虎杖苷对新生小鼠高氧肺损伤的保护作用研究

Protective Effects of Polydatin on Hyperoxic Lung Injury in Neonatal Mice

目的:研究虎杖苷通过SPOCK2表达对新生小鼠高氧肺损伤的保护作用。方法:体外培养小鼠正常肺上皮细胞mlE-12,作为对照组;利用高氧刺激mlE-12细胞,作为高氧组。用50、100和150μmol/ml的虎杖苷处理mlE-12细胞,再用高氧刺激,作为高氧+低剂量组、高氧+中剂量组和高氧+高剂量组。将pcDNA、pcDNA-SPOCK2转染至mlE-12细胞,再用高氧刺激细胞,作为高氧+pcDNA组、高氧+pcDNA-SPOCK2组。将si-NC、si-SPOCK2转染至mlE-12细胞,高氧刺激后,再用高剂量(150μmol/ml)虎杖苷处理细胞,作为高氧+高剂量+si-NC组、高氧+高剂量+si-SPOCK2组。采用化学法检测细胞丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;采用酶联免疫吸附试验法检测细胞肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和白细胞介素6(IL-6)的含量。采用实时荧光定量聚合酶链反应检测细胞SPOCK2 mRNA表达。采用蛋白质印迹法检测SPOCK2蛋白质表达。结果:与对照组比较,高氧组细胞MDA含量显著升高,SOD活性显著降低;与高氧组比较,高氧+低剂量组、高氧+中剂量组和高氧+高剂量组细胞MDA含量显著降低,SOD活性显著升高,差异均有统计学意义(P<0.05)。与对照组比较,高氧组细胞TNF-α、IL-1β和IL-6水平显著升高;与高氧组比较,高氧+低剂量组、高氧+中剂量组和高氧+高剂量组细胞TNF-α、IL-1β和IL-6水平显著降低,差异均有统计学意义(P<0.05)。与对照组比较,高氧组细胞SPOCK2 mRNA和蛋白表达显著降低;与高氧组比较,高氧+低剂量组、高氧+中剂量组和高氧+高剂量组细胞SPOCK2 mRNA和蛋白表达显著升高,差异均有统计学意义(P<0.05)。与高氧+pcDNA组比较,高氧+pcDNA-SPOCK2细胞MDA含量和TNF-α、IL-1β、IL-6水平显著降低,SOD活性显著升高;与高氧+高剂量+si-NC组比较,高氧+高剂量+si-SPOCK2组细胞MDA含量和TNF-α、IL-1β、IL-6水平显著升高,SOD活性显著降低,差异均有统计学意义(P<0.05)。结论:虎杖苷通过上调SPOCK2表达抑制新生小鼠高氧肺损伤,对肺部具有保护作用。

OBJECTIVE To probe into the protective effects of polydatin on hyperoxic lung injury through the expression of SPOCK2 in neonatal mice. METHODS: Normal lung epithelial cells mlE-12 in mice were cultured in vitro and set as the control group;mlE-12 cells were stimulated with hyperoxia and set as the hyperoxia group. The mlE-12 cells were treated with 50,100 and 150 μmol/ml of thujaplicin,and then stimulated with hyperoxia,which were set as the hyperoxia + low dose group,hyperoxia + medium dose group and hyperoxia + high dose group,respectively. The pcDNA and pcDNA-SPOCK2 were transfected into mlE-12 cells,which were stimulated with hyperoxia and set as hyperoxia + pcDNA group and hyperoxia + pcDNA-SPOCK2 group. The si-NC and si-SPOCK2 were transfected into mlE-12 cells,then stimulated with hyperoxia and treated with high dose( 150 μmol/ml) of thujaplicin,which were set as hyperoxia + high dose + si-NC group and hyperoxia + high dose + si-SPOCK2 group.Malondialdehyde( MDA) content and superoxide dismutase( SOD) activity were detected by chemical method;tumor necrosis factor α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) content were detected by enzyme-linked immunosorbent assay. Real-time fluorescence quantitative polymerase chain reaction was used to detect SPOCK2 mRNA expression. Expression of SPOCK2 protein was detected by protein blotting. RESULTS: Compared with the control group,the MDA content increased significantly and SOD activity decreased significantly in the hyperoxia group;compared with the hyperoxia group,the MDA content decreased significantly and SOD activity increased significantly in the hyperoxia + low dose group,hyperoxia + medium dose group and hyperoxia + high dose group,with statistically significant differences( P<0. 05). Compared with the control group,the levels of TNF-α,IL-1β and IL-6 increased significantly in the hyperoxia group;compared with the hyperoxia group,the levels of TNF-α,IL-1β and IL-6 decreased significantly in the hyperoxia + low dose group,hyperoxia + medium dose group and hyperoxia + high dose group,with statistically significant differences( P<0. 05). Compared with the control group,the SPOCK2 mRNA and protein expression decreased significantly in the hyperoxia group;compared with the hyperoxia group,the SPOCK2 mRNA and protein expression increased significantly in the hyperoxia + low dose group,hyperoxia + medium dose group and hyperoxia + high dose group,with statistically significant differences( P < 0. 05). Compared with the hyperoxia + pcDNA group,the MDA content,levels of TNF-α,IL-1β and IL-6 decreased significantly and the SOD activity increased significantly in the hyperoxia + pcDNA-SPOCK2 cells;compared with the hyperoxia + high dose +si-NC group,the MDA content,levels of TNF-α,IL-1β and IL-6 increased significantly and SOD activity decreased significantly in the hyperoxia + high dose + si-SPOCK2 group,with statistically significant differences( P < 0. 05).CONCLUSIONS: Polydatin can inhibit hyperoxic lung injury in neonatal mice through up-regulating the SPOCK2 expression,which has protective effects for lung tissues.