硫化氢对糖尿病大鼠肾脏纤维化的影响及其机制

目的：观察硫化氢（H₂S）对糖尿病大鼠肾脏组织纤维化的影响并探讨其作用机制。方法：雄性SD大鼠随机分为正常对照（NC）组、糖尿病对照（DC）组、糖尿病+硫氢化钠（DM+NaHS）组和糖尿病+炔丙基甘氨酸（DM+PAG）组,每组8只。采用一次性腹腔注射链脲佐菌素的方法制备1型糖尿病大鼠模型。造模成功4周后,DM+NaHS组和DM+PAG组大鼠每天分别腹腔注射56 μmol/kg NaHS和40 mg/kg PAG溶液。处理4周后,测定各组大鼠空腹血糖（FBG）、尿素氮（BUN）和肌酐（SCr）水平;Masson染色观察肾脏组织胶原纤维变化,计算胶原容积分数（CVF）;透射电镜观察肾脏超微结构;利用试剂盒检测肾组织白细胞介素1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）和羟脯氨酸（Hyp）水平;Western blot测肾组织TGF-β1、Smad3、磷酸化Smad3（p-Smad3）和IV型胶原（col-IV）的蛋白表达水平。结果：与NC组比较,DC组FBG、BUN、SCr、CVF、IL-1β、IL-6、TNF-α和Hyp均明显增高,肾脏胶原纤维沉积、超微结构损伤明显加重,TGF-β1、Smad3、p-Smad3、p-Smad3/Smad3和col-IV表达均明显增加。与DC组比较,除FBG外,DM+NaHS组中上述指标均明显改善;而DM+PAG组中上述指标损伤均进一步加重。结论：H₂S可以减轻糖尿病大鼠肾脏纤维化,其机制可能与减少促炎因子释放,下调TGF-β1/Smad3通路,抑制肾脏col-IV过量生成相关。     

SIRT1 inhibits TGF‐β‐induced endothelial‐mesenchymal transition in human endothelial cells with Smad4 deacetylation

Endothelial‐mesenchymal transition (EndMT) plays a pivotal role in organ fibrosis. This study examined the effect of SIRT1 on transforming growth factor beta (TGF‐β)‐induced EndMT in human endothelial cells (ECs) and its probable molecular mechanism. We assessed EndMT by immunofluorescence staining, quantitative real‐time polymerase chain reaction, Western blotting, and migration and invasion assays. Adenovirus was used to overexpress or knockdown SIRT1 in ECs. The regulatory relationship between SIRT1 and Smad4 was analyzed by coimmunoprecipitation assay. We found that SIRT1 was decreased in TGF‐β‐induced EndMT, and SIRT1 inhibited TGF‐β‐induced EndMT through deacetylating Smad4. Our findings suggest that SIRT1 has an important role in inhibiting EndMT by regulating the TGF‐β/Smad4 pathway in human ECs and, thus, protecting against fibrosis.     

Endothelial cells enhance adipose mesenchymal stromal cell‐mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross‐talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full‐thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue‐derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF‐β pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC‐mediated matrix contraction via regulation of fibrosis‐related proteins.     

Drug-Driven Phenotypic Convergence Supports Rational Treatment Strategies of Chronic Infections

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Extracellular pH and lung infections in cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na⁺/K⁺ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.     

Collagen and the myocardium: fibrillar structure,biosynthesis and degradation in relation to hypertrophy and its regression

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.     

Optimization of the technique of cell migration from explants: Application to human synoviocytes on cryoprecipitate coating

The explant technique, though easy to carry out, is unpredictable and thus not readily reproducible. Addition of cryoprecipitate, which is rich in fibronectin, serves to optimize this technique of cell migration from explants by improving adhesion.     

Successful targeting of the mouse cystic fibrosis transmembrane conductance regulator gene in embryonal stem cells

We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are pluripotent and competent to form chimaeras.