Grape Seed Extract's Effect On Omycin-induced Mouse Pulmonary Fibrosis

Objective:Pulmonary fibrosis is caused by all kinds of pathogenic factors including idiopathic pulmonary fibrosis,chronic obstructive pulmonary disease caused by small pulmonary fibrosis and asthma airway remodeling.The patients with idiopathic pulmonary fibrosis(IPF)have a median survival of three to five years from diagnosis,and accompanied with complications in interstitial pneumonia.Due to increasing IPF incidence,as well as the current drug inefficacy and side effects,there is an urgent need for developing new drugs for IPF therapy.Grape seed is a traditional medicine derived from natural product.It contains different bioactive ingredients such as procyanidis and resveratrol.Modern pharmacology researches demonstrate that grape seed extract(GSE)has a variety of bioactivities such as anti-cancer,scavenging free radicals as well as prevent cardiovascular diseases.Furthermore,grapes are widely cultivated with higher yield in China,providing a plenty of resources for extraction and utilization of the grape seed.The present study aims to explore the effect of GSE on Bleomycin(BLM)-induced pulmonary fibrosis in mice and its underlying mechanism.Methods:Part one:The ICR mice were randomly divided into five groups of ten mice each group;Control group(only phosphate buffer saline,PBS),model group(BLM+saline),GSE group(GSE at doses of 50 or 100 mg/kg)and positive control group(DXM at a dose of 0.5 mg/kg).The mice were anesthetized by intraperitoneal injection of 3%sodium pentobarbital anesthesia(10 mL/kg).The pulmonary fibrosis model was performed via intratracheal instillation of BLM(2 mg/kg)dissolved in sterile PBS.In the control group,mice wrer dripped the same amount of saline solution.In the model,mice were treated with saline(0.2 mL/20 g BW)by intragastric administration.In the GSE group,mice were treated with GSE at doses of 50 or 100 mg/kg by intragastric administration(0.2 mL/g BW)daily for three weeks starting at one day after intratracheal instillation of BLM.In the DXM group,mice were treated with DXM at a dose of 0.5 mg/kg daily for three weeks starting at one day after intratracheal instillation of BLM.The body weight of each mouse was measured and recorded during the experimental period.After the experiment,the Buxco animal lung function analysis system was used to determine the pulmonary function of mice.The left lung was ligatured with nylon wire,and the right lung tissues were lavaged with Bronchial alveolar lavage fluid(BALF),the BALF was collected and was used to count the number of white blood cells and detect LDH level.The left lung was sectioned within paraffin-embedded blocks,and stained with Hematoxylin and Eeosin(H&E)and immunohistochemical staining to evaluate inflammatory cell infiltration using light microscopy.Part two:The ICR mice were randomly divided into five groups of ten mice each group;Control group(only phosphate buffer saline,PBS),model group(BLM+saline),GSE group(GSE at doses of 50 or 100 mg/kg)and positive control group(DXM at a dose of 0.5 mg/kg).The mice were anesthetized by intraperitoneal injection of 3%sodium pentobarbital anesthesia(10 mL/kg).The pulmonary fibrosis model was performed via intratracheal instillation of BLM(2 mg/kg)dissolved in sterile PBS.In the control group,mice wrer dripped the same amount of saline solution.In the model,mice were treated with saline(0.2 mL/20 g BW)by intragastric administration.In the GSE group,mice were treated with GSE at doses of 50 or 100 mg/kg by intragastric administration(0.2 mL/g BW)daily for three weeks starting at one day after intratracheal instillation of BLM.In the DXM group,mice were treated with DXM at a dose of 0.5 mg/kg daily for three weeks starting at one day after intratracheal instillation of BLM.The left lung was ligatured with nylon wire,and the right lung tissues were lavaged with Bronchial alveolar lavage fluid(BALF),supernatant used to detection TGF-?1 leve.The left lung was sectioned within paraffin-embedded blocks,and stained with Masson and immunohistochemical staining,using optical microscope observed.Another part of the left lung used for detecting the Hydroxyproline(HYP)content and further real-time quantitative PCR(qRT-PCR)assay.Results:Part one:Compared with model group,oral administration with GSE attenuated BLM-induced increase in Penh,significantly inhibited accumulation inflammatory cells including leukocytes,lymphocytes and neutrophils in BALF.GSE significantly reduced IL-6 and IL-1? mRNA expression in lung tissue.In addition,GSE attenuated BLM-induced alveolar structure destruction and pulmonary parenchyma in lung tissue,decreased the LDH level in BALF.Part two:Oral administration with GSE attenuated BLM-induced significantly decreased the MMP-9 and TIMP-1 mRNA and protein expressions in lung tissue.GSE reduced the deposition of collagen fibers and the level of hydroxyproline in lung tissue.Moreover,GSE decreased the TGF-?1 level in BALF.Besides,GSE adjusted the level of EMT related factors by p38 signaling pathway.Upregulated the level of E-cadherin and downregulated the level of of FN1 and COL1A1,attenuated lung fibrosis.Conclusions:The present study found that oral administration with GSE not only significantly attenuated the BLM-induced accumulation or infiltration of inflammatory cells,but remarkedly inhibited expressions bio-markers of lung fibrosis,such as hydroxyproline,TGF-?,MMP-9,COL1A1 and FN1.In summary,GSE,as a natural antioxidant from plant,manifested effective lung protective action to ameliorate the developing pulmonary fibrosis induced by chronic BLM administration in mice by p38 signal pathway.