Effect of NRP1 gene knockout on process of radiationinduced pulmonary fibrosis and its mechanism / 吉林大学学报(医学版)

Objective: To observe the effect of neuroopilin-1 (NRP1) gene on the process of radiation-induced pulmonary fibrosis (RIPF), and to explore its roles in the occurrence and development of epithelial-mesenchymal transition (EMT) mediated by Wnt/fi-catenin pathway tail identification was performed in and TGF-β1/Smads pathway, and extracellular matrix (ECM) deposition. Methods: The Cre-LoxP recombinase system was used to construct the transgenic C57BL/6J mice with NRP1 gene specific knockout in alveolar type II epithelial cells (AT-II) and the mice. A total of 160 mice were randomly divided into 4-week group, 8-week group, 16-week group and 24-week group. In each group, the mice were randomly divided into wild type (Con) group, wild type+irradiation (IR) group, NRP1 gene-specific knockout (KO-Con) group, NRP1 gene-specific knockout+irradiation (KO+IR) group according to the method of random number table; there were 10 mice per group. In KO-Con and KO + IR groups, the NRP1 gene was specifically knocked out in the AT-II cells by intraperitoneal injection of tamoxifen, and the mouse models of RTPF were established by 20 Gy total thoracic irradiation in IR group and KO+IR group. After the models were constructed, HE staining and Masson staining were used to verify whether the models were successfully constructed. Immunohistochemistry (IHC) method was used to detect the type I collagen (Col I) and crsmooth muscle actin α-SMA) protein expression levels; Western blotting method was performed to detect the NRP1, β-catenin, TGF-β1, and Smad2 protein expression levels in the lung tissue of the mice; Quantitative fluorensence real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression levels of NRP1, Col I, α-SMA, β-catenin, TGF-βl, Smad2, E-cadherin, N-cadherin, and Vimentin mRNA in the lung tissue of the mice. Results: The results of HE and Masson staining showed the RTPF models were successfully established, and the lung tissue of the mice in IR group mainly showed the pathomorphology of radiation pneumonitis. Compared with Con group, the protein and mRNA expression levels of NRP1 in the lung tissue of the mice in IR group were gradually increased with the prolongation of time (P<0.05), and reached the highest at 24 weeks (P<0.01). Compared with Con group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 proteins and mRNA in the lung tissue of the mice in IR group and KO+IR group were increased gradually with the prolongation of time (P<0.05 or P<0.01). Compared with IR group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 protein and mRNA in the lung tissue of the mice in KO+IR group were significantly decreased (P<0.05 or P<0.01), but they were higher than those in Con group (P<0.05 or P<0.01). Compared with Con group, the expression levels of the epithelial cell marker E-Cadherin mRNA in the lung tissue of the mice in IR group and KO+IR group were gradually decreased with the prolongation of time (P< 0.01), and the expression levels of the interstitial cell markers N-Cadherin and Vimentin were increased (P<0.05 or P<0.01), but the expression levels of E-cadhern mRNA in the lung tissue of the mice in KO-IR group were significantly higher than those in IR group (P<0.05 or P<0.01), and the expression levels of N-Cadherin and Vimentin mRNA in the lung tissue of the mice in KO + IR group at each time point were lower than those in IR group (P<0.05 or P<0.01). Conclusion: Knockout of NRP1 gene can inhibit the occurrence and development of RTPF, and its mechanism may be involved in regulating the expressions of Wnt/fi-catenin and TGF-β1/Smads signaling pathways in the lung tissue and inhibiting the EMT process in the mice.

Effect of acupuncture on TGF-β1/Smads pathway in mice with airway remodeling mic / 南方医科大学学报

OBJECTIVE@#To investigate the effect of acupuncture on TGF-β1/Smads signaling pathway in the lung tissue of mice with airway remodeling.@*METHODS@#Thirty specific pathogen-free mice were randomly divided into blank group, model group and acupuncture group (=10). Mouse models of asthma were established in the model group and the acupuncture group, and the mice in the latter group received 7 acupuncture therapies (at bilateral Fei Shu, Da Zhui and Zu Sanli, 20 min each time) every other day, starting on the 10th day after the modeling. At 24 h after the last acupuncture, the mice were subjected to inhalation of 1% OVA for 3 days, and 24 h after the last challenge, the mice were given methacholine chloride (Mch) inhalation at different concentrations for measurement of lung resistance using a noninvasive stroke volume meter. HE staining was used to observe the pathological changes in the lung tissues, and TGF-β1 levels in the the bronchoalveolar lavage fluid (BALF) and serum were detected using ELISA; Western blotting was used to detect the differential protein expressions in the airway smooth muscles between the two groups. The airway smooth muscle cells were isolated from the mice in the acupuncture group and treated with a TGF- β1 inhibitor (LY2157299), and the relative expressions of type-Ⅰ and Smads proteins were detected using Western blotting.@*RESULTS@#The mice in the model showed obvious tracheal fistula with airway pathologies including lumen narrowing, bronchial mucosa thickening, dissociation of the epithelial cells, and thickening of the alveolar septum and airway smooth muscles. These pathological changes were obviously milder in the acupuncture group. The asthmatic mice exhibited significantly increased lung resistance in positive correlation with Mch concentration. Serum TGF-β1 level was significantly elevated in asthmatic mice ( < 0.05); TGF-β1 levels in the serum and BALF were significantly lower in the acupuncture group than in the model group ( < 0.05). In the model group, the expressions of -SMA, TGF-β1 and Smads in the airway smooth muscles were significantly higher than those in the other two groups (both < 0.05). In cultured airway smooth muscle cells, the expressions of type-Ⅰ and Smads were significantly higher in cells treated with LY2157299 than in the control cells (>0.05).@*CONCLUSIONS@#Acupuncture can inhibit airway remodeling by inhibiting the expression of airway TGF-β1 and down-regulating the expression of Smads and -SMA to reduce airway inflammatory response. Airway expressions of type-Ⅰ and Smads proteins remain high after inhibiting TGF-β1. Acupuncture may control asthma progression through the TGF-β1/Smads pathway.

Effects and mechanisms of UCG ameliorating renal interstitial fibrosis by regulating TGF-β1/SnoN/Smads signaling pathway in renal failure rats / 中国中药杂志

This study was aimed to demonstrate preliminarily the effects and mechanisms of uremic clearance granule (UCG) ameliorating renal interstitial fibrosis (RIF) by regulating transforming growth factor (TGF)-β1/SnoN/Smads signaling pathway in vivo. Fifteen rats were randomly divided into 3 groups：the normal group,the model group and the UCG group. The rats with renal failure were induced by intragastric administration of adenine and unilateral ureteral obstruction (UUO). After modeling,the rats in the UCG group and in the other groups were intervened by intragastric administration of UCG and distilled water respectively during 3 weeks. The body weight and 24 h urinary protein excretion (Upro) in all rats were tested after drug administration. All rats were killed after drug administration for 3 weeks,blood and kidneys were collected and weighted,kidney appearance and renal morphological characteristics were observed. In addition,serum biochemical indices and the protein expressions of TGF-β1,SnoN,phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in the kidney were evaluated respectively. The results indicated that,after the intervention of UCG,the general state of health,kidney appearance,serum creatinine (Scr),blood urea nitrogen (BUN),uric acid (UA),albumin (Alb),Upro and renal morphological change in model rats were improved in different degrees,respectively. Moreover,UCG down-regulated the protein expressions of TGF-β1 and p-Smad2/3,and up-regulated the protein expressions of SnoN and Smad7 in the kidney. In conclusion,UCG reduces extracellular matrix (ECM) synthesis and delays the progression of renal failure via possibly multi-targeting at regulating TGF-β1/SnoN/Smads signaling pathway in vivo.