中药复方对急性辐射损伤小鼠睾丸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， 2018， 硕士

【摘要】 随着核能的发展和核技术的运用,人类在享受电离辐射带来便利的同时也正在遭受辐射损伤带来的危害。急性辐射损伤(Acute radiation injury,ARI)发病急骤、病情险恶,极易造成人体各系统或组织器官的辐射损伤。睾丸是一个辐射敏感组织,即使低剂量的电离辐射也能导致睾丸生精细胞凋亡,引发生精功能障碍,严重者甚至可以导致不育。目前,传统的辐射损伤防护剂对睾丸的急性辐射损伤的防护效果不尽如人意,因副作用大、价格昂贵等无法满足临床需求。因此研制一种高效、安全又经济的睾丸辐射损伤防护剂尤为迫切。目的研究急性辐射损伤的中医学病因病机,并据此探讨睾丸辐射损伤的防治方法。通过实验检验中药复方对小鼠睾丸辐射损伤的防护效应,并探讨TLR4和TLR5受体在其中的作用。方法理论研究:通过总结急性辐射损伤发生后的临床症状及证候表现,探讨急性辐射损伤的中医学病因。并根据急性辐射损伤的中医学病因,探讨睾丸辐射损伤的病机,根据病机再探讨适宜的防治方法。实验研究:实验一,雄性Balb/c小鼠按体重随机分为空白、模型两组,模型组以4.0 Gy 60Coγ射线照射,分别在照后14 d、21 d和35d,眼眶取血后断颈处死小鼠,检测各取材时间点小鼠睾丸的损伤情况,以及TLR4、TLR5在睾丸中的表达变化。实验二,初步考察中药复方对小鼠睾丸辐射损伤的治疗作用。实验小鼠分为空白组、模型组、阳性药组、中药组,仍以4.0 Gy剂量照射小鼠,照后给药14 d,然后分别在照后14 d、21 d和35 d处死小鼠,检测小鼠睾丸指数、精子浓度、精子活率、精子活力、正常形态精子百分比以及血清T、FSH、LH等指标。实验三:考察中药复方对小鼠睾丸辐射损伤的防护效应及部分机制。小鼠按体重随机分为空白组、模型组、阳性药组、中药高剂量组和中药低剂量组,给药10 d后以2.0 Gy 60Coy射线照射,分别在照射后7 d和21 d处死小鼠,检测生精细胞凋亡、周期和TLR4、TLR5、MyD88、NF-κB、TNF-α、IL-6 表达变化情况。结果1、理论研究发现,超过一定剂量的电离辐射致病急骤,病情严重,顽固难愈,符合中医毒邪的概念,但该毒邪特点鲜明,与温毒、火毒、光毒等传统毒邪明显不同,因此我们将引起急性辐射损伤的电离辐射命名为电离毒。而男性生殖系统辐射损伤因防治困难、病程长等因素,导致至今没有令人满意的防治措施和药物,以治未病思想指导男性生殖系统的辐射损伤防治,对于提高本病的疗效,改善患者的生活质量具有重要价值。2、4 Gy60Coγ射线对小鼠睾丸损伤严重:(1)4.0 Gy 60Coy射线照射后,Balb/c小鼠体重迅速减轻,在照射后4 d左右其体重开始恢复;照后小鼠睾丸辐射损伤发生,睾丸萎缩,睾丸指数在照后14 d即显著低于空白组小鼠(P<0.001)。(2)小鼠睾丸病理结果显示,受照组小鼠睾丸受损严重,生精细胞大量凋亡坏死,生精小管萎缩,生精上皮破损伤严重,受到的损伤随时间的推移加重。(3)照后14 d时,照射组小鼠血清T水平较空白组小鼠升高(P<0.05);照后21 d时,空白组小鼠血清LH升高;而小鼠血清中FSH水平变化不大。(4)照后14 d,TLR4mRNA水平变化不显著,NF-κBmRNA较空白组显著升高(P<0.05),TLR5、MyD88、TNF-α、IL-6mRNA 水平均显著降低;照后 21 d,TLR4/5、MyD88、NF-κ B、TNF-α、IL-6mRNA水平均显著降低;照后35d,TLR4、MyD88、IL-6mRNA水平较空白组低,TLR5、NF-κB、TNF-αmRNA水平变化不显著。(5)TLR4和TLR5在正常小鼠睾丸中也广泛表达,其中TLR4在睾丸支持细胞和间质细胞上高表达,电离辐射对TLR4在睾丸中的表达影响较小;TLR5在精子细胞上高表达,照射后TLR5在小鼠睾丸中表达增强,但在照后21 d和35 d时,随着精子细胞的数量减少,TLR5的阳性区域面积减小。3、中药复方对小鼠睾丸辐射损伤的治疗效应不显著:(1)Balb/c小鼠受4.0 Gy射线照射后,各受照组小鼠体重迅速减轻,模型组和阳性药组在照后4 d左右降至最低,之后开始缓慢恢复,中药组小鼠体重则持续降低,一直降至给药结束,也就是照后14 d,之后才开始缓慢恢复。照后各照射组小鼠睾丸指数下降(P<0.001),阳性药和中药复方的治疗效应不显著。(2)照后14 d时,各照射组小鼠精子浓度无显著性变化,照后21 d各照射组小鼠精子浓度显著降低(P<0.05),阳性药和中药复方的治疗作用不显著;照后14 d,各照射组小鼠精子活率和精子活力无显著性变化,照后21 d各照射组小鼠精子活率显著降低(P<0.01),精子活力也显著降低(P<0.001),照后35 d,各照射组小鼠精子活率和精子活力几乎为0;照射后14 d,模型组和阳性药组小鼠正常精子形态百分比与空白组比较无显著性差异,中药组小鼠正常形态精子百分比显著降低(P<0.05),照后21 d和35 d,各照射组小鼠正常形态精子百分比均显著降低,阳性药和中药复方的治疗效应不显著。综上,4 Gy 60Coγ射线对小鼠睾丸造成严重损伤,中药复方的治疗效应有待进一步研究。4、中药复方预防性给药能够在一定程度上防护2 Gy60Coγ射线造成的睾丸辐射损伤:(1)Balb/c小鼠受2.0 Gy射线照射前给予中药灌胃10 d,照后小鼠体重迅速下降,在照后4d降至最低,之后开始缓慢恢复。照射后7 d时,模型组小鼠睾丸指数较空白组显著降低(P<0.001),阳性药组和中药高剂量组小鼠睾丸指数显著高于模型组(P<0.05;P<0.01),表现出了一定的防护作用;照后21 d,各照射组小鼠睾丸指数均显著降低(P<0.001),给药组未表现出较好的保护作用。(2)照后7 d,模型组小鼠睾丸生精细胞凋亡率显著升高(P<0.05),各给药组与空白组比较无显著性差异;照后21 d时,模型组、阳性药组、中药低剂量组小鼠生精细胞凋亡率显著高于空白组(P<0.01),中药高剂量组也显著高于空白组(P<0.05),中药高剂量组表现出了较好的保、护作用。(3)照后7 d时,模型组小鼠1 C细胞较空白组显著降低(P<0.01),中药高剂量组显著高于模型组(P<0.05);照后21 d,各照射组小鼠睾丸中1 C细胞百分比均显著降低。照后7 d,中药低剂量组小鼠睾丸中2 C细胞百分比升高(P<0.05);照后21 d,各照射组小鼠睾丸中2 C细胞百分比均显著升高(P<0.001),其中中药高剂量组较模型组显著降低(P<0.05)。照后7 d,各个组小鼠4 C细胞百分比无显著性变化;照后21 d,各照射组小鼠睾丸内4 C细胞百分比较空白组显著升高。(4)照后7 d,模型组和中药低剂量组小鼠睾丸中TLR4mRNA水平较空白组显著降低(P<0.05),阳性药组和中药高剂量组与空白组比较无显著性差异;照后21 d,各照射组TLR4mRNA水平均显著降低。照后7 d,中药高剂量组小鼠睾丸内TLR5mRNA水平较空白组和模型组均显著升高(P<0.05),中药高剂量组MyD88、NF-κ BmRNA在照后7 d时也较空白组显著升高;照后21 d时,各照射组小鼠睾丸中TLR5、MyD88和NF-κ BmRNA水平均显著降低(P<0.001)。照后7 d,阳性药组和中药低剂量组TNF-α水平较空白组升高(P<0.01),各组IL-6mRNA水平无显著性差异;照后21 d,各照射组TNF-α和IL-6 mRNA水平较空白组均显著降低(P<0.001)。(5)免疫组化半定量结果发现电离辐射对小鼠睾丸中TLR4的表达影响不显著,在照后7 d时,中药高剂量组小鼠睾丸中TLR5表达较空白组显著升高(P<0.05);照后21 d,模型组和阳性药组TLR5表达较空白组显著升高(P<0.01),中药低剂量组较模型组TLR5表达显著降低(P<0.05),中药高剂量组较空白组表达降低(P<0.05),较模型组也显著降低(P<0.001)。结论1、通过实验我们发现电离毒致病急骤、损伤严重、治疗困难,因此运用中医“治未病”理论,在电离毒致病前即予以中药防护,以起到减轻睾丸辐射损伤的目的。2、本实验睾丸辐射损伤模型复制成功,照后给药治疗效果不显著,这可能与照射剂量过大有关,其机制尚待进一步研究;而照前给予中药预防能起到较好的保护睾丸的作用,与理论研究结果一致,进一步说明“治未病”理论指导下的中药防护小鼠睾丸辐射损伤具有重要意义。3、电离辐射能够引起小鼠睾丸中TLR4和TLR5的表达变化,中药复方能够在一定程度上调控其变化,但具体机制仍有待进一步探讨。更多还原

【Abstract】 With the development of nuclear energy and the use of nuclear technology,humans are also suffering from radiation damage when they enjoy the convenience of ionizing radiation.The rapid onset and sinister conditions of acute radiation injury(ARI)make it extremely easy to cause radiation damage to various systems or tissues of the human body.The testis is a radiation-sensitive tissue.Even low-dose ionizing radiation can lead to apoptosis of spermatogenic cells in the testes,leading to spermatogenic dysfunction,and severe cases can even lead to infertility.At present,the traditional radiation damage protection agent is unsatisfactory when protecting the testis from acute radiation damage.Because of the large side effects and high cost,it cannot meet the clinical needs.Therefore,it is very urgent to develop an efficient,safe and economical testicular radiation protection agent.Purpose:Study the etiology and pathogenesis of acute radiation injury in Chinese medicine,and investigate the treatments and prescriptions of testicular radiation damage.Examine the protective effect of traditional Chinese medicine compound on radiation damage of testis in mice through experiments,and explore the role of TLR4 and TLR5 signaling pathways.Method:Theoretical research:To investigate the etiology of acute radiation injury in traditional Chinese medicine by summarizing the clinical symptoms and syndrome performance after acute radiation injury.According to the etiology,study the pathogenesis of testicular radiation damage,and then explore the appropriate treatment and prescription.Experimental research:Experiment 1,male mice were randomly divided into blank group and model group according to their body weight.The model group was irradiated with 4.0 Gy dose of 60Coy-rays.The mice were sacrificed at 14d,21 d and 35d after irradiation.We tested the testicular damage in rats and the expression of TLR4 and TLR5 in the testis.Experiment 2,a preliminary study was conducted on the therapeutic effects of Chinese herbal compound on radiation damage of mouse testis.The experimental mice were divided into 4 groups:blank group,model group,positive drug group,and traditional Chinese medicine group.The mice were still irradiated with 4.0 Gy,followed by dosing for 14 days,and then the mice were killed on 14d,21d and 35d after irradiation respectively,and tested for various indicators including mouse testis index,sperm concentration,sperm survival rate,sperm motility,percentage of normal morphological sperm,and the T,FSH,and LH in serum.Experiment 3:study the protective effect and mechanism of traditional Chinese compound prescription on mice’s testicular radiation damage.Mice were randomly divided into blank group,model group,positive drug group,high-dose traditional Chinese medicine group,and low-dose traditional Chinese medicine group according to body weight.After 10 days of dosing,they were irradiated with 2.0 Gy dose of 60Co γ-rays and sacrificed on 7d and 21 d after irradiation respectively.The detection indicators included the apoptosis cycle of spermatogenic cell,and TLR4,TLR5,MyD88,NF-κB,TNF-α,IL-6 mRNA levels.Result:1.Theoretical studies have found that acute radiation injury caused sudden,serious and persistence illness,which is consistent with the concept of poisoning in traditional Chinese medicine.However,the characteristics of this poison are distinct,and are distinctly different from traditional poison such as hot poison,fire poison,and phototoxicity.Therefore,we named it ionizing poison;Because the radiation damage to male reproductive system was difficult to control and the duration of the disease was long,aim at the testicular damage cased by ionizing poisoning,the efficacy of after-irradiation treatment was unsatisfactory,so we used the theory of "preventive treatment of disease" in Chinese medicine,protected before irradiation by using Chinese medicine compound prescription.It was of great value to improve the efficacy of the disease and improve the quality of life of patients.2.4 Gy60Co Gamma Rays Cause Severe Testicular Injury in Mice(1)After 4.0 Gy 60Co γ-ray irradiation,Balb/c mice lost weight quickly,and their weights began to recover around 4 days after irradiation.Irradiation damage to the testes and testicular atrophy occurred.The testis index was significantly lower than that in the Blank group mice(P<0.001)at 14th day after irradiation.(2)The results of mice testicular pathology showed that the mice in the exposed group suffered severe damage to the testes,a large number of apoptotic and necrotic spermatogenic cells,atrophy of the seminiferous tubules,severe damage to the seminiferous epithelium,and the damage increased over time.(3)At 14th day after irradiation,the level of serum T in the irradiated group was higher than that in the blank group(P<0.05).At 21st day,the level of serum LH in the blank group increased,but the level of FSH in the serum did not change significantly.(4)At 14th day after irradiation,the level of TLR4mRNA did not significantly change,NF-κ BmRNA was significantly higher than that in blank group(P<0.05),TLR5,MyD88,TNF-a,IL-6mRNA levels decreased significantly.At 21st day after irradiation,the levels of TLR4/5,MyD88,NF-κB,TNF-α,IL-6mRNA decreased significantly.TLR4,MyD88,and IL-6mRNA levels were lower than those in the blank group at 35th day after irradiation,and the levels of TLR5,NF-κB,and TNF-amRNA did not change significantly.(5)TLR4 and TLR5 also widely expressed in the testis of normal mice.TLR4 highly expressed on Sertoli cells and Leydig cells,and ionizing radiation had little effect on the expression of TLR4 in the testis;TLR5 highly expressed on sperm cells,after irradiation.the expression of TLR5 enhanced in mice testis,but at 21st day and 35th day after irradiation,the positive areas of TLR5 decreased as the number of sperm cells decreased.3.Therapeutic effects of Chinese herbal compound on mouse testicular radiation injury are not significant:(1)After Balb/c mice were irradiated with 4.0 Gy rays,the body weights of mice in each irradiated group rapidly reduced.The weights of model group and positive drug group reduced to a minimum around 4 days after irradiation,and slowly recovered afterwards.The mice in the Chinese medicine group continued to lose weight,until the end of administration,that was the 14th day after irradiation,then it began to recover slowly.After the irradiation,the testis index of the mice in each irradiation group decreased(P<0.001),and the therapeutic effect of the positive drug and the Chinese compound compound was not significant.(2)At 14th day after irradiation,there was no significant change in sperm concentration in each irradiation group,and the sperm concentration in each irradiation group was significantly decreased at 21st day after irradiation(P<0.05).The therapeutic effect of the positive drug and the Chinese compound compound was not significant;At 14th day,there was no significant change in sperm viability and sperm motility in each irradiation group.At 21 st day,the sperm viability decreased(P<0.01)and sperm motility decreased(P<0.001)significantly.At 35th day after irradiation,the sperm viability and sperm motility of each irradiation group were almost 0.At 14th day after irradiation,there was no significant difference in the percentage of normal sperm morphology among the model group,the positive group and the blank group,when the Chinese medicine group significantly decreased(P<0.05).At 21st day and 35th day after irradiation,the percentage of normal sperm in each irradiation group significantly reduced,and the therapeutic effect of the positive drug and Chinese herbal compound was not significant.In summary,4 Gy 60Co γ-rays caused serious damage to the testes of mice,and the therapeutic effects of Chinese herbal compound need to be further studied.4.Preventive administration of traditional Chinese medicine compound can protect the testicular radiation damage caused by 2 Gy60Co gamma rays to some extent:(1)Balb/c mice were given intragastric administration for 10 days before irradiation with 2.0 Gy rays.After the irradiation,the body weights of the mice dropped rapidly.At 4th day,they were reduced to a minimum,and then slowly recovered.(2)At 7th day after irradiation,the testis index of mice in the model group was significantly lower than that in the blank group(P<0.001),and the testis index in the positive drug group and the high-dose Chinese medicine group was significantly higher the model group(P<0.05;P<0.01).A certain protective effect was exhibited;At 21st day,the testis index of mice in each irradiation group significantly decreased(P<0.001),and the administration group did not show a good protective effect.At 7th day after irradiation,the apoptosis rate of spermatogenic cells in the model group significantly increased(P<0.05).There was no significant difference between the administration groups and the blank group.At 21st day,the apoptosis rates of spermatogenic cells in the model group,positive drug group and the low-dose group were significantly higher than that in the blank group(P<0.01).The high-dose group was also significantly higher than the blank group(P<0.05).The high-dose group showed a better Protective effects.(3)At 7th day after irradiation,the 1C cells in the model group were significantly lower than those in the blank group(P<0.01),and the high dose group were significantly higher than the model group(P<0.05);The percentage of 1 C-cells in each group significantly reduced at 21st day.At 7th day after irradiation,the percentage of 2 C cells in the testis of the low-dose group was increased(P<0.05);at 21st day,the percentage of 2 C-cells in each group was significantly higher(P<0.001).Among them,the high dose group of traditional Chinese medicine was significantly lower than that in the model group(P<0.05).At 7th day after irradiation,the percentage of 4C cells in each group had no significant change.At 21st day,the percentage of 4C cells in the testis of each irradiated group was significantly higher than that of the blank group.(4)At 7th day after irradiation,the levels of TLR4mRNA in the model group and the low dose group of traditional Chinese medicine were significantly lower than those in the blank group(P<0.05).There was no significant difference among the positive drug group,the high dose group and the blank group;The TLR4mRNA levels in each irradiation group were significantly lower at 21st day.At 7th day after irradiation,the TLR5mRNA level in the high-dose group was significantly higher than that in the blank group and the model group(P<0.05).MyD88 and NF-κBmRNA in the high-dose group were also higher than the blank group 7th day after irradiation.The levels of TLR5,MyD88,and NF-κBmRNA in each irradiation group significantly decreased(P<0.001)at 21st day.At 7th day after exposure,the levels of TNF-α in the positive drug group and the low dose group were significantly higher than those in the blank group(P<0.01).There was no significant difference in IL-6mRNA levels among these groups.At 21st day after irradiation,the levels of TNF-α and IL-6mRNA in each irradiation group.were significantly lower than that in the blank group(P<0.001).(5)The semi-quantitative results of immunohistochemistry showed that ionizing radiation had no significant effect on the expression of TLR4 in the testis of mice.At the 7th day after irradiation,the expression of TLR5 in high-dose group was significantly higher than that in the blank group(P<0.05).At 21st day after irradiation,the expression of TLR5 in the model group and the positive drug group was significantly higher than that in the blank group(P<0.01).The expression of TLR5 in the low dose group was significantly lower than that in the model group(P<0.05),the high dose group was lower than the blank group(P<0.05),and was also significantly lower than that the model group(P<0.001).Conclusion:1.Through experiments,we have found that ionizing poisons cause sudden illnesses,severe injuries,and difficulties in treatment.Therefore,the theory of "preventive treatment of disease" in Chinese medicine was used to protect before ionizing poisoning,to reduce the damage of radiation in testicular.2.In this experiment,the model of testicular radiation damage was successfully replicated,and the effect of post-irradiation administration was not significant.However,the prevention of traditional Chinese medicine before exposure could play a good role in protecting the testis,which was consistent with the theoretical research results,and it was of great significance that under the guidance of the theory "preventive treatment of disease",protected mice against testicular radiation damage by using traditional Chinese medicine.3.Ionizing radiation can lead changes in the expression of TLR4 and TLR5 in mice testis.Chinese herbal compound can regulate the changes on some extent,but the specific mechanism remains to be further explored.更多还原