中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
7期
481-485
,共5页
杨明美%马月红%李锁%王仕忠%郭盛华
楊明美%馬月紅%李鎖%王仕忠%郭盛華
양명미%마월홍%리쇄%왕사충%곽성화
角蛋白细胞%紫外线%基质金属蛋白酶1%细胞外信号调节MAP激酶类%安石榴苷%基质金属蛋白酶抑制因子
角蛋白細胞%紫外線%基質金屬蛋白酶1%細胞外信號調節MAP激酶類%安石榴苷%基質金屬蛋白酶抑製因子
각단백세포%자외선%기질금속단백매1%세포외신호조절MAP격매류%안석류감%기질금속단백매억제인자
Keratinocytes%Ultraviolet rays%Matrix metalloproteinase 1%Extracellular signal-regulated MAP kinases%Punicalagin%Matrix metalloproteinase inhibitory factor
目的 探讨安石榴苷对中波紫外线(UVB)诱导角质形成细胞损伤的保护机制.方法 培养的HaCaT细胞分为空白对照组、安石榴苷组、UVB组、安石榴苷+UVB组.噻唑蓝(MTF)法检测细胞增殖能力,Hoechst/碘化丙锭(PI)染色和流式细胞仪检测细胞凋亡,RT-PCR法测定金属基质蛋白酶1(MMP1)及其组织抑制因子1 (TIMP1) mRNA表达水平,Western印迹检测丝裂原活化蛋白激酶(MAPK)通路相关蛋白P38、JNK、ERK的磷酸化水平变化.结果 MTT试验示,10～ 40μmol/L安石榴苷对UVB诱导的HaCaT细胞损伤有较佳的预保护作用.UVB组HaCaT细胞强Hoechst和强PI双染细胞较空白对照组增多,安石榴苷+UVB组较UVB组减少.流式细胞仪分析,UVB组凋亡细胞百分率(9.82％±0.11％)高于空白对照(1.24％±0.91％,P＜0.01),而安石榴苷(10、20、40 μmol/L)+UVB组凋亡细胞百分率(分别为6.38％±0.14％、5.24％±0.17％、3.77％±0.11％)较UVB组低,差异有统计学意义(均P＜ 0.01).UVB组MMP1 mRNA相对表达量(12.376±0.602)高于空白对照组(1.007±0.147,P＜ 0.01),而TIMP1 mRNA相对表达量(0.103±0.006)低于空白对照组(1.006±0.139,P＜ 0.01),安石榴苷组MMP1及TIMP1 mRNA与空白对照组比较,差异无统计学意义(均P＞0.05).安石榴苷预处理的HaCaT细胞经30 mJ/cm2 UVB照射后MMP1 mRNA相对表达量较UVB组降低(均P＜ 0.01),而TIMP1 mRNA较UVB组升高(均P＜0.01).Western印迹示,经UVB照射后,HaCaT细胞p-ERK、p-JNK及p-p38表达升高(均P＜ 0.01).安石榴苷组HaCaT细胞p-ERK、p-JNK及p-p38表达没有明显改变(P＞0.05),而安石榴苷+ UVB组有不同程度下降(均P＜0.01).结论 安石榴苷对UVB引起HaCaT细胞损伤有一定的预防作用.
目的 探討安石榴苷對中波紫外線(UVB)誘導角質形成細胞損傷的保護機製.方法 培養的HaCaT細胞分為空白對照組、安石榴苷組、UVB組、安石榴苷+UVB組.噻唑藍(MTF)法檢測細胞增殖能力,Hoechst/碘化丙錠(PI)染色和流式細胞儀檢測細胞凋亡,RT-PCR法測定金屬基質蛋白酶1(MMP1)及其組織抑製因子1 (TIMP1) mRNA錶達水平,Western印跡檢測絲裂原活化蛋白激酶(MAPK)通路相關蛋白P38、JNK、ERK的燐痠化水平變化.結果 MTT試驗示,10～ 40μmol/L安石榴苷對UVB誘導的HaCaT細胞損傷有較佳的預保護作用.UVB組HaCaT細胞彊Hoechst和彊PI雙染細胞較空白對照組增多,安石榴苷+UVB組較UVB組減少.流式細胞儀分析,UVB組凋亡細胞百分率(9.82％±0.11％)高于空白對照(1.24％±0.91％,P＜0.01),而安石榴苷(10、20、40 μmol/L)+UVB組凋亡細胞百分率(分彆為6.38％±0.14％、5.24％±0.17％、3.77％±0.11％)較UVB組低,差異有統計學意義(均P＜ 0.01).UVB組MMP1 mRNA相對錶達量(12.376±0.602)高于空白對照組(1.007±0.147,P＜ 0.01),而TIMP1 mRNA相對錶達量(0.103±0.006)低于空白對照組(1.006±0.139,P＜ 0.01),安石榴苷組MMP1及TIMP1 mRNA與空白對照組比較,差異無統計學意義(均P＞0.05).安石榴苷預處理的HaCaT細胞經30 mJ/cm2 UVB照射後MMP1 mRNA相對錶達量較UVB組降低(均P＜ 0.01),而TIMP1 mRNA較UVB組升高(均P＜0.01).Western印跡示,經UVB照射後,HaCaT細胞p-ERK、p-JNK及p-p38錶達升高(均P＜ 0.01).安石榴苷組HaCaT細胞p-ERK、p-JNK及p-p38錶達沒有明顯改變(P＞0.05),而安石榴苷+ UVB組有不同程度下降(均P＜0.01).結論 安石榴苷對UVB引起HaCaT細胞損傷有一定的預防作用.
목적 탐토안석류감대중파자외선(UVB)유도각질형성세포손상적보호궤제.방법 배양적HaCaT세포분위공백대조조、안석류감조、UVB조、안석류감+UVB조.새서람(MTF)법검측세포증식능력,Hoechst/전화병정(PI)염색화류식세포의검측세포조망,RT-PCR법측정금속기질단백매1(MMP1)급기조직억제인자1 (TIMP1) mRNA표체수평,Western인적검측사렬원활화단백격매(MAPK)통로상관단백P38、JNK、ERK적린산화수평변화.결과 MTT시험시,10～ 40μmol/L안석류감대UVB유도적HaCaT세포손상유교가적예보호작용.UVB조HaCaT세포강Hoechst화강PI쌍염세포교공백대조조증다,안석류감+UVB조교UVB조감소.류식세포의분석,UVB조조망세포백분솔(9.82％±0.11％)고우공백대조(1.24％±0.91％,P＜0.01),이안석류감(10、20、40 μmol/L)+UVB조조망세포백분솔(분별위6.38％±0.14％、5.24％±0.17％、3.77％±0.11％)교UVB조저,차이유통계학의의(균P＜ 0.01).UVB조MMP1 mRNA상대표체량(12.376±0.602)고우공백대조조(1.007±0.147,P＜ 0.01),이TIMP1 mRNA상대표체량(0.103±0.006)저우공백대조조(1.006±0.139,P＜ 0.01),안석류감조MMP1급TIMP1 mRNA여공백대조조비교,차이무통계학의의(균P＞0.05).안석류감예처리적HaCaT세포경30 mJ/cm2 UVB조사후MMP1 mRNA상대표체량교UVB조강저(균P＜ 0.01),이TIMP1 mRNA교UVB조승고(균P＜0.01).Western인적시,경UVB조사후,HaCaT세포p-ERK、p-JNK급p-p38표체승고(균P＜ 0.01).안석류감조HaCaT세포p-ERK、p-JNK급p-p38표체몰유명현개변(P＞0.05),이안석류감+ UVB조유불동정도하강(균P＜0.01).결론 안석류감대UVB인기HaCaT세포손상유일정적예방작용.
Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B (UVB)-induced damage to keratinocytes.Methods Cultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide (PI) staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinase-1 (TIMP1) in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase (MAPK) pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out by t test,one-way analysis of variance,and Dunnett's t-test.Results As the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group (9.82％ ± 0.11％ vs.1.24％ ± 0.91％,P ＜ 0.01),but decreased significantly in the three combination groups (punicalagin (10,20 and 40 μmol/L) + UVB) compared with the UVB group (6.38％ ± 0.14％,5.24％ ± 0.17％ and 3.77％ ± 0.11％ vs.9.82％ ± 0.11％,all P＜ 0.01).Theexpression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group (both P ＜ 0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group (all P ＞ 0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA (P ＜ 0.01),but elevated that of TIMP1 mRNA (P ＜ 0.01) in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group (all P ＜0.01),but experienced no significant changes in the punicalagin groups (all P ＞ 0.05) compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group (all P ＜0.01).Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells.
