The Role of TSP-1-CD47 in ROS-mediated Pulmonary Fibrosis Induced by Paraquat / 中华劳动卫生职业病杂志

Objective@#Taking human A549 cells as the research object, to construct the paraquat-induced pulmonary fibrosis model in vitro, and to explore the role of TSP-1 (Thrombospondin-1, TSP-1) and its receptor CD47 in PQ-induced pulmonary fibrosis.@*Methods@#Human A549 cells were cultured in vitro, divided into normal control group, PQ group, Anti-TSP1 group (PQ with neutralizing anti-TSP1 antibody at a final concentration of 10 μg/ml) . A549 cells were stimulated with different concentrations (50, 100, 200, 400, 600, 800, 1 000 μmol/L) for different time (12, 24, 48 h) , and then CCK8 method was used to detect the cell viability to screen out the concentration and time of half cell viability. The subsequent test will be performed at this concentration point.The morphology of the cells was observed under inverted microscope. The expression levels of Fibronectin (FN) and type I collagen were determined by Enzyme Linked Immunosorbent Assay (ELISA) . Immunocytochemistry (ICC) and Immunofluorescence (IF) were used to observe the expression of TSP-1 and CD47 protein and the co-expression.The mRNA expression of TSP-1 and CD47 was detected by Real Time PCR (RT-PCR) . The protien expression of TSP-1 and CD47 was detected by Western Blot (WB) . The levels of Reactive Oxygen Species (ROS) were measured by flow cytometry.@*Results@#Before neutralizing anti-TSP1 antibody intervention: (1) When the time of PQ was constant, the cell viability decreased with the increase of PQ concentration. (2) The cells in the control group were closely connected, cobble-like, arranged neatly; with the increase of PQ concentration, the cell gap of PQ group gradually increased, spindle shape or long spindle shape. (3) With the increase of PQ concentration, the relative expression of FN and I collagen in PQ group was gradually increased compared with the control group in a concentration-dependent manner, and 200 μmol/L is the most obvious. (4) Compared with the control group, the mRNA level and the protein expression of TSP-1 and CD47 in PQ group was significantly increased, and 200 μmol/L is the most obvious, and Immunofluorescence showed they were co-expression in cytoplasm. (5) Compared with the normal group, the level of ROS in A549 cells was significantly increased at 24 h after PQ stimulation. (6) Compared with PQ group, the cell viability of Anti-TSP1 group was significantly increased, and the morphology changed to normal cell morphology, and the mRNA level and the protein expression of TSP-1 and CD47 decreased, and the overexpression of ROS was inhibited, and the relative expression of FN and I collagen decreased.@*Conclusion@#PQ stimulation induced morphological changes of A549 cells, increased expression of TSP-1, CD47, FN and type I collagen, and increased production of ROS.Neutralizing anti-TSP1 antibodies against TSP-1 can partially improve the above lesions. TSP-1-CD47 may be associated with oxidative stress-mediated PQ-induced pulmonary fibrosis.

The roles of TSP-1 and its receptor CD47 in pathogenesis of paraquat-induced pulmonary fibrosis in rats / 中华劳动卫生职业病杂志

Objective@#To establish a rat model of paraquat (PQ) -induced pulmonary fibrosis and observe the changes in thrombospondin-1 (TSP-1) and its receptor CD47 in lung tissue, and to investigate their roles in the pathogenesis of PQ-induced pulmonary fibrosis.@*Methods@#Fifty-four clean adult male Sprague-Dawley rats were randomly divided into normal control group (n=6) and 2 h, 12 h, 1 d, 3 d, 7 d, and 14 d PQ poisoning groups (n=8 per group). A rat model of PQ poisoning was established by a single gavage of 20 wt.% PQ solution (50 mg/kg). Flow cytometry was used to determine the concentration of reactive oxygen species (ROS) in blood and lung tissue. Enzyme-linked immunosorbent assay was used to determine the concentrations of hydroxyl radicals, malondialdehyde, and hydroxyproline in lung tissue. HE staining and Masson staining were used to observe the pathological damage of lung tissue after PQ poisoning. The expression of TSP-1 and CD47 in lung tissue was measured by Immunohistochemistry.@*Results@#Compared with the normal control group, the 2 h to 7 d PQ poisoning groups showed significant increases in ROS fluorescence intensity in red blood cells and lung tissues and the concentrations of malondialdehyde and hydroxyl radicals in lung tissue (P<0.05) , and the 14 d PQ poisoning group had a significant increase in the concentration of hydroxyproline in lung tissue (P<0.05). HE staining showed that the 2 h to 7 d PQ poisoning groups had significantly higher semiquantitative pathological scores of pulmonary alveolitis than the normal control group (P<0.05). The Masson staining showed that the 7 d and 14 d PQ poisoning groups had significantly higher semiquantitative pathological scores of pulmonary fibrosis than the normal control group (P<0.05). Compared with the normal control group, all PQ poisoning groups (except the 12 h group) had significantly increased expression of TSP-1 in lung tissue (P<0.05) , and all PQ poisoning groups (except the 1 d group) had significantly increased expression of CD47 in lung tissue (P<0.05). Within 2 h after PQ poisoning, the expression of TSP-1 and CD47 was positively correlated with the concentrations of ROS, hydroxyl radicals, and malondialdehyde and the degree of pulmonary alveolitis (P<0.01) ; at 1 d after PQ poisoning, the expression of TSP-1 and CD47 was positively correlated with the concentration of hydroxyproline in lung tissue (P<0.01) .@*Conclusion@#The expression of TSP-1 and CD47 is closely related to oxidative stress and subsequent pulmonary fibrosis, and they may be involved in the development and progression of pulmonary alveolitis and subsequent pulmonary fibrosis in rats with PQ poisoning.

Effects of ginsenoside Rg1 on streptozocin-induced diabetic nephropathy in rats / 生物医学工程学杂志

This study sought to assess the effect of Ginsenoside Rg1 on streptozocin-induced diabetic nephropathy in rats and to unveil the underlying mechanism. Diabetic nephropathy (DN) was induced by intraperitoneal injection of streptozocin (STZ). Eight weeks after drug administration, the rats from each group were sacrificed. Serum creatine (Scr) and 24 hours urine protein, cross reaction protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) were measured at the end of the study. The histological changes of renal interstitial tissues were observed by periodic acid-Schiff staining (PAS). Immunohistochemical method was used to examine the expression levels of ectodermal dysplasia (ED-1). The mRNA of transforming growth factor-beta1 (TGF-beta1) was measured by real-time PCR (RT-PCR), and the protein expression of TGF-beta1 was surveyed by Enzyme-Linked Immunosorbent Assay (ELISA). The renal pathological changes in DN rats given ginsenoside Rg1 treatment were ameliorated, and the expression levels of 24 h urine protein, serum creatinine, CRP, TNF-alpha, ED-1 and TGF-beta1 were significantly lower than those in the diabetic nephropathy group (P < 0.05). So, we reach a conclusion that, in the experiment, Ginsenoside Rg1 obviously reduced TGF-beta1 expression and the already-mentioned inflammatory reaction factors in the renal tissues and improved the renal pathological changes in DN rats.

Effect of Astragalus mongholicus on expression of transforming growth factor- beta1 in SD rats with unilateral ureteral occlusion / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Astragalus mongholicus (AM) on the expression of transforming growth factor-beta1 (TGF-beta1) in SD rats with unilateral ureteral occlusion (UUO) and to elucidate the mechanisms underlying the renoprotective effects of AM.</p><p><b>METHOD</b>Fifty-four Sprague-Dawley rats were randomly divided into 4 groups: sham-operation group, the UUO group and AM treatment group. After administration of AM (10 g kg(-1) d(-1)) for 3, 7 and 14 days, the dynamic histological changes of renal interstitial tissues were observed and renal damage including tubular impairment and interstitial fibrosis were quantified on HE and Masson stained tissue sections. The expression of TGF-beta1 and alpha-smooth muscle actin (alpha-SMA) was measured by immunohistochemistry staining sections. The mRNA of TGF-beta1 and alpha-SMA were reverse transcribed and quantified by real-time PCR. The expression of TGF-beta1 protein were assessed by Western blot.</p><p><b>RESULT</b>Renal damage was exacerbated and the expression of alpha-SMA and TGF-beta1 were all significantly increased in UUO group compared with those of sham-operation group (P<0.05) at each time point. Tubular impairment and interstitial fibrosis were alleviated, and up-regulations of expressions of TGF-beta1 and alpha-SMA were significantly suppressed by AM treatment (P<0.05).</p><p><b>CONCLUSION</b>AM can ameliorate renal interstitial fibrosis induced by UUO in vivo. The mechanisms of its antifibrotic effects might be related with the down-regulation of TGF-beta1 expression and suppression of tubular epithelial myofibroblast transdifferentiation in the progress of renal interstitial fibrosis.</p>

Expression of connective tissue growth factor following Sodium Ferulate in rats with unilateral ureteral obstruction / 中国组织工程研究

BACKGROUND: Connective tissue growth factor (CTGF) is a kind of factor that can mediate downstream action of transforming growth factor-β 1 (TGF- β 1). The upregulation of connective tissue growth factor expression plays an important role in pathological changes of renal interstitial fibrosis.OBJECTIVE: To explore the effect of Sodium Ferulate on the expression of CTGF mRNA and protein in rats with unilateral ureteral obstruction (UUO) and pathological changes of renal interstitial fibrosis, and to compare with Losartan.DESIGN: Randomized and controlled animal trial.SETTING: Department of Nephrology, West China Hospital of Sichuan University, and College of Public Health, Sichuan University.MATERIALS: Twenty-four healthy adult male SD rats were selected from the Experimental Animal Center of Sichuan University. Sodium Ferulate was provided by Sichuan Hengda Pharmacy Co, Ltd (No. 050302); rabbit anti-rat CTGF by Santa Cruz; Western blotting by BioRAD, USA; DNA Engine OpticonTM real-time fluorescent quantitation PCR device by MJ Research, USA.METHODS: The experiment was performed at Research Laboratory of Clinical Medicine (grade BSL-1), College of Public Health, Sichuan University from May to December 2006. Twenty-four healthy rats were randomly divided into 4 groups (n=6): UUO model group, Sodium Ferulate group, Losartan group, and sham-operation group. According to the previous protocol, UUO models were established in UUO model group, Sodium Ferulate group, and Losartan group, and the other rats were subjected to sham operation. From the first day after UUO, Sodium Ferulate group was intragastrically (i.g.)administrated with 150 mg/kg/d Sodium Ferulate; Losartan group was administrated ig. with 20 mg/kg/d. Losartan; UUO and sham operation groups were administrated i.g. with matching normal saline. All rats were executed 14 days after UUO to harvest partial renal tissues. All experimental procedure was accorded with animal ethical standards.MAIN OUTCOME MEASURES: The mRNA and protein expressions of CTGF were quantified by real-time PCR and Western blot. The pathological changes of renal interstitial tissues were observed by hematoxylin/eosin (HE) and Masson staining.RESULTS: Twenty-four rats were included in final analysis. Fourteen days after UUO, CTGF mRNA and protein expressions in UUO model group were significantly increased compared with sham operation group, but the expressions in Sodium Ferulate group were significantly lower than model group (P < 0.05). Compared with Losartan treated group, there was no significant difference (P > 0.05). HE and Masson staining showed inflammatory cell infiltration and tubular and interstitial changes as well as collagen deposition in renal interstitial tissues on day 14 after UUO. Sodium Ferulate obviously improved the renal pathological changes in UUO rats (P < 0.05), and the effect was similar to Losartan (P > 0.05).CONCLUSION: Sodium Ferulate inhibits UUO-induced renal interstitial fibrosis. This action, similar to the effect of Losartan, might be due to downregulation of CTGF expression.

Effect of sodium ferulate on transdifferentiation and extracellular matrix of rat renal tubular epithelial cells induced by transforming growth factor beta 1 / 中国组织工程研究

BACKGROUND: Renal tubulointerstitial fibrosis is mainly featured as the accumulation of extracellular matrix (ECM) in renal interstitium. The tubular epithelial-myofibroblast transdifferentiation (TEMT) is important to the pathogenesy of renal tubulointerstitial fibrosis. OBJECTIVE: To examine the effects of sodium ferulate (SF) on TEMT, and ECM main components such as collagen Ⅰ, collagen Ⅲ and fibronectin, in rat renal tubular epithelial cellsinduced by transforming growth factor-beta 1 (TGF- β1)- DESIGN: Randomized and controlled experimental study based on cells. SETTING: Department of Kidney in West China Hospital of Sichuan University. MATERIALS: Rat renal tubular epithelial cells (NRK-52E) originated from American Type Culture Collection (ATCC), were offered by the laboratory of Department of Nephrology in Australian Monash Medical Center. Cell strain used in this study was cultured at the 36th passage. SF white crystal with water solubility and more than 98.0% purify, was from Chengdu Hengda Pharmaceutical Co., Ltd. Different concentrations of SF (125,250, 500μreel/L) were designed in this study. Rabbit anti-rat α-smooth muscle actin (α -SMA) was produced by Wuhan Boster Company. Enzyme-linked immunosorbent assay (ELISA) kit was the produced of Shanghai Senxiong Science and Technology Co.,Ltd. Human recombinant TGF- β1 was produced by R&D Company. DNA Engine OpticonTM real-time fluorescence quantitative polymerase chain reaction apparatus was the product of MJ Research Company. METHODS: Rat renal tubular epithelial cells (NRK-52E) cultured in vitro were divided into five groups. Control group was added with serum-contained DMEM; TGF-β1-induced group was added with TGF-β1 at final concentration of 5 ng/L; SF at different concentrations groups were added with 125, 250, 500 μ mol/L SF and TGF- β1 at final concentration of 5 ng/L,respectively. MAIN OUTCOME MEASURES: The contrast phase microscope, real-time fluorescence quantitative polymerase chain reaction and ELISA method were used to detect TEMT of NRK52E cells induced by TGF-β1 and levels of collagen Ⅰ, collagen Ⅲ and fibronectin in the supernatant. RESULTS: Morphology of NRK52E cells: Compared with control group, TGF-β1 could induce the transdifferentiation of NRK52E cells, showing fibroblast-like in morphology after 3 days, which were previously the typical road stone-like epithelial cells. In three different concentration SF groups, the morphologic transformation stimulated by TGF-β1 could be partly ameliorated in a dose-dependent manner. Expression of α-SMA mRNA: Compared with control group, 5 ng/L TGF- β1 enhanced expression of α-SMA at 6 hours, and reached a peak at 72 hours; SF depressed the expression in a dose-dependent manner at 72 hours (P < 0.05). Changes of ECM: After induced by 5 ng/L TGF- β1 for 72 hours, the levels of collagen Ⅰ, collagen Ⅲ and fibronectin in the supernatant increased significantly (P < 0.05), whereas SF decreased these levels in a dose-dependent manner (P < 0.05). CONCLUSION: TGF- β1 induces the TEMT, and promotes the secretion of collagen Ⅰ, collagen Ⅲ and fibronectin. SF can inhibit TGF- β1-induced TEMT In a dose-dependent manner.