枸杞多糖通过线粒体途径抑制放射性肠损伤小鼠小肠上皮细胞凋亡

Lycium barbarum polysaccharide inhibits apoptosis of intestinal epithelial cells through mitochondrial pathway in mice with radiation-induced intestinal injury

目的研究枸杞多糖(lycium barbarum polysaccharide,LBP)对辐射损伤小鼠小肠上皮细胞(intestinal epithelial cells,IECs)的辐射防护机制。方法利用直线加速器构建放射性肠损伤小鼠模型,各LBP干预组接受辐射后,通过灌胃分别给予不同剂量LBP(200、400、800 mg/kg),连续7 d,辐射组接受射线处理,并给予等量生理盐水,LBP组仅使用800 mg/kg LBP灌胃不经过X线照射,正常组不作任何处理。镜下观察近十二指肠上段小肠上皮病理变化情况,采用CCK-8检测IECs活力,流式细胞仪检测IECs细胞凋亡率,分光光度法检测IECs细胞抗氧化指标超氧化物歧化酶(superoxide dismutase,SOD)活性及丙二醛(malondialdehyde,MDA)含量, Western blot检测IECs细胞凋亡调节因子Bcl-2(B-cell lymphoma-2)和Bcl-2同源的水溶性相关蛋白Bax(Bcl-2-Associated X)的表达,以探究LBP对肠黏膜的辐射防护机制。结果 X线照射小鼠后7 d,与辐射组相比,经不同剂量LBP处理的各组小鼠肠绒毛长度及隐窝数量均有一定增加,且IECs细胞活力均较高(P<0.05)。随着枸杞多糖剂量增加,经过枸杞多糖处理的各组IECs细胞中SOD活性逐渐升高[(1.22±0.05)vs(1.32±0.06)vs(1.53±0.03) U/mL],凋亡率及MDA含量出现逐渐降低趋势。经枸杞多糖干预的各组IECs细胞较辐射组Bcl-2表达量明显增加(当LBP为800 mg/kg时,Bcl-2表达增加近1倍),Bax表达量降低(当LBP剂量为800 mg/kg时,Bax表达减少近0.5倍)。结论 LBP可能通过作用于凋亡相关蛋白Bcl-2和Bax来减少IECs细胞凋亡,提高SOD活性,减低MDA含量,增强IECs细胞抗氧化能力。

Objective To study the protective effect of Lycium barbarum polysaccharide(LBP) against apoptosis of small intestinal epithelial cells(IECs) in mice with radiation-induced intestinal injury and explore the pathway that mediates this effect. Methods A mouse model of radiation-induced intestinal injury was established using a linear accelerator. In 2 h after the radiation exposure, the mice were treated with 200, 400, or 800 mg/kg LBP by gavage for 7 d(LBP intervention groups) or with normal saline(radiation group), and the mice without any treatment and those treated with 800 mg/kg LBP by gavage without X-ray radiation served as the control groups. After the treatments, the intestinal segment near the duodenum was dissected for pathological examination. The viability of the IECs isolated from the intestines of the mice was evaluated with CCK-8 assay, the cell apoptosis rate was detected with flow cytometry, and superoxide dismutase(SOD) activity and malondialdehyde(MDA) content were determined by spectrophotometry;Western blotting was performed to detect the expression levels of apoptosis regulators Bcl-2 and Bax in the IECs. Results Compared with the mice in the radiation group, those in LBP intervention groups showed greater intestinal villus length and crypt number and higher viability of the IECs at 7 d after the radiation(P<0.05). The increase of LBP concentration significantly increased SOD activity(1.22±0.05 U/mL at 200 mg/kg, 1.32±0.06 U/mL at 400 mg/kg, and 1.53±0.03 U/mL at 800 mg/kg) and lowered the apoptotic rate and MDA content in the IECs. Compared with the IECs in the radiation group, the cells from LBP intervention groups showed significantly increased expression of Bcl-2(nearly doubled at the LBP dose of 800 mg/kg) and lowered expression of Bax(by about 50% at the LBP dose of 800 mg/kg). Conclusion For mice with radiation-induced intestinal injury, LBP treatment suppresses apoptosis of IECs by regulating Bcl-2 and Bax expressions and enhances their antioxidant capacity by increasing SOD activity and decreasing the content of MDA.