Efficacy Evaluation Of Recombinant Thymosin β4 On Radiation Induced Interstitial Pneumonia And The Underlying Mechanisms

Objective and significance:Radiation induced lung injury is a common and refractory complication of thoracic tumor radiotherapy and bone marrow transplantation pretreatment, it also could be found in nuclear accidents. Interstitial pneumonia is the mainly pathological change at the early stage of radiation induced pulmonary injury, while fibrosis is the character of latter stage. At present, there is no effective treatment measure for radiation induced lung injury; the common methods used in clinic include application of cortin, antibiotics and other symptomatic treatment. Cortin has anti-inflammatory effect, but its adverse effects limit its use. Recombined thymosin β(rh-Tβ4) is a recombined protein. It has been found that thymosin β4 has anti-inflammatory effect and it also could reduce apoptosis and collagen formation. Therefore, the efficacy evaluation of recombinant thymosin β4 on radiation induced interstitial pneumonia (RIP) and the underlying mechanisms has important theoretical and practical significance to improve the life quality of patients with RIP, so as to elevate the effect of thoracic tumor radiotherapy and improve the treatment for nuclear accident.Materials and methods:(1) Total 150 C57BL/6 mice were randomly divided into 6 groups, namely, control group, model group, low, middle and high dose of rh-Tβ4 group and dexamethasone group. Each group was of 25 mice. The thoraces of C57BL/6J were irradiated with X ray to establish the model of RIP. Low, middle and high dose of rh-Tβ4 was 1,5,25ug/kg respectively. rh-Tβ4 was administered by intraperitoneal injection, once a day for 30 days. Mice in dexamethasone group were intraperitoneal injected with dexamethasone at the dose of 4.5mg/kg/d for 2 weeks and then the dose was halved. The efficacy of rh-Tβ4 on RIP was evaluated by mortality rate, respiratory function, pathological changes of lung tissue and detection of inflammatory factor. (2) RAW264.7 cells were induced into M1 subtype macrophages by LPS and IFN-gamma, while IL-4 treatment induced RAW264.7 cells into M2 subtype macrophages. rh-Tβ4 at the concentration of 1,10,100 and 1000 μg/ml was used to treat the M1 and M2 macrophages respectively. The proliferation of cells was measured by MTT and the cytokine secreted by macrophage was detected with ELIS A kit.Results:1. The mortality rate of model group was 12%, while the mortality rate of dexamethasone group and high dose of rh-Tβ4 group had no significant difference with the model group. However, the mortality rate of low dose of rh-Tβ4 group (8%) and middle dose of rh-Tβ4 group (4%) was significantly lower than that of the model group.2. Pulmonary function of mice with RIP had a number of abnormal indexes, including tidal volume, minute-ventilation, peak inspiratory flow,50% expiratory flow and peak expiratory, these indexes were significantly decreased. Application of rh-Tβ4 could improve pulmonary function of mice. Tidal volume, minute-ventilation, peak inspiratory flow,50% expiratory flow and peak expiratory were all significantly increased. Moreover, the effect of rh-Tβ4 is superior to dexamethasone. Low and middle dose of rh-Tβ4 had better effects.3. Application of rh-Tβ4 could significantly reduce inflammatory cell infiltration in lung and alleviate alveolar wall thickening so to increase alveolar cavity area significantly. Three doses of rh-Tβ4 were all effective and the effect is better than that of dexamethasone. Low and middle dose of rh-Tβ4 had better effects.4. Application of rh-Tβ4 could significantly change the expression of IL-6, IL-13, TNF-a and TGFβ in lung of RIP mice. IL-6, IL-13, TNF-α and TGFβ positive macrophages could be seen in control group. IL-6 positive macrophages increased significantly in model group. While in low and high dose of rh-Tβ4 group and dexamethasone group, IL-6 positive macrophages were fewer than that of model group. IL-13 positive macrophages increased significantly in model group, dexamethasone group and three dose of h-Tβ4 group. TNF-a positive macrophages increased significantly in model group, dexamethasone group and high dose of rh-Tβ4 group, while TNF-a positive macrophages were fewer in low and middle dose of rh-Tβ4 group than that of model group. TGFβ positive macrophages increased significantly in model group, while TGFβ positive macrophages in three dose of rh-Tβ4 group were all fewer than that of model group.5. The proliferative activity of M1 macrophages was promoted by 1,10 and 100μg/ml rh-Tβ4 at 24h and 72h after administration. While the proliferative activity of M2 macrophages could be promoted by all four doses of rh-Tβ4. Besides that, the proliferation of M2 macrophage was more active than that of M1 macrophages.4. Application of rh-Tβ4 could significantly change the secretion of TNF-α, IL-13, TGFβ and CTGF by M1 and M2 macrophages. The secretion of TNF-α by M1 macrophages increased significantly in all group, especially in 10μg/ml group.. The secretion of CTGF by M1 macrophages treated with 10μg/ml rh-Tβ4 decreased significantly, while the secretion of CTGF in M2 macrophages treated with 1μg/ml rh-Tβ4 decreased, but with no statistical significance. The secretion of TGFβ by M1 macrophages treated with 1μg/ml rh-Tβ4 increased, while the secretion of TGFβ by M2 macrophages treated with lOμg/ml rh-Tβ4 decreased, but with no statistical significance.Conclusions:1.rh-Tβ4 has a therapeutic effect on RIP. rh-Tβcan reduce the mortality rate, improve lung function, alleviate the pathological changes of lung tissue and its efficacy is better than that of dexamethasone.1 and 5μg/ml rh-Tβ4 are more effective on RIP. The alleviation of TH1 and TH2 reaction of rh-Tβ4 is one of the underlying mechanisms.2. rh-Tβ4 could promote the proliferation of M1 and M2 macrophages. Meanwhile rh-Tβ4 could change the secretion of TNF-α, IL-13, TGFβ and CTGF by macrophage. These may serve as a mechanism of rh-Tβ4 efficacy on RIP. The effect of 1 and 10μg/ml rh-Tβ4 are better.