| Objective: (1) To establish the BleomycinA5-induced pulmonary fibrosis model in rats; (2)To investigate the effects of Qidan granule on the body weights and lung coefficients in rats; (3) To observe the effects of Qidan granule on the pulmonary pathology in rats; (4) To study the effects of Qidan granule on expressions of TGF- β 1 and TNF- α in lung tissue of rats; (5)To compare Qidan granule with Hydrocortisone sodium succinate on the body weights , lung coefficients ,pulmonary pathology ,and expressions of TGF- β 1 and TNF- α in lung tissue of rats. Methods: (1) 160 Male Sprague-Dawley rats were used, aged 7 weeks, and weights ranging from 180 gram to 210 gram(197± 10g).The rats were randomly divided into control group, model group, Qidan group one, Qidan group two, hydrocortisone group one and hydrocortisone group two. The rats were anesthetized with abdominal injection of pentobarbital sodium in a dose of 45mg/kg body weight and then were intratracheally dripped with bleomycin A5 hydrochloride (5mg/kg in 0.25ml saline) in experimental groups, while 0.9% saline in control group. (2)Part 1: The preventive group of Qidan granule on bleomycinA5-induced pulmonary fibrosis in rats (Part 1). Treatments were started from day 1 to day 28 with a daily oral Qidan granule ( 3125mg/kg)in Qidan group one, intraperitoneal Hydrocortisone sodium succinate (25mg/kg) in Hydrocortisone group one, and oral saline in control group and model group ,respectively. The action, body figure,inspiration and stools of rats were observed every day, and body weights were measured every week. The rats were killed at day7, day 14 and day28 after bleomycin A5 hydrochloride adminstration and their lungs were removed. The lung volume, figure, color and nodule were observed and then fixed in inflation for 24h with formalin and processed for Hematoxylin and eosin-staining and immunohistochemical analysis. Paraffin sections(5 u m) were stained using Hematoxylin and eosin-staining. The Immunohistochemical methods (SABC) were used to analyze the expressions of TGF- 3 1 and TNF- a .Brown yellow granules deposited in cytoplasm were considered to be positive. (3)Part 2: The therapeutic group of Qidan granule on bleomycinA5-induced pulmonary fibrosis in rats (Part 2). Treatments were started from day 15 to day 42 with a daily oral Qidan granule ( 3125mg/kg )in Qidan group two, intraperitoneal Hydrocortisone sodium succinate (25mg/kg) in Hydrocortisone group two, and oral saline in control group and model group respectively. The action, body figure, inspiration and stools of rats were observed every day, body weights were measured every week. The rats were killed at day28, and day42 after bleomycin A5 hydrochloride adminstration and their lungs were removed. The lung volume, figure, color and nodule were observed and then fixed in inflation for 24h with formalin and processed for Hematoxylin and eosin-staining and immunohistochemical analysis. Paraffin sections(5 u m) were stained using Hematoxylin and eosin-staining. The Immunohistochemical methods (SABC) were used to analyze the expressions of TGF- P 1 and TNF- a . Brown yellow granules deposited in cytoplasm were considered to be positive. SPSS 10.0 statistics software was adopted to process analysis of variance (ANOVA) and Student-Newman-Keuls multiple range test (SNK). Results:1 General observation (1) Part 1: There were no rats died in control group. The action, bodyfigure, inspiration and bowels of rats were well in control group. There were no rats died in model group.The inaction, dyspnea, loss of weight (13/30, 34%) and out of spirit of rats were found in model group.There were no rats died in Qidan group one. The rats'manifestations were similar to those in model group at early stage, but the rats'manifestations were similar to those of control group after treatment. There was one rat died in Hydrocortisone group one. The rats'manifestations were similar to those in model group at early stage, but the rats'manifestations were developed slightly after treatment.(2) Part 2: There were no rats died in control group. The action, body figure, inspiration and bowels of rats were well in normal group. There was one rat died in model group. The inaction, dyspnea, loss of weight and out of spirit of rats were found in model group. There were two rats died in Qidan group two. The rats'manifestations were similar to those in model group at early stage, but the rats'manifestations were similar to those in normal group after treatment. There were no rats died in Hydrocortisone group two. The rats'manifestations were similar to those in model group at early stage , the rats'manifestations were developed slightly after treatment, but loss of weights in three rats were found after three weeks treatment. 2 Body weight2.1 Part 1: The body weight were no significantly differnt before intratracheally dripped with bleomycin A5 hydrochloride in every group(/?>0.05). After intratracheally dripped with bleomycin A5 hydrochloride the body weights were significantly differnt at day7> dayl^ day21 in every group(p<0.01),but there were no significantly differnt at 28day(>>0.05).2.2 Part 2: The body weights were no significantly differnt before intratracheally dripped with bleomycin A5 hydrochloride in every group(/?>0.05). After intratracheally dripped with bleomycin A5 hydrochloride the body weights were significantly differnt at day28^day42 in every group(p<0.01),but there were no significantly differnt at14day(p>0.05).3^ Lung morphology3.1 Part 1: There were no abnormal findings in control group, whose lung profile was clear, smooth, pink and elastic. The dense bit haemorrhage, focal bleeding, nodule change and lobe volume shrink were found in model group. The findings of lung morphology in Qidan group one were similar to those in control group. The findings of lung morphology in hydrocortisone group one were similar to those in model group.Part 2: There were no abnormal findings in control group, whose lung profile was clear, smooth, elastic. The lobe pale, volume shrink, stiffness and nodule changes were found in model group. The findings of lung morphology in Qidan group two were similar to those in control group. The findings of lung morphology in hydrocortisone grouptwo were similar to those in model group.3.2 Lung coefficient:Part 1: The lung coefficients were decreased significantly at day7 and day 14 in every group (p<0.01), but no significant difference were found at day28 (p>0.05).Part 2: The lung coefficients were no significantly differnt at day28 and day42 in every group (p>0.05).3.3 Alveolitis and fibrosisAlveolitis and fibrosis were evaluated with Hematoxylin and eosin-staining and were graded using the following criteria : none(O). no alveolitis or fibrosis; mild(l+), mild alveolitis or fibrosis, less than 20% of the lung and with good preservation of the alveolar architecture; moderate(2+), a more widespread alveolitis or fibrosis involving 20% to 50% of the lung; severe(3+), a diffuse alveolitis or fibrosis involving more than 50% of the lung.Part 1: Alveolitis and fibrosis were significantly differnt at day7, dayl4^ day28 in every group(/?<0.01). There were completely normalparenchymal structures in control group. The alveolar septums and pleura were thin, and the alveolar walls formed a fine pattern. There were moderate or severe alveolitis manifested by a thickening of alveolar walls accompanied by an accumulation of mononuclear cells within the parenchymal structures in model group at day7, day 14. There were moderate or severe fibrosis manifested by a thickening of alveolar walls accompanied by an accumulation of fibroblast, collagen within the parenchymal structures in model group at dayl4, day28. Qidan group one indicated that the alveolar septum was thicken slightly, and the pulmonary tissue were closely normal. The inflammatory cells were decreased significantly in hydrocortisone group one , but the effects of relieving pulmonary fibrosis were significantly different to those in Qidan group one.Part 2: Alveolitis and fibrosis were significantly differnt at day28, day42 in every group(/?<0.01). There were completely normal parenchymal structures in control group. The alveolar septums and pleura were thin, and the alveolar walls formed a fine pattern. There were moderate or severe alveolitis manifested by a thickening of alveolar walls accompanied by an accumulation of mononuclear cells within the parenchymal structures in model group at day28, day42. There were moderate or severe fibrosis manifested by a thickening of alveolar walls accompanied by an accumulation of fibroblast, collagen within the parenchymal structures in model group at day28, day42. Qidan group two indicated that the alveolar septum were thicken slightly, and the pulmonary tissue were closely normal. The inflammatory cells were decreased significantly in hydrocortisone group two, but the effects of relieving pulmonary fibrosis were significantly different to those in Qidan group two.3.4 Expressions of TGF-P 1> TNF-aTGF- 3 1 was expressed in cytoplasm of alveolar macrophages, epithelial cells, endothelial cells and fibroblasts. TNF- a was expressed incytoplasm of alveolar macrophages, lymphocyte, epithelial cells, endothelial cells and interstitial cells. The expressions of TGF- P 1 were showed faintly in cytoplasm of alveolar macrophages, epithelial cells, endothelial cells and fibroblasts in normal group.Part 1: The continuous expression of TGF- 3 1 were significantly increased in model group at day7, dayl4, day28(/?<0.01). The expressions of TGF- P 1 in Qidan group one were significantly decreased than those in model group(p<0.01). The expression of TGF- P 1 in Hydrocortisone group one were significantly decreased, but were significantly increased than those in Qidan group one(p<0.01). The continuous expression of TNF- a were significantly increased in model group at day7, dayl4, day28(p<0.01). The expressions of TNF- a were significantly decreased than those in the model group and in hydrocortisone group one(p<0.01), but were significantly increased than those in Qidan group one(><0.01).Part 2: The expressions of TGF- 3 1 were significantly increased in model group at day28 ,day42. The expressions of TGF- 3 1 in Qidan group two were significantly decreased than those in model group(/?<0.01). The expression of TGF- 3 1 in Hydrocortisone group were significantly decreased, but were significantly increased than those in Qidan group two(jp<0.01). The expression of TNF-a were significantly increased in model group at day28, day42. The expressions of TNF- a were significantly decreased in Qidan group two than those in the model group and in hydrocortisone group two (p<0.01), but were significantly increased than those in Qidan group two(/?<0.01). Conclusions:1 Qidan granule and Hydrocortisone sodium succinate may improve the effects of bleomycinA5-induced loss of weight, but Hydrocortisone sodium succinate may increase the loss of weight at later stage;2 Qidan granule and Hydrocortisone sodium succinate may decrease the lung coefficient at early stage(<2w) in every group,but do not decreasethe lung coefficient at later stage;3 Qidan granule may ameliorate the alveolitis and pulmonary fibrosis, and Hydrocortisone sodium succinate may ameliorate the alveolitis and pulmonary fibrosis also, but there were significantly different to those in Qidan granule;4 Qidan granule may mainly prevent pulmonary interstitial fibrosis from decreasing expressions of TGF- P 1 and TNF- a ,and may be considered as a promising drug in the treatment of pulmonary interstitial fibrosis;5 Qidan granule were safe, effective without side effects. |
PDF Full Text Request