| Backgrounds Radiotherapy for thoracic and abdominal tumors affects heart which locates in the mediastinum and cause damage to the heart, which is known as radiation-induced heart disease (RIHD). The incidence of RIHD has been increasing and is becoming a popular topic for research. However, the pathogenesis of RIHD remains unclear and there are no effective treatments. Radiation induced cardiac damage is described as an outside-in, progressive process of fibrosis and is mainly characterized by an increase in cardiac fibroblasts (CFs) and increase in the synthesis of myocardial collagen. CFs are the main effector cells in cardiac fibrosis and therefore are a rational choice for observing the radioactive fibrosis damage. TGF-β1and collagen, which are synthesized by CFs, are indicators of fibrosis damage as they are directly involved in the fibrotic process. Fbrosis is an multi-factorial diseases in which imbalanced expression of cytokines. Additionally, The endoplasmic reticulum (ER) is an organelle that is responsible for the synthesis, modification and the correct folding of a vast array of proteins. The ER has a strong and stable internal system, however there are many factors such as ROS that can lead to an imbalance in ER homeostasis and ultimately ER stress (ERS). Emerging evidence has indicated that ER stress is involved in the cardiac fibrosis and remodeling. However, the significance and role of ER stress in RIHD induced cardiac fibrosis have yet to be elucidated.According to the evidence-based medicine "we’d better search and use the evidence, or we could create new evidence if we didn’t find the evidence", and now the prevention and treatment of RIHD were based on "no evidence" or "little evidence". We should "create the evidence" on the basis of "searching evidence", and study on pathology molecular mechanism of RIHD for providing its possible clinical intervention targets, Which had urgent practical significance. Traditional Chinese Medicine thought radial as "heat evil", for radiation-induced fibrosis, it was due to clogging of blood, damage of Qi and Yin, long illness into meridians and collaterals, and the stagnation of Qi and blood. Treatments should boost Qi and nourish Yin and benefit Qi for activating blood circulation based on "strengthening the body resistance and eliminating evil" Increasing evidence indicates that Chinese Medicine Astragalus relying on its antioxidant properties to effectively attenuate pulmonary fibrosis. In this study, we explored the effects and mechanisms of AST on pro-fibrosis damage in irradiated CFs in vitro; then raised the experimental basis for using AST to treat radiation-induced cardiac fibrosis in clinic.Objectives1) Observing the X-ray induced fibrosis damage effect in CFs and build a cell model;2) To systematically screen84fibrosis-related molecules expression in irradiated CFs with PCR-array and to explore the possible molecular mechanism of radiation-induced fibrosis damage;3) To observe whether there was ERS response in irradiated CFs, and to demonstrate that the relationship between ERS and radiation-induce fibrosis;4) To observe the effect of AST on X-ray induced fibrosis damage, from the aspects of fibrosis-related molecules’expression and ERS to expore the protective effect of AST on fibrosis damage, which in order to provide the basic theory support for its prevention and treatment of RIHD.Methods1) X-ray induced fibrosis damage molding:After low-dose Xray (0,0.5,1,2,4Gy) irradiation of CFs, firstly we detected cell proliferation activity by MTT and determine the observation time, and roughly screened the molding dose by observing the secretion of fibrosis index (TGF-β1, Col-1and Col-3) with ELISA, and finally determined the molding conditions and verified the success of molding with RT-PCR and Western Blot;2) The new technology "PCR Array" was applied to screen84main genes relate to fibrosis and observed the differential expression in the0Gy group and1Gy group, and preliminary analyzed molecular mechanism of pro-fibrosisof radiation;3) The effect of ERS in Xray induced fibrosis damage:Firstly, We observed the morphological changes of endoplasmic reticulum with transmission electron microscope, and then compared the expression of ERS marker GRP78, ATF6, p-PERK and CHOP in0Gy and1Gy groups with RT-PCR and Western blot. Finally, we used ERS inhibitor tauro ursodesoxy cholic acid (TUDCA) to explore the relationship between ERS and radiation induced fibrosis damage;4) CFs were pretreated with Huangqi injection for12hours, and then observed its effects on ROS with flow cytometry, to observed its effects on ERS and fibrosis molecules expression among0Gy,1Gy and AST+1Gy groups with RT-PCR and Western blot.Results1) Irradiation with X-rays significantly decreased the proliferation rate of CFs in a dose-and time-dependent manner compared with the control group.48h seemed to be the turning point of irradiation effects on cell proliferation. Based on cell proliferation results,48h after irradiation with1Gy and2Gy was selected for developing the CFs fibrosis damage model. During the48 hours with1Gy irradiation, the secretion of TGF-β1and Col-1were most, and mRNA expression of TGF-β1increased by138.64%(1Gy) and88.03%(2Gy)<P<0.01), Col-1increased by109.65%(1Gy) and80.62%(2Gy)(P<0.01), when compared with OGy Group. The relative level of the TGF-β1protein increased by160.05%after the1Gy X-rays irradiation and98.95%after the2Gy compared with control group (P<0.01). Col-1increased by201.37%with the1Gy X-ray and by129.54%with the2Gy (P<0.01). While the expression was down-regulated in CFs irradiated with4Gy Results showed that1or2Gy X-ray had the most obvious pro-fibrotic effect. Based on the comprehensive consideration for the effects of radiation on CFs proliferation and fibrosis molecular expression,1or2Gy X-rays were confirmed as the conditions for CFs fibrosis damage modeling and48h after irradiation was the observed time point. Because under these modeling conditions, CFs proliferation activity was not affected apparently, and fibrosis performance was most significant.2) We screened the fibrosis related genes with PCR Array. Compared with OGy group, in lGy group there was44genes with differences expression,30genes with up-regulated expression,14genes with down-regulated expression. Besides, the expression of Col-1A2and Col-3A1in extracellular matrix were up-regulated, and the MMP14, MMP3, MMP8, Plau and Serpinala in refactoring enzyme gene were up-regulated, but Lox, Plat, Serpinel and TIMP1were down-regulated. For inflammatory cytokines and chemotactic factor genes, the expression of CXCR4,1L-10, IL-13, IL-13ra2, IL-la, IL-lb and TNF were up-regulated, but Ccr2was down-regulated; For TGF-B superfamily genes, the expression of BMP7, Cavl, Dcn, TGF-β1, Smad2, Smad3, Smad4and TGIF1were up-regulated, but Eng, Inhbe, Smad7, TGF-Br1and Thbs1were down-regulated. There was no changes among5anti-fibrosis genes’ expression; the expression of growth factor seemed be mainly down-regulated, and the expression of Fasl gene was up-regulated by2.1.3) Dilatation, obvious proliferation and a disordered arrangement of the ER were observed in irradiated CFs under the electron microscope. Compared with0Gy group, mRNA expression of ERS marker GRP78and CHOP respectively increased by158.66%(1Gy),133.12%(2Gy) and106.23%(1Gy),189.65%(2Gy)(P<0.01). Comparable changes in the relative levels of the GRP78, CHOP, ATF6, p-PERK protein were also detected, GRP78with1.50and1.45-fold increases after irradiation with1Gy and2Gy, CHOP with2.50and2.81-fold increases after irradiation, ATF6with70.27%and23.74%increases after irradiation, p-PERK with42.9%and28.6%increases after irradiation (P<0.01). The above results confirmd that ER stress was activated in the irradiation-induced fibrosis damage model. Administration of0.8mmol/L TUDCA prevented the activation of ER stress and decreased the expression of Col-1, TGF-β1and p-Smad2/3in irradiated CFs, thus weakening the radiation induced fibrosis damage.4) Administration of Huangqi injection reduced ROS production in a dose-dependent manner in irradiated CFs. Meanwile, enhanced TGF-B1and Col-1expression in irradiated CFs was attenuated by AST in a similar manner, shown as the attenuation of20μg/ml AST was more obvious than10μg/ml AST. When compared with1Gy group, the mRNA expression of GRP78, CHOP, TGF-β1and Col-1in ’20μg/ml AST+1Gy" group respectively decreased by40.8%,42.46%,48.12%and39.22%(P<0.01), and their protein expression respectively decreased by54.02%,68.01%,48.14%and64.02%, p-Smad2/3protein expression decreased by38.20%(P<0.01). PCR Array showed that pretreated with20μg/ml AST reversed the majority of genes being changed by irradiation.Conclusions Low-dose X-ray irradiation of CFs have a noticeable pro-fibrosis damage effect. This effect was mediated by multiple molecules. It mainly related to the imbalance of pro-fibrosis and anti-fibrosis, negative regulation of signaling cascade reaction of TGF-B was suppressed and positive regulation was strengthened. Moreover, extrac ellular matrix was increased and inflammatory response and matrix reconstitution systems were activated in the early radial exposure. ERS caused by damage was also activated during the radiation of CFs. ERS inhibitors TUDCA could weaken radiation-induced fibrosis damage obviously, Which seems to indicate that ERS was involved in early fibrosis of RIHD, thus inhibiting ERS may be a possible intervention to reduce the radiation-induced fibrosis. The present study demonstrated that AST could effectively protected irradiated CFs, which due to its inhibition of radiation-induced ERS and attenuation of radiation-induced pro-fibrosis damage. This protective effects maybe relate to its antioxidant properties. The present in vitro findings support the interpretation that AST is a substance that may be beneficial in protecting the myocardium against radiation-induced fibrosis damage. this study about "AST against radiation-induced fibrosis damage" just is a preliminary exploratory research, its effectiveness and the exact molecular mechanism still need to be further confirmed by a verification experiment, in order to complete the basic support for its clinical application. |
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