| Objective:In order to observe the effects of Bangci electro-acupuncture for pressure ulcers in rats during wound healing area changes,the impact of changes in blood flow,microvascular count and vascular endothelial cell count in the skin,Ras protein,c-Raf protein,p-MEK1 protein,MEK1protein,p-ERK1 protein,ERK1 protein,MEKlmRNA and ERK1mRNA expression;To investigate the mechanism of acupuncture treatment of pressure ulcers.Methods:1.Healthy female SD rats 120,Weight 200-250g,at a temperature of 20 ±2 ℃,humidity of 40-50%RH,natural light environments adaptive feeding for a week,single cage feeding,enough food,drinking water during this time.These rats were divided into Id,3d,5d,7d,9d five sub-groups based on the treatment time after the first packet,each sub-group included six rats.2.Given treatments to the rats which got second stage pressure ulcers through using homemade pressure sores modeling equipments.The EA group received Bang-ci treatment,the center of the wound as the first point of the acupuncture,needling depth for 0.2 inches,choose from the wound at the inside edge of 0.5cm for the second entry point,flat piercing the skin,acupuncture needles are consistent with the direction of the leg muscles texture of rats,did not twist the needle,connecting micro-current electrical stimulation instrument to begin treatments(Current:500μA,0.5Hz for 30minutes once a day),where in the negative electrode placed in the center of the skin damage,skin damage outside of the positive connection.EA +inhibitor group received received injection of PD98059 using a micro syringe intravenous before each treatment 1h,after connecting electrical stimulation of acupuncture instrument,but no electricity.The model group received povidone-iodine disinfection process.Other measures of the blank group were the same to the other groups,but no treatment.3.Measured changes in the area of pressure ulcer through using a planimeter and sulfates painted film in moiding finished,1d,3d,5d,7d,9d,treatment finished respectively.4.Detected microvascular count and vascular endothelial cell count each group rats pressure sores in the skin tissue using HE staining method.5.Detected Ras and c-Raf protein expression levels through using immunohistochemistry in rats pressure ulcers.6.Detected MEK1,ERK1 protein expression and phosphorylation levels by Western-blot in rats pressure ulcers.7.Detected differences of the expression levels between MEK1mRNA and ERK1mRNA in rats pressure ulcers by Real-time PCR.Results:1.Pressure Ulcer area measurements:After Id treatment,the model group,the EA group and the EA+inhibitor group compared with each other,there were no significant differences in pressure ulcer healing rate(P>0.05);The remaining points of the time,from 3d to 9d,there was significant difference in pressure ulcer healing rate between the EA group and the EA+inhibitor group(P<0.01);There was significant difference in pressure ulcer healing rate between the EA group and the model group(P<0.01).2.Rats skin blood flow observation in pressure sores:Compared with the blank group,blood flow increased significantly in pressure sores of the model group,which the difference was significant between them from Id to 3d(P<0.05),however blood flow declined significantly in the same area of the model group,which the difference was significant between them from 5d to 9d(P<0.05);Compared with the model group,there were significant changes in the observation area of blood flow of pressure ulcers at each point in the EA group after treatment(P<0.05);Compared with the EA group,there was no difference in the EA + inhibitor group after treatment(P>0.05).3.HE dyeing observation results:Compared with the blank group,including 1d～7d model group,the EA group and the EA + inhibitor group,capillary number declined greatly,there were significant differences between these groups(P<0.05);Compared with the model group,capillary number has significantly increased in the EA group from 3d to 7d,there was significant difference between them(P<0.05);Compared with the EA group,capillary number has significantly increased in the EA + inhibitor group from 3d to 7d,there was significant difference between them(P<0.05);But there was no significant differences between groups in the number of microvessels in 9d(P>0.05).Compared with the blank group,including 1d～7d model group,the EA group and the EA + inhibitor group,vascular endothelial cell count declined greatly,there were significant differences between these groups(P<0.05);Compared with the model group,vascular endothelial cell count has significantly increased in the EA group from 3d to 7d,there was significant difference between them(P<0.05);Compared with the EA group,vascular endothelial cell count has significantly increased in the EA + inhibitor group from 3d to 7d,there was significant difference between them(P<0.05);But there was no significant differences between groups in the number of vascular endothelial cell in 9d(P>0.05).4.Immunohistochemical results:Ras protein expression in rat pressure ulcers skin localized in the cytoplasm,which the color was yellow or brown.Compared with the blank group,there was no significant difference among the model group,the EA group and EA+inhibitor group after 1d treatment.where as Ras protein expression was significantly declined(P<0.05)in the model group from 3d to 9d;Compared with the model group,Ras protein expression was significantly increased after 3d treatment in th EA group,which the difference was significant between them(P<0.01):Compared with the EA group,Ras protein expression was decreased from 3d to 9d in the EA+inhibitor group,and the difference was statistically significant(P<0.05).Compared among the five subgroups of the group,Ras protein expression in model group rised highest after Id treatment,and then gradually declined to the lowest point in 5d,then gradually rised.Ras protein expression levels increased gradually from 1d～9d in the EA group and the EA + inhibitor group.At different time points after the pressure sores occurred,compared with the blank group,there was no significant difference among the model group,the EA group and EA+ inhibitor group after 1d treatment in the expression of c-Raf protein.Where as c-Raf protein expression was significantly declined(P<0.05)in the model group from 3d to 9d;Compared with the model group c-Raf protein expression was significantly increased in the EA group,which the difference was statistically significant(P<0.01)after 3d treatment;Compared with the EA group,c-Raf protein expression declined slightly in 3d～9d on each time point in the EA + inhibitor group,but the difference was no statistically significant(P>0.05).Compared among the five subgroups of the group,c-Raf protein expression in model group rised highest after 1d treatment,and then gradually declined to the lowest point in 3d,then gradually rised.c-Raf protein expression levels increased gradually from 1d～9d in the EA group and the EA + inhibitor group.The expression level of an upward trend has slowed after 7d treatment.5.Western-blot results:Compared with the blank group,there was no significantly increased in the expression leves of MEK1,p-MEK1,ERK1 and p-ERK1 protein among the model group,the EA group,and the EA+inhibitor group after 1d treatment.But the expression significantly increased in the model group,and the difference was statistically significant from 3d to 9d(P<0.05).The peak of the three kinds of protein expression within the group appeared in 5d.Compared with the model group,MEK1,p-MEK1 and p-ERK1 expression levels in the EA group was significantly increased,and the difference was statistically significant(P<0.05),furthermore peak appeared in the 3d within the group;Compared with the EA group,MEK1,p-MEK1 and p-ERK1 expression levels in EA+inhibitor group declined slightly,which was statistically significant(P<0.05).They had the same protein expression trend.But there was no significant difference among these groups in ERK1 protein expression.6.Real-Time PCR results:Compared with the blank group,there was no significantly increased in the expression leves of MEK1mRNA and ERK1mRNA among the model group,the EA group,and the EA+inhibitor group after 1d treatment.Where as MEK1mRNA expression was significantly increased after 3d treatment in the EA group,which the difference was significant between them(P<0.01).The peak of MEK1mRNA appeared in the 5d within it;Compared with the model group,MEK1mRNA expression levels in the EA group significantly increased from the 3d to the 9d,and the difference was statistically significant(P<0.05);Compared with the EA group,MEKlmRNA expression levels in EA + inhibitor group declined slightly at the same time,which was statistically significant(P<0.05).But there was no significant difference among these groups in ERK1mRNA expression.Conclusions:1.EA Bang-ci can effectively promote the healing of pressure sores.2.EA Bang-ci has a significant positive adjustment for skin blood flow of pressure sores.3.MAPK/ERK signaling pathway involved in the repair process of pressure sores,and promoted the number of vascular endothelial cell and microvascular through improving Ras,c-Raf,MEK1,p-MEK1,p-ERKlprotein and MEK1mRNA expression levels is one of the mechanisms of EA Bang-ci treatment of pressure sores. |
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