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

发布时间：2018-05-22 19:17

本文选题：针刺 + 皮肤光老化　；参考：《辽宁中医药大学》2013年博士论文

【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。
[Abstract]:Objective : To investigate the expression of apoptosis and apoptosis - related genes in skin photoaging rats induced by UV irradiation in simulated sunlight , and the role of p38MAPK and NF - 魏B signaling pathway in the mechanism of photoaging .
To study the mechanism of acupuncture therapy in the course of skin photoaging , with a view to developing acupuncture therapy with definite prevention and control effect on photoaging .

Materials and Methods : UVA + UVB light source ( 40W ultraviolet tube : UVB lamp tube 5 , UVA tube 2 ) was used to irradiate the rat back skin to prepare rat skin photoaging model . Each rat was exposed to ultraviolet light for 2 hours at the second week . The expression of oxygen free radical in skin tissue , activity of antioxidant enzymes , apoptosis , matrix metalloproteinase - 1 ( MMP - 1 ) expression and expression of p38MAPK and NF - 魏B signaling pathway were taken as the effect index . The experimental rats were randomly divided into normal group , model blank group , acupuncture group and vitamin E group ( VE group ) .
After 13 weeks , the animals were sacrificed and the skin tissues of the back were taken to detect the following indexes : malondialdehyde ( MDA ) , hydrogen peroxide ( H _ 2O _ 2 ) , superoxide dismutase ( SOD ) , catalase ( CAT ) and glutathione peroxidase ( GSH - Px ) were detected by colorimetry .
The expression of Bcl - 2 and Bax protein and the number of apoptotic positive cells were detected by immunohistochemistry .
The expression of c - Jun , c - fos , p38MAPK , NF - 魏B , MMP - 1 mRNA was detected by RT - PCR .
Western Blot was used to detect the expression of c - Jun , c - fos , p38MAPK , NF - 魏B and MMP - 1 .

Results :

1 . The signs of the rats showed that the whole skin of the normal group had no obvious change in the whole course of the experiment , the fur had the luster , the activity was good , the appetite was good , the appetite was normal . After the 13 week experiment in the model blank group , the luster of the fur decreased , the skin became thicker , the elasticity was lost , and the wrinkles of width and depth appeared . The above expression of the acupuncture group and the VE group had different degrees , among which the acupuncture group had better effect .

2 . The weight of the rats in each group showed no significant difference ( P > 0.05 ) . Compared with the normal group , the weight of the rats in each group was significantly lower than that in the normal group ( P < 0.01 ) .
7 . GSH - Px activity of rat skin tissue : Compared with the normal group , the activity of GSH - Px in the model blank group was significantly decreased and the difference was statistically significant ( P < 0.01 ) .

3 . SOD activity of rat skin tissue : Compared with the normal group , the activity of SOD in the model blank group was significantly lower than that in normal group ( P < 0.01 ) .
Compared with model blank group , SOD activity in acupuncture group and VE group increased significantly ( P < 0.01 ) .

4 . MDA content in skin tissue of rats : Compared with the normal group , the content of MDA in the model blank group increased significantly ( P < 0.01 ) , but there was no significant difference in acupuncture group ( P > 0.05 ) .
Compared with model blank group , the content of MDA in acupuncture group and VE group decreased significantly ( P < 0.01 ) .

5 . The content of H _ 2O _ 2 in rat skin was significantly higher than that in normal group ( P < 0.01 ) .
Compared with the model blank group , the content of H _ 2O _ 2 in the acupuncture group decreased , the difference was statistically significant ( P < 0.01 ) , but there was no significant difference in VE group ( P > 0.05 ) .

6 . CAT activity of rat skin tissue : Compared with the normal group , the CAT activity of the model blank group decreased significantly ( P < 0.01 ) , but there was no significant difference in acupuncture group ( P > 0.05 ) .
Compared with the model blank group , the CAT activity of the acupuncture group increased , the difference was statistically significant ( P < 0.01 ) , but there was no significant difference in VE group ( P > 0.05 ) .

Compared with the model blank group , the body weight of each treatment group increased significantly ( P < 0.01 ) .
Compared with model blank group , the activity of GSH - Px in acupuncture group and VE group increased significantly ( P < 0.01 ) .

8 . Immunohistochemical staining of rat skin tissue :

( 1 ) The mean gray value of Bcl - 2 and Bax protein in skin tissues of rats : Compared with the normal group , the mean gray value of Bcl - 2 protein in the model blank group increased , indicating that the protein content was decreased and the difference was statistically significant ( P < 0.01 ) ;
Compared with the model blank group , the mean gray value of the expression of the acupuncture group and VE group decreased , indicating that the protein content increased and the difference was statistically significant ( P < 0.01 ) .
Bax protein expression was the opposite trend .

( 2 ) The number of apoptosis - positive cells in skin tissues of rats : positive expression of apoptotic cells in normal skin tissues , diffuse positive expression in the blank group of model , and statistical significance between the number of apoptotic positive cells and the normal group ( P < 0.01 ) ;
There was significant difference between the number of positive cells and the number of apoptotic cells in the acupuncture group and the VE group ( P < 0.01 ) .

9 . RT - PCR results : c - Jun , c - fos mRNA , p38MAPK , NF - 魏B and MMP - 1 were detected in normal rat skin tissues .

( 1 ) c - Jun mRNA expression : Compared with the normal group , the expression of c - Jun mRNA increased significantly ( P < 0.01 ) ;
Compared with model blank group , the expression of acupuncture group and VE group decreased significantly ( P < 0.01 ) .
There was no significant difference between acupuncture group and VE group ( P > 0.05 ) .

( 2 ) c - fos mRNA expression : Compared with the normal group , the expression of c - fos mRNA increased significantly ( P < 0.01 ) ;
Compared with model blank group , the expression of acupuncture group and VE group decreased significantly ( P < 0.01 ) .
There was no significant difference between acupuncture group and VE group ( P > 0.05 ) .

( 3 ) p38MAPK , NF - 魏B , MMP - 1 mRNA expression : Compared with the normal group , the expression of p38MAPK , NF - 魏B and MMP - 1 mRNA was significantly increased ( P < 0.01 ) .
Compared with model blank group , the expression of acupuncture group and VE group decreased significantly ( P < 0.01 ) .

10 . Western blot showed that c - Jun , c - fos , p38MAPK , NF - 魏B and MMP - 1 were detected in normal rat skin tissues .

( 1 ) c - Jun protein expression : Compared with the normal group , the expression of c - Jun protein increased significantly ( P < 0.01 ) ;
Compared with model blank group , the expression of acupuncture group and VE group decreased significantly ( P < 0.01 ) .

( 2 ) c - fos protein expression : Compared with the normal group , the expression of the blank group increased significantly ( P < 0.01 ) .
Compared with the model blank group , the expression of the acupuncture group and the VE group decreased significantly ( P < 0.01 ) , and the difference was statistically significant ( P < 0.01 ) .

( 3 ) p38MAPK , NF - 魏B , MMP - 1 protein expression : Compared with the normal group , the expression of p38MAPK , NF - 魏B and MMP - 1 increased significantly ( P < 0.01 ) ;
Compared with model blank group , the expression of acupuncture group and VE group decreased significantly ( P < 0.01 ) .
Compared with VE group , the down - regulation of NF - 魏B and MMP - 1 in acupuncture group was more significant ( P < 0.05 ) .

Conclusion :

1 . The acupuncture can be used for preventing and treating skin photoaging in rats , and can reduce the content of oxygen free radicals in the skin tissues of the photoaging rats and improve the activity of antioxidant enzymes .

2 . Acupuncture could down - regulate the expression of pro - apoptotic gene Bax protein in photoaging skin of rats , up - regulate the expression of apoptosis - inhibiting gene Bcl - 2 protein , thus reducing the number of apoptotic cells , which is one of the main mechanisms for acupuncture to resist skin photoaging .

3 . UV irradiation activated p38MAPK in skin tissue , activated MAPK signaling pathway , which led to the increase of MMP - 1 ;
At the same time , the ROS formed by UV radiation activated the downstream NF - 魏B signal transduction pathway , and also raised the expression of MMP - 1 in the target gene , degrading extracellular matrix components and forming skin photoaging .

4 . One of the ways to fight skin photoaging is to inhibit the expression of p38MAPK and NF - 魏B in skin tissue , and then decrease MMP - 1 production , prevent it from degrading extracellular matrix components , and function as anti - skin photoaging .
【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R245

【参考文献】