外源性硫化氢对肺纤维化大鼠肺组织中MMP-9、TIMP-1表达的影响

发布时间：2018-08-14 12:24

【摘要】：目的:应用博来霉素(bleomycin,BLM)诱导大鼠构建肺纤维化模型,探讨基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)在肺纤维化过程中的表达及应用外源性硫化氢的干预作用。方法:健康雄性SD大鼠共45只,按随机数字表法分为3组(每组15只),即对照组、BLM组、BLM+Na HS(硫化氢供体)组。BLM组、BLM+Na HS组按5mg/kg剂量给予BLM溶液一次性气管内注入建立肺纤维化模型,对照组气管内注入等体积的生理盐水,造模后第一天开始,BLM+Na HS组每天腹腔注射Na HS溶液(28μmol/kg);BLM组和对照组每天腹腔注射等体积生理盐水。造模后分别在第7,14,28天进行取材,各组每次随机处死5只大鼠。取左肺组织放于4%甲醛固定后做HE染色和Masson染色电镜观察肺泡炎和肺纤维化改变,免疫组织化学染色法检测肺组织中MMP-9、TIMP-1蛋白的表达情况;取右肺组织进行RT-PCR检测肺组织中MMP-9、TIMP-1 m RNA的表达情况。结果:1.HE和Masson染色结果显示:对照组各时间段均未见炎症及肺纤维化改变,BLM组7d以炎症反应为主,14d开始出现纤维区,肺泡腔开始出现较多肺成纤维细胞;28d肺泡炎症明显减轻,肺泡腔内可见大量肌成纤维细胞及胶原沉积,肺纤维化模型形成;BLM+Na HS组也出现了肺泡炎和纤维化改变,但较BLM组有明显减轻。2.免疫组化和RT-PCR结果显示:对照组各时期MMP-9、TIMP-1仅有少量表达,而BLM组和BLM+Na HS组MMP-9、TIMP-1表达与对照组比较有显著增加,差异有统计学意义(P㩳0.05),BLM+Na HS处理组MMP-9的表达在各时间阶段较BLM组有不同程度减低,7d时MMP-9表达降低与BLM组比较差异有显著统计学意义(P㩳0.01),14、28d较BLM组比较差异有统计学意义(P㩳0.05),BLM+Na HS组TIMP-1的表达在各时间阶段较BLM组有不同程度的降低,7d时差异有统计学意义(P㩳0.05),14、28d时差异有显著统计学意义(P㩳0.01)。3.MMP-9/TIMP-1平衡比值结果显示:与对照组比较,BLM组MMP-9/TIMP-1平衡比值在7d时较对照组升高,差异有显著统计学意义(P㩳0.01),在14、28d时比值较对照组降低,差异有统计学意义(P㩳0.05);BLM+Na HS组MMP-9/TIMP-1比值在7d时较BLM组下降,差异有统计学意义(P㩳0.05),14、28d较BLM组有升高,差异有统计学意义(P㩳0.05);较对照组比较,BLM+Na HS组MMP-9/TIMP-1比值在7d时有升高,差异有统计学意义(P㩳0.05),14、28d时比值有降低,差异无统计学意义。结论:1.外源性硫化氢能够减轻博来霉素所致大鼠肺泡炎和肺纤维化的程度,具有抗肺纤维化作用。2.外源性硫化氢抗肺纤维化机制之一可能是抑制肺组织中MMP-9、TIMP-1的表达,并在一定程度上调整MMP-9和TIMP-1的比值使其趋于平衡。
[Abstract]:Aim: to investigate the expression of matrix metalloproteinase-9 (matrix metalloproteinase-9) and matrix metalloproteinase-1 (TIMP-1) in pulmonary fibrosis induced by bleomycin (BLM) and the effects of exogenous hydrogen sulfide (H2S) on the expression of matrix metalloproteinase-1 (TIMP-1) in pulmonary fibrosis. Methods: there were 45 healthy male SD rats. According to the random number table method, the rats were divided into three groups (15 rats in each group), namely, the control group was treated with BLM Na HS (hydrogen sulfide donor. The model of pulmonary fibrosis was established by intratracheal instillation of BLM solution in the BLM Na HS group according to the dose of 5mg/kg. The same volume of saline was injected into the trachea of the control group. The same volume of saline was injected intraperitoneally every day in the BLM Na HS group (28 渭 mol/kg) and the control group on the first day after the establishment of the model. After modeling, 5 rats were randomly killed in each group on day 14, 28, respectively. The changes of alveolitis and pulmonary fibrosis were observed by HE staining and Masson staining after the left lung tissue was fixed with 4% formaldehyde. The expression of MMP-9 and TIMP-1 protein was detected by immunohistochemical staining. The expression of MMP-9 and TIMP-1 m RNA was detected by RT-PCR in right lung tissue. Results 1. The results of HE and Masson staining showed that there were no inflammatory and pulmonary fibrosis changes in the control group. In the BLM group, the inflammatory reaction mainly appeared on the 14th day, and the alveolar inflammation began to appear in the alveolar cavity. The alveolar inflammation in the alveolar cavity began to appear more than 28 days, and the alveolar inflammation was obviously alleviated. A large number of myofibroblasts and collagen deposition could be seen in the alveolar cavity, and alveolitis and fibrosis were also found in the model of pulmonary fibrosis in the BLM Na HS group, but the changes of alveolitis and fibrosis were significantly reduced compared with those in the BLM group. The results of immunohistochemistry and RT-PCR showed that there was only a little expression of MMP-9 and TIMP-1 in the control group, while the expression of MMP-9 and TIMP-1 in the BLM group and BLM Na HS group was significantly higher than that in the control group. The expression of MMP-9 in BLM-Na HS group was significantly lower than that in BLM group at 7 days after treatment with BLM Na HS. There was significant difference between BLM group and BLM group (P0. 01) 1428 days after treatment, there was significant difference between BLM group and BLM group. (P0. 05) the expression of TIMP-1 in BLMNa HS group was significantly lower than that in BLM group at day 7 (P0. 05). (P0. 01). The results of MMP-9 / TIMP-1 balance ratio showed that: compared with the control group, the MMP-9/TIMP-1 balance ratio of BLM group was significantly lower than that of control group (P0. 05). On the 7th day, it was higher than that of the control group. The difference was statistically significant (P0. 01). The ratio of MMP-9/TIMP-1 in BLM Na HS group was significantly lower than that in BLM group at day 14 and 28. The ratio of MMP-9/TIMP-1 in BLM Na HS group was significantly lower than that in BLM group at day 7, and the difference was statistically significant (P0. 05) at 1428 days, which was higher than that in BLM group. Compared with the control group, the ratio of MMP-9/TIMP-1 increased at the 7th day, and decreased at 14 ~ 28 days, but there was no significant difference in the ratio between the two groups (P < 0. 05, P < 0. 05), but no significant difference was found between the control group and the control group (P < 0. 05, P < 0. 05). Conclusion 1. Exogenous hydrogen sulfide can reduce the degree of alveolitis and pulmonary fibrosis induced by bleomycin in rats. One of the mechanisms of exogenous hydrogen sulfide against pulmonary fibrosis may be to inhibit the expression of MMP-9 and TIMP-1 in lung tissues and to adjust the ratio of MMP-9 and TIMP-1 to a certain extent to balance them.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

【相似文献】