针刺对皮肤光老化大鼠p38MAPK和NF-κB信号传导通路影响的实验研究
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摘要
目的：本项研究通过体内实验，旨在探讨模拟日光中UV照射所致皮肤光老化大鼠细胞凋亡和凋亡相关调控基因的表达情况，以及p38MAPK和核转录因子kappaB（NF-κB）信号传导通路在光老化发生机制中的作用；并研究针刺疗法在皮肤光老化的干预过程中的作用机制，以期发掘出对光老化有明确防治作用的针刺疗法。
材料与方法：本研究采用UVA+UVB光源（40W紫外线灯管：UVB灯管5根，UVA灯管2根）照射大鼠背部皮肤制备大鼠皮肤光老化模型。每只大鼠均用电推剪剪除大鼠背部5.0cm×5.0cm区域内毛发，放置于自制紫外线灯箱中。适应环境饲养一周后，第2周开始每天照射2h，第3-7周每天照射4h，第8-13周每天照射6h，照射以5天为一个周期，间隔2天，再开始下一个周期，直至造模成功。采用针刺为实验因素，维生素E为阳性对照药，以皮肤组织中氧自由基含量、抗氧化酶类活力、细胞凋亡情况、基质金属蛋白酶-1（MMP-1）表达及p38MAPK、NF-κB信号传导通路中相关基因和蛋白表达为效应指标进行体内实验。实验大鼠随机分为正常组、模型空白组、针刺组、维生素E组（简称VE组），各组于造模前分别进行针刺和外涂相应药物后进行紫外线照射；13周后处死动物，取背部皮肤组织检测以下指标：比色法检测丙二醛（MDA）、过氧化氢（H_2O_2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活力；免疫组化法检测Bcl-2和Bax蛋白表达及凋亡阳性细胞数目；RT-PCR法检测c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1mRNA表达；Western Blot法检测蛋白c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1蛋白表达。
结果：
1.大鼠体征表现：正常组大鼠实验全程皮肤无明显改变，皮毛有光泽，活泼喜动，食欲佳，食量正常。模型空白组大鼠13周实验结束时皮毛光泽度下降，皮肤增厚，弹性丧失，出现宽而深的皱纹。针刺组和VE组上述表现均有不同程度的改善，其中针刺组效果更好。
2.各组大鼠体重变化：实验第1周各组大鼠体重比较，无明显差异（P＞0.05）。在实验进行至第13周末时，各组大鼠体重比较具有明显差异。与正常组比较，各组大鼠体重均明显下降，其中以模型空白组下降最为明显（P＜0.01）；与模型空白组比较，各治疗组体重均明显升高，具有显著差异（P＜0.01）。
3.大鼠皮肤组织SOD活性：与正常组比较，模型空白组大鼠SOD活性明显降低，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组和VE组SOD活性均升高，差异有统计学意义(P＜0.01)。
4.大鼠皮肤组织MDA含量：与正常组比较，模型空白组大鼠MDA含量明显升高，差异有统计学意义(P＜0.01)，而针刺组无显著性差异（P＞0.05）；与模型空白组比较，针刺组和VE组MDA含量均降低，差异有统计学意义(P＜0.01)。
5.大鼠皮肤组织H_2O_2含量：与正常组比较，模型空白组大鼠H_2O_2含量显著升高，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组H_2O_2含量降低，差异有统计学意义(P＜0.01)，而VE组无显著性差异（P＞0.05）。
6.大鼠皮肤组织CAT活性：与正常组比较，模型空白组大鼠CAT活性明显降低，差异有统计学意义(P＜0.01)，而针刺组无显著性差异（P＞0.05）；与模型空白组比较，针刺组CAT活性升高，差异有统计学意义(P＜0.01)，而VE组无显著性差异（P＞0.05）。
7.大鼠皮肤组织GSH-Px活性：与正常组比较，模型空白组大鼠GSH-Px活性明显降低，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组和VE组GSH-Px活性均升高，差异有显著意义(P＜0.01)。
8.大鼠皮肤组织免疫组化染色结果：
（1）大鼠皮肤组织Bcl-2和Bax蛋白平均灰度值：与正常组比较，模型空白组大鼠Bcl-2蛋白表达的平均灰度值增加，表明蛋白含量减少，差异有统计学意义（P＜0.01）；与模型空白组比较，针刺组和VE组蛋白表达的平均灰度值减弱，表明蛋白含量增加，差异有统计学意义（P＜0.01）；Bax蛋白表达呈相反趋势。
（2）大鼠皮肤组织凋亡阳性细胞数：正常组皮肤组织凋亡细胞存在散在阳性表达，模型空白组呈弥散阳性表达，凋亡阳性细胞数与正常组比较差异有统计学意义（P＜0.01）；针刺组和VE组均呈散在阳性表达，凋亡阳性细胞数与模型组比较差异有统计学意义（P＜0.01）。
9.RT-PCR结果：正常大鼠皮肤组织可检测到c-Jun、c-Fos mRNA、p38MAPK、NF-κB、MMP-1表达。
（1）c-Jun mRNA表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较差异无统计学意义（P＞0.05）。
（2）c-Fos mRNA表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较差异无统计学意义（P＞0.05）。
（3）p38MAPK、NF-κB、MMP-1mRNA表达：与正常组比较，模型空白组表达明显升高，（P＜0.01）；与模型空白组比较，针刺组和VE组表达均显著下调，差异有统计学意义（P＜0.01）。
10.Western blot结果：正常大鼠皮肤组织可检测到c-Jun、c-Fos、p38MAPK、NF-κB和MMP-1蛋白表达。
（1）c-Jun蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调，差异有统计学意义（P＜0.01）。
（2）c-Fos蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）差异有统计学意义（P＜0.01）。
（3）p38MAPK、NF-κB、MMP-1蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较，针刺组NF-κB和MMP-1蛋白下调更为显著，差异有统计学意义（P＜0.05）。
结论：
1.针刺可用于防治大鼠皮肤光老化，能降低光老化大鼠皮肤组织氧自由基含量，提高抗氧化酶类活性。
2.针刺可下调大鼠光老化皮肤组织促凋亡基因Bax蛋白表达、上调凋亡抑制基因Bcl-2蛋白的表达，从而减少皮肤细胞凋亡数目，这是实现针刺抗皮肤光老化的主要机制之一。
3.UV照射活化了皮肤组织p38MAPK，激活了MAPK信号传导通路，进而导致MMP-1升高；同时，UV辐射形成的ROS，激活了下游NF-κB信号传导通路，也能上调靶基因MMP-1的表达，使细胞外基质成分降解，形成皮肤光老化。
4.针刺对抗皮肤光老化途径之一是抑制皮肤组织p38MAPK和NF-κB表达，进而降低MMP-1生成，阻止其降解细胞外基质成分，起到抗皮肤光老化的作用。
Purpose：This study through the experiment in vivo, aims to explore theexpression of skin photoaging apoptosis and apoptosis related genes in ratscaused by UV irradiation in simulated solar light, the role of p38MAPK and nuclearfactor kappaB (NF-κB) signaling pathways in the skin photoaging mechanism;Research the mechanism of acupuncture in skin photoaging intervention, in orderto have a clear control effect of acupuncture in the process of skin photoaging.
Material and method：This research adopts the UVA and UVB light source (40w Uv lamp:5UVB light tubes,2UVA light tubes),irradiates the back skin ofrat to prepare skin photoaging model. Cut off each back hair of rat adaptingto the environment after feeding one week in5.0cm x5.0cm area with electrichair cutter, placed in the self-made ultraviolet (uv) light box. The second weekwe begin to irradiate2h every day, The third to seven week are given dailyirradiation for4h, The eight to thirteen week are given daily radiation for6h, irradiation in5days for a cycle, interval of2days, then to start thenext cycle until the building is successful. Choose acupuncture as theexperimental factor, vitamin E as positive control drug, with oxygen free radicalcontent in skin tissue, antioxidant enzyme activity, cell apoptosis, silk crackthe matrix metalloproteinase(MMP-1) expression and p38MAPK and NF-κB signalingpathway related gene and protein expression as effect indexes in vivo.Experimental rats were randomly divided into normal group, blank model group,acupuncture group and vitamin E group, each group had ten rats, prior to the building each group separately is given ultraviolet radiation after acupunctureand outside with corresponding drugs. After13weeks put to death animals, takeback skin tissue totest the following indicators: colorimetric method to detectmalondialdehyd(MDA),hydrogen peroxide(H_2O_2)contents, superoxidedismutase(SOD),catalase(CAT), glutathione peroxidase(GSH-Px) activity; Immunohistochemicalmethod to detect the Bcl-2and Bax protein expression and apoptosis positivecell number; RT-PCR method to detect c-Jun, c-Fos, p38MAPK and NF-κB, MMP-1mRNA expression; Western Blot method to detect protein c-Jun, c-Fos, p38MAPK,NF-κB, MMP-1protein expression.
Results：
1.Physical signs of rats: normal group rat skins had no significant change, allthe skins luster and vibrant, appetite was good and normal. After the thirteenweek blank model group surface gloss of rats declined, skin thickeninged, lossof elasticity, a wide and deep wrinkles. The acupuncture group and VE group allhad different degrees of improvement, the performance of acupuncture group werebetter.
2.Body weight change: In experiment the first week each rat body weight had nosignificant difference (P＞0.05). In the experiment to thirteen week, groupsof body weight had obvious difference. Compared with normal group, each groupof body weight were significantly declined, the decline of blank model groupwas most pronounced (P＜0.01); Compared with blank model group, the treatmentgroup were significantly higher with significant difference (P＜0.01).
3.SOD activity in the skin tissue of rats: compared with normal group, modelblank group SOD activity significantly decreased, the difference wasstatistically significant (P＜0.01); Compared with blank model group,acupuncture group and VE SOD activity were higher, the difference wasstatistically significant (P＜0.01).
4.MDA content in the skin tissue of rats: compared with normal group, model blankgroup MDA content increased significantly, the difference was statisticallysignificant (P＜0.01),acupuncture group had no significant difference (P＞0.05); Compared with blank model group, acupuncture group and VE MDA content were lower,the difference was statistically significant (P＜0.01).
5.H_2O_2content in the skin tissue of rats: compared with normal group, model blankgroup H_2O_2content increased significantly, the difference was statisticallysignificant (P＜0.01); Compared with blank model group, acupuncture group H_2O_2content reduced, the difference was statistically significant (P＜0.01), andVE group had no significant difference (P＞0.05).
6.CAT activity in the skin tissue of rats: compared with normal group, modelblank group CAT activity decreased obviously, the difference was statisticallysignificant (P＜0.01), and acupuncture group had no significant difference (P＞0.05); Compared with blank model group, acupuncture group increased CATactivity, the difference was statistically significant (P＜0.01), and VE grouphad no significant difference (P＞0.05).7.GSH-Px activity in the skin tissue of rats: compared with normal group, blankmodel group activity of GSH-Px decrease, the difference was statisticallysignificant (P＜0.01); Compared with blank model group, the activity of GSH-Pxin acupuncture group and VE group were higher with significant difference (P＜0.01).
8.The immunohistochemical staining results in the skin tissue of rats:
(1) Bcl-2and Bax protein average grey value in the skin tissue of rat: comparedwith normal group, model blank group the average gray values of Bcl-2proteinexpression increased, showed that protein content decreased, the difference wasstatistically significant (P＜0.01); Compared with blank model group,acupuncture group and VE average gray value of protein expression decreased,showed that protein content increase, the difference was statisticallysignificant (P＜0.01).
(2) apoptosis positive cell number in the skin tissue of rat: normal group ofskin tissue apoptosis cells exist in positive expression,blank model groupshowed diffuse positive expression, compared with normal group the differenceof positive apoptosis cell number was statistically significant (P＜0.01); Acupuncture group and the VE group were scattered in the positive expression,compared with the model group the difference of positive apoptosis cell numberwas statistically significant (P＜0.01).
9.RT-PCR results: normal skin tissues of rats can be detected c-Jun, c-Fos mRNA,p38MAPK and NF-κB, expression of MMP-1.
(1)c-Jun mRNA expression: compared with normal group, expression of blank modelgroup increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01);thedifference was not statistically between acupuncture group and VE.
(2)c-Fos mRNA expression: compared with normal group, expression of blank modelgroup increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01); there wasno statistically significant difference (P＞0.05)between acupuncture group andVE.
(3)P38MAPK, NF-κB, MMP-1mRNA expression: compared with normal group,expression of blank model group was obviously higher,(P＜0.01); Compared withblank model group, the expression of acupuncture group and VE group weresignificantly lower, the difference was statistically significant (P＜0.01).10.Western blot results: normal skin tissues of rat can be detected c-Jun, c-Fos,p38MAPK and NF-κB and MMP-1protein expression.
(1)c-Jun protein expression: compared with normal group, expression of blankmodel group increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered, the difference wasstatistically significant (P＜0.01).
(2)c-Fos protein expression: compared with normal group,expression of blankmodel group increased significantly (P＜0.01);Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01),thedifference was statistically significant (P＜0.01).
(3)p38MAPK, NF-κB, MMP-1protein expression: compared with normal group,expression of blank model group increased significantly (P＜0.01); Compared with blank model group, acupuncture group and VE expression significantly lowered(P＜0.01); Compared with VE group, acupuncture group the NF-kappa B and MMP-1protein is more significant, the difference had statistical significance(P＜0.05).
Conclusion：
1.Acupuncture can be used for prevention and treatment of rat skin photoaging,reduce oxygen free radical content of rat skin photoaging tissue, increase theantioxidant enzymes SOD, GSH-Px and CAT activity.
2.Acupuncture can promote the expression of apoptosis gene Bax protein in ratskin photoaging tissue, increase the expression of apoptosis suppressor genesBcl-2protein, thus reduce the number of skin cell apoptosis, which is to realizeone of the main mechanisms of acupuncture resistance to skin photoaging.
3.UV irradiation activated the skin tissue p38MAPK, activated the MAPK signalingpathways, and then led to the rise of MMP-1; At the same time, ROS formed by UV radiationactivated the downstream NF-κ B signaling pathways, also could raise theexpression of target gene MMP-1, led to the degradation of extracellular matrixcomposition and formed skin photoaging.
4.One way of acupuncture against skin photoaging was that reduced the expressionof skin tissue p38MAPK and NF-κB，in turn inhibited synthesis of MMP-1,preventedthe degradation of collagen in the extracellular matrix to make the resistanteffect of skin photoaging.
材料与方法：本研究采用UVA+UVB光源（40W紫外线灯管：UVB灯管5根，UVA灯管2根）照射大鼠背部皮肤制备大鼠皮肤光老化模型。每只大鼠均用电推剪剪除大鼠背部5.0cm×5.0cm区域内毛发，放置于自制紫外线灯箱中。适应环境饲养一周后，第2周开始每天照射2h，第3-7周每天照射4h，第8-13周每天照射6h，照射以5天为一个周期，间隔2天，再开始下一个周期，直至造模成功。采用针刺为实验因素，维生素E为阳性对照药，以皮肤组织中氧自由基含量、抗氧化酶类活力、细胞凋亡情况、基质金属蛋白酶-1（MMP-1）表达及p38MAPK、NF-κB信号传导通路中相关基因和蛋白表达为效应指标进行体内实验。实验大鼠随机分为正常组、模型空白组、针刺组、维生素E组（简称VE组），各组于造模前分别进行针刺和外涂相应药物后进行紫外线照射；13周后处死动物，取背部皮肤组织检测以下指标：比色法检测丙二醛（MDA）、过氧化氢（H_2O_2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活力；免疫组化法检测Bcl-2和Bax蛋白表达及凋亡阳性细胞数目；RT-PCR法检测c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1mRNA表达；Western Blot法检测蛋白c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1蛋白表达。
结果：
1.大鼠体征表现：正常组大鼠实验全程皮肤无明显改变，皮毛有光泽，活泼喜动，食欲佳，食量正常。模型空白组大鼠13周实验结束时皮毛光泽度下降，皮肤增厚，弹性丧失，出现宽而深的皱纹。针刺组和VE组上述表现均有不同程度的改善，其中针刺组效果更好。
2.各组大鼠体重变化：实验第1周各组大鼠体重比较，无明显差异（P＞0.05）。在实验进行至第13周末时，各组大鼠体重比较具有明显差异。与正常组比较，各组大鼠体重均明显下降，其中以模型空白组下降最为明显（P＜0.01）；与模型空白组比较，各治疗组体重均明显升高，具有显著差异（P＜0.01）。
3.大鼠皮肤组织SOD活性：与正常组比较，模型空白组大鼠SOD活性明显降低，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组和VE组SOD活性均升高，差异有统计学意义(P＜0.01)。
4.大鼠皮肤组织MDA含量：与正常组比较，模型空白组大鼠MDA含量明显升高，差异有统计学意义(P＜0.01)，而针刺组无显著性差异（P＞0.05）；与模型空白组比较，针刺组和VE组MDA含量均降低，差异有统计学意义(P＜0.01)。
5.大鼠皮肤组织H_2O_2含量：与正常组比较，模型空白组大鼠H_2O_2含量显著升高，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组H_2O_2含量降低，差异有统计学意义(P＜0.01)，而VE组无显著性差异（P＞0.05）。
6.大鼠皮肤组织CAT活性：与正常组比较，模型空白组大鼠CAT活性明显降低，差异有统计学意义(P＜0.01)，而针刺组无显著性差异（P＞0.05）；与模型空白组比较，针刺组CAT活性升高，差异有统计学意义(P＜0.01)，而VE组无显著性差异（P＞0.05）。
7.大鼠皮肤组织GSH-Px活性：与正常组比较，模型空白组大鼠GSH-Px活性明显降低，差异有统计学意义(P＜0.01)；与模型空白组比较，针刺组和VE组GSH-Px活性均升高，差异有显著意义(P＜0.01)。
8.大鼠皮肤组织免疫组化染色结果：
（1）大鼠皮肤组织Bcl-2和Bax蛋白平均灰度值：与正常组比较，模型空白组大鼠Bcl-2蛋白表达的平均灰度值增加，表明蛋白含量减少，差异有统计学意义（P＜0.01）；与模型空白组比较，针刺组和VE组蛋白表达的平均灰度值减弱，表明蛋白含量增加，差异有统计学意义（P＜0.01）；Bax蛋白表达呈相反趋势。
（2）大鼠皮肤组织凋亡阳性细胞数：正常组皮肤组织凋亡细胞存在散在阳性表达，模型空白组呈弥散阳性表达，凋亡阳性细胞数与正常组比较差异有统计学意义（P＜0.01）；针刺组和VE组均呈散在阳性表达，凋亡阳性细胞数与模型组比较差异有统计学意义（P＜0.01）。
9.RT-PCR结果：正常大鼠皮肤组织可检测到c-Jun、c-Fos mRNA、p38MAPK、NF-κB、MMP-1表达。
（1）c-Jun mRNA表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较差异无统计学意义（P＞0.05）。
（2）c-Fos mRNA表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较差异无统计学意义（P＞0.05）。
（3）p38MAPK、NF-κB、MMP-1mRNA表达：与正常组比较，模型空白组表达明显升高，（P＜0.01）；与模型空白组比较，针刺组和VE组表达均显著下调，差异有统计学意义（P＜0.01）。
10.Western blot结果：正常大鼠皮肤组织可检测到c-Jun、c-Fos、p38MAPK、NF-κB和MMP-1蛋白表达。
（1）c-Jun蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调，差异有统计学意义（P＜0.01）。
（2）c-Fos蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）差异有统计学意义（P＜0.01）。
（3）p38MAPK、NF-κB、MMP-1蛋白表达：与正常组比较，模型空白组表达明显升高（P＜0.01）；与模型空白组比较，针刺组和VE组表达显著下调（P＜0.01）；针刺组和VE组比较，针刺组NF-κB和MMP-1蛋白下调更为显著，差异有统计学意义（P＜0.05）。
结论：
1.针刺可用于防治大鼠皮肤光老化，能降低光老化大鼠皮肤组织氧自由基含量，提高抗氧化酶类活性。
2.针刺可下调大鼠光老化皮肤组织促凋亡基因Bax蛋白表达、上调凋亡抑制基因Bcl-2蛋白的表达，从而减少皮肤细胞凋亡数目，这是实现针刺抗皮肤光老化的主要机制之一。
3.UV照射活化了皮肤组织p38MAPK，激活了MAPK信号传导通路，进而导致MMP-1升高；同时，UV辐射形成的ROS，激活了下游NF-κB信号传导通路，也能上调靶基因MMP-1的表达，使细胞外基质成分降解，形成皮肤光老化。
4.针刺对抗皮肤光老化途径之一是抑制皮肤组织p38MAPK和NF-κB表达，进而降低MMP-1生成，阻止其降解细胞外基质成分，起到抗皮肤光老化的作用。
Purpose：This study through the experiment in vivo, aims to explore theexpression of skin photoaging apoptosis and apoptosis related genes in ratscaused by UV irradiation in simulated solar light, the role of p38MAPK and nuclearfactor kappaB (NF-κB) signaling pathways in the skin photoaging mechanism;Research the mechanism of acupuncture in skin photoaging intervention, in orderto have a clear control effect of acupuncture in the process of skin photoaging.
Material and method：This research adopts the UVA and UVB light source (40w Uv lamp:5UVB light tubes,2UVA light tubes),irradiates the back skin ofrat to prepare skin photoaging model. Cut off each back hair of rat adaptingto the environment after feeding one week in5.0cm x5.0cm area with electrichair cutter, placed in the self-made ultraviolet (uv) light box. The second weekwe begin to irradiate2h every day, The third to seven week are given dailyirradiation for4h, The eight to thirteen week are given daily radiation for6h, irradiation in5days for a cycle, interval of2days, then to start thenext cycle until the building is successful. Choose acupuncture as theexperimental factor, vitamin E as positive control drug, with oxygen free radicalcontent in skin tissue, antioxidant enzyme activity, cell apoptosis, silk crackthe matrix metalloproteinase(MMP-1) expression and p38MAPK and NF-κB signalingpathway related gene and protein expression as effect indexes in vivo.Experimental rats were randomly divided into normal group, blank model group,acupuncture group and vitamin E group, each group had ten rats, prior to the building each group separately is given ultraviolet radiation after acupunctureand outside with corresponding drugs. After13weeks put to death animals, takeback skin tissue totest the following indicators: colorimetric method to detectmalondialdehyd(MDA),hydrogen peroxide(H_2O_2)contents, superoxidedismutase(SOD),catalase(CAT), glutathione peroxidase(GSH-Px) activity; Immunohistochemicalmethod to detect the Bcl-2and Bax protein expression and apoptosis positivecell number; RT-PCR method to detect c-Jun, c-Fos, p38MAPK and NF-κB, MMP-1mRNA expression; Western Blot method to detect protein c-Jun, c-Fos, p38MAPK,NF-κB, MMP-1protein expression.
Results：
1.Physical signs of rats: normal group rat skins had no significant change, allthe skins luster and vibrant, appetite was good and normal. After the thirteenweek blank model group surface gloss of rats declined, skin thickeninged, lossof elasticity, a wide and deep wrinkles. The acupuncture group and VE group allhad different degrees of improvement, the performance of acupuncture group werebetter.
2.Body weight change: In experiment the first week each rat body weight had nosignificant difference (P＞0.05). In the experiment to thirteen week, groupsof body weight had obvious difference. Compared with normal group, each groupof body weight were significantly declined, the decline of blank model groupwas most pronounced (P＜0.01); Compared with blank model group, the treatmentgroup were significantly higher with significant difference (P＜0.01).
3.SOD activity in the skin tissue of rats: compared with normal group, modelblank group SOD activity significantly decreased, the difference wasstatistically significant (P＜0.01); Compared with blank model group,acupuncture group and VE SOD activity were higher, the difference wasstatistically significant (P＜0.01).
4.MDA content in the skin tissue of rats: compared with normal group, model blankgroup MDA content increased significantly, the difference was statisticallysignificant (P＜0.01),acupuncture group had no significant difference (P＞0.05); Compared with blank model group, acupuncture group and VE MDA content were lower,the difference was statistically significant (P＜0.01).
5.H_2O_2content in the skin tissue of rats: compared with normal group, model blankgroup H_2O_2content increased significantly, the difference was statisticallysignificant (P＜0.01); Compared with blank model group, acupuncture group H_2O_2content reduced, the difference was statistically significant (P＜0.01), andVE group had no significant difference (P＞0.05).
6.CAT activity in the skin tissue of rats: compared with normal group, modelblank group CAT activity decreased obviously, the difference was statisticallysignificant (P＜0.01), and acupuncture group had no significant difference (P＞0.05); Compared with blank model group, acupuncture group increased CATactivity, the difference was statistically significant (P＜0.01), and VE grouphad no significant difference (P＞0.05).7.GSH-Px activity in the skin tissue of rats: compared with normal group, blankmodel group activity of GSH-Px decrease, the difference was statisticallysignificant (P＜0.01); Compared with blank model group, the activity of GSH-Pxin acupuncture group and VE group were higher with significant difference (P＜0.01).
8.The immunohistochemical staining results in the skin tissue of rats:
(1) Bcl-2and Bax protein average grey value in the skin tissue of rat: comparedwith normal group, model blank group the average gray values of Bcl-2proteinexpression increased, showed that protein content decreased, the difference wasstatistically significant (P＜0.01); Compared with blank model group,acupuncture group and VE average gray value of protein expression decreased,showed that protein content increase, the difference was statisticallysignificant (P＜0.01).
(2) apoptosis positive cell number in the skin tissue of rat: normal group ofskin tissue apoptosis cells exist in positive expression,blank model groupshowed diffuse positive expression, compared with normal group the differenceof positive apoptosis cell number was statistically significant (P＜0.01); Acupuncture group and the VE group were scattered in the positive expression,compared with the model group the difference of positive apoptosis cell numberwas statistically significant (P＜0.01).
9.RT-PCR results: normal skin tissues of rats can be detected c-Jun, c-Fos mRNA,p38MAPK and NF-κB, expression of MMP-1.
(1)c-Jun mRNA expression: compared with normal group, expression of blank modelgroup increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01);thedifference was not statistically between acupuncture group and VE.
(2)c-Fos mRNA expression: compared with normal group, expression of blank modelgroup increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01); there wasno statistically significant difference (P＞0.05)between acupuncture group andVE.
(3)P38MAPK, NF-κB, MMP-1mRNA expression: compared with normal group,expression of blank model group was obviously higher,(P＜0.01); Compared withblank model group, the expression of acupuncture group and VE group weresignificantly lower, the difference was statistically significant (P＜0.01).10.Western blot results: normal skin tissues of rat can be detected c-Jun, c-Fos,p38MAPK and NF-κB and MMP-1protein expression.
(1)c-Jun protein expression: compared with normal group, expression of blankmodel group increased significantly (P＜0.01); Compared with blank model group,acupuncture group and VE expression significantly lowered, the difference wasstatistically significant (P＜0.01).
(2)c-Fos protein expression: compared with normal group,expression of blankmodel group increased significantly (P＜0.01);Compared with blank model group,acupuncture group and VE expression significantly lowered (P＜0.01),thedifference was statistically significant (P＜0.01).
(3)p38MAPK, NF-κB, MMP-1protein expression: compared with normal group,expression of blank model group increased significantly (P＜0.01); Compared with blank model group, acupuncture group and VE expression significantly lowered(P＜0.01); Compared with VE group, acupuncture group the NF-kappa B and MMP-1protein is more significant, the difference had statistical significance(P＜0.05).
Conclusion：
1.Acupuncture can be used for prevention and treatment of rat skin photoaging,reduce oxygen free radical content of rat skin photoaging tissue, increase theantioxidant enzymes SOD, GSH-Px and CAT activity.
2.Acupuncture can promote the expression of apoptosis gene Bax protein in ratskin photoaging tissue, increase the expression of apoptosis suppressor genesBcl-2protein, thus reduce the number of skin cell apoptosis, which is to realizeone of the main mechanisms of acupuncture resistance to skin photoaging.
3.UV irradiation activated the skin tissue p38MAPK, activated the MAPK signalingpathways, and then led to the rise of MMP-1; At the same time, ROS formed by UV radiationactivated the downstream NF-κ B signaling pathways, also could raise theexpression of target gene MMP-1, led to the degradation of extracellular matrixcomposition and formed skin photoaging.
4.One way of acupuncture against skin photoaging was that reduced the expressionof skin tissue p38MAPK and NF-κB，in turn inhibited synthesis of MMP-1,preventedthe degradation of collagen in the extracellular matrix to make the resistanteffect of skin photoaging.
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