Study On The Protective Effect Of Natural Product Eriocalyxin B On Radiation-induced Hematopoietic Injury And Its Mechanisms

Acute Radiation Sickness(ARS)refers to a systemic disease caused by the body being exposed to high doses of ionizing radiation(IR)in a short period of time.Among them,the type with bone marrow hematopoietic tissue injury as the basic pathological change is called Bone Marrow form of Acute Radiation Sickness(BM-ARS).In recent decades,great progress has been made in the prophylaxis and treatment of acute radiation sickness(ARS),but there are still many problems that have not been resolved,and effective countermeasures for ARS are still scarce.It is imperative to develop novel radioprotective agents with fine anti-radiation effects,high safety,and clear mechanisms.In the process of searching for novel radioprotectants,we discovered for the first time that the natural small molecule compound Eriocalyxin B(EriB)has potential radioprotective efficacy.EriB is a natural diterpenoid isolated from Isodon eriocalyx var.laxiflora.EriB possesses anti-inflammatory and anti-tumor effects,and modulates ROS production and various signaling pathways(such as NF-κB).Hence,we focus on the protective effect of EriB on BM-ARS,and further explore underlying mechanisms of its protective effects on radiation-induced hematopoietic injury.First,the effect of EriB prophylaxis on survival of lethally irradiated mice received 9.0 Gy irradiation was evaluated.Our results indicated pretreatment with EriB significantly increased the survival rate and prolonged mean survival time in the lethally irradiated mice.Compared with the IR-control group,the survival of EriB-treated group increased by 70%.Furthermore,prophylactic administration of EriB significantly increased the number of peripheral blood leukocytes,red blood cells and platelets in mice irradiated with a sublethal dose of 6.5Gy.Above all,EriB showed exact protective effects on BM-ARS mice.Further studies showed that prophylactic administration of EriB prominently improved the number of hematopoietic stem cells(HSCs),especially long-term hematopoietic stem cells(LT-HSCs)in irradiated mice.Colony forming units(CFUs)assay exhibited that EriB administration strikingly enhanced the colony-forming ability of hematopoietic progenitor cells.Also,competitive bone marrow transplantation experiments uncovered that EriB improved the ability of HSCs to reconstitute hematopoiesis,and ameliorated the long-term suppression of bone marrow hematopoietic function in irradiated mice,which heightened the long-term hematopoietic re-establishment capability of hematopoietic stem cells.These results revealed that EriB had a critical radio-protective effect on the numbers and function of murine hematopoietic stem and progenitor cells(HSPCs).Mechanistic research disclosed that EriB relieved the imbalanced cell cycle and apoptosis in HSPCs,and simultaneously alleviated ROS generation and DNA double-strand breakages(DSBs)in HSCs,suggesting the radio-preventive effects of EriB on HSPCs by above regulatory role.During our investigation for the molecular mechanism,the effects of EriB on the Nrf2/ARE signaling transduction pathway were identified and confirmed.Experimental results from the murine RAW 264.7 cell line demonstrated that EriB activated Nrf2/ARE pathway in a dose-dependent manner,upregulating the transcriptional and translational level of its downstream target genes(such as HO-1and NQO1),especially propelling the IR-activated Nrf2/ARE signal.Furthermore,transcriptional enhancement of Nrf2 downstream genes after EriB administration was observed in diverse murine cell lines,such as 32 D cells,marrow mesenchymal stem cell and primary hematopoietic progenitor cell,suggesting the capacity of EriB to activate Nrf2/ARE signaling pathway in marrow hematopoietic cell.To further explore the role of Nrf2 signaling pathway in the radioprotective effect of EriB,Nrf2-knockout mice were used as a radiation model to monitored the EriB efficacy.The results indicated that EriB administration showed similar protective effect on the survival of wildtype and Nrf2-knockout mice after IR.Furthermore,the loss of the Nrf2 gene did not affect the radio-protective effect of EriB on peripheral blood cell counts of irradiated mice.In summary,all results come to the following conclusions:(1)EriB has an established radio-protective function in BM-ARS mice;(2)EriB administration outstandingly increases the number of HSPCs in radiated mice,enhancing the ability of HSCs to reconstitute hematopoiesis,thereby mitigating the hematopoietic injury in BM-ARS mice;(3)mechanistic study identified that EriB activates and modulates its downstream target genes of Nrf2 signaling pathway.Nevertheless,the radioprotective effect of EriB has nothing to do with this pathway.