霉酚酸酯对糖尿病肾病大鼠肾组织TGF-β1和CTGF的影响

目的探讨免疫抑制剂霉酚酸酯对糖尿病肾病大鼠肾组织转化生长因子-β1（TGF-β1）和结缔组织生长因子（CTGF）表达的影响和肾保护作用。方法应用STZ造成糖尿病肾病模型,将SD大鼠随机分为健康对照组、糖尿病肾病对照组和糖尿病肾病霉酚酸酯干预组。观察血糖、肾功能、24h尿蛋白排泄量变化。2月后处死,免疫组化检测肾脏TGF-β1、CTGF的表达。结果与健康对照组（A组）相比,DN对照组（B组）和霉酚酸酯干预组（C组）的血糖、肾重／体重比、尿蛋白水平明显上升。应用免疫组化方法可见TGF-β1在小球中有散在表达,在B组和C组小球中的表达均明显高于A组。C组较B组显著下降。在小管中成片表达,B组和C组亦较A组明显增多,C组较B组亦呈显著下降趋势。CTGF在正常小球中几乎不表达,B组和C组小球中表达明显增多。小管中各组CTGF均明显表达,B组和C组均较A组显著增加,C组较B组有所下降。TGF-β1、CTGF的表达与尿蛋白、肾重体重比呈正相关,且两因子之间亦呈正相关。结论1、MMF可抑制糖尿病肾病大鼠模型TGF-β1、CTGF在肾脏的过度表达;2、MMF可减少尿蛋白,减轻肾脏肥大,在一定程度上改善糖尿病肾病病理改变,其改善作用可能和抑制TGF-β1、CTGF在肾脏的过度表达有关。     

红芪多糖对早期糖尿病肾病db/db小鼠肾间质纤维化的影响

目的通过研究红芪多糖(HPS)对早期糖尿病肾病(DN)db/db小鼠肾组织中转化生长因子-β1(TGF-β1)与结缔组织生长因子(CTGF)表达的影响,探讨HPS对早期DN小鼠肾脏的保护作用机制。方法 SPF级6周龄雄性小鼠60只,其中db/db小鼠50只,用随机数字表法分为5组:HPS高、中、低剂量组(200、100、50 mg/kg HPS溶液灌胃)、替米沙坦组(5 mg/kg替米沙坦溶液灌胃)、模型组(等体积蒸馏水灌胃),每组10只;db/m小鼠10只,作为正常对照组,给予等体积蒸馏水灌胃。连续灌胃8周,于治疗前及治疗后每2周检测血糖浓度,第4周末及第8周末收集小鼠24 h尿液,ELISA法检24 h尿微量白蛋白水平。框后静脉取血,血清用于检测血清肌酐(Scr)、尿素氮(BUN)。处死小鼠,剥离肾脏,左肾皮质用于HE染色,观察肾脏病理学变化;右肾皮质用于RT-PCR、Western blotting检测肾组织TGF-β1及CTGF mRNA和蛋白的表达量。结果与正常组比较,模型组小鼠血糖、Scr、BUN 24 h尿微量白蛋白水平显著升高(P0.01);肾小球肥大,系膜区明显增宽,毛细血管基底膜增厚;TGF-β1及CTGF mRNA和蛋白的表达水平显著升高(P0.01)。与模型组比较,除HPS低剂量组外(P0.05),其余各治疗组小鼠血糖、Scr、BUN和24 h尿微量白蛋白水平均明显下降(P0.05);肾小球肥大,系膜区明显增宽,毛细血管基底膜增厚情况均有不同程度改善;TGF-β1及CTGF mRNA和蛋白的表达水平明显降低(P0.01);HPS中剂量组疗效明显优于替米沙坦组。结论 HPS能够防治db/db小鼠早期DN,其机制可能与HPS抑制TGF-β1及CTGF mRNA在肾脏的表达有关。     

老年慢性心力衰竭患者血浆CTGF、iPTH、甲状腺激素水平与心功能的相关性

 目的 探讨老年慢性心力衰(chronic heart failure,CHF)竭患者血浆CTGF、iPTH、甲状腺激素、NT-proBNP及LVMI与心功能的相关性。方法 选取≥60岁CHF患者120例,分为心功能Ⅱ级、Ⅲ级、Ⅳ级组,各40例;选取同期住院体检的健康老人40例作为对照组。比较四组血浆CTGF、iPTH、甲状腺激素水平、NT-proBNP及LVMI的差异,采用Spearman等级相关分析CHF患者血浆CTGF、iPTH、甲状腺激素水平、NT-proBNP、LVMI与心功能分级的相关性。结果 CHF心功能Ⅱ级组、Ⅲ级组、Ⅳ级组血浆CTGF、iPTH、NT-proBNP、LVMI水平明显高于对照组(P＜0.01),且随心功能分级的升高,血浆CTGF、iPTH、NT-proBNP、LVMI水平明显升高 (P＜0.05);心功能Ⅱ、Ⅲ、Ⅳ级三组患者FT3水平明显均低于对照组(P＜0.01),且随心功能分级的升高,FT3水平明显降低 (P＜0.05)。血浆CTGF、iPTH、NT-proBNP、LVMI与心功能分级呈正相关(ρ>0.6,P＜0.05);FT3与心功能分级呈负相关 (ρ<-0.6,P＜0.05)。CHF患者血浆CTGF、NT-proBNP、iPTH均与LVMI呈正相关(r>0.6, P＜0.05);FT3与LVMI呈负相关(r<-0.6, P＜0.05)。结论 血浆CTGF、iPTH、FT3、NT-proBNP及LVMI与老年CHF患者心功能分级相关性较强,可作为CHF临床诊断和病情评估的重要指标。     

木犀草素通过CTGF/EGFR通路对人结肠癌细胞LoVo增殖和凋亡的影响

目的探讨木犀草素通过结缔组织生长因子(CTGF)/表皮生长因子受体(EGFR)通路对人结肠癌细胞LoVo增殖和凋亡的影响。方法MTT法确定木犀草素半数抑制质量浓度、作用时间。将细胞实验分为阴性对照组、阳性对照组、木犀草素低、中、高剂量组。阴性对照组细胞不进行处理,阳性对照组用40 μg/L西妥昔单抗干预,木犀草素低、中、高剂量组分别用20、40、80 μg/L木犀草素干预。采用流式细胞术检测各组细胞凋亡率,蛋白印迹法检测细胞CTGF、EGFR、蛋白质丝氨酸苏氨酸激酶(Akt)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸酶3(Caspase-3)蛋白水平。结果MTT法确定木犀草素半数抑制质量浓度40 μg/L,半数抑制时间24 h。随着木犀草素质量浓度增加,LoVo细胞增殖率降低(P＜0.05)。与阴性对照组比较,其余各组细胞凋亡率、Bax和Caspase-3蛋白均增加,细胞CTGF、p-EGFR/EGFR、Akt和Bcl-2蛋白均降低(P＜0.05)。与阳性对照组比较,木犀草素各剂量组细胞凋亡率、Bax和Caspase-3蛋白均降低,细胞CTGF、p-EGFR/EGFR、Akt和Bcl-2蛋白均增加,且随木犀草素剂量增加呈剂量效应关系(P＜0.05)。结论木犀草素抑制LoVo细胞增殖,诱导LoVo细胞凋亡,其机制可能与抑制CTGF/EGFR通路的激活有关。     

Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts

Purposes: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl₂ to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. Materials and methods: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. Results: The real-time PCR results show that mechanical stretch or CoCl₂ treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl₂-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl₂ treatment group alone. Western blot analysis and ELISA also confirmed these results. Conclusions: Our results demonstrated that mechanical stretch and CoCl₂-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.     

Loss of tumour suppressor PTEN expression in renal injury initiates SMAD3‐ and p53‐dependent fibrotic responses

Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)‐mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK‐2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI‐1, vimentin, α‐SMA and fibronectin expression, compared to HK‐2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53^Ser15 phosphorylation, with an accompanying decrease in population density and an increase in epithelial G₁ cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI‐1 rescued the PTEN loss‐associated epithelial proliferative arrest. Moreover, TGFβ1‐initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53‐dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3‐ and p53‐mediated fibrotic gene induction, with accompanying PAI‐1‐dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Acellular dermal matrix scaffolds coated with connective tissue growth factor accelerate diabetic wound healing by increasing fibronectin through PKC signalling pathway

The regional injection of connective tissue growth factor (CTGF) for diabetic wound healing requires multiple components and results in a substantial loss of its biological activity. Acellular dermal matrix (ADM) scaffolds are optimal candidates for delivering these factors to local ischaemic environments. In this study, we explored whether CTGF loaded on ADM scaffolds can enhance fibronectin (FN) expression to accelerate diabetic wound healing via the protein kinase C (PKC) signalling pathway. The performance of CTGF and CTGF + PKC inhibitor, which were loaded on ADM scaffolds to treat dorsal skin wounds in streptozotocin‐induced diabetic mice, was evaluated with naked ADM as a control. Wound closure showed that ADM scaffolds loaded with CTGF induced greater diabetic wound healing in the early stage of the wound in diabetic mice. Moreover, ADM scaffolds loaded with CTGF obviously increased the expression of FN both at the mRNA and protein levels, whereas the expression of FN was significantly reduced in the inhibitor group. Furthermore, the ADM + CTGF group, which produce FN, obviously promoted alpha‐smooth muscle actin and transforming growth factor‐beta expression and enhanced neovasculature and collagen synthesis at the wound sites. ADM scaffolds loaded with CTGF + PKC inhibitor delayed diabetic wound healing, indicating that FN expression was mediated by the PKC signalling pathway. Our findings offer new perspectives for the treatment of diabetic wound healing and suggest a rationale for the clinical evaluation of CTGF use in diabetic wound healing.     

CTGF和TIMP1双基因体外联合转染恒河猴腰椎间盘细胞对II型胶原和蛋白多糖的影响

目的 建立恒河猴腰椎间盘细胞体外培养模型,研究腺相关病毒（adeno-associated virus,AAV）介导的结缔组织生长因子（connective tissue growth factor,CTGF）和基质金属蛋白酶组织抑制因子1(tissue inhibitor of metalloproteinases 1,TIMP1) 双基因体外联合转染恒河猴椎间盘细胞较单基因对II型胶原和蛋白多糖合成的影响的变化,以探讨体外延缓椎间盘细胞退变的方法。方法 应用酶消化法培养恒河猴腰椎间盘髓核细胞,以感染复数（MOI） 为106的rAAV2-CTGF-IRES-TIMP1及rAAV2-CTGF、rAAV2-TIMP1分别感染髓核细胞,应用Western blot鉴定和RT-PCR检测II型胶原及蛋白多糖mRNA的表达,35S整合法性行蛋白多糖合成率的测定,SP-ABC免疫组化法检测Ⅱ型胶原含量。结果 rAAV2-CTGF-IRES-TIMP1双基因及rAAV-CTGF、rAAV-TIMP1单基因能够转染恒河猴椎间盘髓核细胞并在其内表达细胞因子。与对照组相比,CTGF可以促进II型胶原及蛋白多糖的合成,TIMP1可以促进蛋白多糖的合成,对II型胶原的合成未见到明显作用;双基因联合感染可以显著促进髓核细胞蛋白多糖和II型胶原的合成。结论 CTGF和TIMP1 单基因转染均可以促进蛋白多糖的合成,CTGF 对 II型胶原的合成亦有促进作用;双基因联合感染可以明显促进蛋白多糖和II型胶原的合成,其效果优于单基因,为多基因治疗椎间盘退变奠定了良好的基础。