黑枸杞水提物对中波紫外线辐射后人角质形成细胞增殖与凋亡及凋亡相关蛋白表达水平的影响研究
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摘要
目的研究黑枸杞水提物对中波紫外线(UVB)辐射后人角质形成(HaCaT)细胞增殖与凋亡及凋亡相关蛋白[P38丝裂原活化蛋白激酶(P38MAPK)、P53、半胱氨酸蛋白酶-8(caspase-8)、半胱氨酸蛋白酶-3(caspase-3)、B细胞淋巴瘤因子2(Bcl-2)]表达水平的影响,以期探讨黑枸杞水提物减轻UVB损伤HaCaT细胞的作用机制。方法 2015年3月—2016年1月,提取黑枸杞水提物,体外培养HaCaT细胞,取对数生长期的HaCaT细胞,采用随机数字表法分为对照组(无辐射)、UVB组(30 m J/cm~2UVB辐射40 min)、黑枸杞水提物组(30 mJ/cm~2UVB辐射40 min+黑枸杞水提物2 mg/ml)。采用MTS法检测各组细胞增殖活力,流式细胞仪检测各组细胞凋亡率,Western blotting法检测各组P38MAPK、P53、caspase-8、caspase-3、Bcl-2表达水平。结果 UVB组细胞增殖活力小于对照组(P<0.05);黑枸杞水提物组细胞增殖活力小于对照组,大于UVB组(P<0.05)。UVB组细胞凋亡率大于对照组(P<0.05);黑枸杞水提物组细胞凋亡率大于对照组,小于UVB组(P<0.05)。UVB组P38MAPK、P53、caspase-8、caspase-3表达水平高于对照组,Bcl-2表达水平低于对照组(P<0.05);黑枸杞水提物组P38MAPK、P53、caspase-8、caspase-3表达水平低于UVB组,Bcl-2表达水平高于UVB组(P<0.05)。结论黑枸杞水提物可促进UVB辐射后HaCaT细胞的增殖,同时阻止其凋亡,其机制可能为黑枸杞水提物能够下调P38MAPK、P53、caspase-8、caspase-3表达水平及上调Bcl-2表达水平,进而减轻HaCaT细胞的辐射损伤。
Objective To research the effect of black wolfberry water extracts on proliferation and apoptosis and P38 mitogen activated protein kinase( P38MAPK),P53,cysteine proteinase 8( caspase-8),cysteine proteinase 3( caspase-3)and B cell lymphoma factor 2( Bcl-2) expression of HaCaT cells after ultraviolet B( UVB) radiation,in order to explore the mechanism of black wolfberry water extracts reducing the damage of HaCaT cells after UVB radiation. Methods From March2015 to January 2016,black wolfberry water extracts was extracted and HaCaT cells were cultured in vitro. HaCaT cells in logarithmic growth phase were collected and randomly divided into 3 groups by random number table method: control group( without radiation),UVB group( 30 mJ/cm~2 UVB radiation for 40 min) and black wolfberry water extracts group( 30 mJ/cm~2 UVB radiation for 40 min + black wolfberry water extracts 2 mg/ml). Cell proliferation in each group was detected by MTS,apoptotic rate in each group was assessed by flow cytometry. The expression levels of P38 MAPK,P53,caspase-8,caspase-3and Bcl-2 in each group were detected by Western blotting. Results Cell proliferation in UVB group was lower than that in control group( P < 0. 05); cell proliferation in black wolfberry water extracts group was lower than that in control group but higher than that in UVB group( P < 0. 05). Apoptotic rate in UVB group was higher than that in control group( P < 0. 05);apoptotic rate in black wolfberry water extracts group was higher than that in control group but lower than that in UVB group( P <0. 05). The expression levels of P38 MAPK,P53,caspase-8,caspase-3 in UVB group were higher than those in control group,but the expression level of Bcl-2 in UVB group was lower than that in control group( P < 0. 05); the expression levels of P38 MAPK,P53,caspase-8,caspase-3 in black wolfberry water extracts group were lower than those in UVB group,but the expression level of Bcl-2 in black wolfberry water extracts group was higher than that in UVB group( P < 0. 05). Conclusion The black wolfberry water extracts can promote the proliferation of HaCaT cells after UVB radiation and prevent their apoptosis. The mechanism might be that the black wolfberry water extracts can down-regulate the expression levels of P38 MAPK,P53,caspase-8 and caspase-3 and up-regulate the expression level of Bcl-2,so as to reduce the radiation damage of HaCaT cells.
Objective To research the effect of black wolfberry water extracts on proliferation and apoptosis and P38 mitogen activated protein kinase( P38MAPK),P53,cysteine proteinase 8( caspase-8),cysteine proteinase 3( caspase-3)and B cell lymphoma factor 2( Bcl-2) expression of HaCaT cells after ultraviolet B( UVB) radiation,in order to explore the mechanism of black wolfberry water extracts reducing the damage of HaCaT cells after UVB radiation. Methods From March2015 to January 2016,black wolfberry water extracts was extracted and HaCaT cells were cultured in vitro. HaCaT cells in logarithmic growth phase were collected and randomly divided into 3 groups by random number table method: control group( without radiation),UVB group( 30 mJ/cm~2 UVB radiation for 40 min) and black wolfberry water extracts group( 30 mJ/cm~2 UVB radiation for 40 min + black wolfberry water extracts 2 mg/ml). Cell proliferation in each group was detected by MTS,apoptotic rate in each group was assessed by flow cytometry. The expression levels of P38 MAPK,P53,caspase-8,caspase-3and Bcl-2 in each group were detected by Western blotting. Results Cell proliferation in UVB group was lower than that in control group( P < 0. 05); cell proliferation in black wolfberry water extracts group was lower than that in control group but higher than that in UVB group( P < 0. 05). Apoptotic rate in UVB group was higher than that in control group( P < 0. 05);apoptotic rate in black wolfberry water extracts group was higher than that in control group but lower than that in UVB group( P <0. 05). The expression levels of P38 MAPK,P53,caspase-8,caspase-3 in UVB group were higher than those in control group,but the expression level of Bcl-2 in UVB group was lower than that in control group( P < 0. 05); the expression levels of P38 MAPK,P53,caspase-8,caspase-3 in black wolfberry water extracts group were lower than those in UVB group,but the expression level of Bcl-2 in black wolfberry water extracts group was higher than that in UVB group( P < 0. 05). Conclusion The black wolfberry water extracts can promote the proliferation of HaCaT cells after UVB radiation and prevent their apoptosis. The mechanism might be that the black wolfberry water extracts can down-regulate the expression levels of P38 MAPK,P53,caspase-8 and caspase-3 and up-regulate the expression level of Bcl-2,so as to reduce the radiation damage of HaCaT cells.
引文
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[13]毛德文,陈月桥,王丽,等.Caspase-8及Caspase-3与细胞凋亡[J].辽宁中医药大学学报,2008,10(10):148-150.DOI:10.3969/j.issn.1673-842X.2008.10.091.MAO D W,CHEN Y Q,WANG L,et al.Relationship of caspase-8 and caspase-3 to apoptosis[J].Journal of Liaoning University of Traditional Chinese Medicine,2008,10(10):148-150.DOI:10.3969/j.issn.1673-842X.2008.10.091.
[14]WU J,ZHAO S,ZHANG J,et al.Over-expression of survivin is a factor responsible for differential responses of ovarian cancer cells to S-allylmercaptocysteine(SAMC)[J].Exp Mol Pathol,2016,100(2):294-302.DOI:10.1016/j.yexmp.2016.02.003.
[2]陈忱,彭景,孙桂菊,等.枸杞多糖提取工艺的研究进展[J].粮食与食品工业,2013,20(4):54-57.DOI:10.3969/j.issn.1672-5026.2013.04.015.CHEN C,PENG J,SUN G J,et al.Research progress on extraction technology of lycium barbarum polysaccharide[J].Cereal and Food Industry,2013,20(4):54-57.DOI:10.3969/j.issn.1672-5026.2013.04.015.
[3]加杨娥,任立汆,燕华玲,等.黑枸杞水提物对中波紫外线辐射人永生化角质形成细胞抗氧化的作用[J].中国皮肤性病学杂志,2017,31(3):251-254.DOI:10.13735/j.cjdv.1001-7089.201609092.JIA Y E,REN L C,YAN H L,et al.Anti-oxidant effects of black wolfberry water extracts on Ha Ca T cells damaged by UVB radiation[J].Chin J Derm Venereol,2017,31(3):251-254.DOI:10.13735/j.cjdv.1001-7089.201609092.
[4]BRAVO B S,AZULAY D R,LUIZ R R,et al.Oral isotretinoin in photoaging:objective histological evidence of efficacy and durability[J].An Bras Dermatol,2015,90(4):479-486.DOI:10.1590/abd1806-4841.20153703.
[5]杜然然,毕宏生,郭俊国,等.枸杞多糖对体外培养视网膜神经节细胞的保护作用[J].眼科新进展,2014,34(2):119-122.DOI:10.13389/j.cnki.rao.2014.0030.DU R R,BI H S,GUO J G,et al.Protective effects of lycium barbarum polysaccharides on cultured retinal ganglion cell[J].Recent Advances in Ophthalmology,2014,34(2):119-122.DOI:10.13389/j.cnki.rao.2014.0030.
[6]郭砚,孙娟,王丽雯.藏雪莲水提取物对中波红斑效应紫外线辐射后人角质形成细胞中蛋白表达的影响研究[J].中国全科医学,2015,18(24):2929-2933.DOI:10.3969/j.issn.1007-9572.2015.24.012.GUO Y,SUN J,WANG L W.Influence of saussurea tridactyla schbip water extracts on the protein expression of Ha Ca T cells after UVB radiation[J].Chinese General Practice,2015,18(24):2929-2933.DOI:10.3969/j.issn.1007-9572.2015.24.012.
[7]刘丽君,彭建新,洪华珠,等.线粒体在细胞凋亡中的变化与作用[J].细胞生物学杂志,2005,27(2):117-120.DOI:10.3969/j.issn.1674-7666.2005.02.005.LIU L J,PENG J X,HONG H Z,et al.Mitochondrial changes and role in apoptosis[J].Chinese Journal of Cell Biology,2005,27(2):117-120.DOI:10.3969/j.issn.1674-7666.2005.02.005.
[8]贾文斌.脂肪间充质干细胞抑制烧伤创面内残存角质形成细胞凋亡的实验研究[D].西安:第四军医大学,2014.JIA W B.Inhibitory effect of adipose derived mesenchymal stem cells on the apoptosis of residual keratinocytes in burn wounds[D].Xi'an:The Fourth Military Medical University,2014.
[9]HACKER G,PASCHEN S A.Therapeutic targets in the mitochondrial apoptotic pathway[J].Expert Opin Ther Targets,2007,11(4):515-526.DOI:10.1517/14728222.11.4.515.
[10]MATSUSHITA K,WU Y,QIU J,et al.Fas receptor and neuronal cell death after spinal cord ischemia[J].J Neurosci,2000,20(18):6879-6887.
[11]HUANG H,XU S,LI F,et al.Clinical application of computed tomography-guided(125)I seed interstitial implantation for head and neck cancer patients with unmanageable cervical lymph node metastases[J].Eur J Med Res,2016,21:18.DOI:10.1186/s40001-016-0213-1.
[12]SCATENA R,BOTTONI P,BOTTA G,et al.The role of mitochondria in pharmacotoxicology:a reevaluation of an old,newly emerging topic[J].Am J Physiol Cell Physiol,2007,293(1):C12-21.DOI:10.1152/ajpcell.00314.2006.
[13]毛德文,陈月桥,王丽,等.Caspase-8及Caspase-3与细胞凋亡[J].辽宁中医药大学学报,2008,10(10):148-150.DOI:10.3969/j.issn.1673-842X.2008.10.091.MAO D W,CHEN Y Q,WANG L,et al.Relationship of caspase-8 and caspase-3 to apoptosis[J].Journal of Liaoning University of Traditional Chinese Medicine,2008,10(10):148-150.DOI:10.3969/j.issn.1673-842X.2008.10.091.
[14]WU J,ZHAO S,ZHANG J,et al.Over-expression of survivin is a factor responsible for differential responses of ovarian cancer cells to S-allylmercaptocysteine(SAMC)[J].Exp Mol Pathol,2016,100(2):294-302.DOI:10.1016/j.yexmp.2016.02.003.