The Experimental Study On The Effect Of Acupuncture Against Skin Photoaging Rats In P38MAPK And NF-κB Signaling Pathways

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（H₂O₂）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.H₂O₂content in the skin tissue of rats: compared with normal group, model blankgroup H₂O₂content increased significantly, the difference was statisticallysignificant （P＜0.01）; Compared with blank model group, acupuncture group H₂O₂content 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.