Ionization Radiation On The Molecular Mechanism Of The Influence Of The Skin Wound Healing In Mice

Research background:Wound healing is a complex process involving several stages that includeinfammation, formation of granulation tissue, reepithelialization, matrix formation andremodeling.Several growth factors play an important role in wound healing. In the acutewound, the healing process is completed by these growth factors resulting in thereestablishment of the skin’s barrier function. In chronic wound, the generation of aproteolytic environment by infammatory cells infltrating the wound site leads todegradation of these growth factors and inhibit wound healing.It has been reported thatsystemic radiation damage could cause the number and function of hematopoietic cells andrepaired cell, and destroy the healing process, slow wound healing. The radiation damagewas always accompanied with burns, trauma, infection and other complication, andeventuality leading to delayed wound healing, even refractory.The current study found thatabnormal cytokine expression levels may be exist in this kind of wound. Thus, the rightamount and reasonable usage of cytokine could promote wound healing: not only promotetissue repair, but also could improve the quality of wound repair. However, despite thereare many local cytokine treatment in the previous study, but the kind and amount ofcytokine remain controversy. In addition, due to refractory of ionizing radiation, theinflammatory cells would be invade continuously, leading to the environment of aproteolytic enzyme and exogenous cytokines could be degraded easily. It is difficult forthese cytokines to play a normal roles in the healing process. Therefore, the effectivecontrol for local inflammatory, appropriate cytokine, cytokine dose and delivery are veryimportant to ionizing radiation combined trauma wound healing.Cytokine is a kind of glycoproteins or small molecule biological active polypeptidewhich is able to transmit information between cells and regulate immunefunctions.Cytokines have pleiotropic, antagonistic, overlap, and synergies, the interactionof different cytokines could be involved in a variety of cellular process in wound healing,including cell proliferation, cell migration, extracellular matrix synthesis and secretion. Ithas reported that VEGF-A, PDGF-BB, TNF-α and GM-CSF have an important roles in the regulation of wound healing, and different expression level in different stage of woundhealing. However, the study about the relationship of these cytokines and radiation woundis relatively rare. GM-CSF, as an important cytokine, could increase the number andfunction of neutrophils in the inflammatory stage of wound healing and promoteinflammatory response in wound.And in the process of angiogenesis, GM-CSF is able topromote the migration and proliferation of endothelial cells. It also could promotekeratinocytesdirectly or indirectly to increase keratinocyte proliferation and improvere-epithelialization. Local GM-CSF treatment could be a effective way in promoting thehealing of chronic wound, so it is possible to be a new therapy way in chronic radiationwound.Therefore, based on the previous study, we conduct the present study including twoparts:(1) we detect the relatively expression level of VEGF-A, PDGF-BB, TNF-α,GM-CSF in the process of wound healing.Firstly, the mice under6Gy60Coγ radiationcombined with wound on the back was experimental group, while the mice withoutradiation damage was control group. Then we examined the dynamic expression level ofthese four cytokinesthrough RT-PCR and immunohistochemistryto study the expression ofvarious cytokines in the wound healing.(2) we explore the therapeutic effect of GM-CSFin the wound healing. Inthe mouse model of ionizing radiation combined with injuryabsence of the skin on the back, we determine the effect ofa mixture of different doses ofGM-CSF in wound healing using the detection of wound residual area and masson stainingof collage fibers in day14. Thus we could observe the therapeutic effect of different dosesof GM-CSF in radiation wound and seek the proper approach and doses.Part I: Detecting the expression levels of VEGF-A、PDGF-BB、TNF-α、GM-CSF inback skin of IR-injured mice skin during the defect wound healing processObjective: To explore the effect of different cytokines on the detect wound healing process,we detected the dynamic expression levels of VEGF-A、 PDGF-BB、 TNF-α、GM-CSFmRNAandProtein in back skin of IR-injured mice skin during the defect woundhealing process.Methods: The mice wasunder6Gy60Co γ whole body ionizing radiation, and 1.5cm×1.5cm of skin defects was produced in back skin of the mice to establish the backskin defect wound model. At the1day,3day,5day,7day after injury, the dynamicexpression levels of VEGF-A、PDGF-BB、TNF-α、GM-CSF mRNA were detected usingRT-PCR method, while the protein levels of these cytokines were detected usingImmunohistochemistry method.Results: The result obtained from RT-PCR showed that the relative expression levels ofVEGF-A、PDGF-BB、TNF-α、GM-CSF m RNA was significant different from the normalwound. Compared with the normal wound, the expression level of PDGF-BB was lowerthat control group at the time point including1day,3day,5day,7day. And PDGF-BB wassignificant lower than control group at3day(P<0.01), which showed that PDGF-BB wasdecreased in the IR-injured mice. VEGF-A reach the peak at5day in both experimentalgroup and control group, but the control group was significantly higher than theexperimental group(p<0.05). The expression of VEGF-A in control group decreasedquickly and was significantly lower than experimental group(p<0.01), which demonstratedthat ionizing radiation will lead to the lag of VEGF-A expression level in wound tissue.The results showed that the expression level of TNF-alpha was higher in6Gy experimentalgroup than control group during the entire wound healing process, and the mRNAexpression were significantly higher at7day than control group (P <0.01), suggesting thatIR injury wound lead to excessive inflammation TNF-alpha expression during the woundhealing process of mice back skin defect wound; GM-CSF expression levels inexperimental group were lower than control group at the the day1,3,5, and at the day3, theexperimental group is significantly lower than the control group (P <0.01). Besides, at theday7after injury the significant differences disappeared between the two groups,suggesting that the low expression of GM-CSF may seriously affect the IR injury mouseback skin defect wound healing process.Similarly, theresults ofimmunohistochemistryshowed thatVEGF-A, PDGF-BB, TNF-alpha, GM-CSFproteinexpression werein accordance with the results of mRNAexpression. Conclusion: IR injury can causea variety of cytokinesexpressiondisordersin skindefectwound, includingthe delay or decrease of growth factor expression andlead directlytotheinhibition ofgranulation tissue, angiogenesis; Besides, the inflammatorycytokineexpressiondisorder would cause the delay of inflammation orextendthecycle. Thecytokineexpressiondisorderis not only theIRresults, but also the reason of RCIhealingdelay.Part II. Evaluation the efficacy ofdifferent doses ofrhGM-CSFin IR-injured miceduring skin defect wound healing processObjective: To study the effect of different doses of rhGM-CSF chitosan-collagenhydrogel (1500ng/ml,6000ng/ml and10000ng/ml) on the wound healing of the back skinin IR-injured in mice, and then find the optimal dose of actionMethods: Based on the result of part.1, we selected rhGM-CSF as a drug intervention invivo, we use the different doses of rhGM-CSF(1500ng/ml,6000ng/ml and10000ng/ml)topical administration for5days,1time/day. And we also userhGM-CSF-freechitosan-collagen hydrogel as control group, and then evaluated the wound healingevaluation and cytokine expression was also detected.(1):the evaluation of woundhealing:using the Image Pro Plus v1.5software system to detect the residual area in bothexperimental treatment group and control group, and measure, analyze the skin defectresidual area change during the wound healing process.The Masson staining detectionshowed the skin wound collagen fiber content at14days.(2) the detection of relevantendogenous cytokine: using RT-PCR method to detect the m RNA expression level ofPDGF-BB、VEGF-A、TNF-α and analyze the effect of different dose rhGM-CSF on woundhealing, further find the most suitable treatment dose under the present condition.Results:(1)At day14, the wound residual area in back skin of IR-injured mice wassignificant smaller in rhGM-CSF1500ng/ml、rhGM-CSF6000ng/ml groups than controlgroup(P<0.01), but that of rhGM-CSF10000ng/ml groups was elarger than control(P<0.05). The results also showed that the wound healing was better in rhGM-CSF6000ng/ml groupswhen compared withrhGM-CSF1500ng/ml group, which revealed that local administrationof exogenous rhGM-CSF gel could significantly improve defect wound healing process inIR-injured mice. And the therapeutic efficacy was proportional to the concentration of therhGM-CSF within a certain dose range(GM-CSF6000ng/mlgelgroup was superiortoGM-CSF1500ng/ml). However, the wound healing would be inhibited when the dose ofrhGM-CSF was10000ng/mL. The result from the Masson staining at day14, the collagenfiber content was better in rh-GM-CSF6000ng/mL than control group, and the result ofthis group showed the deep collagen coloring, and the area is more widely distributed andarranged closely.(2) At day1, day3, day5, day7, the RT-PCR result displayed VEGF-A,PDGF-BB was significantly lower in experimental group than control group, suggestingthat the high dose rhGM-CSF((10000ng/ml) gel will inhibit the expression of othercytokines such as growth factors.Conclusion: Cytokines was the key factor of regulating the healing of RCI would. Theexogenous GM-CSF could improve the would healing process in IR-injured mice. Thepresent study showed that The local administration of exogenous rhGM-CSF gel couldsignificantly improveRCI wound healing process and thetherapeutic efficacy isproportional to the concentration of rhGM-CSF within a certain dose range of itsconcentration with (GM-CSF6000ng/ml gel group was superior to rhGM-CSF1500ng/ml). As for wound healing, he exogenous administration of rhGM-CSF concentrationshould be controlled within a certain range, which is related the wound type and the modeof administration. Specifically, under the conditions of the present model, GM-CSF of theoptimum concentration or dose need to be furtherconfirmed.