| Objective:To clarify the effects of Bangci electro-acupuncture in promoting wound healing at mouse skin; to explore the regulatory mechanism of Bangci electro-acupuncture in mouse skin wound healing.Methods:Mouse back skin excision wound model was used in this study, C57BL/6 mice were randomly divided into five groups:normal group, model group, Bangci EA group, EA inhibitor group, inhibitor group. Each group was give iodine disinfection before the intervention. Model group received conventional therapy of iodine; Bangci electro-acupuncture group:two needles(0.20mm × 13mm) were punctured in the skin, one in the wound center first, the other one in the normal skin 0.5 cm away from the wounded skin edge, and then connected with micro-current electrical stimulation apparatus (MICRO Plus TM, BioMedical Life Systems, Inc)with 500uA,0.5Hz for 30 min, once a day. Bangci EA+inhibitor group and inhibitor group:specific PI3K inhibitor wortmannin (1.4mg · kg-1) was given to mouse by intraperitoneal injection before modeling or before each acupuncture treatment,. First experiment, planimeter was used to measure the wound area of the mice came from model group, Bangci EA group and Bangci EA+inhibitor group on timepoint of 1d,3d,7d, 10d,14d.h According to the data obtained, we calculated the rate of wound healing of each subgroup. Then, the healing time was recorded and compared;the second experiment, immunohistochemistry method was used to detect two downstream signaling molecule of PI3K/AKT signaling pathway, eNOS protein and phosphorylated caspase9 in skin tissue near the wound. IOD values of two proteins expression in immunohistochemical pictures were obtained with the application of Image-pro plus6.0 software, with which we did a statistical analysis; Experiment 3, Realtime-PCR method was used to detect the expression of eNOSmRNA, caspase9mRNA and AktmRNA in skin tissue came from five groups of mice. β-actin was chose as internal control gene. Make sure that the amplification efficiency of the target gene and internal reference gene close to 100%, and difference within 5%. the 2-ΔΔCT calculation method was used to measure the relative expression of the target gene in each sample group; Fourth experiment, western-blot method was used to detect the t-Akt, p-Akt, eNOS, p- caspase9 (Ser196) protein expression and β-actin was selected as an internal control protein, IOD values of bands were obtained using ImageJ software to calculate the ratio of the target protein and internal reference protein, by which wo did a statistical analysis.Results:The results of the first part of this study found that Bangci EA group was significantly better than the model group on the healing time and healing rate. After the application of specific inhibitor of PI3K/Akt signal transduction pathway wortmannin, healing time was extended and healing rates declined on varying degrees. The foundation indicated that Bangci electro-acupuncture for skin wounds in mice have the exact effect, and PI3K/Akt signaling pathway was involved in the restoration and promotion of mouse skin wounds. In order to observe the relationship between PI3K/Akt signal transduction pathway and Bangci electro-acupuncture treatment, we performed experiments on the organizational level, the genetic level, and protein levels as follows.The second part of the study found that:Compared with the model group, EA improved the expression of endothelial nitric oxide synthase (eNOS). eNOS is the important downstream cytokine of PI3K/Akt signal transduction pathway, which can produce NO, and NO is a very powerful medium to promote angiogenesis [5],which improves blood circulation that means for providing important material foundation to wound healing. Meanwhile, p-caspase9 (Ser196) protein levels significantly increased in Bangci electro-acupuncture group.caspase-9 is a pro-apoptotic factor, a downstream cytokine of PI3K/ Akt signal transduction pathway. Activated Akt can lead to make Ser 196 sites of caspase-9 protein phosphorylation. The increasing of phosphorylation levels of caspase9 may cause apoptosis be weaken, which means the protective effect of the damaged tissue. The application of PI3K/Akt signaling pathway specific inhibitors wortmannin makes EA’s role is diminished.Realtime-PCR results, the third part of the study, showed that:compared with the model group, Bangci electro-acupuncture treatment inhibited the expression level of caspase-9 gene, there is a significant difference (P<0.05); and gene expression levels of eNOS was significantly increased in Bangci electro-acupuncture group, rather than the model group (P<0.05), compared with Bangci electro-acupuncture group, caspase-9 gene expression level increased in EA+inhibitor group,and the gene expression levels of eNOS were significantly decreased (P<0.05).western-blot results, the forth part of the study, showed that:Compared with the model group, the protein expression levels of p-Akt (Ser473) expression increased, the difference were statistically significant (P<0.05), while the endothelial nitric oxide synthase (eNOS) expression levels increased significantly, the expression levels of p - caspase-9 were increased, the difference was statistically significant (P<0.05).Conclusion1. EA effectively promoted the wound-healing of mouse skin; and the time required for wound healing was shorten.2. PI3K/Akt signal transduction pathways involved in and promote the repair of skin wounds in mice, with the application of PI3K/Akt signal transduction pathway inhibitor wortmannin, wound healing time will be extended.3. EA can activate PI3K/Akt signaling pathway, which by phosphorylation of Akt, a downstream factor promoting endothelial nitric oxide synthase (eNOS) expression, and thus play a role in promoting wound healing; Akt in activation can promote the expression of p-caspase-9, playing the anti-apoptotic, protecting damaged tissue.4. PI3K/Akt signaling pathway is the important target of Bangci electro-acupuncture and Bangci electro-acupuncture can promotes skin wound repair in mice by activating the PI3K/Akt signaling pathway to achieve. |
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