本文選題：針刺 + 皮膚光老化��； 參考：《遼寧中醫(yī)藥大學(xué)》2013年博士論文

【摘要】：目的：本項(xiàng)研究通過體內(nèi)實(shí)驗(yàn)，旨在探討模擬日光中UV照射所致皮膚光老化大鼠細(xì)胞凋亡和凋亡相關(guān)調(diào)控基因的表達(dá)情況，以及p38MAPK和核轉(zhuǎn)錄因子kappaB（NF-κB）信號(hào)傳導(dǎo)通路在光老化發(fā)生機(jī)制中的作用；并研究針刺療法在皮膚光老化的干預(yù)過程中的作用機(jī)制，以期發(fā)掘出對(duì)光老化有明確防治作用的針刺療法。 材料與方法：本研究采用UVA+UVB光源（40W紫外線燈管：UVB燈管5根，UVA燈管2根）照射大鼠背部皮膚制備大鼠皮膚光老化模型。每只大鼠均用電推剪剪除大鼠背部5.0cm×5.0cm區(qū)域內(nèi)毛發(fā)，放置于自制紫外線燈箱中。適應(yīng)環(huán)境飼養(yǎng)一周后，第2周開始每天照射2h，第3-7周每天照射4h，第8-13周每天照射6h，照射以5天為一個(gè)周期，間隔2天，再開始下一個(gè)周期，直至造模成功。采用針刺為實(shí)驗(yàn)因素，維生素E為陽性對(duì)照藥，以皮膚組織中氧自由基含量、抗氧化酶類活力、細(xì)胞凋亡情況、基質(zhì)金屬蛋白酶-1（MMP-1）表達(dá)及p38MAPK、NF-κB信號(hào)傳導(dǎo)通路中相關(guān)基因和蛋白表達(dá)為效應(yīng)指標(biāo)進(jìn)行體內(nèi)實(shí)驗(yàn)。實(shí)驗(yàn)大鼠隨機(jī)分為正常組、模型空白組、針刺組、維生素E組（簡(jiǎn)稱VE組），各組于造模前分別進(jìn)行針刺和外涂相應(yīng)藥物后進(jìn)行紫外線照射；13周后處死動(dòng)物，取背部皮膚組織檢測(cè)以下指標(biāo)：比色法檢測(cè)丙二醛（MDA）、過氧化氫（H_2O_2）含量及超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）活力；免疫組化法檢測(cè)Bcl-2和Bax蛋白表達(dá)及凋亡陽性細(xì)胞數(shù)目；RT-PCR法檢測(cè)c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1mRNA表達(dá)；Western Blot法檢測(cè)蛋白c-Jun、c-Fos、p38MAPK、NF-κB、MMP-1蛋白表達(dá)。 結(jié)果： 1.大鼠體征表現(xiàn)：正常組大鼠實(shí)驗(yàn)全程皮膚無明顯改變，皮毛有光澤，活潑喜動(dòng)，食欲佳，食量正常。模型空白組大鼠13周實(shí)驗(yàn)結(jié)束時(shí)皮毛光澤度下降，皮膚增厚，彈性喪失，出現(xiàn)寬而深的皺紋。針刺組和VE組上述表現(xiàn)均有不同程度的改善，，其中針刺組效果更好。 2.各組大鼠體重變化：實(shí)驗(yàn)第1周各組大鼠體重比較，無明顯差異（P＞0.05）。在實(shí)驗(yàn)進(jìn)行至第13周末時(shí)，各組大鼠體重比較具有明顯差異。與正常組比較，各組大鼠體重均明顯下降，其中以模型空白組下降最為明顯（P＜0.01）；與模型空白組比較，各治療組體重均明顯升高，具有顯著差異（P＜0.01）。 3.大鼠皮膚組織SOD活性：與正常組比較，模型空白組大鼠SOD活性明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)；與模型空白組比較，針刺組和VE組SOD活性均升高，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。 4.大鼠皮膚組織MDA含量：與正常組比較，模型空白組大鼠MDA含量明顯升高，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)，而針刺組無顯著性差異（P＞0.05）；與模型空白組比較，針刺組和VE組MDA含量均降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。 5.大鼠皮膚組織H_2O_2含量：與正常組比較，模型空白組大鼠H_2O_2含量顯著升高，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)；與模型空白組比較，針刺組H_2O_2含量降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)，而VE組無顯著性差異（P＞0.05）。 6.大鼠皮膚組織CAT活性：與正常組比較，模型空白組大鼠CAT活性明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)，而針刺組無顯著性差異（P＞0.05）；與模型空白組比較，針刺組CAT活性升高，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)，而VE組無顯著性差異（P＞0.05）。 7.大鼠皮膚組織GSH-Px活性：與正常組比較，模型空白組大鼠GSH-Px活性明顯降低，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)；與模型空白組比較，針刺組和VE組GSH-Px活性均升高，差異有顯著意義(P＜0.01)。 8.大鼠皮膚組織免疫組化染色結(jié)果： （1）大鼠皮膚組織Bcl-2和Bax蛋白平均灰度值：與正常組比較，模型空白組大鼠Bcl-2蛋白表達(dá)的平均灰度值增加，表明蛋白含量減少，差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）；與模型空白組比較，針刺組和VE組蛋白表達(dá)的平均灰度值減弱，表明蛋白含量增加，差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）；Bax蛋白表達(dá)呈相反趨勢(shì)。 （2）大鼠皮膚組織凋亡陽性細(xì)胞數(shù)：正常組皮膚組織凋亡細(xì)胞存在散在陽性表達(dá)，模型空白組呈彌散陽性表達(dá)，凋亡陽性細(xì)胞數(shù)與正常組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）；針刺組和VE組均呈散在陽性表達(dá)，凋亡陽性細(xì)胞數(shù)與模型組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）。 9.RT-PCR結(jié)果：正常大鼠皮膚組織可檢測(cè)到c-Jun、c-Fos mRNA、p38MAPK、NF-κB、MMP-1表達(dá)。 （1）c-Jun mRNA表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)顯著下調(diào)（P＜0.01）；針刺組和VE組比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 （2）c-Fos mRNA表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)顯著下調(diào)（P＜0.01）；針刺組和VE組比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。 （3）p38MAPK、NF-κB、MMP-1mRNA表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高，（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)均顯著下調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）。 10.Western blot結(jié)果：正常大鼠皮膚組織可檢測(cè)到c-Jun、c-Fos、p38MAPK、NF-κB和MMP-1蛋白表達(dá)。 （1）c-Jun蛋白表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)顯著下調(diào)，差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）。 （2）c-Fos蛋白表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)顯著下調(diào)（P＜0.01）差異有統(tǒng)計(jì)學(xué)意義（P＜0.01）。 （3）p38MAPK、NF-κB、MMP-1蛋白表達(dá)：與正常組比較，模型空白組表達(dá)明顯升高（P＜0.01）；與模型空白組比較，針刺組和VE組表達(dá)顯著下調(diào)（P＜0.01）；針刺組和VE組比較，針刺組NF-κB和MMP-1蛋白下調(diào)更為顯著，差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論： 1.針刺可用于防治大鼠皮膚光老化，能降低光老化大鼠皮膚組織氧自由基含量，提高抗氧化酶類活性。 2.針刺可下調(diào)大鼠光老化皮膚組織促凋亡基因Bax蛋白表達(dá)、上調(diào)凋亡抑制基因Bcl-2蛋白的表達(dá)，從而減少皮膚細(xì)胞凋亡數(shù)目，這是實(shí)現(xiàn)針刺抗皮膚光老化的主要機(jī)制之一。 3.UV照射活化了皮膚組織p38MAPK，激活了MAPK信號(hào)傳導(dǎo)通路，進(jìn)而導(dǎo)致MMP-1升高；同時(shí)，UV輻射形成的ROS，激活了下游NF-κB信號(hào)傳導(dǎo)通路，也能上調(diào)靶基因MMP-1的表達(dá)，使細(xì)胞外基質(zhì)成分降解，形成皮膚光老化。 4.針刺對(duì)抗皮膚光老化途徑之一是抑制皮膚組織p38MAPK和NF-κB表達(dá)，進(jìn)而降低MMP-1生成，阻止其降解細(xì)胞外基質(zhì)成分，起到抗皮膚光老化的作用。
[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 .
【學(xué)位授予單位】：遼寧中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R245

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