益气解毒方药对辐射致小鼠支持细胞铁死亡的防护效应及机制研究

【作者】 王磊；

【导师】 胡素敏；

【作者基本信息】 北京中医药大学 ， 中医临床药学， 2022， 博士

【摘要】 背景随着科技的发展,电离辐射技术在科学研究和临床诊疗过程中的应用日趋广泛。与此同时,电离辐射对人体造成的损害也不可避免。睾丸对电离辐射高度敏感,低剂量(0.03 Gy)的电离辐射即可导致精原细胞数量减少,随着辐射剂量增大,睾丸生精功能损伤随之加重,严重者可以导致无精症而引起男性不育。并且目前临床上尚没有针对睾丸辐射损伤的防护药物。近年来,辐射损伤的中医药防护研究取得了不少进展。本课题组前期对急性辐射损伤的中医学病因、病机、治法和方药进行了一系列探索;通过实验研究发现电离辐射可以导致小鼠睾丸生精细胞的严重损伤,并且诱导细胞铁死亡的发生,由益气解毒中药组成的益气解毒方对电离辐射导致的睾丸损伤具有较好的防护效果。但是中医药防治辐射致睾丸生精功能损伤的理论研究尚不够深入,以及电离辐射是否诱导小鼠睾丸支持细胞铁死亡至今尚未见报道。因此本研究拟在前期研究基础上,基于藏象学说探讨从补肾健脾、益气解毒论治辐射致睾丸生精功能损伤的中医药理论;并通过体内和体外实验研究辐射致支持细胞铁死亡的效应,以及探讨益气解毒方药对小鼠睾丸支持细胞铁死亡的防护效应和机制。目的在理论研究方面,基于藏象学说,探讨生精细胞和支持细胞的功能,然后结合辐射致睾丸生精功能损伤的中医病机,将补肾健脾、益气解毒的整体辨证论治思路拓展到微观细胞层面,从而为辐射致睾丸生精功能损伤的中医药防护提供理论依据。在实验研究方面,首先在体外复制支持细胞辐射损伤模型,探讨电离辐射对支持细胞的损伤以及益气解毒方的防护效应,以及电离辐射导致支持细胞铁死亡的效应和益气解毒方的防护机制;然后通过体内实验,进一步验证益气解毒方调控辐射诱导的支持细胞铁死亡的效应和机制。方法理论研究:(1)基于藏象学说探讨生精细胞的功能,并将其归属于藏象学说中肾的功能系统,同时基于藏象学说探讨支持细胞的功能,并将其归属于藏象学说中脾的功能系统;(2)探讨电离辐射致睾丸生精功能损伤的中医病机;(3)通过将生精细胞和支持细胞功能分别归属于藏象学肾和脾的功能系统,可将整体辨证论治的理法方药体系拓展至生精和支持细胞层面。然后通过探讨益气解毒方药防治睾丸辐射损伤的治法和组方分析,为益气解毒方防治睾丸辐射损伤提供理论依据。实验研究:体外实验:(1)筛选复制TM4细胞辐射损伤模型的照射剂量。(2)免疫荧光检测12.0 Gy 60Co γ射线对TM4细胞DNA的氧化损伤情况和细胞骨架蛋白损伤情况,检测照后TM4细胞通透性模拟电离辐射对血睾屏障的损伤情况,检测SOD和GSH水平以评估辐射对细胞抗氧化能力的损伤,以及益气解毒方的防护效应。(3)使用铁死亡诱导剂Erastin作对照,通过流式细胞实验观察12.0 Gy 60Co γ电离辐射诱导TM4细胞铁死亡以及益气解毒方的防护效应。(4)通过PCR和Western blot实验,探索益气解毒方通过Keap1/Nrf2途径调控电离辐射致TM4细胞铁死亡的机制。体内实验:6.0Gy 60Coγ射线一次性全身照射Balb/c小鼠,照后24h观察小鼠睾丸支持细胞(Sertolicell)的损伤、Sertoli细胞发生铁死亡的情况以及益气解毒方的防护效应。并检测Keap1/Nrf2途径相关蛋白的表达,探索益气解毒方对辐射致Sertoli细胞铁死亡的防护机制。结果理论研究:(1)本研究基于藏象学说探讨生精细胞和支持细胞的功能:1)以中医藏象学说为基础,结合现代医学对生精细胞功能的认识,分析生精细胞的功能,并与藏象学肾的功能对比分析,发现生精细胞的功能是肾藏精和肾主生殖、发育功能在睾丸中的体现,因此可将生精细胞的功能归属于藏象学说中肾功能系统。从而为从肾论治睾丸生精功能障碍,选用具有补肾益精功效的中药提供理论依据。2)以中医藏象学说为基础,结合现代医学对支持细胞功能的认识,分析支持细胞的功能,并与藏象学脾的功能相比对,发现两者之间联系紧密。当我们把机体的局部器官睾丸作为一个整体来看时,睾丸中的支持细胞的生理功能与脾在人体中发挥的主运化、主升清和主卫外的功能相近。因此可将支持细胞的功能归属于藏象学说中脾的功能系统,从而为从脾论治并选用健脾中药防治支持细胞辐射损伤,提供理论依据。(2)通过对电离辐射致睾丸生精功能损伤特点的分析,发现其中医病机可以概括为:1)肾虚精亏,生精乏源;2)脾失健运,气血化生乏源;3)毒热炽盛,气阴两虚。(3)生精细胞的功能可归属于肾藏象的功能系统,支持细胞的功能可归属于脾藏象的功能系统。因而可以将脾和肾在整体辨证论治过程中的理、法、方、药拓展至睾丸中生精细胞和支持细胞的微观层面。根据辐射致睾丸生精功能损伤的中医病机,提出从补肾健脾、益气解毒论治辐射致睾丸生精功能损伤的治法。并对益气解毒方进行组方分析:该方以枸杞、淫羊藿补肾益精;以黄芪、茯苓、山楂健脾益胃;同时以西洋参、马齿苋益气解毒,并配伍当归、白芍养血。诸药共奏补肾健脾、益气解毒养血之功,可以达到防护辐射致睾丸生精功能损伤的效果。实验研究:体外实验:(1)电离辐射可以抑制TM4细胞活力,而益气解毒方高、中、低剂量均可以增强12 Gy γ射线辐射后TM4细胞的活力。(2)12Gy 60Coγ射线照射后TM4细胞DNA氧化损伤明显,细胞骨架蛋白明显受损,细胞抗氧化能力降低;益气解毒方可以通过促进DNA损伤修复,保护细胞骨架蛋白和提高细胞抗氧化能力而发挥细胞保护作用。(3)12 Gy 60Coγ射线照射后,TM4细胞内动态铁池水平和脂质过氧化物水平显著上升,与Erastin诱导的TM4细胞铁死亡动态铁池水平和脂质过氧化物水平变化一致,表明辐射诱导了 TM4细胞铁死亡,益气解毒方可能通过抑制铁过载,降低细胞脂质过氧化物的积累,减轻辐射诱导的TM4细胞铁死亡。(4)益气解毒方通过激活Keap1/Nrf2信号通路,促进抗铁死亡因子GPX4的表达,抑制脂质过氧化过程中的关键催化酶ACSL4、LPCAT3和LOX的表达减轻脂质过氧化物的积累,而发挥抑制TM4细胞铁死亡的作用。体内实验:6.0 Gy 60Coγ射线照后24h可以导致小鼠睾丸Sertoli细胞损伤,表现为ZO-1和Occludin蛋白表达水平降低;睾丸中MDA和Fe含量升高以及GSH含量降低符合铁死亡的生化特征,并且Sertoli细胞线粒体结构发生变化,符合铁死亡细胞线粒体形态,表明Sertoli细胞发生铁死亡;应用益气解毒方可以抑制辐射诱导的Sertoli细胞铁死亡。在体内益气解毒方可能通过激活Keap1/Nrf2通路,促进GPX4的表达,抑制ACSL4、LPCAT3和LOX的表达而发挥抑制Sertoli细胞铁死亡的作用。结论理论研究表明,基于藏象学说,睾丸生精细胞和支持细胞的功能可分别归属于肾和脾的功能系统,从而可将整体辨证论治的方法应用到微观的细胞层面。结合辐射致睾丸生精功能损伤的中医病机,确定了从补肾健脾、益气解毒法论治睾丸生精功能辐射损伤的治法,并对益气解毒方进行组方分析,可以为进一步探讨防护辐射致睾丸生精功能损伤的中医药理论提供依据。实验研究表明电离辐射可以诱导Sertoli细胞发生铁死亡,益气解毒方可以有效抑制Sertoli细胞铁死亡;其机制可能是益气解毒方可能通过激活Keap1/Nrf2信号通路,促进GPX4表达,抑制ACSL4、LPCAT3和LOX的表达减轻脂质过氧化物的积累而发挥抗铁死亡的作用。更多还原

【Abstract】 BackgroundWith the development of science and technology,radiology technology has been widely used in scientific research and clinical diagnosis and treatment.At the same time,the damage caused by ionizing radiation to the human body is inevitable.The testis is highly sensitive to ionizing radiation.A low dose(0.03 Gy)of ionizing radiation can reduce the number of spermatogonial cells.With the increase of radiation dose,the injury of testicular spermatogenesis function will increase,and serious cases can lead to azoospermia and male infertility.There are no clinically available protective drugs for radiation damage to the testicles.In recent years,research on the protection of traditional Chinese medicine against radiation injury has made a lot of progress.In the early stage,our research group carried out a series of explorations on the traditional Chinese medicine etiology,pathogenesis,treatment,and prescriptions of acute radiation injury.And found that ionizing radiation can cause serious damage to spermatogenic cells in mice testes,and induce ferroptosis in cells.The Yiqi Jiedu decoction has a good protective effect on testicular injury caused by ionizing radiation.Therefore,on the basis of previous research and based on the theory of Zang Xiang,this study intends to explore the TCM theory of treating radiation induced testicular spermatogenesis function damage from the perspective of tonifying the kidney and spleen,supplementing qi and detoxifying.It also investigates the effect of ferroptosis in Sertoli cells caused by radiation through in vivo and in vitro experiments,and explores the protective effect and mechanism of Yiqi Jiedu decoction on ferroptosis in Sertoli cells of mouse testes.ObjectiveIn terms of theoretical research,based on the Zang Xiang theory,this paper discusses the functions of spermatogenic cells and Sertoli cells,and attributes their functions to the functional system of the kidney and spleen respectively.Then,combined with the TCM pathogenesis of testicular radiation injury,the overall syndrome differentiation,and treatment idea of Tonifying the kidney and strengthening the spleen is extended to the microcellular level,so as to provide a theoretical basis for the TCM protection of testicular radiation injury.In terms of experimental research,firstly,the radiation injury model of Sertoli cells was replicated in vitro to explore the damage of ionizing radiation to Sertoli cells and the protective effect of Yiqi Jiedu decoction,as well as the effect of ionizing radiation on ferroptosis of Sertoli cells and the protective mechanism of Yiqi Jiedu decoction;Then,the effect and mechanism of Yiqi Jiedu decoction on the regulation of radiation-induced Sertoli cell ferroptosis were further verified through in vivo experiments.MethodsTheoretical research:(1)The function of spermatogenic cells is discussed based on the theory of Zang Xiang,and it is attributed to the functional system of the kidney in the Zang Xiang theory,while the function of Sertoli cells is discussed based on the theory of Zang Xiang,and it is attributed to the functional system of the spleen in the theory of Zang Xiang;(2)To explore the TCM pathogenesis of testicular spermatogenic function injury caused by ionizing radiation;(3)By attributing the functions of spermatogenic cells and Sertoli cells to the functional systems of the kidney and spleen of Zangxiang theory,the system of principles,methods,and prescriptions for overall syndrome differentiation and treatment can be expanded to the level of spermatogenic cells and Sertoli cells.Then,through the discussion of the therapeutic methods and the analysis of the combination of Yiqi Jiedu decoction for the prevention and treatment of testicular radiation injury,it provides a theoretical basis for Yiqi Jiedu decoction for the prevention and treatment of testicular radiation injury.Experimental study:In vitro experiments:(1)Screening the irradiation dose of replicating the radiation injury model of TM4 cells.(2)Immunofluorescence detection 12.0 Gy 60Co γ the oxidative damage of TM4 cell DNA and cytoskeleton protein caused by radiation,the damage of blood testis barrier caused by simulated ionizing radiation to TM4 cell permeability after radiation,the levels of SOD and GSH were detected to evaluate the damage of radiation to cell antioxidant capacity and the protective effect of Yiqi Jiedu decoction.(3)Using ferroptosis inducer Erastin as control,12.0 Gy 60Co was observed by flow cytometry γ the ferroptosis of TM4 cells induced by ionizing radiation and the protective effect of Yiqi Jiedu decoction.(4)Through PCR and Western blot experiments,the mechanism of Yiqi Jiedu decoction regulating ferroptosis of TM4 cells induced by ionizing radiation through the Keap1/Nrf2 pathway was explored.In vivo experiment:Balb/c mice were irradiated with 6.0 Gy 60Co γ-ray with a single wholebody irradiation.The damage of Sertoli cells in mice testis,the ferroptosis of Sertoli cells and the protective effect of Yiqi Jiedu decoction were observed 24 h after irradiation.The expression of proteins related to Keap1/Nrf2 pathway was also examined to explore the protective mechanism of Yiqi Jiedu decoction against ferroptosis in Sertoli cells caused by radiation.ResultsTheoretical research:(1)This study discusses the functions of spermatogenic cells and Sertoli cells based on the theory of Zang Xiang:1)Based on the theory of Zang Xiang of traditional Chinese medicine and in combination with the understanding of the function of spermatogenic cells in modern medicine,the function of spermatogenic cells is analyzed and compared with the function of kidney in the study of visceral manifestations,Therefore,the function of spermatogenic cells can be attributed to the kidney function system in the Zang Xiang theory.So as to provide a theoretical basis for the treatment of testicular spermatogenesis dysfunction from the perspective of the kidney,and the selection of traditional Chinese medicine with the effect of tonifying kidney and essence.2)Based on the theory of Zang Xiang of TCM,combined with the understanding of the function of Sertoli cells in modern medicine,the function of Sertoli cells was analyzed,and compared with the function of the spleen in visceral manifestation,it was found that the two were closely related.When we look at the local organ of the body,the testis as a whole,the physiological function of the Sertoli cells in the testis is very similar to that of the spleen in the human body,which plays a leading role in transportation,purification,and external defense.Therefore,the function of Sertoli cells can be attributed to the functional system of the spleen in the theory of Zang Xiang,which provides a theoretical basis for the treatment of the spleen and the selection of spleen-strengthening herbs to prevent and treat the radiation injury of Sertoli cells.(2)Through the analysis of the characteristics of testicular spermatogenesis function damage caused by ionizing radiation,we found that the pathogenesis of traditional Chinese medicine can be summarized as 1)kidney deficiency and deficiency of essence,lack of source of spermatogenesis;2)The spleen loses its healthy function,and qi and blood are transformed into energy and blood;3)The poison is hot and the qi and yin are weak.(3)The function of spermatogenic cells can be attributed to the functional system of the kidney visceral state,and the function of Sertoli cells can be attributed to the functional system of the spleen visceral state.Therefore,the principles,methods,prescriptions,and drugs for the spleen and kidney in the process of overall syndrome differentiation and treatment can be extended to the micro level of spermatogenic cells and Sertoli cells in the testis.According to the TCM pathogenesis of testicular radiation injury,we treat testicular radiation injury by tonifying the kidney and strengthening the spleen.The prescription analysis of Yiqi Jiedu decoction:the prescription uses medlar and epimedium to invigorate the kidney and replenish essence;Astragalus,tuckahoe,and hawthorn are used to strengthen the spleen and stomach;At the same time,American ginseng,angelica,white peony,purslane,and other herbs are used to replenish qi,nourish blood and detoxify.All drugs play the functions of Tonifying the kidney and spleen,supplementing qi and detoxifying,which can achieve the effect of preventing testicular radiation damage.Experimental study:In vitro experiments:(1)Ionizing radiation can inhibit the vitality of TM4 cells in a dose-dependent manner.High,medium,and low doses of Yiqi Jiedu decoction can enhance the vitality of TM4 cells after radiation.(2)The oxidative damage to DNA,cytoskeletal proteins,and antioxidant capacity of TM4 cells were significantly damaged after 12 Gy 60Co γ-irradiation;Yiqi Jiedu decoction could play a cytoprotective role by promoting DNA damage repair,protecting cytoskeletal proteins and improving the antioxidant capacity of cells.(3)The labile iron pool and lipid peroxide level in TM4 cells increased significantly after 12 Gy 60Co γ-irradiation,which was consistent with the changes in the labile iron pool and lipid peroxide level in TM4 cells induced by Erastin,indicating that radiation-induced ferroptosis in TM4 cells and that Yiqi Jiedu decoction may reduce the accumulation of lipid peroxide in TM4 cells by inhibiting iron overload and reducing radiation-induced ferroptosis.(4)Yiqi Jiedu decoction inhibits ferroptosis in TM4 cells by activating the Keap1/Nrf2 signaling pathway,promoting the expression of anti-ferroptosis factor GPX4,and inhibiting the expression of ACSL4,LPCAT3,and LOX,which are key catalytic enzymes in the process of lipid peroxidation,to reduce the accumulation of lipid peroxides.In vivo experiment:6.0 Gy 60Co γ-irradiation caused damage to Sertoli cells in mice testes 24 h after irradiation,as evidenced by reduced expression levels of ZO-1 and Occludin proteins;increased MDA and Fe content and reduced GSH content in testes,and changes in the mitochondrial structure of Sertoli cells,consistent with the mitochondrial morphology of ferroptosis cells,indicating that ferroptosis occurred in Sertoli cells;application of The application of Yiqi Jiedu decoction could inhibit the radiation-induced ferroptosis in Sertoli cells.In vivo,Yiqi Jiedu decoction may activate the Keap1/Nrf2 pathway,promote the expression of GPX4,inhibit the expression of ACSL4,LPCAT3 and LOX and exert an inhibitory effect on ferroptosis in Sertoli cells.ConclusionTheoretical research shows that based on the theory of Zang Xiang,the functions of testicular spermatogenic cells and Sertoli cells can be attributed to the functional system of the kidney and spleen respectively.The TCM pathogenesis of testicular radiation injury is kidney deficiency and essence deficiency,and spleen dysfunction.Therefore,testicular radiation injury can be treated from the method of tonifying kidney and spleen;Combined with the pathogenesis and treatment of testicular radiation injury,the prescription analysis of Yiqi Jiedu decoction can provide a theoretical basis for further discussion of traditional Chinese medicine protection theory and prescription of testicular radiation injury.Experimental studies show that ionizing radiation can induce ferroptosis in Sertoli cells,and Yiqi Jiedu decoction can inhibit ferroptosis by regulating iron metabolism and reducing the accumulation of lipid peroxidation in cells;The mechanism may be that Yiqi Jiedu decoction promotes the activation of Keap1/Nrf2 pathway,plays an anti-ferroptosis role by promoting the expression of its downstream anti-ferroptosis factors HO-1 and GPx4,and inhibiting the expression of ferroptosis promoting factors ACSL4,LPCAT3,and LOX.更多还原