| ObjectiveT lymphocytes can be synergistically activated and the anti-tumor effect can be enhanced with PD-1 inhibitors combined with radiotherapy.However,combined chest radiation therapy has an increased risk of myocardial damage caused by radiation.In this study,a mouse model of Radiation Induced Heart Disease(RIHD)was established to observe the effects of PD-1 inhibitors on RIHD,and to explore its potential mechanism from the perspective of immune microenvironment.MethodsTotally 20 C57BL/6 mice were divided into 4 groups by completely random method,including group A as normal control,group B receiving intraperitoneally injected with anti-PD-1 antibody,group C receiving full thoracic x-ray irradiation with15 Gy in one fraction,Group D receiving anti-PD-1 as the same as group B and receiving irradiation as group C.1 month after irradiation,the mice were anesthetized and sacrificed,and the cardiac tissue was collected.The morphological changes of myocardial tissue were observed by HE staining.The degree of myocardial fibrosis was evaluated by Masson staining and the collagen volume fraction(CVF)of myocardial collagen was calculated.CD3+,CD3+CD4+,CD3+CD8+ lymphocyte subsets were detected by flow cytometry.Detecting myocardial tissue factor(IL-4,IL-6,IL-17 a,TNF-α,TGF-β 1,IFN-γ)level by flow cytometry CBA method.Detecting the apoptosis of cardiomyocytes by TUNEL method,and the apoptosis index(AI)was calculated.Results1 month after irradiation,no obvious myocardial injury was observed in group B by HE staining,edema and rupture of myocardial cells were observed in group C and D,and the injury in group D was more serious than that in group C.Masson staining showed no obvious myocardial fibrosis in group B,while collagen fibers were found distributed in the interstitium of myocardial cells in group C and D.The results of semiquantitative analysis showed that cardiac collagen volume fraction(CVF)in groups A,B,C and D were(1.97±0.36)%,(2.83±1.03)%,(5.39±0.77)% and(7.72±1.43)%,respectively.The CVF of group A was similar to that of group B(P =0.314),and the differences of CVF among other groups were statistically significant(P < 0.05).The percentages of CD3+T lymphocytes in group A,B,C and D were(9.32±1.05)%,(15.90±1.55)%,(15.8±0.67)% and(23.17±3.33)%,respectively.The absolute values of CD3+T lymphocytes in each group were(114±8)count /ul,(142±10)count /ul,(166±4)count /ul and(213±10)count /ul,respectively.Compared with group A,the absolute value and percentage of CD3+T lymphocytes in groups B,C and D were increased(P< 0.01),and those in group D were higher than those in group B and C(P < 0.001).The percentage and absolute value of CD3+CD4+T lymphocytes were similar among all groups,and there was no statistical difference between the groups(P > 0.05).The percentages of CD3+CD8+T lymphocytes in each group were(34.32±2.30)%,(33.72±2.26)%,(41.12±1.81)% and(56.47±3.13)%,respectively.The percentages in group B(P=0.758)and C(P=0.432)were similar to those in group A,and those in group D were higher than those in group A,B and C(P < 0.001).The absolute values of CD3+CD8+T lymphocytes were(21±2)count /ul,(62±3)count /ul,(89±2)count /ul,and(129±7)count /ul,respectively.Compared with group A,the absolute numbers of CD3+CD8+T lymphocytes in group B(P < 0.001),C(P < 0.001)and D(P < 0.001)were increased,and those in group D were also higher than those in group B(P < 0.001)and C(P < 0.001).The expression levels of IL-6 in group A,B,C and D were(27.67±4.33)pg/ml,(79.52±3.83)pg/ml,(129.91±14.92)pg/ml and(207.18±6.43)pg/ml,respectively.The expression levels of IL-17 A were(1.33±0.97)pg/ml,(20.26±3.49)pg/ml,(54.75±6.98)pg/ml and(122.21±4.80)pg/ml,respectively.TGF-β1 expression levels were(95.51±4.34)pg/ml,(180.78±4.33)pg/ml,(373.28±5.32)pg/ml,and(543.63±7.63)pg/ml,respectively.The levels of IL-6,IL-17 A and TGF-β1 in group D were higher than those in group A,and the differences were statistically significant(all P < 0.001).Apoptosis index of each group was(2.16±0.22)%,(5.03±0.24)%,(9.37±0.35)%,(15.62±0.48)%,respectively,and there was statistically significant difference in the apoptotic index among all groups(P <0.001).ConclusionsPD-1 inhibitors combined with cardiac irradiation can aggravate myocardial injury.The mechanism may be related to the fact that PD-1 inhibitors combined with cardiac irradiation can cause a large number of CD8+T lymphocytes to aggregate to the myocardium,resulting in local tissue immune inflammatory response.Among them,a variety of cytokines released by inflammatory cells,such as IL-6,IL-17 A and TGF-β1,further promote the myocardial inflammatory response. |
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