| Objectives:To define the effect of moxibustion in promoting wound healing of pressure ulcer.To clarify the regulation of molecules mechanism of moxibustion which can contribute to the angiogenesis of pressure ulcer tissue.All that mentioned above will provide theoretical and experimental basis for moxibustion in the clinical treatment of bedsore as well as skin ulcers.Methods:The models of rats with pressure ulcers which belonged to 2?3 stage were established by using self-made devices of pressure ulcers.The models of seventy SD female rats with skin ischemia reperfusion injury were randomly divided into model group and moxibustion group.There were 35 rats in each group.Each group was divided into five subgroups including 1d,3d,5d,7d and 10d according to the length of intervention time.Seven experimental rats were included in each subgroup.The blank group was given shaving disinfection,tied together with other groups of rats given only conventional iodophor intervention.The model group was only given conventional iodophor intervention which had the same binding mode as the other two groups.The moxibustion group was given iodophor intervention at first and direct moxibustion intervention to the local tissue of pressure ulcer with cyclotron operation.The distance between the moxa burning and wound of bedsore was around 3cm,with moxibustion once a day and 15 minutes each time.It was required to attach great importance to adjusting the distance between the moxa burning and wound of bedsore according to the non-contact electronic temperature instrument timely.The display value of the thermometer was controlled in a suitable range from 40 ? to 42?,which was quantified in the temperature range.The skin surface temperature of the wound was measured every 30 seconds.The distance between the temperature detector and bedsore wound was about 3cm.The interventions were given to the experimental rats with pressure ulcers from the fifth day of model preparation.The wound area and average wound blood perfusion of bedsore were measured respectively after five time-points(first,third,fifth,seventh,tenth day after intervention)before drawing.HE staining and transmission electron microscopy were used to observe the pathological changes and the ultrastructure of the pressure ulcer.The numbers of CD31+PCNA+ which indicate new vessels of the wound tissues of pressure ulcers were observed by laser confocal microscope at different time-points with immunofluorescence double labeling technique.The protein expression levels of VEGF,RAS and P-ERK1/2 in the wound tissues of rats with pressure ulcers were detected by immunohistochemistry at different time-points.The mRNA expression levels of VEGF,VEGFR2,RAS,RAF-1 and ERK in the wound tissues of rats with pressure ulcers were detected with real-time quantitative PCR technology from gene aspect at different time-points.The protein expression levels of VEGF,VEGFR2 and RAS which originated from wound tissues in rats with pressure ulcers were measured with Western Blot technique at different time-points.And accordingly,the phosphorylated levels of RAF-1 and ERK1/2 proteins were detected from protein aspect simultaneously.Results:1.The wound healing rates were compared between different groups of rats at different time-points.There were no significant differences in the wound healing rates between the groups in the first day after intervention(P>0.05).Compared with the model group,the wound healing rates of moxibustion group were significantly higher at the four time-points including the third,fifth,seventh and tenth day after treatment.At the same time-point,there were significant differences between the two groups(P<0.01),with statistical significances.2.After the injuries of pressure ulcers,the average local blood flow volumes of wound surfaces were significantly higher than those of normal skins.After the moxibustion treatment,the average local blood perfusions of moxibustion group increased at first and then decreased,which had the same trend as the model group.The average local blood perfusions of two groups both reduced to the lowest volumes of at seventh day after intervention.While the average local blood perfusions of the moxibustion group at tenth day after treatment were close to those of normal skins.However,the time required for the recovery of blood flow from the model group to normal skins was significantly more than 10 days.Compared with the blank group,the average local volumes of blood flow in the model group were significantly higher before the first,third and fifth day after iodophor intervention.There were significant differences between the two groups(P<0.01).While the average local volumes of blood flow in the model group distinctly decreased before the seventh and tenth day after iodophor intervention.There were significant differences between the two groups at the mentioned above time-points(P<0.01).Compared with the model group,the average local volumes of blood flow in the moxibustion group significantly reduced before the third and fifth day after treatment.There were significant differences between the two groups at the mentioned above time-points(P<0.01).The wound blood perfusions of moxibustion group were higher before the first,seventh and tenth day after treatment.There were no significant differences before and immediately after iodophor intervention of the mentioned above time-points on the wound blood perfusions of the model group(P>0.05).In the comparison of rats with pressure ulcers of the moxibustion group,there were significant differences before and immediately after the treatment of the mentioned above time-points on the wound blood perfusions.The average local wound blood perfusions of immediate blood flow after treatment were distinctly higher than those of before.3.Compared with the model group,the granulation tissues in the wounds of rats which belonged to moxibustion group with pressure ulcers had obvious advantages both in the time-point and the growth states.There were granulation tissues at the third day after treatment in the moxibustion group,while those of model group appeared about at the seventh day after iodophor intervention.Simultaneously,the wound contraction rates of pressure ulcers were faster in the moxibustion group which had more obvious area reductions.Pathological changes of pressure ulcers were observed by HE staining that the peaks of the inflammatory infiltrations which existed in the moxibustion group had appeared in advance compared with the model group.Meanwhile,Fibroblasts appeared earlier,having more numbers and obvious degrees of hyperplasia.The occurrence time of granulation tissues was earlier in the moxibustion group.Hence,neovascularization which belonged to moxibustion group formed earlier than that of model group.Moxibustion treatment can make epidermal structures recover in advance.4.The effects of moxibustion on the ultrastructures of the wounds of rats with pressure ulcers were observed by transmission electron microscope.The results showed that the epidermis was partly lost and the normal skin structures changed after model preparation,compared with the blank group.Compared with the model group,the moxibustion group had obvious advantages on the repair of epidermal structures.The structures of the epidermis were partially intact which alone contained the basal laminas and spinous layers in parts.The whole structures of the epidermis which included the basal layers,spinous layers,granular layers,transparent layers and stratum corneums existed at the seventh day after treatment.While the basal and spinous layers of epidermis structures can be found at the tenth day after intervention without granular layers,transparent layersand stratum corneums in the model group.At the same time,the mitochondria of basal cells and prickle cells had changed in the structure,morphology and quantity with the accumulation of moxibustion stimulation at the fifth,seventh and tenth day after treatment in the moxibustion group.The mitochondria of basal cells and prickle cells can be seen more abundant with higher quantities and clear structures so as to promote the process of repair and healing of wound tissues with pressure ulcers.It presented the basic pathological changes of wound healing and self repair of pressure ulcers at different time-points in the model group.The mitochondria of basal cells and prickle cells still showed swelling,lower quantities with big and empty morphological characteristics up to the tenth day after iodophor intervention.The process from injury to self repair was relatively slow in the model group.Microscopically,there were substantial granulocyte infiltrations at the first day,which decreased at the third day and changed to stale states at the fifth day after treatment.Phagocytes and lymphocytes existed at the seventh day,with massive lymphocytes at the tenth day after treatment.It reflected the changes of the wound tissue with pressure ulcer from the high degree of acute inflammation to chronic inflammation.The sates of granulocytes also altered from fresh to old.While the stage of acute inflammation focused on the fifth day after iodophor intervention in the model group.Compared with the model group,the moxibustion group had the acute inflammatory infiltration peak in advance as a whole in order to shorten the repair time from the overall process.5.The results of immunofluorescence double staining showed that the numbers of CD31+PCNA+(angiogenesis)in the wound tissues which belonged to the model group were significantly higher than that of the blank group from the first to tenth day after iodophor intervention(P<0.01),with statistical significances.Compared with the model group,the numbers of CD31+PCNA+(angiogenesis)which existed in wound tissues with pressure ulcers of the moxibustion group increased from the first to seventh day after treatment.The degree of increase varied at the time points mentioned above.It had remarkable significance in the first day,which altered to the highly notable from the third to fifth day after treatment.However,there was no significant difference in the seventh day after treatment.While the numbers of CD31+PCNA+(angiogenesis)in the wound tissues which belonged to the moxibustion group were lower than that of the model group in the tenth day after treatment,without statistical significances.The numbers of CD31+PCNA+(angiogenesis)in the wound tissues which belonged to both the moxibustion and model group had the same variation tendency that increased first and then decreased.However,the two groups were different in the climax time of numbers of CD31+PCNA+(angiogenesis).The climax time of the moxibustion group appeared in the fifth day,while that of model one emerged in the seventh day after iodophor intervention.6.The immunohistochemistry results showed that the positive expression of VEGF protein in the skin tissues of pressure ulcer were orientated in the cytoplasm which expressed in epithelial cells and macrophages.Compared with the blank group,the expressions of VEGF protein in the model group were significantly higher from the first to tenth day after intervention.Compared with the model group,the expressions of VEGF protein in the wound tissues with pressure ulcers of the moxibustion group were significantly increasing from the third to fifth day after treatment.There were significant differences between the two groups.While the expression levels of VEGF protein which existed in the wound tissues with pressure ulcers were lower than those of the model group from the seventh to tenth day after treatment,but there were no significant differences between the two groups.The positive expression of RAS protein in the skin was located in the cytoplasm.Compared with the blank group,the expression levels of RAS protein in the model group were significantly lower from the first to tenth day after intervention.Compared with the model group,the expression levels of RAS protein which existed in the wound tissues with pressure ulcers in the moxibustion group were distinctly higher,with significant differences between the two groups.The RAS protein expressions in the two groups of moxibustion and model group both showed a rising trend.The positive expressions of phosphorylated ERK1/2 protein in the skin of pressure ulcers were located in the cytoplasm.Compared with the blank group,the expression levels of P-ERK1/2 protein in the model group were significantly higher from the third to tenth day after intervention.Compared with the model group,the expression levels of P-ERK1/2 protein which existed in the wound tissues of the pressure ulcers in the moxibustion group were increasing to different extents,which was slightly higher than those of the model group from seventh to tenth day after treatment.While the expression levels of P-ERK1/2 protein were significantly higher from the third to fifth day after treatment.The expression levels of P-ERK1/2 protein in the two groups of the moxibustion and the model group both showed the trend of the first increasing and then decreasing.However,there was a significant difference between the two groups in the time-point which high peak of expression levels emerged.The highest peak in the moxibustion group appeared in the third day after treatment,while that of model group occurred in the fifth day after iodophor intervention.7.The results of Real-time PCR showed that the expression level of VEGF mRNA was distinctly weak positive in normal skins.Compared with the blank group,the expression levels of VEGF mRNA in the model group were significantly higher from the first to tenth day after intervention.Compared with the model group,the expression levels of VEGF mRNA which existed in the wound tissues with pressure ulcers of the moxibustion group significantly increased from the third to fifth day after treatment.There were significant differences between the two groups.However,the expression levels of VEGF mRNA in the wound tissues with pressure ulcers which belonged to the moxibustion group were lower than those of the model group,but there were no significant differences between the two groups.The results of VEGFR2 mRNA seemed to be similar to VEGF mRNA as a whole,which can reflect that the premise of the biological effect of VEGF may be the combination with its receptor.VEGF can have specific binding with its receptor VEGFR2,which could promote the proliferation and division of vascular endothelial cells as well as angiogenesis and neovascularization.Compared with the blank group,the expression levels of RAS mRNA in the model group were significantly lower from the first to tenth day after intervention.Compared with the model group,the expression levels of RAS mRNA in the wound tissues with pressure ulcers which belonged to the moxibustion group were significantly increasing from the third to tenth day after treatment,with significant differences between the two groups.The expression levels of RAS mRNA in the moxibustion and the model group both showed a rising trend.After the formations of pressure ulcers,there were no obvious changes of RAF-1mRNA and ERK1/2 mRNA in the wound tissues.And accordingly,there were no significant changes in each group from the first to tenth day after treatment.8.The results of Western Blot showed that the expressions of VEGF protein were extremely weak positive in normal skin tissues.Compared with the blank group,the expression levels of VEGF protein which belonged to the model group were significantly higher from the first to tenth day after intervention.Compared with the model group,the expression levels of VEGF protein which existed in the wound tissues of the pressure ulcers in the moxibustion group were distinctly higher from the third to fifth day after intervention.There were significant differences between the two groups.While expression levels of VEGF protein in the wound tissues of pressure ulcers which belonged to moxibustion group altered the change direction.They were slightly lower than those of the model group,without significant differences between the two groups.The results of VEGFR2 protein levels tended to resemble VEGF on the whole,which can reflect that the premise of the biological effect of VEGF may be the combination with its receptor.VEGF can have specific binding with its receptor VEGFR2,which could promote the proliferation and division of vascular endothelial cells as well as angiogenesis and neovascularization.Compared with the blank group,the expression levels of RAS protein and phosphorylated levels of RAF-1 protein in the wound tissues which belonged to model group were significantly lower.The expression levels of RAS protein and phosphorylated levels of RAF-1 protein which existed in the wound tissues of pressure ulcers in the moxibustion group were extremely higher than those of the model group from the third to tenth day after intervention.There were significant differences between the two groups.The expression levels of RAS protein and phosphorylated levels of RAF-1 protein were gradually increasing in the model group from first to tenth day after intervention.The variation tendency of RAS protein levels also increased gradually in the moxibustion group.The phosphorylated levels of RAF-1 protein were increasing from the first to seventh day after treatment and decreasing from the seventh to tenth day after treatment.Compared with the blank group,the phosphorylated levels of ERK1/2 protein in the model group was distinctly higher from the third to tenth day after intervention.Compared with the model group,the phosphorylated levels of ERK1/2 protein which existed in the wound tissues of the pressure ulcers in the moxibustion group increased to different degrees,which were extremely higher from the third to fifth day after treatment and slightly increased from the seventh to tenth day after treatment.The phosphorylated levels of ERK1/2 protein in both groups of the moxibustion and model showed a trend of increasing at first and then decreasing.However,there was a significant difference at the time-point of high peak of expression levels between the two groups.The highest peak in the moxibustion group appeared in the third day after treatment,while that of model one arised in the fifth day after iodophor intervention.Conclusions:1.Moxibustion can promote the wound healing effectively.2.Moxibustion does have the regulating effect on the average local blood flow volume of wound tissue of rat with pressure ulcer which is both bidirectional and benign.Moxibustion intervention can significantly increase the average volume of local blood perfusion in rat with pressure ulcer as immediate effect.3.Moxibustion intervention can effectively promote the proliferation of vascular endothelial cells,increasing the numbers of new blood vessels and boosting the angiogenesis of pressure ulcer.4.Moxibustion intervention can increase the expression levels of both VEGF and its receptor VEGFR2 in the wound tissue of pressure ulcer from the level of protein as well as gene.5.RAS/RAF/ERK signaling pathway does participate in the repair process of pressure ulcer.Moxibustion intervention can raise the expression levels of protein and mRNA which belong to RAS and promote the phosphorylated levels of both RAF-1 and ERK1/2 protein by activating RAS/RAF/ERK signaling pathway.Hence,it is moxibustion intervention that enhances the activity of the RAS/RAF/ERK signaling pathway and promotes the proliferation of vascular endothelial cells as well.6.RAS/RAF/ERK signaling pathway is a significant target of moxibustion intervention,which may be one of the mechanisms of moxibustion promoting wound healing of pressure ulcer. |
PDF Full Text Request