Effects of bilberry anthocyanins on MMP2 and collagen I in human fetal scleral fibroblast in vitro

AIM:To investigate the effects of bilberry anthocyanins on matrix metalloproteinase 2 (MMP2) and collagen type I (collagen I) expression in human fetal scleral fibroblasts (HFSF) in vitro and to provide experimental data for the prevention and treatment of myopia by bilberry anthocyanins. METHODS:HFSF were treated with bilberry anthocyanins at different concentrations (0, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2, 10^-1 and 1 g/L). The effects of bilberry anthocyanins on the viability of HFSF for different time (6 h, 8 h, 12 h, 24 h and 48 h) were measured by MTT assay. The cell cycle distribution and apoptosis of HFSF with highest viability (10^-1 g/L bilberry anthocyanins for 12 h) were analyzed by flow cytometry. The mRNA expression of MMP2, COL1A1 and COL1A2 in the HFSF was detected by RT-qPCR. The protein expression of MMP2 and collagen I was determined by Western blot. RESULTS:The cell viability was the highest after treatment with bilberry anthocyanins at a concentration of 10^-1 g/L for 12 h. Compared with blank control group, 10^-1 g/L bilberry anthocyanin group showed increased cell numbers in S and G₂ phases (P<0.05), but no significant difference of the apoptotic rate was observed. The mRNA expression of MMP2 was decreased (P<0.05), and that of COL1A1 was increased (P<0.05). No significant difference of COL1A2 expression was seen. The protein expression of MMP2 was decreased (P<0.05), and collagen I protein was increased (P<0.05). CONCLUSION:Bilberry anthocyanins inhibit the expression of MMP2 and increase the expression of collagen I in the HFSF.     

Expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and collagen type IV in lung of rats with multiple organ dysfunction syndrome

AIM: To determine the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of me-talloproteinase-1 (TIMP-1) and collagen type IV (IV-C) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. METHODS: Adult male Sprague-Dawley (SD) rats (n=40) were randomly divided into sham control group and cecal ligation and puncture (CLP) model group. The rats in CLP group were divided into 4 subgroups as different intervals (6 h, 12 h, 24 h and 48 h), and there were 8 rats in each group. The rat model of MODS was established by CLP. All rats were sacrificed at various intervals. The functions of the liver, kidney and lung were determined by blood biochemical and blood gas analysis. The morphological changes of the lung tissues were observed with HE staining. The serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, MMP-9 and TIMP-1 were measured by ELISA. The expression of MMP-9 and TIMP-1 in the lung tissues was detected by RT-PCR and immunohistochemistry, and the expression of IV-C in the lung tissues was detected by immunofluorescence and Western blot. RESULTS: Compared with sham control group, the functions of the liver, kidney and lung were damaged at different degrees in model groups. No histopathological change in the lung tissues of sham control group was found, and the lung injury was serious in model groups. Compared with sham control group, the serum levels of TNF-α, IL-1β, MMP-9 and TIMP-1 in model groups increased significantly (P<0.05) and peaked at the interval of 12~24 h after modeling (P<0.01). The expression of MMP-9 and TIMP-1 in the lung tissues of model groups increased, and peaked at 12 and 24 h, respectively (P<0.01). The protein level of IV-C in MODS 6 h group was not changed as compared with control group, while that at the interval of 12~48 h after modeling was significantly decreased and dropped to the lowest at 24 h (P<0.01). CONCLUSION: MMP-9 and TIMP-1 play important roles in lung injury of MODS rats by regulating the synthesis and decomposition of IV-C which is the main component of extracellular matrix.     

霞水母胶原蛋白活性肽对小鼠免疫功能的影响

1材料与方法 1．1材料 1．1．1霞水母胶原蛋白活性肽。由霞水母酶解提取、经冷冻干燥得到,为淡棕色粉末。由江南大学医药学院药物化学与药物分析实验室制备（具体方法参见已向国家申请的专利200710171188．6）。     

饲料组成对中国对虾肌肉组织中胶原蛋白、肌原纤维和失水率的影响↑（*）

用不同饲料喂养中国对虾,结果表明,饲料成分是影响肌肉组织中胶原蛋白含量、肌原纤维耐折断力和失水率大小的重要因素。鲜活饲料和维生素Ｃ能促进胶原蛋白的形成,加强对虾运动,也可提高肌肉组织中胶原蛋白的含量,野生虾肌肉胶原蛋白的含量大于养殖虾。饲料成分对胶原蛋白含量的影响规律和对肌原纤维耐折断力的影响相同,而对失水率的影响相反。肌原纤维耐折断力和失水率与胶原蛋白含量分别是正相关和负相关关系,其回归直线分别为＾Ｙ＝－５６７＋３０６６Ｘ和＾Ｙ＝６７３６－１５９６８Ｘ。上述３者影响肌肉组织柔韧性,即胶原蛋白含量越高,肌原纤维耐折断力越好,失水率越小,肌肉组织的柔韧性越强。     

Effect of Different Flavonoids on Collagen Synthesis in Human Fibroblasts

In this study, we discovered that flavonoids belonging to the subclasses: (flavanone, flavone, and flavonol) display differential effects on the synthesis of collagen in human dermal fibroblasts. At 80 μg/ml flavonoids quercetin-3,3′,4′, 5,7-pentahydroxyflavone, 3-methyl quercetin, and 7-hydroxyflavone significantly decreased the total protein concentration which was a direct consequence of their cytotoxic effect, while naringenin exhibited no effect on total collagen and total protein concentration. Quercetin-3,3′4′,7-tetramethyl ether, 4′-hydroxyflavanone, flavanone, and fisetin significantly decreased collagen concentration while morin, rutin, and chrysin increased collagen concentration without changing the overall protein concentration. The initial screening performed in this study enables the identification of compounds that exert significant effects on fibroblast function and show potential as starting material for pharmaceutical preparations targeted against various disorders centered around disturbed collagen metabolism.     

武昌鱼鱼鳞胶原蛋白提取工艺的研究

[目的]探索武昌鱼鱼鳞胶原蛋白的最佳提取工艺条件。[方法]以武昌鱼鱼鳞作为原料,采用不同浓度的盐酸作为脱钙溶液,用0.5 mol/L乙酸-乙酸钠缓冲溶液提取其胶原蛋白。以胶原蛋白提取率为指标,选择脱钙时间、脱钙酸浓度、固液比和提取时间4个因素为影响因子,通过正交试验确定武昌鱼鱼鳞胶原蛋白的最佳提取工艺条件。[结果]正交试验表明,当提取温度确定为12℃时,4个因素对武昌鱼鱼鳞胶原蛋白提取率的影响程度依次为提取固液比﹥脱钙时间﹥脱钙酸浓度﹥提取时间。提取鱼鳞胶原蛋白的最佳工艺条件为:固液比1∶25,脱钙时间3 h,提取时间每次1.5 d,脱钙酸浓度为0.4 mol/L,在此条件下,胶原蛋白提取率为1.142%。[结论]该研究为鱼鳞胶原蛋白的开发和应用提供参考依据。     

Inhibitory effect of sorafenib on collagen synthesis in human hepatic stellate cells

AIM: To investigate the effects of sorafenib on collagen synthesis in human hepatic stellate cells (HSCs). METHODS: HSC cell line LX-2 was used in vitro in this study. -proline incorporation assay was performed to measure the collagen synthesis. Immunocytochemistry was applied to detect type I collagen and real-time PCR was used to determine the mRNA expression of collagen α1 (I). RESULTS: Stimulation with platelet-derived growth factor (PDGF) induced the increase in type I collagen synthesis, while treatment with sorafenib (10.0 μmol/L) for 24 h markedly decreased the collagen synthesis. Sorafenib resulted in dose-dependent and time-dependent decrease in collagen synthesis in LX-2 cells in the absence or presence of PDGF by -proline incorporation assay. The inhibition rates were 22.69%, 37.52% and 71.74%, respectively, when LX-2 cells was treated with sorafenib at 10.0 μmol/L for 12 h, 24 h and 48 h. Sorafenib dose-dependently blocked the mRNA expression of collagen α1 (I) in LX-2 cells stimulated with PDGF. Sorafenib at the concentrations of 2.5 μmol/L, 5.0 μmol/L and 10.0 μmol/L down-regulated the mRNA expression of collagen α1 (I) in LX-2 cells by 58.66%, 67.06% and 81.64%, respectively. CONCLUSION: Sorafenib inhibits the collagen synthesis and blocks the expression of type I collagen at mRNA and protein levels in vitro in LX-2 cells. Therefore, sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.     

Comparison of vascular remodeling between small artery and aorta in spontaneous hypertensive rats

AIM: To examine the difference of vascular remodeling between aorta and small artery in sponta-neous hypertensive rats (SHR) and control rats.METHODS: Male SHR (20-week-old) were used as experiment group, and age matched male Wistar-Kyoto (WKY) rats were used as control group. The systolic blood pressure and body weight were measured once a week. At 43 weeks old, the rats were anaesthetized, blood samples were collected, and thoracic aorta and mesenteric small artery tissue were harvested. The morphological changes of the arterial tissue were observed with HE staining. The collagen and elastine fibers were detected by the Sirius red-Victoria blue staining. The protein expression of type I and Ⅲ collagens were analyzed by confocal laser-scanning microscopy and Western blot. The changes of the vascular ultrastructure were imaged by transmission electron microscopy. The expression of proliferating cell nuclear antigen (PCNA) and the cell apoptosis in the arterial wall were examined by immunohistochemical method and TdT-mediated dUTP nick and labeling (TUNEL) detection.RESULTS: The inner diameter (ID) and luminal cross-sectional area (LCSA) of mesenteric small artery were decreased, whereas ratio of wall thickness (WT) to ID (WT/ID) and ratio of wall cross-sectional area (WCSA) to LCSA (WCSA/LCSA) were increased. Meanwhile, adventitia fibroblast migrated to the media, with overload collagens, especially collagen Ⅲ. Proliferation index (PI) and apoptotic index (AI) of the mesenteric small artery wall cells were increased. The ID, LCSA, WT/ID and WCSA/LCSA of the aorta were increased. Moreover, the vascular smooth muscle cells (VSMCs) showed hypertrophy and hyperplasia, with overload collagens. The PI and AI of the aortic wall cells were increased.CONCLUSION: The difference of vascular remodeling between the aorta and small artery is significant. The small artery mainly appears hyperplasia of matrix, especially the adventitial collagen Ⅲ. Meanwhile, the cell apoptosis in the small artery wall is increased. The aorta mainly appears hyperplasia and hypertrophy of media VSMCs.     

Effect of oleanolic acid on expression of TNF-α and collagen in silicotic rats in vivo

AIM: To observe the effect of oleanolic acid (OA) on the expression of Tumor necrosis factor-α (TNF-α) and collagen in silicotic rats in vivo and its possible mechanism. METHODS: Male Wistar rats were divided into 4 groups according to the randomized block design: control group, model group, OA group and solvent control group (20 rats in each group). Except control group, the rats in other groups were induced by intratracheal instillation of silicon di-oxide (SiO₂; 250 mg/kg). The rats in OA group were intragastrically administered with OA (60 mg/kg) from the second day of giving SiO₂. The rats in solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose solution (10 mL/kg). The rats in control group were given normal saline under the same condition for 56 consecutive days. All rats were killed at the 7th, 14th, 28th and 56th days. The lung coefficient was detected and the morphological changes were observed. The serum contents of TNF-α were detected by ELISA. The content of total collagen in the lung tissue was measured. The protein level of nuclear factor-κB (NF-κB) in the lung tissue was determined by immunohistochemical method. RESULTS: (1) According to the morphological changes, the silicosis model was successfully established. Compared with control group, the lung coefficient and total collagen increased obviously in model group and solvent control group. The lung coefficient and total collagen content in OA group at each time point reduced compared with those in model group and solvent group, and increased compared with those in control group at the corresponding time points. (2) The serum contents of TNF-α in model group and solvent control group significantly increased, peaking at the 14th day, slightly decreasing afterward, and showing statistically significant difference at each time point compared with those in control group. No significant difference between model group and solvent group at different time points was observed. OA had inhibitory effect on the contents of TNF-α compared with model group and solvent group at the corresponding time points. (3) NF-κB in model group and solvent control group significantly increased, peaking at the 28th day, and showing statistically significant difference at each time point compared with those in control group. The NF-κB expression in OA group was similar to model group, but significantly decreased compared with control group at each time point. CONCLUSION: OA inhibits the expression of TNF-α and collagen and attenuates the silicosis fibrosis, which may be related to the NF-κB pathway.