| Radioactive lung injury is one of common complications in the chest tumorradiotherapy, radiation pretreatment before bone marrow transplants and nuclearaccidents. As the tumor incidence increases year by year, patients undergoingradiation treatment also increased dramatically. Notwithstanding the radiation therapytechnology and equipments progress significantly in recent years, which have reducedthe damage of normal tissue around the tumor by irradiation, but the incidence ofradioactive lung injury remains high. Owing to lack of effective prevention andcontrol of radioactive lung injury in clinical practice currently， it becomes mainprimary dose limiting factor which influences tumor radiotherapy in patients.Therefore, the prevention and control of radioactive lung injury becomes the problemof which radiation medicine and clinical oncology presses for solution.In recent years, a large number of studies have shown that oxidative stress playsan important role in radioactive lung injury and fibrosis formation. Several scientificresearch institutions have found that persistent existence of oxidative stress andhypoxia in normal tissue is closely related to the development of radioactive lunginjury, and claim it may be the cause of pulmonary sustained damage. When lungexposes to radioactive ray, the ionization effect of water molecules and moleculartargets can lead to reactive oxygen species quickly explosive generation. Moment ofgenerate ROS can damage directly parenchymal cells of lung tissue, on the other hand,the successive activation of inflammatory cells also increases the damage of lungtissue. Increase of oxygen free radicals by exposure can cause the sensitive kinase andtranscription factors rapid activation,thereby the increased production of inflammatorycytokines. Alveolar macrophages are also activated after exposure, in the process ofwhich play a crucial role in removal of necrotic tissue can produce large amounts ofROS. Furthermore alveolar macrophage can also lead to more immune effector cells (lymphocyte, neutrophil, etc.) infiltration damage lung tissue through chemotaxis, andthese biological effects cells can also produce a large number of ROS. EndogenousROS generated mainly in mitochondria where the electronic leakage happens in themitochondrial electron transport chain, due to its part of the reduction of O2. Besides，part of the ROS also comes from the catalytic product catalyzed by endogenousflavoenzyme (such as: xanthine oxidase), fatty oxidase, cyclooxygenase and serousrelated oxidase (such as phagocytes NAPDH oxidase) in cells. These large generateROS will soon deplete the body’s antioxidant ability, lead to lung parenchyma celldamage, and cause lung’s persistant inflammation.NrF2-ARE signaling pathway is considered to be the most important antioxidantstress signaling pathways presently. NrF2-ARE signaling pathway gradually arisespeople’s attention due to its important role playing in process of anti-tumor,anti-inflammatory and anti apoptosis. When cells are attacked by ROS andelectrophilic substances, chemical modification of cysteine residues of Keap1molecular can cause Keap1conformation change and lead to NrF2-Keap1complexdissociation, and then NrF2moleculars enter the nucleus. Meanwhile, the mitogenactivated protein kinases (MAPKs), protein kinase C (PKC) and phosphatidyl inositol3kinase (PI3K) moleculars are activated, which can make NrF2moleculesphosphorylated and activated. Activated NrF2molecules combine with Maf proteincomponents into heterodimers can recognize ARE element and start the genetranscription ofⅡstage detoxification enzymes and antioxidant enzyme, which canimprove the ability of cell resistance to oxidative stress.The antioxidant enzymesystem regulated by NrF2-ARE signaling pathways nicludes heme oxygenase-1(HO-1), gamma-glutamine cysteine synthetase (γ-GCS), NAD (P) H: quinoneoxidoreductase-1(NQO-1), superoxide dismutase (SOD), etc. NrF2moleculars arehighly expressed in lung tissue, probably due to its explosure to external environmentand easy to attack by reactive oxygen species and toxins. Therefore, raising the levelof NrF2expression may play an important role in regulating the lung’s oxidation-antioxidation balance.Numerous studies showed that epigallocatechin-3-gallate (EGCG) has manybiological activities and pharmacological effects such as antioxidant, anti lipidperoxidation, scavenging free radicals, anti mutation and antitumor etc. EGCG’s structure contains multiple phenolic hydroxyl and can be used as a hydrogen donorand H+. Many experiments in vivo and in vitro confirmed that the EGCG was stronghydrogen donor, antioxidant and free radical scavenger. EGCG can also chelate withmetal ions which is as coenzyme in catalysis of oxidation reaction and enhance theactivity of antioxidant enzyme. EGCG can mediate the STAT signal pathway toregulate transcription factors T-bet and vitamin A orphan receptor gamma/alpha(RORγ/α), selectively inhibit CD4+T cells generating IFN-γand IL-17, directinhibit Th1and Th17cell differentiation, and inhibit the synergistic stimulationfunction of antigen presenting cells (APC) through changing the CD80and CD86expression, which confirmed that EGCG has significant anti-inflammation effectthrough inhibiting inflammatory factors release by immune cells and immuneregulation. EGCG can also induce the activation of Nrf2-Keap1signaling pathway toenhance endogenous antioxidant stress ability. Confocal microscope, western blot andRT-PCR studies confirmed that EGCG can significantly induce the up-expression ofNrF2and promote the NrF2-Kaep1complex dissociation, induce the NrF2moleculesentering the nucleus to increase the expression of antioxidant enzymes and thedetoxification enzyme. In addition, EGCG can inhibit the NF-kappa B’s expression toexert anti-inflammatory activity.EGCG’s effect of anti oxidative stress may have potential of treating andpreventing radioactive lung injury and control pulmonary fibrosis development. Whileat present，related research is rare. Therefore, we use Co60source of γray toestablish lung injury model in rats, with EGCG intervention, to research its protectionof radioactive lung injury and the related mechanisms, so as to provide new way forprevention and control of radioactive lung injury.Part One: Establishment and Identification of radioactive lung injury in rats modelPurpose: Use Co60gamma-ray to irradiate rats whole lung inducing radioactive lunginjury rats model.By continuous pathology observation to explore the lesions in law.Investigate the role of oxidative stress in radioactive lung injury. Provide animalmodel and theoretical basis for the research of prevention of radioactive lung injury.Methods: Use Co60source gamma-ray22Gy singlely to irradiate SD rats whole lung.Randomly chosen6rats once were sacrificed at0,1，7，15，21，30，60and120daysafter irradiation. Body and whole lung weight of rats were measured to calculate lung coefficient. Right lung were extracted to observe the pathological changes andcollagen fiber deposition of lung tissue by HE, Masson and Sirius red staining. Leftlung were extracted to measure Hydroxyprolin content of lung tissue. Serum wasextracted from venous blood to measure MDA content,Total-SOD activity and TGF-β1level. Use immunohistochemical method to detect the expression of alpha-SMAand SPB in lung tissue. Eighty male SD rats were assigned randomly to controlgroup-received sham-irradiation, n=10and treatment.Results:(1) Obvious macro changes in the rats begin with15days. Main changesmanifest: hair removal and skin ulcers in illuminated area, lung swelling, sporadicbleeding point on lung surface. These characteristic changes deteriorate graduallyover time and the30days after irradiation the lesions becomes the most serious. Lungcollapse and nodular and strip funicular pale fibrosis lesions is visible on the surfaceof lung at60days and120days after irradiation. The lung coefficient (5.864±0.148)increased significantly since15days after irradiation, compared with normal controlgroup (5.098±0.445), P <0.05and it increased gradually over time. According to60days to120days after irradiation the lung coefficient of rats becomes slightlylower but it is still significantly higher than the normal control group (P <0.05).(2)Early pathological histology changes of lung characterize interstitial exudativeinflammation since from7days to30days after irradiation and gradually aggravateand extend over time. The alveolar inflammation score began to increase significantlyat7days (8.833±1.329) after irradiation (compared with normal control group, P <0.001). The alveolar inflammatory score at30days (22.333±2.066) after irradiationwas the highest, while according to60days (21.167±2.317) and120days (21.333±3.266) the alveolitis score become slightly lower, but compared with30days afterirradiation there is no significant statistical difference (P>0.05).(3) The pathologicalhistology changes of lung from60days to120days after irradiation mainly manifesttissue cells hyperplastic inflammation, and fibrosis lesion formation. Pulmonaryfibrosis score increased significantly since15days (8.833±2.137) after irradiation,compared with normal control group, P=0.001). Compared the15days’ fibrosisscore to21days’ there is no obvious statistical difference (P>0.05), the pulmonaryfibrosis score of30days to120days’ progressively increase (compared betweengroups, P <0.05).(4) The HYP content in lung tissue significantly increased from15 days (0.442±0.056) after irradiation, there is significant statistical differencecompared with normal control group (0.168±0.021, P <0.05), and its contentgradually increased with the extension of time.(5) The Total-SOD activity in serum transiently increased at1day(104.168±8.975)to7days(109.075±12.719) after irradiation, compared with normal control group(79.613±10.210), the1day’ P=0.003, the7days’ P <0.001. According to15daysafter irradiation the T-SOD activity significantly decreased, and the low levelmaintained to120days after irradiation (compared with normal control group, P>0.05).(6) The MDA content in serum significantly increased at1day after irradiation(4.055±0.617), and compared with normal control group (2.491±0.653), P=0.007.Its content is gradually increased over time, and compared with normal control group,P <0.001.(7) The content of TGF-β1in the rat serum increased significantly since15days (2816.52±191.142) after irradiation, and compared with normal controlgroup (2150.31±127.768), P <0.001. Its content gradually increased over time.(8)The expression of alpha-SMA in lung tissue began to increase at15days(0.085±0.014) after irradiation, but compared with normal control group (0.048±0.013), P>0.05. From60days to120days its expression significantly increased, and comparedwith normal control group, P <0.05.(9) The expression of SPB in lung tissuesignificantly decreased at7days (0.313±0.074) after irradiation, and compared withnormal control group (0.479±0.078), P <0.05. With the extension of time, itsexpression level decreased further, while from60days to120days after irradiation itsexpression increased slightly, but still significantly lower than normal control group,P <0.05).Conclusion:(1) Obvious inflammation of the lung tissue appears at7days afterirradiation. Characteristic pathology change manifest：small vessels dilating, alveolarpartition broadening, inflammation effusion and inflammatory cells infiltration inpulmonary interstitial tissue, and the inflammation extension of the lung tissueaggravate gradually over time, when30days after irradiation the inflammation of lungtissue becomes most serious.(2) Collagen deposition in lung tissue begin with15days after irradiation, and it increase gradually over time. Until60days and120daysafter irradiation lung’s inflammation relieves slightly, while the main pathological changes in this period manifest pulmonary fibrosis and the composition of fibrosislesions is mainlytypeⅠcollagen deposition.(3) The TGF-beta1level which is seenas one of important serological indicators reflecting the severity of radioactive lunginjury began to rise apparently at15days after irradiation, and rise more apparentlyover time.It indicates that the lung injury in rats aggravates gradually and its levelchange is consistent with pathological alteration.(4) The extent of oxidative stress inrats aggravated significantly since15days after irradiation, aggravating and persistingover time, and is consistent with the degree of lung injury. Serum T-SOD vitalitychange indicates that endogenous antioxidant stress system can only compensate towithstand the condition of oxidative stress in the early stage，while it becomesdecompensative in the late stage so that lung tissue continously subjects to oxygenfree radicals.(5) The expression level of alpha SMA in lung tissue increasedsignificantly at60days and120days after irradiation.It is showed that myofibroblastsmay play an important role in the late stage of radioactive lung injury.(6) Thereduction of typeⅡalveolar cells after irradiation, which seriously affects the lungfunction may be associated with the damage of oxidative stress. Abnormalhyperplasia of typeⅡalveolar cells were observed at60days and120days afterirradiation, and the cells may participate in the adanced fibrosis development ofradioactive lung injury.Part Two: Therapeutic effect of Epigallocatechin-3-gallate(EGCG) on radiationinduecd lung injury in rats and related mechanisms research.Purpose: To study the therapeutic effects and related mechanisms of EGCG onradiation induced lung injury in rats.Methods:160SD rats were assigned randomly to control group(receivedsham-irradiation, n=40）、model group(received irradiation on the thoraces with singlefraction22gray γ rays from Co60，n=40)、Dexamethasone group(receivedDexamethasone therapy in intratracheally injecting way after irradiation on thethoraces，n=40) and EGCG group(received EGCG therapy in intratracheally injectingway after irradiation on the thoraces，n=40). Randomly chosen6rats from each grouponce were sacrificed at15、30、60and120days after irradiation.Observe and evaluatethe ameliorating effect of EGCG on radiation pneumonitis and fibrosis by lungcoefficient assessment, HE staining, Masson staining, Sirius red staining and HYP content determination. Examine TGF-β1level in rats serum. Examine the total SODactivity and MDA content in rats serum so as to evaluate the improvement ofoxidative stress in model rats by EGCG. Use immunohistochemical method to detectthe expression of alpha-SMA and SPB in lung tissue so as to observe the effect ofEGCG on muscle fibroblasts and typeⅡalveolar cells. Examine the inflammationfactors: IL-6, IL-10, IFN-γ, TNF-α in rats serum by Cytometric bead array.Assessthe expression of NrF2, HO1and NQO1in rats lung tissue by Western Blotting.Results:(1) Macro changes：after treatment with EGCG, hair removal and skin ulcerin rats improved significantly; Hyperemia edema obviously alleviated compared withmodel group and dexamethasone group;Lung coefficient from15days to60daysafter irradiation is significantly lower than model group (P <0.05), and30days to120days’ lung coefficient is significantly lower than dexamethasone group (P <0.05).(2) There is obvious improvement of inflammation exudation and inflammatory cellsinfiltration of lung tissue in EGCG treatment group15days and30days. Thecomparation of alveolar inflammatory score with model group, P <0.05; but the15days’ comparison with dexamethasone group there is no significantly statisticalsignificance, P>0.05.While the lung inflammation of EGCG treatment group60daysand120days improved significantly compared with model group and thedexamethasone group (statistical analysis of alveolar inflammatory score, P <0.05).(3) The lung interstitial collagen fibers deposition and fibrosis lesions of EGCGtreatment group30days to120days were significantly better than that of model groupand dexamethasone group.There is significant statistical difference (P <0.05) ofpulmonary fibrosis score compared with model group and dexamethasone group.(4)The HYP content in lung tissue of EGCG treatment group15days to120daysdecreased significantly compared with the model group and there is statisticallysignificant difference (P <0.05). And that of60days and120days are significantlylower than dexamethasone group (P <0.05).(5) The TGF-beta1content in serum ofEGCG treatment group15days to120days was significantly lower than model group,(P <0.05). And that of30days to120days was significantly lower thandexamethasone group (P <0.05).(6) The MDA content in serum of EGCG treatmentgroup15days to120days were significantly lower than model group (P <0.05). Andthat of60days and120days were significantly lower than dexamethasone group (P < 0.05).(7) The total-SOD activity in serum of EGCG treatment group15days to120days were significantly higher than the normal control group, model group anddexamethasone group (P <0.05).(8) Treated by EGCG15days later, the levels ofIL-6and TNF-alpha in rats serum elevated slightly, and there is no statisticallysignificant difference compared with normal control group (P>0.05), but haveobvious statistical difference compared with the model group (P <0.05); IL-10andIFN-gamma levels increased significantly, compared with normal control group withsignificant statistical difference (P <0.05), but compared with the model group anddexamethasone group there is no statistically significant difference (P>0.05). As30days to120days the levels of inflammatory cytokines elevated continously, but weresignificantly lower than the model group and dexamethasone group (P <0.05).(9)Treated by EGCG15days and30days later, alpha-SMA expression is rare in lungtissue, and there was no significant statistical difference compared the OD value withthe normal control group (P>0.05); As60days and120days later the alpha-SMA’distribution is of scattered in lung tissue and the expression increased obviouslycompared with before.There was significant statistical difference compared OD valuewith normal control group (P <0.05), but it was significantly lower than model group(P <0.01) and dexamethasone group (P <0.05).(10) The SPB expression in lungtissue decreased though treated with EGCG for15days and30days, compared theOD value with normal control group there was remarkable difference (P <0.05).But there were notable increases from the model group and dexamethasone group, ODvalue has significant difference compared with model group and dexamethasonegroup (P <0.05). As60days and120days after treatment with EGCG, SPBexpression is significantly higher than that of model group and dexamethasone group(OD value, P <0.05). Besides abnormal hyperplasia of alveolar typeⅡcells is notobvious, and normal alveolar typeⅡcells were still visible distributing in the alveolarwalls.(11) The expression level of Nrf2, NQO1and HO1in lung tissue is obviouslyincreased treated with EGCG after15days.Their relative optical densitys hadsignificantly statistical difference compared with normal control group, model groupand dexamethasone group (P>0.05); As30days to120days after the Nrf2, NQO1and HO1expression in lung tissue continued to increase and maintained at a highlevel, and their relative optical density values were significantly higher than those ofmodel group and dexamethasone group (P <0.05). Conclusion:(1) EGCG can alleviate inflammation in the early stage of radioactivelung injury and delay the pulmonary fibrosis development in the late stage;(2) thecontinuity of EGCG ‘s effect on the anti-inflammatory and anti fibrosis is superior todexamethasone.(3) EGCG can reduce the MDA level in rats serum and improve theSOD vitality so as to improve the state of oxidative stress in rats and lighten thecontinuous damage on lung tissue by ROS. The antioxidant capacity of EGCG may bea primary mechanism of EGCG in the role of lung protection;(4) EGCG can protectalveolar typeⅡcells and suppress the abnormal growths, of which may be associatedwith the oxidative stress resistance; EGCG can supress lung fibroblasts proliferationand transformation, and thus reduce the pulmonary fibrosis lesions;(5) EGCG canreduce inflammatory cytokines levels such as IL-6, IL-10, IFN-gamma, TNF-alpha inrats serum, which represents the strong ability of anti inflammation in the treatment ofradioactive lung injury by EGCG.(6) EGCG can improve the rats’ antioxidantcapacity through the activation of NrF2-ARE signaling pathway, which enhances theexpression of Ⅱphase antioxidant enzymes and the detoxification enzymes andactivity and proves EGCG ‘s important role in maintaining the balance of oxidationantioxidation system in irradiated rats.Anyhow, EGCG can protect lung parenchyma cell damage by removing oxygenfree radicals, reducing excessive generation of oxygen free radical, activating theendogenous antioxidant stress mechanism, immune regulation and other molecularmechanisms, through which can reduce lung inflammation and fibrosis lesions. Theproofs provides new clues for the prevention and control of radioactive lung injury. |
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