Studies On The Therapeutic Effect And Mechanism Of RH005 On Acute Radiation-induced Non-infectious Inflammation

Acute radiation syndrome（ARS）is a systemic disease caused by large doses（>1Gy）of ionizing radiation in the body within a short period of time.According to the different doses,it is generally divided into bone marrow type（>1Gy）,intestinal type（>10Gy）and brain type（>100Gy）radiation diseases.Severe radiation sickness induced by more than 8 Gy rays irradiation is still difficult to treat,which is an urgent problem to be solved.At present,radioprotectants are often used in the clinic to prevent ARS,but they need to be administered before irradiation so to be effective,and they cannot be used as therapeutic drugs for radiation induced damage.The treatment of ARS is mainly to promote hematopoiesis and improve immunity.Radiation causes severe damage to a variety of cells in the body.After cell damage,heat shock proteins,oxygen free radicals,excitatory amino acids,and extracellular matrix degradation products can produce"endogenous danger signals".These danger signals can initiate inflammatory reactions.Radiation induced non-infectious inflammation occurs in multiple organs such as lung,intestine,and brain,so ARS might be alleviated by inhibiting such non-infectious inflammation.RH005 is a self-developed recombinant protein drug.Its pharmacy,general pharmacology,toxicology and pharmacokinetic research have been basically completed,and its safety is high.Preliminary pharmacodynamic studies have shown that RH005 has anti-inflammatory and anti-apoptotic effect,it also can attenuate the secretion of TNF-αby macrophages,suggesting that it may inhibit acute radiation induced non-infectious inflammation and have a therapeutic effect on ARS.In this study,the efficacy of the drug was first evaluated and the optimal administration regimen was determined.（1）Dose-effect investigation and optimal dose determination:236 male SPF C57BL/6N mice were randomly divided into:control group（Con group）,model group（IR group）,dexamethasone group（DEX group,4.5mg/kg）,amifostine group（W group,200mg/kg）,RH005 low dose group（L group,1μg/kg）,RH005 medium dose group（M group,5μg/kg）and RH005 high dose group（H group,25μg/kg）.For the survival rate experiment,there were 20 mice in each group,while for physiological and pathological observation,there were 6 mice in the Con group and 15 mice in each of the other groups.Mice in W group were administered half an hour before the irradiation,and mice in other groups received intraperitoneal injection with RH005 24h after receiving 8Gy ⁶⁰Coγ-rays.Mice in Con and IR groups were administered intraperitoneally with normal saline.According to the 30-day survival rate and the average survival time of the mice,lung function,EEG analysis,detection of inflammatory cytokines in brain,lung and intestine by radioimmunoassay,routine blood examination,and pathological changes of brain,lung,and intestine at 8 days after irradiation,the therapeutic effect of different doses of RH005 on acute radiation induced inflammation and the optimal dose were determined.（2）Optimal administration frequency investigation:236 male SPF C57BL/6N mice were randomly divided into:control group（Con group）,model group（IR group）,dexamethasone group（DEX group,4.5mg/kg）,RH005 1d group（M（1d）group,5μg/kg）,RH005 3d group（M（3d）group,5μg/kg）,RH0057d group（M（7d）group,5μg/kg）,and RH005 14d group（M（14d）group,5μg/kg）.There were 20 mice in each group in the survival rate experiment,and for the cytokine detection there were 15 mice in each group.Each administration group received intraperitoneal injection with RH005 24h after irradiated by 8Gy ⁶⁰Coγ-rays,and the mice in Con and IR groups received intraperitoneal injection with saline.The survival rate was observed and recorded within 40 days after irradiation;brain,lung and intestine of mice were taken at 8 days after irradiation for inflammatory cytokines in brain,lung and intestine by radioimmunoassay.The optimal frequency of administration was determined by two key indicators,namely,the survival rate and inflammatory cytokines.（3）Studies on time effect:120 male SPF C57BL/6N mice were randomly divided into 4 groups:control group（Con group）,model group（IR group）,dexamethasone group（DEX group,4.5mg/kg）and RH005 group（T group,5μg/kg）,10 mice per group.At 2d,4d,8d and 11d after irradiation,the time-effect relationship of RH005 in the treatment of acute radiation inflammation was explored through pulmonary function evaluation,brain MRI analysis and inflammatory cytokines in brain,lung and intestine by radioimmunoassay.Through the above three aspects of research,the the optimal administration regimen for RH005 was finally determined.（4）And then the mechanism underlying the therapeutic effect of RH005 for acute radiation inflammation was studied:30 male SPF C57BL/6N mice were randomly divided into three groups:control group（Con group）,model group（IR group）and RH005 group（T group,5μg/kg）,10 mice per group.Brain,lung,and intestine of mice were taken 11 days after irradiation,and mice Inflammasome PCR chips were used to detect the expression of inflammasome-related genes before and after RH005 administration,so to explore the mechanism underlying the therapeutic effect of RH005 for acute radiation induced inflammation.The results obtained through the above experiments were as follows:（1）Dose-response and optimal dose:RH005 could improve the survival rate and prolong the average survival time of mice after irradiation.The survival rate of mice in IR group within 30 days after irradiation was 0%.After RH005 administration,the survival rate of mice in M group was 10%at the end of 30 days.The average survival time of mice in the IR group was（8.50±2.94）days,and that of the M group was（14.40±2.20）days.Compared with the IR group,RH005 could significantly improve the survival rate of mice after irradiation,delay the time of death and prolong the survival time（P<0.01）.RH005 could improve lung function of mice after irradiation:mice in IR group had significantly reduced tidal volume,minute ventilation,peak inspiratory and peak expiratory after irradiation.RH005 significantly increased the tidal volume,minute ventilation,and peak inspiratory value of mice after irradiation,and the effect of M dose was the most significant（P<0.05）.RH005 could improve brain electrical abnormalities in mice after irradiation.RH005 significantly reduced the content of inflammatory cytokines TNF-αand TGF-βin brain,lung and jejunum after irradiation,and the effect of M dose was most definite（P<0.05）.Routine blood examination results showed that RH005 had no effect on the peripheral blood of mice after irradiation,indicated that it did not affect the hematopoietic system.Besides these,RH005 markedly alleviated the pathological changes in irradtied mice,nuclear pyknosisin in brain,alveolar exudation of lung,and cell necrosis and villus rupture in jejunum were all decreased,while the effect of M dose was the most notable.（2）Optimal Dosing frequency:The administration of RH005 for 3days（1 time/day）could significantly improve the survival rate and prolong the average survival time of mice after irradiation（P<0.05）:the survival rate of mice in IR group was 0%,while the survival rate of mice in M（3d）group within 40 days was 50%.The average survival time of mice in the IR group was（9.80±2.94）d,and that of mice in the M（3d）group was（27.30±8.03）d.The above results showed that 3 times of administration of RH005 could significantly improve the survival rate and prolong the survival time of irradiated mice.Inflammatory cytokines results showed RH005 significantly reduced the content of TNF-αand TGF-βin brain,lung and jejunum after irradiation,3 times administration worked best（P<0.05）.（3）Time-effect of RH005:RH005 could improve the lung function of mice at 3 days after irradiation,and the effect was more significant at 8 and11 days after irradiation（P<0.05）.RH005 could alleviate brain liquefactive necrosis in irradiated mice at 11 days after irradiation,which was showed by brain MRI.Low-density shadows appeared in the hippocampus,cortex,striatum,and hydrangea of mice in IR group,but this appearance was not found in the T group.In addition,RH005 significantly reduced the content of TNF-αand TGF-βat 3 days after administration,which was especially markedly at 7 and 10 days after administration（P<0.05）.（4）Mechanism underlying the therapeutic effect of RH005 for acute radiation-induced inflammation:After screening by mice inflammasome PCR chip,there were 11 differential genes in the brain tissue of mice in IR group compared with mice in Con group after irradiation.The up-regulated genes included Ifng,Il-6,Mefv,Naip1,Nlrp4b,Nlrp4e,Nlrp5,Nlrp9b,Tnfsf11 and Tnfsf4.Ciita was down-regulated.After RH005administration,there were 20 differential genes detected in the mice in T group compared with the IR group,all of which were down-regulated genes:Cd40lg,Ifnb1,Ifng,Il-12b,Il-1b,Il-6,Irf4,Krt14,Mefv,Naip1,Naip5,Nlrp12,Nlrp4b,Nlrp4e,Nlrp5,Nlrp9b,Tnf,Tnfsf11,Tnfsf14 and Tnfsf4.As for lung tissue,there were 9 differential genes in mice of IR group compared with the Con group after irradiation.Ccl12,Cflar,Chuk,Mapk8,Mok,Nod2 and Notch1 were up-regulated;Ccl5 and Tnfsf14 were down-regulated.After RH005 administration,there were 4 differential genes,Il-6,Nlrp12,Nlrp9b and Tnf,detected in the T group compared with the IR group.All were up-regulated For the jejunum tissue,there were 13differential genes in mice of the IR group compared with the Con group after irradiation.The Ccl12,Ccl7,and Il-18 were up-regulated;while Bcl2l1,Birc3,Ccl15,Chuk,Irf4,Mapk3,Mapk8,Nlrc4,P2rx7 and Xiap were down-regulated.After RH005 administration,Ciita was detected as a differentially down-regulated gene in the T group compared with the IR group.From the above experimental results,the following conclusions could be drawn:8Gy⁶⁰Coγ-rays could cause acute severe radiation injury in mice,RH005 had a therapeutic effect on radiation induced non-infectious inflammation and so to alleviate acute severe bone marrow radiation sickness in mice.The optimal administration regimen was intraperitoneal injection 24h after irradiation at 5μg/kg,once a day,for 3consecutive days.One of the mechanisms underlying the therapeutic effect of RH005 for acute radiation induced inflammation was that RH005 significantly reducing the elevated levels of TNF-α,TGF-βand IL-6 and significantly increasing the decreased levels of IFN-γin blood and tissues.