Role And Mechanism Of α-MSH/MC5R Signal Axis On Injury And Repair Of Bone Marrow Hematopoietic System After Irradiation

Nuclear energy and related technologies have been widely used in military industry,energy generation,medical treatment and other fields.It significantly improves social productivity but greatly increases the risk of the body’s exposure to ionizing radiation（IR）.As an irradiation-sensitive tissue,exposure of bone marrow to radiation can lead individuals to develop varying degrees of hematopoietic system damage,eventually causing bleeding,infection and anemia.Studies have found that the body can repair itself in a certain way under the lower doses of irradiation,while higher irradiation doses can cause irreversible and serious damage to tissues and organs.However,the relevant regulatory pathways have not been fully revealed.Therefore,exploring the potential mechanism of bone marrow cell damage and repair after irradiation can provide more guidance for related protection and treatment measures.The realization of long-term hematopoietic function of bone marrow mainly depends on hematopoietic stem cells（HSCs）with self-renewal and multidirectional differentiation.Mouse HSCs are mainly enriched in Lineage^-Sca1⁺c-Kit⁺cells（LSKs）.In addiotion,more specific markers can subdivide subsets of hematopoietic cells with different functions for further study.Under stress,a large number of HSCs change from the quiescent state to the proliferative state,rapidly differentiating into various hematopoietic daughter cells to promote the recovery of the hematopoietic system.Ionizing radiation not only directly damages the DNA in nucleus,but also produces a large amount of reactive oxygen species,which can destroy the protein structure and disrupt the cell metabolism,eventually causing the aging and apoptosis of cells.HSCs are strictly regulated in the process of hematopoietic reconstruction,such as switching cell states,controlling cell production rate,and maintaining differentiation potential.At present,researchers have found a series of treatment solutions,including reducing DNA damage,enhancing cell repair ability and scavenging active oxygen.A variety of hormones have been shown to be involved in the repair of HSCs after radiation damage,such as growth hormone,insulin-like growth factor and gonadotropins.The specific regulatory functions of hormones depend on the distribution their receptors.Melanocortin is a peptide hormone produced by proteolytic digestion of Pro-opiomelanocortin（POMC）,including adrenocorticotropic hormone（ACTH）,α-melanocytes stimulating hormone（α-MSH）,β-MSH,γ-MSH and other cleavage products,performing various regulatory functions after binding to specific melanocortin receptors（MCRs）.It is known that there are five subtypes of MCRs（MC1R-MC5R）,all of which are G protein-coupled receptors（GPCRs）.However,they have certain differences in molecular structure,tissue distribution and ligand affinity of each subtype,so as to play different regulatory roles in melanin formation,inflammation,energy metabolism,reproductive function and gland secretion.As the last discovered and cloned melanocortin receptor,MC5R is expressed in adrenal glands,exocrine glands,skin and other tissues.Compared with other hormone ligands,MC5R is more easily activated byα-MSH.After receiving the ligand signal,it can further activate c AMP-PKA,PI3K/AKT,β-arrestin1/2/c-Raf and JAK2/STAT1 and participate in various physiological processes,such as exocrine gland secretion,fat metabolism and glucose uptake.In recent years,MC5R is found to be expressed in the bone marrow,spleen and lymph node,and has regulatory functions on macrophages,dendritic cells and T cells,which hint that MC5R is closely related to hematopoiesis and immune system.Recent studies have shown that after transplantation of Lewis tumors,the serum level ofα-MSH in tumor-bearing mice was significantly increased,leading to bone marrow cell proliferation and myeloid bias differentiation.Thus,we speculate that the melanocortin signaling axis formed byα-MSH and MC5R may have potential regulatory role in the physiological and pathological processes of HSCs.In this study,qPCR,immunofluorescence and other techniques were used to detect the expression level of MC5R in the bone marrow.To explore the role of MC5R and its ligandα-MSH in murine HSCs,the differences in hematopoietic systems between WT and MC5R^-/-mice during steady-state and acute radiation injury were analyzed by ELISA,flow cytometry,bone marrow transplantation,etc.Furthermore,RNA-seq,HSPC culture and other technical methods were used to explore the mechanism ofα-MSH/MC5R signaling axis in regulating HSC biology.Finally,the treatment ofα-MSH and related pathway inhibitors verified the role and mechanism ofα-MSH/MC5R signaling axis in hematopoietic regulation,which enhanced the practical application significance.The main findings and conclusions of the study are as follows:1.MC5R is significantly enriched in hematopoietic stem progenitor cells（HSPCs）,but is not necessary to maintain homeostatic hematopoiesis.1.1 Flow cytometry,immunofluorescence and qPCR results showed that MC5R was significantly enriched in HSPCs in comparison to mature bone marrow cells,which was consistent with the results of public databases and previous gene expression profiles in our research group,suggesting that MC5R may play a specific role in HSCs.1.2 The results of flow cytometry showed that there was no significant change in the proportion and number of mature bone marrow cells and HSPCs in mice after loss of MC5R,indicating that MC5R is not essential for the regulation of HSC function under steady-state conditions.1.3 Ki67/Hoechst 33342 co-staining and Brdu incorporation assays confirmed that MC5R knockout did not affect the cell cycle state and the proliferation ability of HSCs in mice in steady-state.2.MC5R deficiency impairs the proliferation and hematopoietic reconstruction ability of HSCs after irradiation injury.2.1 ELISA results showed that the levels ofα-MSH in the serum and bone marrow cell supernatant of mice were elevated after irradiation,and qPCR results confirmed that the expression of MC5R in murine HSPCs was increased following irradiation exposure,suggesting that theα-MSH/MC5R signal axis may be involved in the hematopoietic recovery process post radiation injury.2.2 Blood routine test and flow cytometry data showed that MC5R deficiency caused cell cycle arrest of HSCs after irradiation injury,leading to a slowdown in the recovery of hematopoietic function,but there was no significant difference in apoptosis,suggesting that MC5R is involved in the regulation of HSC proliferation following irradiation exposure.2.3 The results of bone marrow transplantation,HSPC culture,homing experiments and other results showed that the expansion and differentiation ability of HSCs in mice was decreased after MC5R knockout,whileα-MSH could promote the proliferation of HSCs in vitro,indicating that theα-MSH/MC5R signaling axis promotes the reconstruction of hematopoietic system.3.Theα-MSH/MC5R signal axis regulates the proliferation of HSCs through the PI3K/AKT and MAPK pathways:3.1 The results of RNA-seq,qPCR and flow cytometry showed that the quiescent-related genes were significantly upregulated and the proliferation-related genes were downregulated in HSCs with MC5R deficiency,accompanied by the decrease in cell metabolism level and protein synthesis rate,indicating that MC5R plays a role in promoting recovery of HSCs after radiation injury.3.2 The results of flow cytometry and immunofluorescence showed that the loss of MC5R impaired the activation of PI3K/AKT and MAPK pathways in murine HSCs after irradiation,suggesting thatα-MSH/MC5R signal axis regulates HSC function via the PI3K/AKT and MAPK.4.Activation of theα-MSH/MC5R signaling axis promotes hematopoietic recovery in mice after irradiation:4.1 Injection ofα-MSH increased the phosphorylation levels of AKT and ERK1/2,therefore accelerating the cell cycle progress and the amplification HSPC pool.These phenomena were abolished when MC5R was knocked out.These further demonstrate that theα-MSH/MC5R signal axis regulates the proliferation of HSCs through the PI3K/AKT and MAPK pathways.4.2 Afterα-MSH treatment,irradiated mice showed accelerated recovery of peripheral blood cells and improved overall survival rate,and these effects were disappeared after loss of MC5R.It indicates that activating theα-MSH/MC5R signaling axis can promote the recovery of the murine hematopoietic system when suffering from radiation injury.In summary,this study reveals the critical role of theα-MSH/MC5R signal axis in the regulation of HSC function after radiation injury,and further explores the molecular mechanism.These findings provide a new treatment strategy to promote the recovery of the hematopoietic system after radiation injury.