Preliminary Study On The Correlation Between HMGB1 Pathway And Myocardial Injury Induced By Irradiation Combined With PD-1 Inhibitors

Objective:Radiotherapy is the mainstay of treatment for thoracic tumours such as lung and esophageal cancers.Combining PD-1 inhibitors may improve the efficacy of treatment,but there is also a risk of exacerbating radiation-induced myocardial injury(RIMI).The immune inflammatory response plays an important role in myocardial injury induced by both PD-1 inhibitors and irradiation.The role of high mobility group box-1 protein(HMGB1),a potent inflammatory stimulator,maintain unclear in myocardial injury caused by irradiation combined with PD-1 inhibitors.In this study,we constructed a mouse RIMI model to observe the effects of PD-1 inhibitors on myocardial inflammatory microenvironment and injury,and preliminarily explored the correlation between HMGB1 and myocardial injury.Methods:Forty C57BL/6 mice were randomly divided into 4 groups of 10 mice each;group A was the control group;group B was the PD-1 inhibitor;group C was the single heart irradiation group with a dose of 15Gy;group D was the PD-1 inhibitor +single heart irradiation group.Five mice were anesthetized and executed at 1 and 3months after irradiation Hematoxylineosin staining(HE)observed the morphology and pathology of heart tissue;Masson staining assessed the heart fibrosis which was semi-quantified by collagen volume fraction(CVF);Tunel staining evaluated heart apoptosis which was semi-quantified by apoptotic index(AI);Flow cytometry detected CD3+,CD4+,and CD8+ T lymphocytes in heart tissues;Western blot(WB)and quantitative real-time polymerase chain reaction(q PCR)detected the expression of protein and m RNA of HMGB1,TLR-4,and NF-κB p65 respectively;Enzyme linked immunosorbent assay evaluated(ELISA)measured the expression of IL-1β,IL-6,and TNF-ɑ of heart tissue.Results:According to HE staining,the myocardial injury in group B was mild,and the myocardial injury in groups C and D was severe,while the injury in group D was the most obvious.There was no obvious fibrosis in group B 1 month after irradiation,and slight fibrosis in myocardial tissue was observed in group B 3 months after irradiation;1 month and 3 months after irradiation,obvious myocardial fibrosis was seen in both groups C and D,especially in group D,and it was more obvious at 3months after irradiation than at 1 month after irradiation.1 month after irradiation,the CVF of groups A,B,C,and D gradually increased,(1.56±0.26)%,(2.52±0.61)%,(5.46±0.87)%,(7.85±0.69)%,respectively.The CVF between groups A and B was similar(P>0.05),and there were statistical differences among the other groups(P<0.05);3 months after irradiation,the CVF in group D was still the highest,and there was a statistical difference in CVF between group A and group B(P<0.05),and the statistical differences in CVF could be found among the other groups(P<0.05);Compared with 1 month after irradiation,CVF was more obvious in group C(P<0.05)and group D(P<0.01)3 months after irradiation.At 1 month and 3 months after irradiation,cardiomyocyte apoptosis in group D was the most obvious,and the differences between groups A,B and C were statistically significant.1 month after irradiation,the absolute count and percentage of CD3+ T lymphocytes in group D were higher than those in group B(absolute count: P<0.0001,percentage: P<0.001)and group C(absolute count: P<0.01,percentage: P<0.01);After 3 months,the absolute count and percentage of CD3+ T lymphocytes in group D were still higher than those in group B(absolute count: P<0.0001,percentage:P<0.0001)and group C(absolute count: P<0.0001,percentage: P<0.05);The absolute counts and percentages of CD3+ T lymphocytes in groups B,C,and D 3 months after irradiation were all lower than those at 1 month after irradiation,and the differences were statistically significant.The change trend of CD8+ T lymphocytes was similar to that of CD3+ T lymphocytes.The absolute count and percentage of CD8+ T lymphocytes in group D were the highest at 1 month and 3 months after irradiation,which were higher than those in A,B,In group C(both P<0.05).3 months after irradiation,the absolute count(P<0.0001)and percentage(P<0.0001)of CD8+ T lymphocytes in group D were still lower than those 1 month after irradiation.1 month after irradiation,the absolute counts(P>0.05)and percentages(P>0.05)of CD4+ T lymphocytes in groups C and D were similar;3 months after irradiation,there was a statistical difference in the absolute counts(P<0.05)in group C were still similar to those in group D while the percentages(P>0.05)of CD4+ T lymphocytes were still similar.1 month after irradiation,the expression of HMGB1 protein in group D was higher than that in group A(P<0.0001),B(P<0.0001)and C(P<0.0001);3 months after irradiation,the expression of HMGB1 protein in group D was still the highest in group A,B,and C(all P<0.0001),and was lower than that of 1 month after irradiation(P<0.0001).The protein expression level of NF-κB p65 was similar to that of HMGB1 in each group and over time.There was no significant difference in the expression level of TLR4 protein among groups A,B,C,and D(P>0.05).The changes in the expression of HMGB1,TLR4,and NF-κB p65 m RNA in groups A,B,C,and D were consistent with the protein expression.1 month and 3 months after irradiation,the expression levels of IL-1β,IL-6,and TNF-α in the myocardial of group D were the highest,and compared with groups A,B,and C,the differences were statistically significant(all P<0.01);the expression levels of IL-1β,IL-6,and TNF-α in groups B,C,and D 3 months after irradiation were all lower than those at 1month after irradiation(all P<0.01),and the differences were statistically significant.Conclusions:Using PD-1 inhibitors alone has little toxicity to the myocardial about experimental,and irradiation can cause their myocardial injury and fibrosis;PD-1inhibitors can increase the infiltration of CD8+ T lymphocytes in the myocardium of experimental mice,and the expression of HMGB1,NF-κB P65 and inflammatory factors IL-1β,IL-6,and TNF-α,which expanded the immune inflammatory response to aggravate radiation myocardial injury and fibrosis.The inflammatory response was more obvious in the early stage(1 month)than late stage(3 months).