水母雪莲多糖对中波紫外线诱导的皮肤成纤维细胞凋亡的研究

Study on the Apoptosis of Skin Fibroblasts Induced by UVB in Saussurea Medusa Maxim Polysaccharides

目的:研究水母雪莲多糖对中波紫外线刺激下人皮肤成纤维细胞凋亡的影响及可能的机制。方法:利用蒸馏水回流提取+醇沉分离法提取水母雪莲中的多糖成分;使用包皮切除术后皮肤,处理后在6孔板中定量接种经10%胎牛血清DMEM培养基传代培养的细胞。细胞体外培养并分为空白对照组、紫外线照射组、10mg/ml水母雪莲多糖干预组和40mg/ml水母雪莲多糖干预组。除空白对照组外,其他组细胞分别在低辐射UVB(L-UVB,30mJ/cm2辐射1h)和高辐射UVB(H-UVB,60mJ/cm2辐射1h)两种条件下接受紫外线照射。在暴露于UVB光前,10mg/ml水母雪莲多糖干预组和40mg/ml水母雪莲多糖干预组细胞以终浓度为10mg/ml和40mg/ml水母雪莲多糖培养1h,紫外线组不干预。照射1h后,将细胞在37℃,10%胎牛血清DMEM培养基中温育18h。测定SOD、GSH、CAT及MDA水平,流式细胞术检测细胞凋亡,Westernblot测定p38、p53、Bcl-2、Bax蛋白及caspase-3的β肌动蛋白含量变化。结果:水母雪莲多糖可以提高经中波紫外线损伤的成纤维细胞中SOD、CAT的活性和GSH含量,并可减少MDA含量,通过上调p38,p53和Bcl-2的表达,同时下调Bax和活性caspase-3的表达。与非干预组相比,无论暴露于低剂量还是高剂量辐射下,10mg/ml和40mg/ml水母雪莲多糖组的成纤维细胞凋亡数目都要更少。结论:水母雪莲多糖可以通过上调人成纤维细胞p38,p53和Bcl-2的表达、下调Bax和活性caspase-3的表达、增加SOD、CAT的活性和GSH的含量、减少MDA含量,从而抑制中波紫外线照射所致的细胞凋亡。

Objective To investigate the effect of Saussurea medusa Maxim polysaccharides on apoptosis of human fibroblasts exposed to UVB and to study possible mechanisms. Methods Distilled water was used for reflux extraction and alcohol precipitation filtration method was used to extract Saussurea medusa Maxim polysaccharides. After skin was removed by foreskin, treated with 10% fetal bovine serum DMEM medium and inoculated in 96-well plates and 6-well plates for later use. Cells were cultured in vitro and divided into blank control group, ultraviolet irradiation group, 10mg/ml jellyfish jellyfish intervention group, and 40mg/ml jellyfish polysaccharide intervention group. Each group of cells was exposed to ultraviolet light under two conditions of low-irradiation UVB (L-UVB, 30mJ/cm2 radiation 1h) and high-radiation UVB (H-UVB,60mJ/ cm2 radiation 1h). Before exposed to UVB light, cells of the experimental group were cultured for 1 hour at a final concentration of 10mg/ml and 40mg/ml Saussurea medusa Maxim polysaccharides. The UV group did not interfere, and the blank control group did not receive irradiation. After 1 hour of irradiation, cells were incubated in 10% fetal bovine serum DMEM medium for 18 hours at 37℃. The levels of SOD, GSH, CAT and MDA were measured. Apoptosis was detected by flow cytometry. The changes of β-actin in p38, p53, Bcl-2, Bax and caspase-3 were determined by western blot. Results The Saussurea medusa Maxim polysaccharides can increase the activity of SOD, CAT and the content of GSH in fibroblasts damaged by UVB, and decrease the content of MDA. The expression of p38, p53 and Bcl-2 was up-regulated, and the expression of Bax and active caspase-3 was down-regulated at the same time. No matter when exposed to low or high doses of radiation, the number of fibroblasts apoptosis in 10mg/ml and 40mg/ml Saussurea medusa Maxim polysaccharides group was lower than that in the control group. Conclusion Saussurea involucrata can up-regulate p38, p53 and Bcl-2, down-regulate the expression of Bax and active caspase-3, and inhibit the expression of MDA by increasing SOD activity, GSH content, CAT activity and reducing MDA content. Irradiation leads to apoptosis of fibroblasts.