Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type Ⅳ

AIM： To investigate the regulation of activin receptor-interacting protein 2 （ARIP2） expression and its possible relationships with collagen type Ⅳ （collagen Ⅳ） in mouse hepatoma cell line Hepal-6 cells. METHODS： The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate （PMA）, forskolin and A23187. After pcDNA3- ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor （ActRⅡ） and collagen Ⅳ expression were evaluated. RESULTS： The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation （2 or 4 h）. The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 （25.3% ± 5.7% vs 48.1% ± 3.6%, P 〈 0.01）. ARIP2 overexpression could remarkably suppress the expression of ActRⅡA mRNA in dose-dependent manner, but has no effect on ActRⅡB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION： These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

The sex determination gene doublesex regulates expression and secretion of the basement membrane protein Collagen Ⅳ

The highly conserved doublesex(dsx) and doublesex/mab-3 related(Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx^(M) in males and Dsx^(F) in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane(BM) protein Collagen Ⅳ in the fat body. We find that Dsx isoforms regulate the expression of Collagen Ⅳ in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase(dPlod) gene, which is involved in the biosynthesis of Collagen Ⅳ, as a direct target of Dsx. We further show that Dsx regulates Collagen Ⅳ through d Plod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen Ⅳ and remotely establish sexual dimorphism.

基于上皮细胞-间充质细胞转分化(EMT)理论的艾灸配合化纤Ⅳ号方对实验大鼠Collagen Type Ⅲ和PDGF干预作用实验研究

目的:基于上皮细胞-间充质细胞转分化(EMT)学说观察化纤Ⅳ号方、艾灸以及二者相配合治疗肺纤维化大鼠Collagen TypeⅢ(Ⅲ-C)和PDGF的变化,探讨其治疗效应及生物学机制。方法:将鼠龄约为6周的SD大鼠随机分为空白组、模型组、化纤Ⅳ号方组、艾灸组、化纤Ⅳ号方与艾灸配合治疗组(简称为"灸药组"),治疗30 d后处死观察其肺组织病理改变,并检测其Collagen TypeⅢ、PDGF的基因和蛋白表达情况。结果:实时荧光定量结果显示:与空白组相比,各组Ⅲ-C和PDGF m RNA表达增高(P<0.05)。与模型组相比,各组的Ⅲ-C和PDGF m RNA表达有明显降低(P<0.01)。而各组中,灸药组疗效最明显,Ⅲ-C和PDGF的表达最低。蛋白免疫印迹法检测结果显示:与模型组相比各组的Ⅲ-C蛋白表达有差异。结论:1艾灸、化纤Ⅳ号方均可减轻博莱霉素诱导肺纤维化大鼠的肺纤维化程度。2艾灸配合化纤Ⅳ号方可减轻博莱霉素诱导肺纤维化大鼠的肺纤维化程度,且其效果优于单用艾灸或单用化纤Ⅳ号方。3艾灸、化纤Ⅳ号方及其二者配合使用不同程度阻抑博莱霉素诱导肺纤维化大鼠肺纤维化进程的效应机制,可能与通过调控其EMT过程中的Ⅲ-C和PDGF表达环节紧密相关。

1型糖尿病大鼠肾脏病变与ADPN、TGF-β1、collagen Ⅳ、ICAM-1的关系

目的探讨脂联素、TGF-β1、collagenⅣ、ICAM-1在1型糖尿病肾脏病变发生及发展中的变化及作用。方法大鼠随机分为正常对照组和糖尿病组,每组分别于4周和8周,测24h尿白蛋白排泄量(UPro/24h)、空腹血浆葡萄糖、血肌酐、尿素氮、血清脂联素(ADPN)、肾重/体重,观察肾脏TGF-β1、Ⅳ型胶原蛋白(collagenⅣ)、ICAM-1的表达。结果糖尿病大鼠UPro/24h增高、TGF-β1、collagenⅣ、ICAM-1的表达增加、脂联素水平降低。结论 TGF-β1、collagenⅣ、ICAM-1在肾脏组织中表达水平与血清ADPN呈负相关。随着ADPN水平降低,TGF-β1、collagenⅣ、ICAM-1在肾脏组织的表达增加。

Collagen type Ⅳ对周围神经中再生轴突及非神经元细胞的作用和影响

本文用抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ对抗体阻断ｃｏｌｌａｇｅｎｔｙｎｅⅣ的方法，研究了ｃｏｌｌａｇｅｎｔｙｐｅⅣ失活的移植神经段（长１０ｍｍ）植入大鼠坐骨神经后对再生轴突和非神经元细胞的作用和影响．实验结果显示：在移植神经段近端距近侧吻合口１ｍｍ处，术后１０ｄ抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突数为对照组的５４％，术后１５ｄ增加到６６％，术后３０ｄ高达９４％．在移植神经段远侧距近侧吻合口９ｍｍ处，术后３０ｄ抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突数为对照组的５８％。表明抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突的生长启动和生长速度明显慢于对照组．巨噬细胞在移植神经段内的滞留数量抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组明显多于对照组．这些结果揭示ｃｏｌｌａｇｅｎｔｙｐｅⅣ在神经损伤和再生中对促进轴突的生长和维持神经微环境的平衡起着积极的作用．本文对ｃｏｌｌａｇｅｎｔｙｐｅⅣ在神经再生中的作用机制作了初步的分析和探讨。

血管球瘤中FLi-1、h-caldesmon及collagen Ⅳ的表达及临床病理分析

目的探讨FLi-1、h-caldesmon及collagenⅣ在血管球瘤中的表达及不同组织学类型与临床病理指标的关系。方法采用免疫组化En Vision法检测35例血管球瘤中FLi-1、h-caldesmon及collagenⅣ的表达。分析35例血管球瘤不同组织学分型与临床病理参数间的关系。结果镜下见肿瘤细胞圆形或多边形,呈片状分布在血管之间或呈环状围绕在血管周围,瘤细胞边界清晰,外形规则,有时可见瘤细胞与梭形平滑肌细胞移行过渡。免疫表型:血管球瘤可表达FLi-1、h-caldesmon及collagenⅣ,阳性率分别为58.6%、97.1%和100%。血管球瘤的不同类型与患者性别和肿瘤体积无关,与患者年龄相关(P=0.01)。35例血管球瘤中vimentin和SMA均呈(+),2例CD34呈局灶(+),1例desmin呈(+),EMA、S-100、Cg A、CD68及CD99均呈(-)。结论 h-caldesmon和collagenⅣ可作为血管球瘤诊断的标志物,FLi-1可作为一种辅助参考标志物,有助于血管球瘤的诊断。

Sublytic C5b-9诱导肾小球系膜细胞产生的TSP-1和TGF-β1对其合成FN和collagenⅣ的影响

目的:探讨亚溶解型(sublytic)C5b-9复合物刺激大鼠肾小球系膜细胞(glomerular mesangial cell,GMC)后,诱生的血小板反应蛋白-1(thrombospondin-1,TSP-1)与转化生长因子-β1(transforming growth factor-β1,TGF-β1)对其促进细胞外基质(ex-tracellular matrix,ECM),如纤维连接蛋白(fibronectin,FN)和Ⅳ型胶原蛋白(collagenⅣ)合成的影响。方法:体外培养大鼠GMC,进行不同分组处理并给予sublytic C5b-9刺激,然后分别检查受刺激的GMC合成TSP-1和TGF-β1因子水平,同时检测GMC分泌FN和collagenⅣ的水平。此外,应用人工合成的TSP-1封闭肽段(GGWSHW)和TGF-β1中和抗体处理培养的大鼠GMC,研究TSP-1和TGF-β1在sublytic C5b-9诱导的GMC分泌上述ECM中的作用及其相互关系。结果:培养的大鼠GMC在sublytic C5b-9刺激后18 h,其FN和collagenⅣ表达水平均明显升高。同时,TSP-1蛋白表达和TGF-β1分泌(包括活化的TGF-β1含量)也显著增多。用TSP-1封闭肽段(GGWSHW)处理大鼠GMC后亦能显著抑制由sublytic C5b-9诱导的TGF-β1活化,并减少FN、collagenⅣ的产生。同样用TGF-β1中和抗体处理GMC后也能明显抑制由sublytic C5b-9导致的FN、collagenⅣ的分泌。结论:体外用sublytic C5b-9刺激大鼠GMC,能诱导其ECM分泌与TSP-1和TGF-β1的合成及活化,而sublytic C5b-9促进GMC分泌ECM的机制可能与其诱导TSP-1合成及活化的TGF-β1存在一定的关系。

芡实对糖尿病肾病大鼠肾组织MMP-9、TIMP-1及Collagen Ⅳ表达的影响

目的:探讨芡实对糖尿病肾病大鼠肾组织MMP-9、TIMP-1及CollagenⅣ表达的影响。方法:健康雄性Wistar大鼠65只,随机选取10只为正常对照组(N组);其余一次性腹腔注射链脲佐菌素(STZ)制作DN模型,造模成功后,随机分为DN组、芡实低、中、高剂量组及氯沙坦钾组,均采用灌胃给药,N组和DN组给予等体积蒸馏水。12周后检测大鼠生化指标;HE、Masson、PAS染色观察肾脏病理变化;免疫组化法测定MMP-9、TIMP-1及CollagenⅣ在肾组织表达情况;采用实时荧光定量PCR技术检测肾组织中MMP-9、TIMP-1及CollagenⅣ基因的表达。结果:实验12周末,与N组比较,DN组大鼠肾小球肥大,细胞外基质积聚,血糖、24 h尿蛋白定量、血尿素氮(BUN)、肌酐(Scr)、尿白蛋白排泄率(UAER)、尿蛋白/尿肌酐(Up/Ucr)比值显著升高(P<0.05),肾组织MMP-9蛋白和mRNA表达降低(P<0.05),TIMP-1、CollagenⅣ蛋白和mRNA表达升高(P<0.05);与DN组比较,EM组、EH组及LP组大鼠病理改变减轻,24 h尿蛋白定量、Scr、BUN、UAER、Up/Ucr显著降低(P<0.05),肾组织MMP-9蛋白和mRNA表达显著升高(P<0.05),TIMP-1、CollagenⅣ蛋白和mRNA表达显著减少(P<0.05)。结论:芡实可能通过调节DN大鼠肾脏MMP-9、TIMP-1的表达,减少细胞外基质积聚,从而减轻肾小球硬化,减少蛋白尿,保护肾功能,延缓DN病变的进展。

未知病变类型脑血管中β-amyloid、α-actin、collagen Ⅳ的含量

目的研究未知病变类型脑血管病变的结构特征。方法通过刚果红染色、免疫组织化学染色、计算机图像分析技术对未知病变类型脑血管病变的β-amyloid、α-actin、collagenⅣ的含量进行研究。结果未知病变类型脑血管壁α-actin、collagenⅣ呈少量阳性染色,与正常脑血管存在显著差异(P<0.05);β-amyloid染色呈阴性,与正常脑血管无差异(P>0.05)。病变血管壁中上述三种蛋白的表达特点与脑血管淀粉样变(cerebralamyloidangiopathy,CAA)及小动脉硬化玻璃样变不同。结论未知病变类型脑血管病变具有不同于CAA的病变特征。

Serum hyaluronic acid, procollagen type Ⅲ and Ⅳ in histological diagnosis of liver fibrosis

OBJECTIVE: To assess the significance of serum hyaluronic acid (HA), proeollagen type Ⅲ (PCⅢ), collagen type Ⅳ (CⅣ) in the histological diagnosis of liver fibrosis. METHODS: The concentrations of serum HA, PCⅢ, CⅣ in 253 patients with chronic liver diseases were measured by radioimmunoassay. Liver biopsies were performed in all patients at the same time. The liver was pathologically evaluated by a pathologist according to a scoring system. Combined with the results of liver pathological diagnosis, the accuracy of serum HA, PCⅢ, CⅣ in diagnosing patients with hepatic fibrosis (staging≥S_2) or cirrhosis (S_4) was assessed using the receiver operating curve (ROC). RESULTS: The cutoff values of serum HA, PCⅢ and CⅣ for identifying patients with hepatic fibrosis (≥S_2) or cirrhosis (S_4) were determined. The cutoff values of serum HA, PCⅢ and CⅣ for detecting patients with fibrosis (stage≥S_2) were 90μg/L, 90μg/L, 75μg/L, respectively; their sensitivity (Se) was 80.4%, 82%, 63.1%; their specificity (Spe) was 70.2%, 60.8%, 83.8%; their positive predictive values (PPV) were 86.7%, 83.5%, 90.4%; their negative predictive values (NPV) were 59.8%, 58.4%, 48.4%, respectively. The cutoff values for detecting patients with liver cirrhosis were 210μg/L for HA, 96.2% for Se, 85.3% for Spe, 65.4% for PPV, 98.8% for NPV; 150μg/L for PCⅢ, 76.4% for Se, 68.7% for Spe, 40.4% for PPV, 91.3% for NPV; 90μg/L for CⅣ, 80% for Se, 75.8% for Spe, 47.8% for PPV, 93.2% for NPV, respectively. CONCLUSIONS: Serum HA, PCⅢ and CⅣ can be determined for an accurate diagnosis of hepatic fibrosis in various stages. HA is the best for screening liver cirrhosis.

Dynamic changes of activator protein 1 and collagen I expression in the sclera of myopia guinea pigs

AIM: To investigate the dynamic changes of activator protein 1(AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6 wk(FDM group). Normal control group(n=25) were untreated. Changes in refractive power and axial length(AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction(RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6 wk, and 4/-1 wk of form-deprivation, the diopter in the FDM group was gradually changed(2.08±0.31,-1.23±0.68,-4.17±0.58,-7.07±0.55, and-2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased(5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated(all P<0.05);the mRNA expressions of them were also gradually down-regulated(all P<0.05);and there was positive correlation between them. The control group had no obvious change in each index(all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.

Effects of gentiana scabra bage on expression of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with liver fibrosis

Objective:To exploit- the effects of gentiana scabra bage on the expression of hepatic collagen proteins in Paragonimus skrjabini rats with liver fibrosis.Methods:Immunohistochemical technique was used to observe the changes of content of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with Liver fibrosis before and after the gentiana scabra bage treatmeat.Results:Comparing with the model group,changes of hepatic tvpe Ⅰ and type Ⅲ collagen proteins in gentiana scabra bage treated group were significantly weakened.Conclusions:Gentiana scabra bage treatment can reduce the content of hepatic type Ⅲ and type Ⅰ collagen protein significantly in Paragonimus skrjabini rats with liver fibrosis,thereby,playing the role against hepatic fibrosis.

Adhesive properties of hepatoma cells to collagen Ⅳ coated surfaces

Objectives: To quantitatively study the adhesive pro- perties of hepatoma cells to collagen Ⅳ coated artifi- cial basement membrane and to investigate the rele- vance of cell adhesive forces to the concentration of collagen Ⅳ. Methods: Synchronous G1 and S phase cells were a- chieved using thymine-2-desoxyriboside and cochicine sequential blockage method and double thymine-2- desoxyriboside blockage method respectively. The adhesive forces of hepatoma cells were investigated by micropipette aspiration technique. Results: The adhesive forces of hepatoma cells to ar- tificial basement membrane were (107.78±65.44) ×10^(-10)N, (182.60±107.88)×10^(-10)N, (298.91± 144.13)×10^(-10)N when the concentration of the membrane coated by 1, 2, 5μg/ml collagen Ⅳ re- spectively (P<0.001). The adhesive forces of G1 and S phases hepatoma cells to artificial basement membrane were (275.86±232.80)×10^(-10)N and (161.16±120.40)×10^(-10)N respectively when the concentration of the membrane coated by 5μg/ml collagen Ⅳ (P<0.001). Conclusions: The adhesive forces of hepatoma cells to artifical basement membrane in direct proportion to the concentration of collagen Ⅳ suggests that the in- crease of basement membrane might be conducive to the chemotactic motion and adhesiveness of tumor cells. G1 phase cells are more capable of adhering to basement membrane than S phase cells. Hepatoma cells, especially G1 phase cells, may survive in blood circulation, and sequest and adhere in microcircula- tion, and get through basement membrane for re- mote metastasis.

Expression of Collagen Ⅳ, Fibronectin, Laminin in Non-small Cell Lung Cancer and Its Correlation with Chemosensitivities and Apoptosis~*

Objective： To study the expression of extracellular matrix （ECM） proteins including Collagen Ⅳ （Co Ⅳ）, Fibronectin, Laminin in human non-small cell lung cancer （NSCLC） specimens and the relationship between ECM and cell adhesion, proliferation, apoptosis and drug sensitivity in NSCLC cell line. And to investigate the role of phosphatidylinositol 3-kinase （PI3-K） in signal transduction of Co Ⅳ in NSCLC. Methods： The expression of ECM proteins was detected by using immunohistochemical staining （Envision＇s）. Adherent cells were stained with 1% methylene blue. Cell proliferation and cytotoxic effects were monitored by MTT assay. Cell apoptosis was analyzed by FITC-Annexin V/PI double staining variables flow cytometry （FCM）. Results： The expression rate of Co Ⅳ （93%） was the highest compared to others in NSCLC stroma. After treated with Co Ⅳ, the adhesion of H1299 cells was increased and the cytotoxicity of cis-platinum （DDP） against H1299 cells was decreased compared to the control （P〈0.05）. After treated with Co Ⅳ both survival and proliferation rates were higher and apoptosis rate was lower than without Co Ⅳ （P〈0.05）. PI3-K inhibitor LY294002 decreased both survival and proliferation rates （82.7%±2.0% and 75.2%±6.8%, respectively）, even on Co Ⅳ-coated surface （92.2%±2.8% and 84.6%±9.2%, respectively）. And it also helped DDP increase apoptosis. Conclusion： ECM remodeling existed in NSCLC. Co Ⅳ protected NSCLC cells from DDP-induced apoptosis and weakened the cytotoxicity of DDP. PI3-K pathway might be the crucial mechanism of apoptosis impairment and drug resistance.

Serum type Ⅳ collagen level is predictive for esophageal varices in patients with severe alcoholic disease

AIM： To determine factors predictive for esophagea varices in severe alcoholic disease （SAD）. METHODS： Abdominal ultrasonography （US） was performed on 444 patients suffering from alcoholism. Forty-four patients found to have splenomegaly and/ or withering of the right liver lobe were defined as those with SAD. SAD patients were examined by upper gastrointestinal （UGI） endoscopy for the presence of esophageal varices. The existence of esophageal varices was then related to clinical variables. RESULTS： Twenty-five patients （56.8%） had esophageal varices. A univariate analysis revealed a significant difference in age and type Ⅳ collagen levels between patients with and without esophageal varices. A logistic regression analysis identified type Ⅳ collagen as the only independent variable predictive for esophageal varices （P = 0.017）. The area under the curve （AUC） for type Ⅳ collagen as determined by the receiver operating characteristic （ROC） for predicting esophageal varices was 0.78. CONCLUSION： This study suggests that the level of type Ⅳ collagen has a high diagnostic accuracy for the detection of esophageal varices in SAD.

Pre-hepatectomy type Ⅳ collagen 7S predicts post-hepatectomy liver failure and recovery

BACKGROUND Liver resection is an effective treatment for benign and malignant liver tumors.However,a method for preoperative evaluation of hepatic reserve has not yet been established.Previously reported assessments of preoperative hepatic reserve focused only on liver failure in the early postoperative period and did not consider the long-term recovery of hepatic reserve.When determining eligibility for hepatectomy,the underlying pathophysiology needs to be considered to determine if the functional hepatic reserve can withstand both surgery and any postoperative therapy.AIM To identify pre-hepatectomy factors associated with both early postoperative liver failure and long-term postoperative liver function recovery.METHODS This study was a retrospective cohort study.We retrospectively investigated 215 patients who underwent hepatectomy at our hospital between May 2013 and December 2016.Early post-hepatectomy liver failure(PHLF)was defined using the International Study Group of Liver Surgery’s definition of PHLF.Long-term postoperative recovery of liver function was defined as the time taken for serum total bilirubin and albumin levels to return to levels of<2 mg/dL and>2.8 g/dL,respectively,and the time taken for Child-Pugh score to return to Child-Pugh class A.RESULTS Preoperative type IV collagen 7S was identified as a significant independent factor associated with both PHLF and postoperative long-term recovery of liver function.Further analysis revealed that the time taken for the recovery of Child-Pugh scores and serum total bilirubin and albumin levels was significantly shorter in patients with type IV collagen 7S≤6 ng/mL than in those with type IV collagen 7S>6 ng/mL.In additional analyses,similar results were observed in patients without chronic viral hepatitis associated with fibrosis.CONCLUSION Preoperative type IV collagen 7S is a preoperative predictor of PHLF and longterm postoperative liver function recovery.It can also be used in patients without chronic hepatitis virus.

Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp(Ctenopharyngodon idella)

Background:Nutrient regulation has been proven to be an effective way to improve the flesh quality in fish.As a necessary nutrient for fish growth,protein accounts for the highest proportion in the fish diet and is expensive.Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content,the mechanism for this effect has not been deeply explored.Moreover,few studies have explored the protein requirements of sub-adult grass crap(Ctenopharyngodon idella).Therefore,the effects of different dietary protein levels on the growth performance,nutritional value,muscle hardness,muscle fiber growth,collagen metabolism and related molecule expression in grass carp were investigated.Methods:A total of 450 healthy grass carp(721.16±1.98 g)were selected and assigned randomly to six experimen-tal groups with three replicates each(n=25/replicate),and were fed six diets with 15.91%,19.39%,22.10%,25.59%,28.53%and 31.42%protein for 60 d.Results:Appropriate levels of dietary protein increased the feed intake,percentage weight gain,specific growth rate,body composition,unsaturated fatty acid content in muscle,partial free amino acid content in muscle,and muscle hardness of grass carp.These protein levels also increased the muscle fiber density,the frequency of new muscle fibers,the contents of collagen and IGF-1,and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase,and decreased the activity of matrix metalloproteinase-2.At the molecular level,the optimal dietary protein increased col-lagen type Iα1(Colα1),Colα2,PI3K,Akt,S6K1,La ribonucleoprotein domain family member 6a(LARP6a),TGF-β1,Smad2,Smad4,Smad3,tissue inhibitor of metalloproteinase-2,MyoD,Myf5,MyoG and MyHC relative mRNA levels.The levels of the myostatin-1 and myostatin-2 genes were downregulated,and the protein expression levels of p-Smad2,Smad2,Smad4,p-Akt,Akt,LARP6 and Smad3 were increased.Conclusions:The appropriate levels of dietary protein promoted the growth of sub-adult grass carp and improved muscle hardness by promoting the growth of muscle fibers,improving collagen synthesis and depressing collagen degradation.In addition,the dietary protein requirements of sub-adult grass carp were 26.21%and 24.85%according to the quadratic regression analysis of growth performance(SGR)and the muscle hardness(collagen content),respectively.

Expression and role of specificity protein 1 and collagenⅠin recurrent pterygial tissues

AIM:To investigate the expression profiles of the transcription factor specificity protein 1(Sp1)and collagenⅠin recurrent pterygial tissues.What is more,to compare the changes of Sp1 and collagen I among primary pterygial tissue,recurrent pterygial tissue and conjunctival tissue.METHODS:In the prospective study,we collected the pterygial tissues of 40 patients who underwent resection of primary pterygial tissue and recurrent pterygial tissue,and the conjunctival tissues of 10 patients with enucleation due to trauma.The relative expression levels of Sp1 and collagen I were analyzed by reverse transcription quantitative-polymerase chain reaction and Western blot.Paired t-test was performed to compare the Sp1 and collagen I of recurrent pterygial tissues,as well as the primary pterygial tissues and conjunctival tissues.In further,Pearson’s hypothesis testing of correlation coefficients was used to compare the correlations of Sp1 and Collagen I.RESULTS:The content of Sp1 and collagen I m RNA and protein was significantly greater in recurrent pterygial tissue than that was in primary and conjunctival tissue(P<0.05).There was a positive correlation between the m RNA and protein levels of Sp1 and collagen I in recurrent pterygial tissues(protein:r=0.913,P<0.05;m RNA:r=0.945,P<0.05).CONCLUSION:Sp1 and collagen I are expressed in normal conjunctival,primary,and recurrent pterygial tissues,but expression is significantly greater in the latter.Sp1 and collagen I may be involved in the regulation of the development of recurrent pterygium.

All-trans Retinoic Acid Diminishes Collagen Production in a Hepatic Stellate Cell Line via Suppression of Active Protein-1 and c-Jun N-terminal Kinase Signal

Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.

Effects of Calcium Dobesilate on Glomerulus TIMP1 and Collagen Ⅳ of Diabetic Rats

Summary ： To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 （TIMP1）, collagen Ⅳ , and ultrastrueture of glomerular basement mem- brane in diabetic rats, rats model of diabetes was established by unilateral nephreetomy and intraperitoneal injection of 1% STZ （55 mg/kg）, and rats were administered calcium dobesilate 100 mg/ kg （DD group） or distilled water （DM group） respectively. 12 weeks later, the changes in the renal uhrastrueture and ereatinine clearance rate （Cer） were examined in each group. The expression of glomerular TIMP1 and collagen Ⅳ were studied by immunohistoehemieal staining. Our results showed that after 12 weeks, the Cer in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane （GBM） in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance betweeh the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen Ⅳ in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the overaccumulation of collagen Ⅳ and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.