The Therapeutic Effects Of Ghrelin On Combined Radiation-burn Injury Animals And Its Possible Mechanisms

Combined radiation injury often occur in nuclear war and nuclear accidents or otheraffairs, which threaten the lives of most people who suffers from it, especially the combinedradiation-burn injury(CRBI). CRBI is a sort of combined injury that mainly suffer from theradiation injury, following with the burn injury simultaneously or consecutively. Comparedwith single injury(radiation or burn injury), the condition of CRBI seems to be more complexand hard to control. After suffering from this injury, the following several pathologicalprocesses will happen, namely: shock and acute stress, hematopoietic dysfunction, immunedisfunction(mostly immune depression), infection, acute inflammatory response and thedelayed union or disunion of burn wounds. Among which, hematopoietic dysfunction,immune depression and systemic acute inflammatory response are the most important causesleading to death. Nowdays, the therapy of CRBI mainly focus on the symptomatic treatmentinstead of etiological treatment, such as blood transfusion, bone marrow transplantation,antibiotics for infecton, blood purification and skin-grafting. But these therapies stillencounter various problems more or less, such as costly, great side effects and high-risk, ect.However, our preliminary studies had illuminated that cervical sympathetic block(CSB)can obviously improve the survival rate of CRBI animals, the possible mechanisms involvedthe reduction of the severity of hematopoietic inhibition, down-regulation of the acuteinflammatory response and acceleration on the union of burn wounds. The technology of CSBhas been widely used in clinical, mainly in the therapy of chronic pain instead of trauma orburn injury for some reasons. Ghrelin is a kind of endogenous brain-gut peptide, consisting of28amino acids, mainly secreted from the X/A like cells at the bottom of the stomach. Ghrelinhas many physiological effects, for example, promotes the secretion of growth hormone(GH)primarily thus regulates the energy metabolism. It can also regulate the immunity, etc. Manystudies have shown that Ghrelin has a therapeutic effect on combined radiation and sepsis animals by possibly impacting the excitability of vegetative neverous system，this mechanismis similar to that of CSB. Additionally, Ghrelin has shown some therapeutic effects in thetreatment of many relative clinical diseases. Importantly, it seems to be more convenient toadministrate with less toxic side-effects compared with CSB. Thus, in order to explore that theGhrelin has a therapeutic effect on CRBI animals as well and to develop new type of drugs forthe treatment of CRBI, we conducted this study.First of all, we established the model of CRBI, the method are summarized as follows:firstly, animals were irradiated with5Gy dose of γ radiation, then15%total body surfacearea(TBSA) of animals is subjected to three degree burn injury in the dorsal part by utilizingbromine tungsten lamp within2hours after irradiation. Subsequently, based on the modelestablished, different studies were executed:①the therapeutic effects on CRBI rats comparedwith CSB. The animals were divided into5groups, namely the control group, the CRBI group,the CRBI group treated with CSB or Ghrelin and the group treated with CSB and Ghrelin.The experimental contents mainly involved with the evaluation of weigh change, woundhealing, hematopoiesis and mortality.②the study of possible mechanisms. The animals weremainly divided into4groups, namely the control group with or without Ghrelin treatment, theCRBI group with or without Ghrelin treatment, mainly involved with the evaluation ofhematopoiesis, the control of inflammatory response, the neuroendocrine regulation and organprotection, etc.③The in vitro study: mainly focus on the partial biological effects of Ghrelinon macrophages and hematopoietic stem cells(HSCs). In the whole experimental process: thehemogram analysis, mainly observed the change of WBC and PLT at certain time-point afterinjury. Wound healing and mortality were both observed for30days. And the concentritionsof TNF-α and IL-6in serum were both analysised by using the method of enzyme linkedimmunosorbent assay(ELISA). As to the neuroendocrine regulation, the contents of variousneurotransmitters or hormones in serum, such as acetylcholine(Ach), catecholamine andcortisol(Cor), are mainly detected by using corresponding methods. Haematoxylin and eosinstain(H.E) and immunohistochemistry(IHC) were performed to study the protective effects ofGhrelin on some organs. Additionally, the livability and relative secreteing functions ofmacrophages(treated with or without LPS) were also detected through the establishment of“combined radiation-burn injury model” in vitro. Lastly, we used the technology ofimmunomagnetic beads to isolate marrow HSCs, and the effects of Ghrelin on HSC proliferation were then studied. The results are summarized as follows:1. Ghrelin can improve the survival rate of CRBI rats about30%(compared with CRBIgroup, P<0.05)as well as CSB while the combination treatment does not(compared withCRBI group, P>0.05). In the aspect of hemogram, both CSB and Ghrelin can promote theelevation of WBC and PLT(compared with CRBI group at certain time-point, P<0.05), mainlyat the7thand14thday. However, the combination treatment doesn’t have a obvious influenceon that(compared with CRBI group, P>0.05). Yet there are no significant differences on thewound healing or weight change among each group.2. Then in the experiment studying the influence of Ghrelin on autonomic nervoussystem, considering that the hypothalamus is one of the parts in which Ghrelin’s receptorGHS-R1a distribute and the combination of which can affect the excitability of autonomicnervous system, thus we detected the expression of GHS-R1a in the arcuate nucleus(Arc) ofhypothalamus. The results indicated that Ghrelin can elevate the expression of GHS-R1a inthis section compared with CRBI group. In addition, through the detection of serumconcentritions of neurotransmitters or hormones related to stress, we find that theconcentrition of epinephrine(E),5-hydroxy tryptamine(5-HT), acetylcholine(Ach) andcortisol(Cor) in the serum of CRBI animals treated with Ghrelin are dramaticaly decreased(compared with CRBI group, P<0.05). It indicated that Ghrelin can regulate the excitability ofautonomic nervous system(mainly surpress the sympathetic nerve) and surpress the excessiveactivation of HPA.3. The inflammatory mediators, such as TNF-α and IL-6in the serum of CRBI animalstreated with Ghrelin are also decreased(compared with CRBI group, P<0.05). In addition,although Ghrelin doesn’t have a significant influence on the livability or the NOS/NOsecretion of macrophages treated with LPS(compared with LPS treatment group, P>0.05),Ghrelin can reduce the secretion of TNF-α in macrophages(compared with LPS treatmentgroup, P<0.05). It indicated that Ghrelin can also regulate the acute inflammatory responseinduced by CRBI.4. Ghrelin can distinctly improve the number of bone marrow nucleated cells(BMNC)10days after CRBI(compared with CRBI group, P<0.05). And by the means of histopathologicalstudy, we also find that Ghrelin effectively inhibit the dramatically decrease of bone marrowhematopoietic cells. However, the in vivo study reveal that Ghrelin could not promote the proliferation of hematopoietic stem cells, the effect of hematopoietic protection may bemediated by other possible pathways.To sum up, through the establishment of CRBI model and the comparision of therapeuticeffects on CRBI between Ghrelin and CSB, yet the subsequent mechnism study, we find thatGhrelin has a therapeutic effect on CRBI animals, the possible mechnisms may be that:regulate the excitability of autonomic nervous system, weaken the early acute stress reaction,control the inflammatory response and alleviate the degree of hematopoietic inhibition.