 ± 4.65)% in group A, (70.47 ± 5.25)% and (60.86 ± 3.42)% in group B, and (59.02 ± 3.41)% and (56.93 ± 4.21)% in group C. With the worsening of the disease, FEV 1%pre and FEV 1/FVC were gradually decreased. FEV 1%pre and FEV 1/FVC in group C were significantly lower than those in group A ( t = 21.08, 12.62, both P < 0.001) and group B ( t = 12.27, 4.86, both P < 0.001). FEV 1%pre and FEV 1/FVC in group B were significantly lower than those in group A ( t = 9.05, 9.25, both P < 0.001). Spearman correlation analysis showed that serum IL-6, IL-8 and TNF-α levels were negatively correlated with FEV1%pre and FEV 1/FVC ( r = -0.50, -0.28, -0.42, -0.61, -0.34, -0.51, all P < 0.05). Conclusion:There is a correlation between chronic periodontitis and pulmonary ventilation function. Inflammatory mediators may be involved in chronic periodontitis as internal systemic factors.

The pan-PPAR agonist lanifibranor reduces development of lung fibrosis and attenuates cardiorespiratory manifestations in a transgenic mouse model of systemic sclerosis.

BACKGROUND: The TßRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFß signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype. METHODS: PPAR pathway gene and protein expression differences from TG and WT sex-matched littermate mice were determined at baseline and following administration of one of two doses of lanifibranor (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. The prevention of bleomycin-induced lung fibrosis and SU5416-induced pulmonary hypertension by lanifibranor was explored. RESULTS: Gene expression data were consistent with the downregulation of the PPAR pathway in the TßRII∆k-fib mouse model. TG mice treated with high-dose lanifibranor demonstrated significant protection from lung fibrosis after bleomycin and from right ventricular hypertrophy following induction of pulmonary hypertension by SU5416, despite no significant change in right ventricular systolic pressure. CONCLUSIONS: In the TßRII∆k-fib mouse strain, treatment with 100 mg/kg lanifibranor reduces the development of lung fibrosis and right ventricular hypertrophy induced by bleomycin or SU5416, respectively. Reduced PPAR activity may contribute to the exaggerated fibroproliferative response to tissue injury in this transgenic model of scleroderma and its pulmonary complications.

Pig lung fibrosis is active in the subacute CdCl2 exposure model and exerts cumulative toxicity through the M1/M2 imbalance.

Environmental pollutant cadmium (Cd) can cause macrophage dysfunction, and the imbalance of M1/M2 is involved in the process of tissue fibrosis. In order to explore the effect of subacute CdCl2 exposure on pig lung tissue fibers and its mechanism, based on the establishment of this model, ICP-MS, H&E staining, Masson staining, Immunofluorescence, RT-PCR, and Western Blot methods were used to detect related indicators. The results found that lung tissue fibrosis, Cd content significantly increased, lung tissue ion disturbance, miR-20a-3p down-regulation, M1/M2 imbalance, LXA4/FPR2 content decreased, MDA content increased, NF-κB/NLRP3, TGFß pathway, PPARγ/Wnt pathway activated, and the expression of fibrosis-related factors increased. The above results indicate that subacute CdCl2 exposure increase Cd content in the pig lungs, which leads to M1/M2 imbalance and down-regulates the content of LXA4/FPR2, further activates the oxidative stress/NF-κB/NLRP3 pathway, thereby activating the TGFß and PPARγ/Wnt pathways to induce fibrosis. This study aims to reveal the toxic effects of CdCl2 and will provide new insights into the toxicology of Cd.

Polystyrene microplastics-induced ROS overproduction disrupts the skeletal muscle regeneration by converting myoblasts into adipocytes.

The environmental problem of Microplastics (MPs) pollution poses a great threat to human and animal health, which has attracted global attention. The physiological integrity of skeletal muscle is extremely important for the survival of animals. Here, we investigated the effect of two size polystyrene microplastics (PS-MPs, 1-10 µm and 50-100 µm) on the growth of anterior tibial (TA) muscle and repair after injury in mice. Results showed that the regeneration of skeletal muscle was delayed by PS-MPs exposure and was negatively correlated with particle size. H&E staining and Oil red O staining showed that PS-MPs exposure reduced the average cross-sectional area (CSA) and diameter of the muscle fibers, increased lipid deposition. Further mechanistic research displayed that though PS-MPs treatment did not affect cell viability of myoblast, it aggravated intracellular ROS generation and oxidative stress, inhibited myogenic differentiation by decreasing the phosphorylation of p38 MAPK, and promote adipogenic differentiation by increasing the expression of NF-κB, which could be alleviated by NAC. In brief, our data demonstrated that the ROS overproduction caused by PS-MPs disturbed the regeneration of skeletal muscle and directed the fate of satellite cells in mice.

The antagonistic effect of selenium on lead-induced necroptosis via MAPK/NF-κB pathway and HSPs activation in the chicken spleen.

Recent studies identified a novel programmed and regulated cell death that was characterized by a necrotic cell death morphology, termed necroptosis. Lead (Pb) is known as a persistent inorganic environmental pollutant that affects the health of humans and animals worldwide. However, there are no detailed reports of Pb-induced necroptosis of immune tissue. Selenium (Se) is a trace element that antagonizes the toxicity of heavy metals. Here, chickens were randomly divided into four groups, treated with Pb ((CH3OO)2Pb, 150 mg/kg) and/or Se (Na2SeO3, 2 mg/kg), aim to study the effect and mechanism of necroptosis in Pb-induced spleen injury and the antagonistic effects of Se on Pb toxicity. Our results showed that Pb exposure evidently increased the accumulation of Pb in spleen and caused necroptosis by upregulating the expression of RIP1, RIP3 and MLKL, and decreasing Caspase8 expression. Meanwhile, Pb treatment inhibited the activities of SOD, GPX, and CAT, caused the accumulation of NO and MDA, and induced oxidative stress, which promoted the expression of MAPK/NF-κB pathway genes (ERK, JNK, P38, NF-κB and TNF-α) and activated HSPs (HSP27, HSP40, HSP60, HSP70 and HSP90). However, the increased content of Pb in spleen and Pb-caused necroptosis were inhibited by Se cotreatment. Overall, we conclude that Se can prevent Pb-induced necroptosis by restoring antioxidant functions and blocking the MAPK/NF-κB pathway and HSPs activation in chicken spleen.

Toll-like receptors 2 and 6 mediate apoptosis and inflammation in ischemic skeletal myotubes.

Critical limb ischemia (CLI) is associated with skeletal muscle damage. However, the pathophysiology of the muscle damage is poorly understood. Toll-like receptors (TLR) have been attributed to play a role in ischemia-induced tissue damage but their role in skeletal muscle damage in CLI is unknown. TLR2 and TLR6 expression was found to be upregulated in skeletal muscle of patients with CLI. In vitro, ischemia led to upregulation of TLR2 and TLR6 by myotubes, and activation of the downstream TLR signaling pathway. Ischemia-induced activation of the TLR signaling pathway led to secretion of the pro-inflammatory cytokine interleukin-6 and muscle apoptosis, which were abrogated by neutralising TLR2 and TLR6 antibodies. Our study demonstrates that TLR2 and TLR6 are upregulated in ischemic muscle and play a role in ischemia-induced muscle damage. Thus, manipulating the TLR pathway locally may be of potential therapeutic benefit.

Hydrogel and membrane scaffold formulations of Frutalin (breadfruit lectin) within a polysaccharide galactomannan matrix have potential for wound healing.

Plant lectins are carbohydrate-binding proteins, which can interact with cell surfaces to initiate anti-inflammatory pathways, as well as immunomodulatory functions. Here, we have extracted, purified and part-characterized the bioactivity of Jacalin, Frutalin, DAL and PNA, before evaluating their potential for wound healing in cultured human skin fibroblasts. Only Frutalin stimulated fibroblast migration in vitro, prompting further studies which established its low cytotoxicity and interaction with TLR4 receptors. Frutalin also increased p-ERK expression and stimulated IL-6 secretion. The in vivo potential of Frutalin for wound healing was then assessed in hybrid combination with the polysaccharide galactomannan, purified from Caesalpinia pulcherrima seeds, using both hydrogel and membrane scaffolds formulations. Physical-chemical characterization of the hybrid showed that lectin-galactomannan interactions increased the pseudoplastic behaviour of solutions, reducing viscosity and increasing Frutalin's concentration. Furthermore, infrared spectroscopy revealed -OH band displacement, likely caused by interaction of Frutalin with galactose residues present on galactomannan chains, while average membrane porosity was 100 µm, sufficient to ensure water vapor permeability. Accelerated angiogenesis and increased fibroblast and keratinocyte proliferation were observed with the optimal hybrid recovering the lesioned area after 11 days. Our findings indicate Frutalin as a biomolecule with potential for tissue repair, regeneration and chronic wound healing.

Current status of professional identity among undergraduates majoring in public affairs management and the related influencing factors / 中华医学教育探索杂志

Objective@#To investigate the current status of professional identity among the undergraduates majoring in public affairs management and the related influence factors.@*Methods@#A stratified cluster sampling method was used to conduct a questionnaire survey among the 115 undergraduates majoring in public affairs management in a medical university in Harbin, China, and the contents of the questionnaires included general status and professional identity. The t-test, one-way analysis of variance, and multiple linear regression analysis were used to analyze the influencing factors of professional identity. In-depth interviews were conducted for some students to explore the profound influencing factors for professional identity among the students majoring in public affairs management.@*Results@#The overall level of professional identity was low among the undergraduates majoring in public affairs management in the medical university in Harbin, and the score of professional identity was 3.25±0.56. The scores of cognitive dimension, emotional dimension, behavioral dimension, and applicable dimension were 3.41±0.69, 3.21±0.78, 3.24±0.71, and 3.13±0.72, respectively. The multivariate analysis showed that grade, academic performance, major selection, and children number in family had significant influence on their professional identity. The students who were at grade four, who had high academic record (b=-0.276, P<0.01), who chose this major independently (b=-0.277, P<0.01), and who were the only child (b=-0.233, P<0.05) had a high score of professional identity. As for the undergraduates at different grades, the undergraduates at grades two and three had a significantly lower score of professional identity than those at grade four (b=-0.296 and -0.369, P<0.05), while there was no significant difference between the undergraduates at grades one and four (b=-0.008, P>0.05). Furthermore, the students with a high score of professional identity had higher possibility to pursue the job related to this major than those with a low score (3.39±0.53 vs. 2.92±0.49, t=4.414, P<0.01).@*Conclusion@#Universities should strengthen the publicity of the major of public affairs management, optimize the curriculum, and add job experience, so as to enhance professional identity among students with this major and provide talent for the field of public health management.

Current status of professional identity among undergraduates majoring in public affairs management and the related influencing factors / 中华医学教育探索杂志

Objective To investigate the current status of professional identity among the undergraduates majoring in public affairs management and the related influence factors.Methods A stratified cluster sampling method was used to conduct a questionnaire survey among the 115 undergraduates majoring in public affairs management in a medical university in Harbin,China,and the contents of the questionnaires included general status and professional identity.The t-test,one-way analysis of variance,and multiple linear regression analysis were used to analyze the influencing factors of professional identity.In-depth interviews were conducted for some students to explore the profound influencing factors for professional identity among the students majoring in public affairs management.Results The overall level of professional identity was low among the undergraduates majoring in public affairs management in the medical university in Harbin,and the score of professional identity was 3.25 ± 0.56.The scores of cognitive dimension,emotional dimension,behavioral dimension,and applicable dimension were 3.41 ± 0.69,3.21 ± 0.78,3.24 ± 0.71,and 3.13 ± 0.72,respectively.The multivariate analysis showed that grade,academic performance,major selection,and children number in family had significant influence on their professional identity.The students who were at grade four,who had high academic record (b =-0.276,P＜ 0.01),who chose this major independently (b=-0.277,P＜0.01),and who were the only child (b=-0.233,P＜ 0.05) had a high score of professional identity.As for the undergraduates at different grades,the undergraduates at grades two and three had a significantly lower score of professional identity than those at grade four (b=-0.296 and-0.369,P＜0.05),while there was no significant difference between the undergraduates at grades one and four (b=-0.008,P＞0.05).Furthermore,the students with a high score of professional identity had higher possibility to pursue the job related to this major than those with a low score (3.39 ± 0.53 vs.2.92 ± 0.49,t=4.414,P＜0.01).Conclusion Universities should strengthen the publicity of the major of public affairs management,optimize the curriculum,and add job experience,so as to enhance professional identity among students with this major and provide talent for the field of public health management.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration.

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Effect of phosphorus deficiency on erythrocytic morphology and function in cows

The aim of this study was to evaluate the influence of phosphorus (P) deficiency on the morphological and functional characteristics of erythrocytes in cows. Forty Holstein-Friesian dairy cows in mid-lactation were randomly divided into two groups of 20 each and were fed either a low-P diet (0.03% P/kg dry matter [DM]) or a control diet (0.36% P/kg DM). Red blood cell (RBC) indices results showed RBC and mean corpuscular hemoglobin decreased while mean corpuscular volume increased significantly (p < 0.05) in P-deficient cows. Erythrocyte morphology showed erythrocyte destruction in P-deficient cows. Erythrocytes' functional characteristics results showed total bilirubin and indirect bilirubin concentrations and aspartate transaminase and alanine transaminase activity levels in the serum of P-deficient cows were significantly higher than those in control diet-fed cows. Activities of superoxide dismutase and glutathione peroxidase in erythrocytes were lower, while the malondialdehyde content was greater, in P-deficient cows than in control diet-fed cows. Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were lower in P-deficient cows than in control diet-fed cows; however, Ca²⁺-ATPase activity was not significantly different. The phospholipid composition of the erythrocyte membrane changed and membrane fluidity rigidified in P-deficient cows. The results indicate that P deficiency might impair erythrocyte integrity and functional characteristics in cows.

Epigenetic regulation of cyclooxygenase-2 by methylation of c8orf4 in pulmonary fibrosis.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

Systematic Review of Acupuncture-moxibustion for Deglutition Disorders after Cerebral Stroke / 上海针灸杂志

Objective To evaluate the clinical efficacy of acupuncture-moxibustion in treating deglutition disorders after cerebral stroke.Method By computer and manual literature retrieval, eligible randomized controlled trials (RCTs) were collected for systematic review by using the Cochrane method, and the meta analysis was performed by using Revman 5.2.Result Nine studies were recruited, covering 577 subjects of deglutition disorders due to cerebral stroke. The total effective rate of the treatment group was significantly higher than that of the control group [RR=0.36, 95%CI (0.25,0.50), Z=5.80 (P<0.00001)]. The treatment group was superior to the control group in improving the water drinking test (χ2=108.73,P<0.00001).Conclusion Acupuncture can produce a content therapeutic efficacy in treating deglutition disorders due to cerebral stroke, which still requires high-quality large-sample-size clinical RCTs for further verification.

Effect of Different Needle-retaining Lengths on the Therapeutic Efficacy of Acupuncture in Treating Post-stroke Shoulder Pain / 上海针灸杂志

ObjectiveTo observe the effect of different needle-retaining lengths on the clinical efficacy of acupuncture in treating post-stroke shoulder pain.MethodSixty patients were randomized into a 30 min group and a 60 min group, 30 cases in each group. The same acupuncture treatment protocol was adopted in the two groups, and the acupuncture treatment was given once a day, 5 sessions a week, for 20 times in total. The Visual Analogue Scale (VAS), shoulder pain frequency, and Fugl-Meyer Assessment (FMA) were observed before treatment and after 4-week treatment.ResultThe scores of each index were significantly improved in both groups after treatment (P<0.01), and there were significant differences in comparing each index score between the two groups after treatment (P<0.05,P<0.01).ConclusionIn the treatment of post-stroke shoulder pain with acupuncture, 30 min needle-retaining is better than 60 min in comparing the analgesic effect.

A Role of Myocardin Related Transcription Factor-A (MRTF-A) in Scleroderma Related Fibrosis.

In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis.

INTRODUCTION: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. METHODS: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. RESULTS: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. CONCLUSIONS: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.