| The incidence of cutaneous wounds is high,and it is easy to cause problems such as poor healing due to the complicated repair process.It has a huge economic and social burden and seriously affects the quality of life of patients.The cutaneous wounds are routinely treated with debridement,antibiotic-assisted anti-inflammatory and other methods to fight infection.It is easy to bring serious adverse consequences caused by the abuse of antibiotics,eventually delay the healing or always difficult to heal.In response to the above problems,basic researchers and clinicians have conducted extensive research and exploration from animal experiments to clinical RCTs,attempt to find a more economical,practical,and easy-to-operate treatment program to promote wound healing.Moxibustion is often used in the treatment of refractory trauma such as pressure ulcerby promoting the local microcirculation of trauma.Therefore,this study will explore the efficacy and mechanism of moxibustion on wound healing in full-thickness skin excisional wound model rats from the perspective of morphology and molecular biology.The efficacy of moxibustion is closely related to the interaction between the nervous and immune system.CGRP and SP are the most important neuropeptides originally released from the C fiber terminals,which can induce neurogenic inflammation,initiate an anti-inflammatory immune response through axonal reflexes to accelerate healing process.Wound healing is a process withhemostasis,inflammation,cell proliferation and tissue remodeling.The inflammation is the most critical phase,including the recruitment of inflammatory cells,the synthesis and release of cytokines,etc.The wound healingwill turn to be chronic and impaired,or to cause continued unhealing or eventually more scarring if the inflammatory phase is interrupted or prolonged(more than 3 weeks).Neuro-immune regulation can rapidly activate a large number of leukocytesinvolved in the immune response.Immunomodulation based on neural levels may differ from traditional ways with indirectly applied to immune organs and cells,therefore it may exhibit particular advantages.If the inflammatory response can be strengthenedby regulating the reaction of inflammatory cells and related cytokines,it will accelerate the inflammatory reaction process and complete the processin advance,and finally promote and accelerate wound healing.1 ObjectiveTo explore the effect of moxibustion on wound healing in rats with full-thickness cutaneous excision,and further to clarify the mechanism of moxibustion promoting wound healing by observing its effect on the expression of cytokines involved in the inflammatory phase of wound healing.2 MethodsThe study consists of two sections.The first section mainly observes the effect of moxibustion on wound healing in rats with full-thickness skin excision.A total of 38 adult SD male rats were randomly assigned to model and moxibustion group.The skin wound model was established by removal of a piece of full-thickness skin from the median line of the rats' back(about 2 cm below the shoulder blade).Moxibustion intervention was applied to the surrounding area of the wounds for 25 min,once daily for continuous6 days,and the model ratswere anesthetizedonly without intervention.The wound healing was observed and photographed after each moxibustion intervention.The wound tissues were harvested after transcardiac perfusion with 4%paraformaldehyde solution on the 2nd and 7th day after modeling,respectively.The tissues were cut into sections and stained with HE and Masson methods for displaying histopathological changes and collagen fiber growth status,respectively.The aim of the second section is to explore the mechanisms underlying the promoting wound healing effect of moxibustion.A total of 102 adult SD male rats were randomly assigned to normal,model and moxibustion group.The method of model preparation and the treatment methods are the same to section 1.The venous blood and local skin tissuesof all groups were collected at 1,2,3 and 5days after modeling,respectively.The relative protein expression of cytokines IL-1??IL-1??IL-2?IL-6?IL-4?IL-7?IL-12?IL-13?G-CSF?M-CSF?GM-CSF?MIP-1?and VEGF in serum and skin tissue of rats were observed with Bio-Plex method.CGRP relative protein expression in local skin tissue of wounds wasdetected with Elisa.The skin was collected at day 1,2,3 and 5 after modeling after cardiac perfusion with 4%paraformaldehyde to observe the positive expression of SP,CGRP,CD31 and macrophage M1 and M2 by fluorescent immunohistochemical stainingand analysis the angiogenesis and reconstruction in traumatic tissues of rats in different periods.3 Results3.1 Effect of moxibustion on wound healing in rats with full-thickness skin excision 3.1.1 Moxibustion promotes wound healing in traumatic model ratsCompared with the model group,the wound area of rats significantly reduced after 2 interventions in the moxibustion group(P<0.01,P<0.05).The healing area of the moxibustion group wasnotablelyincreased following moxibustion(P<0.01).The number of inflammatory cells and new collagen fibers in the wounded tissue of rats in the moxibustion group weresignificantlyincreased(P<0.01,P<0.05).The number of fibroblasts and the amount of collagen fibers in the moxibustion group were remarkablely increased compared with the model groupafter 6-timemoxibustion.The cells and collagen fibers were arranged more closely,the number of new blood vesselswere larger,and the skin of the damaged part was thicker in the moxibustion group.3.1.2 Effect of moxibustion on Local morphology of traumatic tissueOn the 2nd day after modeling,the recruitment and infiltration of inflammatory cells in the wound tissues of the moxibustion group were increased(P<0.01),and most of the inflammatory cells recruited there were neutrophils,mononuclear/macrophages and mast cellsaccording to the morphology of cells.On the 7th day after modeling,the local tissue structure of the wound in the moxibustion group was more dense,mainly composed of fibroblasts and collagen fibers.The cells were various in shape,closely and orderly arranged,and the skin scars at the damaged parts were significantly thickened.3.1.3 Moxibustion promotes collagen reconstruction in traumatic model ratsCompared with the model group,on the 2nd day after modeling,the amount of collagen fiber proliferation in the wound tissue of the moxibustion group weresignificantly increased(P<0.05)and the feature of distribution was from the surface of the wound,while the model group appeard in the deep part of the skin dermis.It suggested that fibroblasts begin to migrate and proliferate locally at thisstage due to the collagen fibers are produced by fibroblasts.On the 7th day after modeling,the collagen fibers in the wound tissue of the moxibustion group were more closely arranged,and the number of new blood vessels increased.3.2 Mechanism of moxibustion intervention promoting wound healing in rats with full-thickness skin excision3.2.1 Effect of moxibustion on serum inflammatory cytokines in traumatic model ratsThe contents of serumIL-1?,IL-1?,IL-2 and IL-6 of the model groupwere increased first and then decreased among 5 days after modeling,and the peaksappeared on the 3rd day.Compared with the model group,the expression curves of IL-1?,IL-1?,IL-2 and IL-6 in the moxibustion group shifted to the left 1-5 days aftermodeling,and peakedon the 2nd day.Compared with the model group,the contents of serumIL-l? and IL-6 of moxibustion group were notably increased on the 1st day aftermodeling(P<0.05),contents of IL-2 were obviously increasedon the 2nd day(P<0.05).While the contents of IL-1?were significantly increased on the 1st and 2nd day(P<0.05).The contents of IL-l?,IL-1?p,IL-2 and IL-6 in the serum of rats in the moxibustion group decreased to the initial level(dayl)after modeling on the 3rd day,while the model group on the 5th day.There was no significant difference in the contents of IL-7 and IL-12 in the serum of the two groups,but the trend was consistent with the expression of IL-l?,IL-1?,IL-2 and IL-6.The contents of serum IL-4,IL-10 and IL-13 were also increased first and then decreased in the model group among 5 days aftermodeling.The peak of contentsofIL-4 appeared on the 2nd day,while IL-10 and IL-13 appeared on the 3rd day.Compared with the model group,the expression curves of IL-4,IL-10 and IL-13 in the moxibustion group also shifted to the left among 5 days after modeling.In comparison with model group,the contents of IL-4 in the moxibustion group was significantly up-regulated on the 1st day after modeling(P<0.05),andIL-10 was on the 1st and 2nd(P<0.05,P<0.01).The contents of IL-4 and IL-10 in serumobviously decreased on the 3rdday after modeling(P<0.05).There was no notable difference in the contents of IL-13 in the serum of both model and moxibustion grouprats after trauma,but it showed the same tendency as IL-10.3.2.2 Effect of moxibustion on serumchemokines in traumatic model ratsThe serum levels of G-CSF,M-CSF and GM-CSF in the model group were increased first and then decreased among 5daysafter modeling,and the peak level ofG-CSF and GM-CSF appeared on the 2nd day,M-CSF on the 3rdday.Compared with model group,the contents of G-CSF and GM-CSF in the serumof moxibustion group were increased on the 1st and 2ndday after modeling,and decreased on the 3rdand 5thday.The contents of M-CSF were decreased on 2ndand 3rd day,and increased on the 5trh day,suggesting that the inflammatory response in the model group may not be end.In comparision with the model group,the contents of MIP-1? in the moxibustion group were up-regulated on the 1 to 3 days after modeling,and significantly up-regulated on the 2nd day(P<0.05),thendecreased on the 5thday.The contents of MIP-la inwounded tissues of both groups showed a gradual increase.The contents of MIP-la in the moxibustion group were significantly higherthan that in the model group on the 2nd and 5thday(P<0.05,P<0.01).3.2.3 Effect of moxibustion on the expression of M1 and M2 in the wound tissue of traumatic ratsCompared with the model group,the expression of M1 macrophages in the moxibustion group reduced,the expression of M2 was increased,and the ratio of M1/M2 became lower.It suggested that moxibustion may promote the differentiation of macrophages from M1 to M2.3.2.4 Effect of moxibustion on the contents of serum and tissue VEGF in traumatic ratsCompared with the model group,the contents of serum VEGF in themoxibustion group were increased on the 1st to 5thday after modeling,and it increasedsignificantly on the 1st and 2ndday(P<0.05,P<0.01),remained at a high level on the 3rdand 5th day.In comparison with model group,the contents of VEGF in the wound tissue ofmoxibustion group did not change significantly on the 1st to 3rdday after modeling,and increased significantly on the 5thday(P<0.01).The fluorescence immunohistochemistry results of CD31-labeled vascular endothelial cells showed that there was no obvious expression of CD31 in the first 3 days after modeling suggesting no notable angiogenesis.Compared with the model group,the positive expression of CD31 inmoxibustion group were increased significantly on the 5thday after modeling,suggesting that the number of new blood vessels in the wound tissue increased significantly,and the results were consistent with the quantitative results of VEGF.3.2.5 Effect of moxibustion on the expression of CGRP in local tissues in traumatic ratsCompared with model group,expression of CGRP in the moxibustion group was increased on the 1st day after modeling,and its expression was expressed in the epidermis,epidermal-dermal junction,and superficial dermis,while the model group only expressed a small amount in the dermis.Compared with model group,expression of CGRP in the moxibustion group was increased significantly on the 3rdday after modeling,and its expression was characterized by a large amount of expression in the epidermis,epidermal-dermal junction,around the hair follicle and in the superficial dermis,while the model group was only in the dermis.In comparison with model group,the contents of CGRP in the local tissues of themoxibustion group were increased on the 1st and 3rdday after moxibustion,andincreased significantly on the 3rdday(P<0.01).3.2.6 Effect of moxibustion intervention on SP expression in local tissues in traumatic ratsCompared with model group,expression of SP in the moxibustion group was increased on the lstday after modeling,and its expression was expressed in the epidermis,epidermal-dermal junction,and superficial dermis,while the model group only expressed a small amount in the dermis.In comparison with model group,expression of SP in the moxibustion group wasincreased significantly on the 3rdday after modeling,and its expression was characterized by a large amount of expression in the epidermis,epidermal-dermal junction,around the hair follicle and in the superficial dermis,while the model group was only in the dermis.4 Conclusion1 Moxibustion significantly promotes wound repair in rats with skin trauma and shortens the process of wound healing.2 Moxibustion can significantly promote the dynamic expression curve shift to the left by promoting proinflammatory cytokines IL-1?,IL-1?,IL-2 and IL-6 and anti-inflammatory cytokines IL-4 and IL-10 expression in the serum of trauma model rats,promote the inflammatory reaction in the period of traumatic inflammation,and shorten the inflammatory phase to promote wound healing into the cell proliferative phase.3 Moxibustion can increase the levels of chemokines G-CSF,GM-CSF and M-CSF which are closely related to macrophages in the serum of traumatic model rats,and regulate the transformation of local macrophages from M1 to M2.It is suggested that moxibustion promotes the inflammatory reaction by affecting macrophage phenotype,shortens the inflammatory phase and accelerates the healing process of wounds.1,4 Moxibustion can increase the content of VEGF in serum and surrounding skin tissue around the wound,promote the expression of CD31 positive cells in vascular endothelial cells,and promote the regeneration and reconstruction of traumatic local blood vessels during trauma repair.5Moxibustion can increase the expression of neuropeptide SP/CGRP in the tissues surrounding the wound which mainly expressed under the epidermis and around the blood vessels,and induce the occurrence of neurogenic inflammation.It is suggested that moxibustion promotes the release of neuropeptides and induces downstream neuroimmune reactions to rapidly enter the repair phase to promote healing of trauma. |
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