| Objective:To investigate whether bangcielectro-acupuncture will help heal the wound of pressure ulcer model in rats(Ischemia-reperfusion)through experiments;Make use of PI3K/Akt signaling pathway inhibitor "wortmannin" to block this pathway and probe the possible mechanisms in which bangcielectro-acupuncture promote wound healing by dynamically and continuouslyobserving the changing levels of the PI3K/Akt signal pathway downstream factors in terms of gene and protein.Method:The first part:the effect of pressure ulcer model wound contraction and healing time caused by bangcielectro-acupuncture:divide 18 SD rats,according to random number table,into model group(Model group,M),bangcielectro-acupuncture group(EA group,EA)and electro-acupuncture inhibitor group(EA+ wortmannin group,EA+W),each of them 6 rats.Next,use rat skin ischemia-reperfusion injury to build rat pressure ulcer models.Before M group modeling and 10mins before daily intervention,give the rats the same amount of normal 0 saline by intraperitoneal injection,seize and fix them for 30mins in the same condition and then give iodine wipe.For EA group,give the rats the same amount of normal saline by intraperitoneal injection before EA modeling and ten minutes before bangci-electro-acupuncture treatment,and then seize and fix the rats to give bangci electro-acupuncture treatment.Do one needle acupuncture in the wound center and the 0.5cm from the wound edge respectively,then connect electro-acupuncture device to both needles(negative electrode inner,positive electrode outer)with the current strength of 500?A.Give one treatment every 30mins and once a day.For EA?W group,give them the Intraperitoneal injection of wortmannin 1.4mg/Kg[106]before modeling and ten minutes before bangci electro-acupuncture treatment,which is the same as EA group.Evaluate models after modeling,continuously and dynamically observe,measure and then compare the rats wound contraction as well as the wound healing time of each group;Part two:discuss the possible mechanismof bangci electro-acupuncture regulating the pressure ulcers models healing based on PI3K/AKT signal pathway:150 SD rats are randomly divided into blank group(Blank group B),model group(Model group,M),bangci electro-acupuncture group(EA group,EA),EA inhibitor group(EA+wortmannin group,EA+W)and inhibitor(wortmannin group,W),a total of five groups of 30 rats.30 rats per group.Group M,EA,EA+W as same as the part one.Group B:give the rats the same amount of normal saline by intraperitoneal injection,seize and fix them for 30mins under the same condition as that of the other groups.Before W group daily intervention and 10mins before modeling,give the rats the Intraperitoneal injection of wortmannin 1.4mg/Kg,seize and fix them for 30mins under the same condition as the other groups and then give iodine wipe.Each experimental group is further divided into five subgroups according to different intervention length of time 1d,3d,5d,7d,9d.Corresponding intervention measures are given respectively.After the treatment,the wound tissue is taken by hematoxylin-eosinstaining,immunohistochemical technique,qRT-PCR detection method,Western Blot detection method.Results:Wound contraction rates among the groups:no obvious difference among the groups after 1 day's treatment and no statistical significance.Compared with M group,EA group wound contraction rates in 3d?5d are obviously better than M group(jP<0.05).7d,9d wound contraction group was prominently better than the M group(P<0.01).EA?W group wound contraction rate was lower than EA group.There is no statistical significance in 1d while others are statistically significant(P<0.01).Wound healing time comparison:EA wound healing time(8.83 ± 0.88)is obviouly less than M group(11.25±1.33)d,with statistical significance(P<O.O1).The EA+W Group(13.42±1.96)d prolongs healing time,compared with the EA group.The observation of pressure ulcer model healing process by hematoxylin-eosinstaining reveals that compared with other groups,EA group local wound neutrophils decrease faster,the number of fibroblast proliferation is more and the granulation tissue appears earlier.At the same time,scab and scab skin off are earlier than the other experimental groups as well.The skin tissue of group B eNOS protein expression quantity is less.It shows a balanced and stable expression without obvious changes after the 9d continuous observation.M group shows a slow increase expression and the 5d group is the strongest,the 7,9d reduced compared with the previous expression amount.Compared with group B,M group expression was significantly higher than that in group B(P<0.01).For EA group,after the treatment of 1d,eNOS protein expression gradually increases.On 3d it reaches the peak,and remains at high level,7d and 9d,the expression gradually reduces.EA group and M group comparison,its eNOS protein expression peak reaches in advance,and maintain a high expression level,which is higher than that of group M(P<0.05).eNOS protein expression of EA?w group increases slowly,the expression peakappears in 5d?each time point expression is significantly lower than that in EA group(P<0.01).eNOS protein of group W at each time point is not only significantly lower than that in group M(P<0.05).The expression of p-bads136 protein in the cytoplasm of B group was very little.Each time point shows no significant change.pgbads136 protein of M group in 1d,3d,5d show increasing expression,7d begins to decrease.Overall,compared with B group,the M group is significantly higher than that of the B group(P<0.01).The expression of p-bads136 protein of 1d,3d in EA group gradually increases.After the 3d group reaches the peak,it still remains high expression while 7d,9d group decreases.Compared with M group,each of its time points is higher(P<0.05).For EA +w group,p-bads136 protein shows a slow increased expression but 5d group protein expression goes down.The p-bads136 protein at each time point are significantly lower than the EA group(P<0.01)W group has a slow upward trend,but compared with M group,p-bads136 protein at each time point maintain a low expression,and each of them is lower than M group(P<0.05).In group B,a small amount of p-akts473 protein expression and no significant change is observed after being continuously watched.In M group,the p-akts473 protein shows an increased expression.After reaching the peak at 5d,it begins a continued slowdown.The protein expression at each time point are significantly higher than those in the B group(P<0.05).In group EA,P-Akts473 protein expression increases,after reaching the peak at 3d,and still sustains high expression while 7d,9d descends.Id,3d,7d,9d group is obviously higher than that of M group(P<0.01or P<0.05).In EA+w group,p-akts473 protein reaches the peak at 3d,5d and then begin to drop.The protein at each time point are significantly lower than those in EA group(P<0.01).In comparison with group M,at each time point p-akts473 protein expression in W group are all decreased and lower(P<0.05).Group B has little expression of eNOSmRNA in skin tissue,which is balanced and stable and has no statistical significance after 9 days'continuous observation.In M group,eNOSmRNA shows an increased expression.But after reaching the peak at 5d,it begins a slow down.Compared with group B,M group is significantly higher(P<0.05).The EA group shows a progressive rise and still continues high expression after reaching the peak at 3d.the expression of group 7d was lower than earlier.1d,3d,5d,7d,9d group are all significantly higher than that of M group(P<0.05).In group EA+W,the expression of eNOSmRNA presents a slowly increased trend after treatment.Compared with the EA group,it is a relatively small rise and the expression is obviously lower(P<0.05).In W group,eNOSmRNA expression at each time point all decrease and are statistically significant in comparison with M group(P<0.05).Group B badmRNA shows little expression and has no obvious change at each time point in the continuous observation.Group M badmRNA decreases after presenting a continuous high expression at 1d,3d.Compared with group B,badmRNA expression in M group is obviously higher and each time point(P<0.01).In EA group,Id,3d,5d,7d,9d maintain a low and continuous badmRNA expression.Compared with group M,EA group badmRNA expression is lower,and each time point is significantly lower than M group(P<0.01).In EA+W group,badmRNA at each time point is all high expression.7d begins to drop.Each time point is significantly higher than EA group(P<0.01);Compared with group M,badmRNA at each time point In W group is relatively highly expressed(P<0.05).In group B,the phosphorylated eNOS protein expression is balanced in the skin tissue,and no significant change is observed at all time points after continuous observation.the p-eNOSs1177 in group M is found to show a slow upward trend,and the expression of 9d shows a decreasing trend after continuous observation.Compared with the B group,the expression of M group is significantly higher than that of B group(P<0.05).The expression amount of p-eNOSs1177 in EA group is significantly higher than that of M group,and the high expression tendency is maintained at each time point.Compared with M group,the expression at each time point is all higher than that in M group(P<0.05).The expression of p-eNOSs1177 protein in EA?W group increases slowly,and there has been a continuous decrease since 5d.The expression level of each time point is significantly lower than that of EA group(P<0.05).The expression changing trend of p-eNOSs1177 protein in W group is the same as that of M group,but the amountis obviously lower than that of M group at different time points(P<0.05).In B group,Akt and P-akts473shows a small amount and balanced expression.there is no significant difference in the expression of P-akts473/akt at different time points.The relative expression of P-akts473/akt in M group increases gradually.After reaching the peak at 5d,the amount declines continually.Compared with B group,P-akts473/akt is highly expressed,and all time points are statistically significant(P<0.05).In EA group,p-akts473/akt shows a progressive high expression.3d group increases the most significantly,reaches the peak and still remains high afterwards.Compared with group M,the expression at each time point obviously improves(P<0.05).EA+W group p-akts473/akt relative expression of continued low expression is found in the continuous observation.There is a transient increase,then comes a reduced expression.Compared with the EA group,the relative expression of p-akts473/akt is obviously decreased at each time point with statistical significance.And the relative expression of W is significantly lower in comparison with M group(P<0.01).The Bad and p-bads136 protei.in B group at each time point maintain dynamic balance,which can regulate the apoptosis and survival of cells in an orderly way,and the relative expression of p-bads136/bad at each time point is constant,with statistical significance.M group shows high expression of Bad protein,while p-bads4136 protein expression is less.P-bads136/bad relative expression is significantly lower than that of B group,and is statistically significant compared with B group(P<0.05).The p-bads136 protein in EA group presents a continuous high expression.(P<0.01),The relative expression of p-bads136/bad protein is obviously higher than that of M group.The expression of p-bads136 protein in EA+w group is small,and the relative expression of p-bads136/bad at each time point is lower than that in EA group(P<0.01).Compared with M group,the expression of p-bads136 protein in W group significantly reduces,and the relative expression of p-bad136/bad is obviously lower.All the time points are statistically significant(P<0.05).Conclusion:l.Bangci electro-acupuncture can promote the healing of pressure ulcers wound and effectively shorten the wound healing time required.2.The use of PU3K specific inhibitor wortmannin prolongs the healingtime of pressure ulcer wound in rats.This shows that PI3K/Akt signaling pathway is involved in the repair of pressure ulcers in rat model,from the early stage of pressure ulcer formation to wound healing.3.Bangci electro-acupuncture may stimulate the activation of PI3K/AKT signaling pathway,promote the activity of eNOS,and participate in vascular regeneration,and thus promote the wound healing.4.Bangcielectro-acupuncture may activate the PI3K/Akt signaling pathway and activate Akt.P-Akt phosphorylates the pro apoptotic factor bad,inhibits thepro apoptotic factor bad,inhibits the role of bad in promoting apoptosis,and thus plays a role in anti apoptosis. |
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