The Wound Healing Effects And Possible Mechanisms Of Ghrelin In Combined Radiation-burn Injury

Combined radiation-burn injury(CRBI)is a kind of combined radiation injury(CRI),manifested as major radiation injury,and followed by burn or thermal injury simultaneously or subsequently.CRBI belongs to special military medicine,which often happens at battle fields or nuclear accidents,and rarely occurs in common life.CRBI shows great harmfulness mainly because of radiation injury,whereas burn injury increases the disease complexity that primarily exhibits as “accentuation effect”.As to moderate and severe CRBI,it is hard to treat because of various pathological injuries such as early acute stress response and shock,systemic inflammatory response,hematopoietic dysfunction,immune suppression,gastrointestinal impairment,and delayed or impaired wound healing,and so on.Endogenous and exogenous infection largely contributes to the death of CRBI animals,however,delayed or impaired wound healing can primarily lead to gradually exaggerated exogenous infection.In light of this,accelerating CRBI wound healing may be helpful for postponing disease progress,increasing survival and improving prognosis.Currently,most studies about CRBI are limited to animal levels,of which the wound healing research seems to be rare and lack systematicness.Although many physical(i.e.crusta cutting and wound nursing),chemical(i.e.iodophor and phenytoin)and biological(i.e.mesenchymal stem cells)treatments have improved the wound healing conditions in some degree,however contributed little for improving the whole animal disease states.Some treatments not only showed lower cost-performance ratio,but also brought certain side effects,thus these disadvantages limited their clinical application.Ghrelin is an endogenous multifunctional brain-gut peptide.Previous study has shown that ghrelin could improve survival of CRBI rats by attenuating acute stress response,alleviating acute inflammatory response and enhancing hematopoietic restoration.Compared with other ghrelin-related references,this study proved again that ghrelin could be further used in comprehensive CRBI therapy.Ghrelin can be simply chemically synthesized and harvested with less cost,and also be conveniently administrated;its therapeutic effect on CRBI animals may partly result from accelerating wound healing except for the above stated mechanisms.Therefore,we performed this study from several parts as follows to explore the wound healing effects and possible mechanisms of ghrelin in CRBI rats:In the first part,effect evaluation of ghrelin in enhancing wound healing in CRBI rats,to verify whether ghrelin possesses this ability or not.Three experimental groups were set: normal group,CRBI group and ghrelin treatment group.Rats received 5 Gray(Gy)total body ? irradiation(TBI),followed with 10~15% total body surface area(TBSA)and third-degree(?°)burn injury in the back within 2 hours(h).The ghrelin concentrations were determinated in various time points within 30 days(d).Exogenous rat ghrelin was subcutaneously(s.c.)infused for consecutive 7 d(24h×7)in a dosage of 50nmol·kg-1·d-1,100 nmol·kg-1·d-1 or 200 nmol·kg-1·d-1,respectively refers to low,middle and high dosage.The count changes of red blood cell(RBC),white blood cell(WBC)and platelets(PLT)were observed at 3,7 and 14 d after injury.The body weight were recorded every other 2 d for 30 d.The wound photos were taken every other 2 d for 30 d,and the average wound healing rate and wound closure time were calculated at late wound healing stage.In the second part,histological analysis for ghrelin improves wound healing in CRBI rats.This part contains 4 major groups: single burn injury group,CRBI group,ghrelin treatment group and [D-Lys3]-GHRP-6 pretreated-before ghrelin treatment group.Rats received 5 Gy ? irradiation and 6~8% TBSA ?° burn injury.[D-Lys3]-GHRP-6 and ghrelin were both s.c.administrated: the former was injected 6 h before each ghrelin infusion in a dose of 5mg ·kg-1 for once per day for consecutive 7 d;the latter was consecutively infused in a dose of 200 nmol·kg-1·d-1 for 7 d.At 10,20 and 30 d after injury,the granulation tissues were harvested for following analysis: content evaluation of DNA,collagen,hexosamine,nitrite and nitrate(namely total NO[nitric oxide]);Sirius red and Masson stain for collagen;immunological histological chemistry(IHC)stain for newborn microvessels;haematoxylin-eosin(H.E.)stain for normal pathologic evaluation;growth hormone secretagogue receptor 1a(GHS-R1a)IHC stain;Western blot(WB)analysis for transforming growth factor beta1(TGF-?1)and vascular endothelial growth factor(VEGF).These indicators assessing microvessels formation and collagen deposition can directly reflect the granulation tissue maturity and wound healing ability.In the third part,the possible inflammatory mechanisms that ghrelin enhances wound healing.Ghrelin is well known for its anti-inflammation property,whereas CRBI wound healing degree shows high correlativity with inflammation degree,thus we decide to explore the wound healing effect from inflammatory scope.As to acute inflammatory response often happens and changes at the early stage of CRBI,so we choose the first 7 d post injury as the research period.Macrophages play an important role in the progression of inflammation,thus we select the sufficient and easily-harvested-rat peritoneal macrophages as the target cells for mechanism study.This part of experiment was performed by two stages.The first experiment stage consisted of three groups: normal group,CRBI group,and ghrelin treatment group.Rats received 5 Gy ? irradiation and 10~15% TBSA ?° burn injury.Ghrelin was s.c.infused for consecutive 7 d in a dose of 200nmol·kg-1·d-1.Enzyme-linked immuno sorbent assay(ELISA)was used to determinate the serum proinflammatory mediators such as tumor necrosis factor alpha(TNF-?),interleukin 1beta(IL-1?)and IL-6,at 1,4,7 d post injury.Meanwhile,the rat peritoneal macrophages from normal and CRBI rats were harvested for total protein abstraction,which would be used for WB analysis of some target inflammatory molecules as follows: p38 mitogen activated protein kinase(p38MAPK),jun N-terminal kinase(JNK),extracellular signal-regulated kinase(ERK),p65 nuclear transcription factor kappa B(p65NF-?B),and glucocorticoid receptor(GR)from glucocorticoid(GC)anti-inflammatory signal pathways.The optimal measurement time point was chosen according to the above protein expression levels,and the influence of ghrelin on the phosphorylation or total expression levels of these molecules was further evaluated as well.The second experiment stage was designed to further verify the hypothesis that ghrelin could improve wound healing through inflammatory pathway,and involved eight groups: normal group,CRBI group,ghrelin treatment group,p38 MAPK blocker(SB203580)treatment group,JNK blocker(SP600125)treatment group,SB203580+SP600125 treatment group,[D-Lys3]-GHRP-6 and ghrelin treated-group and rat TNF-? antibody treated-group.Methods for CRBI model establishment,[D-Lys3]-GHRP-6 and ghrelin administration kept the same as previously stated.SB203580 and SP600125 were all s.c.infused for consecutive 7 d in a dose of 15mg·kg-1·d-1;polyclonal antibody for TNF-? was intraperitoneally(i.p)injected 2 times per d for sequential 7 d in a dose of 10 mg·kg-1·d-1.Serum TNF-? was determinated again at that optimal measurement time point,and the macrophage proteins were also abstracted for WB analysis.In addition,the wound pictures were taken every other 2 d for 30 d and the average wound healing rate and wound closure time were computed at late stage of CRBI.In the fourth part,wound fibroblasts from CRBI rats were selected as the target cells to study whether ghrelin and proinflammatory mediator(TNF-?)could affect its biological functions,and if so,its possible mechanisms.Fibroblasts presents as key repairing cells in granulation tissue maturation and wound healing;however,high concentrations of TNF-? can inhibit wound repair and regeneration by influencing fibroblasts' biological functions such as collagen secretion.After the isolation and culture of wound fibroblasts from CRBI rats,various experiments were performed as follows: immunocytochemistry(ICC)stain for GHS-R1 a in the membrane,and ?-smooth muscle actin(?-SMA)in the cytoplasm;cell viability evaluation for fibroblasts treated with ghrelin or/and TNF-?;wound scratch assay for assessing “migration promotion” ability of ghrelin;phosphorylation alternations of drosophila mothers against decapentaplegic protein-type 3(Smad3)after single stimulation with TNF-? or TGF-?,confirming the optimal time point for Smad3 measurement;phosphorylation change after jointly treated with ghrelin and TGF-?;the influence and interrelation of ghrelin,TNF-? and TGF-? on phosphorylation expression of p38 MAPK,JNK and Smad3.This experiment was designed to preliminarily explore whether ghrelin could promote the collagen secretion from CRBI fibroblasts,and to further elucidate the wound healing effect of ghrelin at cellular and molecular level.The above 4 research contents reflect the contribution of ghrelin to wound repair and regeneration in CRBI rats from animal,histological,cellular,and molecular aspects.Many positive results have been harvested and shown as following:1.The serum ghrelin levels significantly decreased especially at 7 d(by ~50%)after CRBI,and then gradually recovered to normal levels.At 24 d post CRBI,the average wound healing rate of 50,100 and 200nmol·kg-1·d-1ghrelin treated-rats was respectively 60%,50% and 30%(P<0.05,compared to single CRBI);the average wound closure time was also shortened by about 3~8 d(P<0.05,compared to single CRBI).As to body weight change,200nmol·kg-1·d-1 ghrelin increased body weight of CRBI rats,most by 5%(about 11g).Ghrelin also obviously increased WBC and PLT count 3,7,and 14 d post CRBI(P<0.05,compared to single CRBI).2.As with the gradual wound healing,the content of DNA,collagen(hydroxyproline),hexosamine and total NO in wound granulation tissues from different groups increased at first and then declined;it reached to peak at 20 d after injury.The content of DNA,collagen(hydroxyproline),hexosamine and total NO decreased significantly(P<0.05,compared to single BI),which was elevated by 200 nmol·kg-1·d-1 ghrelin therapy at corresponding time points(P<0.05,compared to single CRBI): DNA content respectively decreased by 67%,56% and 63% at 10,20 and 30 d after CRBI,and increased by 138%,98% and 136% after ghrelin treatment;collagen content respectively decreased by 36% and 32% at 10,20 d after CRBI,and improved by 36% and 36% by ghrelin;hexosamine content respectively declined by 34% and 36% at 10 and 20 d after CRBI,and elevated by 26% and 46% after ghrelin therapy;total NO content respectively decreased by 77% and 50% at 10,20 d after CRBI,and increased by 200% and 33% by ghrelin treatment.Sirius red and Masson stain for collagen in granulation tissues of CRBI rats also showed that the collagen content was lower than that in BI rats,however,ghrelin elevated its secretion level(P<0.05);similarly,vessel IHC stain showed that microvessels count in CRBI granulation tissues was also obviously less than that in BI rats,whereas ghrelin increased this(P<0.05).WB assays found that growth factors TGF-? and VEGF level in granulations of BI,CRBI and ghrelin treated-CRBI rats also presented as an “inverted triangle” changing trend.Additionally,CRBI induced less WBC penetration in granulation tissues(P<0.05,compared to single BI),however,ghrelin increased WBC penetration especially granulocyte neutrophils and monocytes.Interestingly,GHS-R1 a expression in granulation tissues decreased in CRBI rats,which was up-regulated by ghrelin infusion(P<0.05,compared to single CRBI).The application of [D-Lys3]-GHRP-6 largely blunted or completely inhibited those positive effects caused by ghrelin.3.The serum levels of proinflammatory mediators such as TNF-?,IL-1? and IL-6 significantly increased in CRBI rats at various time points(P<0.05,compared to normal rats),which reduced a lot after ghrelin treatment(P<0.05,compared to single CRBI).TNF-? is the most important inflammatory mediator influencing cutaneous wound healing.The serum TNF-? concentration in CRBI rats increased by 6,24 and 42 fold(P<0.05,compared to normal rats)at 1,4,and 7 d post CRBI respectively;and decreased to 46% and 61% of original concentration at 4 and 7 d after ghrelin treatment(P<0.05,compared to CRBI rats).Through WB analysis of total protein abstracted from rat peritoneal macrophages in each group,we found that: 4 d after injury,p38 MAPK,JNK and p65NF-?B showed strongest phosphorylation,whereas GR tended to be down-regulated;ghrelin blunted the phosphorylation degree of p38 MAPK,JNK and p65NF-?B,and up-regulated the expression levels of GR.By using the corresponding protein blocker,at the same time point,WB showed that: single administration of SB203580 or SP600125 can respectively inhibit phosphorylation of p38 MAPK and JNK,and coadministration of SB203580 and SP600125 can inhibit both phosphorylation of p38 MAPK and JNK;SB203580,SB203580+SP600125 both inhibited the phosphorylation activation of p65NF-?B,whereas single or combined administration of these two blockers could increase GR expression(coadministration exhibited better effect);anti-TNF-? could also inhibit excessive phosphorylation activation of p38 MAPK,JNK and p65NF-?B and elevate GR level,however this effect was similar to but weaker than that of ghrelin.At the same time point,ghrelin,SB203580,SB203580+SP600125 and anti-TNF-? all reduced serum TNF-? level in CRBI rats.Indicators reflecting wound healing showed that CRBI rats treated with ghrelin,SB203580,SB203580+SP600125 or anti-TNF-? possessed higher average wound healing rate(35%~65%;compared to single CRBI,P<0.05)and less average wound closure time(shortened by 3~8 d;compared to single CRBI,P<0.05),however,ghrelin was more effective.The application of [D-Lys3]-GHRP-6 almost completely suppressed those positive actions mediated by ghrelin.4.GHS-R1 a and ?-SMA highly expressed and distributed in membrane and cytoplasm of fibroblasts isolated from CRBI granulation tissues,respectively.Different doses of ghrelin(1-104ng/mL)or TNF-?(1-100ng/mL)couldn't increase or decrease cell viability;combined administration of ghrelin and TNF-? couldn't influence its proliferation or apoptosis as well.Cell scratch assay indicated that ghrelin(1-104ng/mL)yet couldn't promote cell migration or scratch healing.In the possible mechanism study that ghrelin promoted collagen secretion by fibroblasts,some phenomena were found: TGF-?(5ng/mL)could significantly enhance Smad3 phosphorylation at 0.5~1 h after stimulation(P<0.05),while TNF-?(5ng/mL)suppressed it at 1~2 h after stimulation(P<0.05);ghrelin(1,100ng/mL)exerted no effect on the enhanced Smad3 phosphorylation induced by TGF-?;TNF-? resulted in enhanced phosphorylation of p38 MAPK and JNK,and attenuated Smad3 phosphorylation,while ghrelin treatment reverted these effects caused by TNF-?,showing an increased Smad3 phosphorylation(P<0.05).In conclusion,ghrelin could accelerate the formation and maturation of CRBI granulation tissues,thus dose-dependently promote wound healing;the possible major mechanisms include: improving hematopoietic function,increasing penetration of WBC and secretion of related growth factors in granulation tissues,promoting neovascularization and collagen deposition;inhibiting MAPK signaling pathways,increasing GR expression or activation,decreasing secretion of pro-inflammatory mediators,thus attenuating systemic inflammatory response;suppressing MAPK signaling pathways,enhancing Smad3 phosphorylation,alleviating the inhibition in collagen secretion activity of CRBI fibroblasts that caused by TNF-?.