Zinc Supplementation Attenuates High Glucose-Induced Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells

Zinc (Zn) plays an important role in preventing many types of epithelial-to-mesenchymal transition (EMT)-driven fibrosis in vivo. But its function in the EMT of the peritoneal mesothelial cells (PMCs) remains unknown. Here, we studied the Zn effect on the high glucose (HG)-induced EMT in the rat PMCs (RPMCs) and the underlying molecular mechanisms. We found that Zn supplementation significantly inhibited TGF-??1 and ROS production, and attenuated the HG-induced EMT in the RPMCs, likely through inhibition of MAPK, NF-??B, and TGF-??/Smad pathways.     

Selenium Suppresses Lipopolysaccharide-Induced Fibrosis in Peritoneal Mesothelial Cells Through Inhibition of Epithelial-to-Mesenchymal Transition

Peritoneal fibrosis resulting from long-term clinical peritoneal dialysis has been the main reason of dropout from peritoneal dialysis. Peritonitis as a common complication of peritoneal dialysis treatment may lead to the occurrences of peritoneal fibrosis. We cultured peritoneal mesothelial cells with lipopolysaccharides (LPS) in order to stimulate the environment of peritonitis and investigate whether lipopolysaccharides could induce epithelial-to-mesenchymal transition (EMT). Oxidative stress could stimulate fibrogenesis while selenium has antioxidant properties. So, this study also explored whether selenium supplementation affects lipopolysaccharide-induced EMT and fibrosis. We found that lipopolysaccharides could activate EMT changes such as the loss of E-cadherin and the increase of α-smooth muscle actin (α-SMA), collagen I, vimentin, and fibronectin (FN), while selenium inhibits EMT by modulating reactive oxygen species (ROS) generation and ROS/MMP-9 signaling pathways in peritoneal mesothelial cells. Moreover, it was revealed that selenium decreased the EMT events of peritoneal mesothelial cells via inhibition of PI3k/AKT pathways. In conclusion, these findings enable a better understanding of the mechanism of peritoneal fibrosis and explore a new idea for the prevention and treatment.     

Fibrin-Induced Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells as a Mechanism of Peritoneal Fibrosis: Effects of Pentoxifylline

Excessive fibrin deposition in the peritoneum is thought to be involved in the development of encapsulating peritoneal sclerosis (EPS), an important cause of morbidity and mortality in peritoneal dialysis patients. We investigated fibrin-induced epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) as a possible mechanism of fibrin involvement in EPS. In vitro, fibrin overlay of PMCs altered their morphology; increased α-smooth muscle actin, fibronectin, fibroblast specific protein-1, and α_vβ₃ integrin expression; and decreased cytokeratin 18 and E-cadherin expression. Fibrin overlay also increased focal adhesion kinase and Src kinase phosphorylation. Fibrin-induced changes were inhibited by treating the cells with α_vβ₃ integrin antibody or pentoxifylline (PTX). In a rat model, intraperitoneal injection of Staphylococcus aureus and fibrinogen induced severe EPS features, which were attenuated by PTX treatment. PTX-treated rats also showed preserved peritoneal ultrafiltration function and lower concentrations of cytokines than the untreated rats. S. aureus- and fibrinogen-injected rats had higher percentage of cytokeratin-positive cells in the omentum fibrotic tissue than controls; this was also reduced by PTX treatment. Our results suggest that fibrin induces EMT of PMCs by engaging α_vβ₃ integrin and activating associated kinases. Our EPS animal model showed that fibrin-induced EMT was involved in the pathogenesis of peritoneal fibrosis and was inhibited by PTX.     

Zinc Inhibits High Glucose-Induced Apoptosis in Peritoneal Mesothelial Cells

Zinc (Zn) plays an important role in influencing many types of apoptosis. However, its function in apoptosis in peritoneal mesothelial cells (PMCs) remains unknown. Here, we studied the effects of Zn on high glucose (HG)-induced apoptosis in rat PMCs (RPMCs) and examined the underlying molecular mechanisms. We found that Zn supplementation inhibited HG-induced RPMC apoptosis significantly, by attenuating reactive oxygen species (ROS) production, inhibiting HG-induced sFasR and sFasL over-expression, caspase-8 and caspase-3 activation, and inhibiting release of cytochrome c from mitochondria to the cytosol. Further analysis revealed that Zn supplementation facilitated cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway and MAPK/ERK pathways. These results indicate that Zn can inhibit apoptosis in HG-induced RPMCs by several independent mechanisms, including an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-3 activation.     

分层管理对大面积烧伤患者早期康复治疗的临床意义

目的：重度烧伤早期易合并多种并发症且病情复杂、死亡率高,严重影响患者的生存周期和生活质量。本研究分析并探讨不同干预措施对重度烧伤患者早期并发症的效果,为临床研究提供可借鉴的方法。方法：选取2011年1月．2012年11月我院收治的重度烧伤患者66例,随机分为常规护理组和分级管理组,每组各33例。常规组采取基本护理模式对患者进行康复期护理,分级管理组根据病情对患者进行分级,分配不同专业技能等级的护理人员进行护理。观察并比较两组患者的临床效果及对护理服务的满意度。结果：分层管理组患者并发症发生率为42.3％,明显低于常规护理组69．7％（P〈0．05）;分层管理组的护理质量、患者依从性及对护理服务的满意度均显著高于常规护理组,差异具有统计学意义（P〈0．05）。结论：对大面积烧伤患者实施分层管理的护理模式具有积极的作用,不仅有效降低并发症发生率,而且能够合理分配护理人员,优化服务质量,提高患者满意度,值得在临床护理实践中推广应用。     

高糖诱导腹膜间皮细胞发生上皮问质转化中microRNA的异常变化

目的：定腹膜间皮细胞在高糖引起上皮间质转化（Epithelial—mesenchymnal transition,EMT）过程中microRNA的表达差异。方法：常规培养PMC细胞,利用高糖培养液刺激诱导腹膜间皮细胞发生EMT,倒置显微镜观察各组细胞形态学变化,实时定量PCR检测EMT标志基因变化,以此确定高糖诱导EMT的发生。利用特异茎环结构的引物合成microRNA的cDNA,实时定量PCR检测重要microRNA的表达变化。结果：高糖培养液培养腹膜间皮细胞48hr后,细胞形态呈梭形改变,同时EMT标记基因E—cadherin表达明显减低（P〈0．01）,Vimentin表达显著升高（P〈0．01）,说明高糖诱导腹膜间皮细胞发生了EMT。利用microRNA特异的茎环结构引物,实时定量PCR检测结果发现高糖刺激后miR-193a的表达明显上调（P〈0．01）;miR-15a和let-7e的表达明显降低（P〈0．01）;miR-16和miR-21的表达无明显变化（P〉0．05）,同时检测发现高糖刺激后miR-193a的表达水平随刺激时间表达升高。结论：异常变化的microRNA可能对高糖诱导的腹膜间皮细胞EMT具有重要调控作用。     

高糖诱导腹膜间皮细胞发生上皮间质转化中microRNA的异常变化

目的:定腹膜间皮细胞在高糖引起上皮间质转化(Epithelial-mesenchymal transition,EMT)过程中microRNA的表达差异。方法:常规培养PMC细胞,利用高糖培养液刺激诱导腹膜间皮细胞发生EMT,倒置显微镜观察各组细胞形态学变化,实时定量PCR检测EMT标志基因变化,以此确定高糖诱导EMT的发生。利用特异茎环结构的引物合成microRNA的cDNA,实时定量PCR检测重要microRNA的表达变化。结果:高糖培养液培养腹膜间皮细胞48hr后,细胞形态呈梭形改变,同时EMT标记基因E-cadherin表达明显减低(P0.01),Vimentin表达显著升高(P0.01),说明高糖诱导腹膜间皮细胞发生了EMT。利用microRNA特异的茎环结构引物,实时定量PCR检测结果发现高糖刺激后miR-193a的表达明显上调(P0.01);miR-15a和let-7e的表达明显降低(P0.01);miR-16和miR-21的表达无明显变化(P0.05),同时检测发现高糖刺激后miR-193a的表达水平随刺激时间表达升高。结论:异常变化的microRNA可能对高糖诱导的腹膜间皮细胞EMT具有重要调控作用。     

The Effect of Statin on Epithelial-Mesenchymal Transition in Peritoneal Mesothelial Cells

Background

Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT).

Methods

In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment.

Results

Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin.

Conclusion

This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.     

Fuzi Attenuates Diabetic Neuropathy in Rats and Protects Schwann Cells from Apoptosis Induced by High Glucose

Radix aconite lateralis preparata (Fuzi), a folk medicine, has long been used for the treatment of diabetes and paralysis in China. We examined the effect of Fuzi alone on diabetic rats and Schwann cells in high glucose and the components responsible for its activity. The major constituents of FZE were identified by HPLC-MS/MS data. Male Sprague Dawley rats (n = 36) were randomly divided into control, diabetic, FZE 1.75 g/kg, FZE 3.50 g/kg, FZE 7.00 g/kg, and methylcobalamin groups. After two weeks treatment, nerve conduction velocity and paw withdrawal latency were measured. In vitro, the Schwann cells were grouped according to exposure: normal glucose (NG), normal glucose plus mannitol (NG+M), high glucose (HG), and HG plus different concentrations of FZE (0.1 µg/ml, 1.0 µg/ml, and 10.0 µg/ml). Oxygen free radicals and apoptosis were evaluated through DCFH₂DA, DHE and annexin-PE/7-AAD assay, respectively. Apoptosis factors (Bax, Bcl-2, CytoC, caspase-3, and caspase-9) were analyzed using immunofluorescence. Nine alkaloids were identified. The results from animal model showed that FZE was effective in accelerating nerve conduction velocity and shortening paw withdrawal latency in diabetic rats. And in vitro, FZE was also found to protect Schwann cells against high glucose injury. FZE could significantly decrease the apoptotic ratio, superoxide anion and peroxide level. Furthermore, the apoptosis factors, including Bax, Bcl-2, CytoC, caspase-3, and caspase-9 were ameliorated in FZE treated groups. The HPLC-MSⁿ method is simple and suitable for the identification of alkaloids in Fuzi. FZE has a protective effect in diabetic neuropathic rats, which is probably achieved by the antiapoptotic effect of FZE on Schwann cells. Apoptosis factor data imply that FZE protected Schwann cells through the mitochondria pathway. Alkaloids are major components contributing to the protective effect.     

Tamoxifen Ameliorates Peritoneal Membrane Damage by Blocking Mesothelial to Mesenchymal Transition in Peritoneal Dialysis

Mesothelial-to-mesenchymal transition (MMT) is an auto-regulated physiological process of tissue repair that in uncontrolled conditions such as peritoneal dialysis (PD) can lead to peritoneal fibrosis. The maximum expression of peritoneal fibrosis induced by PD fluids and other peritoneal processes is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. Tamoxifen, a synthetic estrogen, has successfully been used to treat retroperitoneal fibrosis and EPS associated with PD. Hence, we used in vitro and animal model approaches to evaluate the efficacy of Tamoxifen to inhibit the MMT as a trigger of peritoneal fibrosis. In vitro studies were carried out using omentum-derived mesothelial cells (MCs) and effluent-derived MCs. Tamoxifen blocked the MMT induced by transforming growth factor (TGF)-β1, as it preserved the expression of E-cadherin and reduced the expression of mesenchymal-associated molecules such as snail, fibronectin, collagen-I, α-smooth muscle actin, and matrix metalloproteinse-2. Tamoxifen-treatment preserved the fibrinolytic capacity of MCs treated with TGF-β1 and decreased their migration capacity. Tamoxifen did not reverse the MMT of non-epitheliod MCs from effluents, but it reduced the expression of some mesenchymal molecules. In mice PD model, we demonstrated that MMT progressed in parallel with peritoneal membrane thickness. In addition, we observed that Tamoxifen significantly reduced peritoneal thickness, angiogenesis, invasion of the compact zone by mesenchymal MCs and improved peritoneal function. Tamoxifen also reduced the effluent levels of vascular endothelial growth factor and leptin. These results demonstrate that Tamoxifen is a therapeutic option to treat peritoneal fibrosis, and that its protective effect is mediated via modulation of the MMT process.     

腹透液相关浓度葡萄糖对人腹膜间皮细胞端粒长度的影响及葛根素的拮抗作用

目的:研究腹透液相关浓度葡萄糖(1.5%、2.5%)对人腹膜间皮细胞端粒长度的影响及葛根素的拮抗作用.方法:人腹膜间皮细胞在含糖(1.5%、2.5%)、含糖含葛根素(葛根素终浓度为100μg/ml)的无血清培养基培养24h后,收集细胞,提取DNA,采用Southerm杂交测定细胞端粒长度.结果:经含糖1.5%和2.5%培养基培养24h后腹膜间皮细胞大多出现肥大.高浓度葡萄糖组细胞端粒长度缩短,2.5%组和1.5%组无显著差别(P>0.05).葛根素加入后含糖组腹膜间皮细胞端粒长度延长(P<0.05).结论:高糖可使间皮细胞端粒缩短,且可能与高糖作用下细胞肥大有关,葛根素可拮抗高糖对间皮细胞端粒长度的影响.     

MicroRNA-7 Inhibits Epithelial-to-Mesenchymal Transition and Metastasis of Breast Cancer Cells via Targeting FAK Expression

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix integrin signaling, cell motility, cell proliferation and cell survival. Increased FAK expression is observed in a variety of solid human tumors and increased FAK expression and activity frequently correlate with metastatic disease and poor prognosis. Herein we identify miR-7 as a direct regulator of FAK expression. miR-7 expression is decreased in malignant versus normal breast tissue and its expression correlates inversely with metastasis in human breast cancer patients. Forced expression of miR-7 produced increased E-CADHERIN and decreased FIBRONECTIN and VIMENTIN expression in breast cancer cells. The levels of miR-7 expression was positively correlated with E-CADHERIN mRNA and negatively correlated with VIMENTIN mRNA levels in breast cancer samples. Forced expression of miR-7 in aggressive breast cancer cell lines suppressed tumor cell monolayer proliferation, anchorage independent growth, three-dimensional growth in Matrigel, migration and invasion. Conversely, inhibition of miR-7 in the HBL-100 mammary epithelial cell line promoted cell proliferation and anchorage independent growth. Rescue of FAK expression reversed miR-7 suppression of migration and invasion. miR-7 also inhibited primary breast tumor development, local invasion and metastatic colonization of breast cancer xenografts. Thus, miR-7 expression is decreased in metastatic breast cancer, correlates with the level of epithelial differentiation of the tumor and inhibits metastatic progression.     

促红细胞生成素对腹膜间皮细胞凋亡的保护作用

目的:观察重组人促红细胞生成素(recombinant human erythropoietin,rHuEPO)对腹膜间皮细胞(PMCs)的抗凋亡作用及其机制.方法:体外分离小鼠PMCs并给予rHuEPO干预,观察起对JAK2,STAT5,ERK1/2和P38磷酸化的影响.体内试验采用5000U/kg的rHuEPO顸处理后再予以4.25%的腹透液腹腔内注射后4小时,取小鼠脏层腹膜组织检测活性凋亡蛋白酶-3表达.结果:小鼠PMCs上表达EPO受体mRNA和蛋白,5 U/mL的rHuEPO可诱导PMCs的JAK2,STAT5和ERK1/2磷酸化上调.rHuEPO预处理1小时后再予腹透液处理的PMCs较单独予腹透液处理的PMCs,凋亡蛋白酶-3活化降低,DNA碎裂减少.rHuEPO干预可提高ERK1/2磷酸化水平而抑制腹透液诱导的P38磷酸化.特异性的ERK活化抑制剂PD98059可完全阻断rHuEPO的保护作用.通过检测凋亡蛋白酶-3证实rHuEPO可在小鼠体内降低腹透液引起的PMCs凋亡水平.结论:本研究为rHuEPO在腹透临床治疗中的临床应用开辟了新途径,rHuEPO可通过ERK1/2依赖途径减少PMCs的凋亡发挥保护性作用.rHuEPO及其衍生物可能成为维护腹膜完整性的新治疗手段.     

高浓度葡萄糖诱导人晶状体上皮细胞发生EMT

目的:观察高浓度葡萄糖诱导人晶状体上皮细胞发生上皮-间质转分化(epithelial-to-mesenchymal transition,EMT)。方法:将人晶状体上皮细胞HLE-B3系分别培养在正常葡萄糖浓度(5.5 mmol/L)DMEM培养基和高浓度葡萄糖(35.5 mmol/L)的DMEM培养基中24小时,于培养的0 h、3 h、6 h、12 h、24 h在倒置显微镜下观察细胞形态学变化,采用免疫荧光染色检测晶状体上皮细胞中EMT相关蛋白E-cadherin及α-SMA的表达变化。结果:与正常糖浓度组相比,随着时间的延长高糖组细胞逐渐丢失上皮细胞形态,细胞变细、变长,向纤维细胞的形态转变;同时随着时间的延长,高糖组晶状体上皮细胞中E-cadherin染色的荧光强度在各时间点均低于正常糖浓度组,而α-SMA的荧光强度却明显高于正常糖浓度组,在6 h和12 h时差异显著,有统计学意义(P0.01)。结论:高浓度葡萄糖诱导人晶状体上皮细胞发生上皮-间质转分化。     

High Adhesion of Tumor Cells to Mesothelial Monolayers Derived from Peritoneal Wash of Disseminated Gastrointestinal Cancers

The role of the mesothelial layer in the peritoneal spreading of cancer cells is only partially clarified. Here we attempted to better define the mesothelial contribution to the tumor cell adhesion using a direct adhesion test applied to human primary cultures of mesothelial cells (HPMCs) derived from the peritoneal washes of patients with gastric and colorectal cancers. Gastric and colon carcinoma cells were seeded on different mesothelial monolayers and quantitative fluorescence analysis was performed to analyze their growth and adhesive properties. The adhesion of the cancer cells was not affected by the origin of the HPMCs when derived from patients with different cancers or with benign disease. In contrast, the high levels of ICAM1 expression and ROS production, which characterize these senescent mesothelial cells, enhanced the tumor cell adhesion. These results suggest that the mesothelial adhesive properties are dependent on the cell senescence, while are not affected by the tumor environment. The use of peritoneal washes as a source to isolate HPMCs provides a practical and reliable tool for the in vitro analysis of the mesothelial conditions affecting the peritoneal carcinomatosis.     

miR-9 在卵巢癌细胞上皮间质转化中的初步研究

目的:研究miR-9在卵巢癌细胞上皮间质转化(EMT)中的作用。方法:上调或者下调miR-9后,在RNA水平上通过RT-qPCR检测卵巢癌细胞系SKOV3和A2780中上皮指标E-cadherin表达变化;在蛋白水平,通过western blotting方法检测2株细胞系中上皮指标E-cadherin和间质指标vimentin蛋白表达变化。生物信息学预测可能靶向E-cadherin 3'UTR的miR NA,双荧光素酶报告系统进一步验证miR-9靶向结合E-cadherin的3'UTR区。结果:上调miR-9后,卵巢癌细胞系中E-cadherin表达受到明显抑制,vimentin表达明显增加;反之,下调miR-9后,E-cadherin表达明显增高,vimentin表达明显降低。通过生物信息学预测发现miR-9可以直接靶向E-cadherin的3'UTR区,荧光素酶报告系统验证预测结果正确。结论:miR-9促进卵巢癌细胞上皮间质转化。     

Mitochondrial Permeability Transition Induced by Different Concentrations of Zinc

Zinc is one of the required trace elements in animals, and it serves an important role in biological systems. However, high levels of zinc are poisonous to organisms. So far, there exist conflicting reports about zinc ions-induced mitochondrial permeability transition (MPT). We analyzed the effects of Zn²⁺ on MPT by monitoring mitochondrial swelling with the ultraviolet–visible light absorption spectrum, characterizing the fluidity of the membrane with fluorescence anisotropy, detecting the transmembrane potential (Δψ) with fluorescence intensity, and observing mitochondrial ultrastructure with transmission electron microscopy. Data reveal that low concentrations of zinc ions can trigger MPT while high levels of zinc ions cannot, which implies that zinc ions’ toxicity cannot be the result of a single simple mechanism.     

Protective Effects of Salvianolic Acid B on Schwann Cells Apoptosis Induced by High Glucose

Diabetic peripheral neuropathy (DPN) is one of the most common and debilitating microvascular complications of diabetes, and there is no effective therapy for the prevention or treatment of DPN. Oxidative stress triggers several pathways of injury and may be the unifying factor of hyperglycemia. The aim of this study was to investigate protective effect of Salvianolic acid B (Sal B) on the high glucose (HG)-induced oxidative stress-induced mitochondrial pathway activation and Schwann cells (SCs) apoptosis in vitro. We found that Sal B inhibited the HG-induced oxidative stress by reducing ROS and 8-hydroxy-2-deoxy Guanosine (8-OHdG) production, and mitochondrial depolarization and apoptosis in SCs in a dose-dependent manner. Furthermore, Sal B down-regulated the HG-mediated Bax expression and AIF nuclear translocation and the release of cytochrome c, but up-regulated the HG-induced BcL-2 expression in SCs. In addition, Sal B attenuated the HG-induced activation of caspase 3 and 9 and minimized the cleavage of PARP in SCs. Our results indicated that Sal B antagonized the HG-induced oxidative stress, activation of the mitochondrial pathway and apoptosis in SCs.