木豆叶提取物对急性放射性皮炎的作用及其机理研究
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摘要
核辐射引起的放射性急、慢性损伤和致癌作用严重危害着人体的健康。日本的核泄漏再次引起社会对放射性危害防护的重视。本研究将主要针对放射性所致的急性放射性皮炎展开研究。
急性放射性皮炎是放射治疗恶性肿瘤常见的副反应,电离辐射产生自由基和活性氧损伤基底层细胞,其生物效应主要表现为对细胞DNA分子损伤。面对放射损害机制的复杂性和对机体损失的严重性,现今使用药物不能满足病患者的需要,其中很多药物对健康细胞的远期效应还有待研究,中医理论主张清热解毒主治放射性的火热之邪,而对治疗放射性损伤致皮炎症所需的抗菌消炎、镇痛和促进创面愈合等药理作用,可因中药的活性成分多、作用靶点多、给药途径多、不良反应少,从而达到治疗效果。因此,中医药表现出巨大的优势和潜力。
本校对木豆叶有较为深入的研究,袁浩、方永奇和唐勇等分别通过不同的研究发现其有清热解毒、抗菌消炎和促进皮肤创面愈合的疗效,本研究通过木豆叶提取物对急性放射性皮炎的药效学和机制研究,有望将其发掘并开发成一种防治急性放射性皮炎有效的药物。
目的：
通过木豆叶提取物对β射线致放射性损伤大鼠的急性放射性皮炎模型的药效学研究,观察创面发生、愈合情况和创面组织的病理变化以确定疗效,检测木豆叶对Ku70, Ku80mRNA的影响变化,探讨木豆叶影响放射性皮炎的作用机制。
方法：
1.木豆叶提取物对急性放射性皮炎的药效学研究
1.1实验动物的分组及处理
健康雌性SD大鼠40只随机分成4组,每组10只,分别为空白对照组、放射模型组、木豆叶组和阳性药(比亚芬)组。空白对照组不做辐射处理；放射模型组、木豆叶组和阳性药组均于大鼠臀部皮肤放射造成急性放射性皮炎模型。放射后第2天,木豆叶组和阳性药组大鼠于照射部位予以生理盐水清创后,每天两次均匀涂抹相应的药物lg/kg;放射模型组单纯以生理盐水清创；空白对照组不予给药处理。
1.2急性放射性皮炎模型的建立
实验大鼠采用饱和硫化钠与等量滑石粉制成的糊剂对大鼠照射部位脱毛,用0.3%戊巴比妥钠10mL/kg的剂量进行腹腔麻醉后,用直线加速器发射的6MeVβ射线对大鼠臀部皮肤单次照射,照射距离1m,剂量率为200cGy/min,照射时间为15min,各组照射剂量为30Gy,照射面积2cm×4cm,周围部位以10cm厚铅板屏蔽。
1.3观察各组大鼠的活动情况,皮肤创面发生和愈合的情况；照射后第15、30、45、60天,测量各组大鼠创面愈合面积,换算成愈合率,并对创面进行外观评分；照射后第30天对大鼠诊断分级；记录愈合时间。
2.木豆叶提取物对急性放射性皮炎的作用机理研究：
2.1实验动物的分组及处理
健康雌性SD大鼠100只,随机抽取10只分为空白对照组,另外90只随机分成3组,每组30只,分别为放射模型组、木豆叶组和阳性药组。放射造模和给药方法同上1.1～1.2。辐射后,除空白组在第2、8、15、30、45天各处死2只,其他组各处死大鼠6只。
2.2取材检测
取各时期各组照射野皮肤创面组织3块,第1块HE染色后光镜观察细胞形态结构；第2块放电镜液中低温冷藏,以备透射电镜观察；第3块于液氮中储存,用于实时荧光定量PCR检测Ku70、Ku80mRNA.采用实时荧光定量PCR的方法,检测放射后第30天各组Ku70、Ku80mRNA的表达量。
33.统计学处理
使用SPSS16.0软件进行统计学分析,多组间比较用方差分析；两组比较采用q检验。检验水准α=0.05。
结果：
1.放射后,与放射模型组相对而言,木豆叶组出现创面的时间较为延缓;同时期皮肤损伤和创面溃疡的程度较轻,愈合率较大；愈合时间缩小；创面愈合过程中,大鼠的活动情况比较正常。与放射模型组相比较,木豆叶组在放射后第30、45、60天的愈合率均有统计学意义(P＜0.05)；第45、60天的创面外观评分均有统计学意义(P＜0.05)；愈合时间亦具有统计学意义(P＜0.05)。
2.放射后各时期,在光镜和透射电镜下,观察到木豆提取物组的成纤维细胞、土皮细胞、血管内皮细胞、真皮结缔组织及胶原纤维数量明显多于放射组,细胞的超微结构的损伤较放射组轻；且木豆提取物组第30天大鼠创面组织中Ku70、 Ku80mRNA(?)的表达均高于放射组,结果有统计学意义(P＜0.05)
结论：木豆提取物能够通过改善放射损伤皮肤组织的作用,延缓细胞凋亡,减轻放射损伤产生创面的严重程度,同时还促进毛细血管的新生和组织的自我修复,促进皮肤创面的愈合,缩短愈合时间;而检测到创面皮肤中Ku70、Ku80mRNA的高表达,与放射损伤引起DNA断裂的修复有关,木豆叶提取物可能通过提高DNA修复蛋白的表达,促进胶原蛋白的合成而促进创面的愈合。
The studies about radioresistance are increasing as the universal attention of radialization endangering the normal life of people.Nuclearleak of Janpan has aroused our regard again.This study was researching about the acute radiodermatitis chiefly.
Radiodermatitis is the common side reaction of actinotheraphy to malignancy. Basal layer cell should be injured by free radicals and active oxygen which are produced by ionizing radiation.The biological effect is expressing as the DNA damnification.The mechanism of damnification is complicated and the damage of organism is serious.The foreign medicine used for treatment can't fulfil the patients' requirements.Nowadays, Many researchers attach importance to study of Chinese Pharmacy. The active constituents of Chinese Pharmacy are abundance,so there are many acting targets, The multiway methods of administration and the less adverse reaction are also the advantages of Chinese Pharmacy. Our university have researched Cajanus cajan leaf for many years, Hao Yuan,Yongqi
Fang and Yong Tang had discovered that it was effective for Cajanus cajan leaf improving the healing of skin wound and geting rid of the skin inflammation.In this study,we using the extraction of Cajanus cajan leaf for experimentation, make sure the effect of the extraction to prevent and therapy acute radiodermatitis;and research the function and relativity with the DNA double strand break(DSB) repair protein Ku70/80,to explore the mechanism of Cajanus cajan leaf influencing radiodermatitis.So Cajanus cajan leaf is expected to be a effective developed medicine to prevent and cure acute radiodermatitis.
Purpose:From observating the occurrence and healing of wound and wound tissue pathological changes of Acute radiation dermatitis pharmacological rat models,which was injured by beta ray radiation and daubed with the cajan leaf extract,we would determine the pharmacodynamics efficacy of the cajan leaf extract;And from detecting Ku70, Ku80mRNA,we would explore the mechanism of cajanus cajan effecting of radioactive dermatitis.
Method:
1. Pharmacodynamic study of Cajanus cajan leaf extract to acute radiation dermatitis
1.1Grouped and treated the rats Divided40SD female rats into4groups randomly, each group of10, as blank control group, radiation pattern group, Cajanus cajan leaf group and positive control(biafine)group,respectively.Blank control group without radiation treatment; The rats of radiation pattern group, Cajanus cajan leaf group and positive control group should be gave radiation injury on the buttock skin.The second day after the radiation, The rats of Cajanus cajan leaf group and positive control group should be daubed with the corresponding drug1G/kg in the irradiated site alter saline debridement, two times a day, keeping the medicine60days after radiation.The radiation pattern group was given saline debridement only. The blank control group was not given any medicine treatment.
1.2Built the cute radiation dermatitis model
Removed the hair of irradiated site of rats with paste consisted of saturated sodium sulfide and equivalent amount of talcum powder, Abdominal anaesthesia with0.3%pentobarbital sodium at the dose of10mL/kg,and doing a single irradiation6MeV bata ray fired by linear accelerator on the gluteal skin. Irradiation distance of1m, the dose rate of200cGy/min, the irradiation time for15min, the dose of30Gy, irradiated area of2cm X4cm, around the site shielded with leading of10cm.
1.3Observing the activity and the appearance of the wound of each groups rats; In the15,30,45,60days after irradiation, measured healing area of each rat's wound, and converted to the healing rate. Rated the wound appearance and diagnostic classification; recorded the healing time.
2. Mechanism research of Cajanus cajan leaf extract effecting on acute radiation dermatitis
2.1Grouped and treated the rats Divided10from100SD female rats randomly, as blank control group; Divided the other90rats into3groups randomly,each group of30, as radiation pattern group, Cajanus cajan leaf group and positive control group, respectively. The method of radiation model and administration was as1.1.Sacrificed6rats of each groups in2,8,15,30,45clays after radiation, except the blank control group should be sacrificed2rats.
2.2Sampled and detected Cut3pieces of buttock skin wound tissues from each rats of the radiation field,the first for light microscopic observation;the second stored in low temperature for transmission electron microscopic observation;and the third for detecting the content of Ku70, Ku80mRNA using real time fluorescence quantitative PCR method.
3. Statistical process Used SPSS16.0software to conduct statistical analysis, using analysis of variance for multiple groups comparison;Used Q test for comparison of the two groups. Inspection standard of a=0.05.
Result:
1. Compared with the rats of radiation pattern group, of the rats of Cajanus cajan leaf group,the time of emergence of wound was delayed, the skin injury wound and ulcer was a lesser degree, the wound healing rate was larger and healing time was reduced; In the process of wound healing, the activity of rat is more normal. Compared with radiation pattern group, The wound healing rate of Cajanus cajan leaf group in the30,45,60days were statistically significant(P<0.05);the score of wound appearance of Cajanus cajan leaf group in the45,60days were statistically significant(P<0.05);. The healing time was also statistically significant(P<0.05).
2. Each period after the radiation,Used light microscopy and transmission electron microscopy,more fibroblasts, epithelial cells, endothelial cells, dermal connective tissue and collagen fiber can be observed during the skin wound tissue of rats of Cajanus cajan leaf extract group than the rats of radiation group. And cells ultrastructure was damaged lighter than the radiation group. And the Ku70, Ku80mRNA expression in wound tissue of Cajanus cajan leaf extract group were higher than the radiation group's, the results were statistically significant(P<0.05).
Conclusion:Cajanus cajan leaf extract can improve the radiation injured epidermal tissue,delay the apoptosis of cell, relieve the severity of radiation injured wound. At the same time promote capillary angiogenesis and repair tissues of itself. The high expression of Ku80mRNA in Ku70, may be associated with radiation injury caused by DNA fracture repair. Cajanus cajan leaf extract promote the synthesis of collagen and promote wound healing possibly by increasing DNA repair protein expression.
急性放射性皮炎是放射治疗恶性肿瘤常见的副反应,电离辐射产生自由基和活性氧损伤基底层细胞,其生物效应主要表现为对细胞DNA分子损伤。面对放射损害机制的复杂性和对机体损失的严重性,现今使用药物不能满足病患者的需要,其中很多药物对健康细胞的远期效应还有待研究,中医理论主张清热解毒主治放射性的火热之邪,而对治疗放射性损伤致皮炎症所需的抗菌消炎、镇痛和促进创面愈合等药理作用,可因中药的活性成分多、作用靶点多、给药途径多、不良反应少,从而达到治疗效果。因此,中医药表现出巨大的优势和潜力。
本校对木豆叶有较为深入的研究,袁浩、方永奇和唐勇等分别通过不同的研究发现其有清热解毒、抗菌消炎和促进皮肤创面愈合的疗效,本研究通过木豆叶提取物对急性放射性皮炎的药效学和机制研究,有望将其发掘并开发成一种防治急性放射性皮炎有效的药物。
目的：
通过木豆叶提取物对β射线致放射性损伤大鼠的急性放射性皮炎模型的药效学研究,观察创面发生、愈合情况和创面组织的病理变化以确定疗效,检测木豆叶对Ku70, Ku80mRNA的影响变化,探讨木豆叶影响放射性皮炎的作用机制。
方法：
1.木豆叶提取物对急性放射性皮炎的药效学研究
1.1实验动物的分组及处理
健康雌性SD大鼠40只随机分成4组,每组10只,分别为空白对照组、放射模型组、木豆叶组和阳性药(比亚芬)组。空白对照组不做辐射处理；放射模型组、木豆叶组和阳性药组均于大鼠臀部皮肤放射造成急性放射性皮炎模型。放射后第2天,木豆叶组和阳性药组大鼠于照射部位予以生理盐水清创后,每天两次均匀涂抹相应的药物lg/kg;放射模型组单纯以生理盐水清创；空白对照组不予给药处理。
1.2急性放射性皮炎模型的建立
实验大鼠采用饱和硫化钠与等量滑石粉制成的糊剂对大鼠照射部位脱毛,用0.3%戊巴比妥钠10mL/kg的剂量进行腹腔麻醉后,用直线加速器发射的6MeVβ射线对大鼠臀部皮肤单次照射,照射距离1m,剂量率为200cGy/min,照射时间为15min,各组照射剂量为30Gy,照射面积2cm×4cm,周围部位以10cm厚铅板屏蔽。
1.3观察各组大鼠的活动情况,皮肤创面发生和愈合的情况；照射后第15、30、45、60天,测量各组大鼠创面愈合面积,换算成愈合率,并对创面进行外观评分；照射后第30天对大鼠诊断分级；记录愈合时间。
2.木豆叶提取物对急性放射性皮炎的作用机理研究：
2.1实验动物的分组及处理
健康雌性SD大鼠100只,随机抽取10只分为空白对照组,另外90只随机分成3组,每组30只,分别为放射模型组、木豆叶组和阳性药组。放射造模和给药方法同上1.1～1.2。辐射后,除空白组在第2、8、15、30、45天各处死2只,其他组各处死大鼠6只。
2.2取材检测
取各时期各组照射野皮肤创面组织3块,第1块HE染色后光镜观察细胞形态结构；第2块放电镜液中低温冷藏,以备透射电镜观察；第3块于液氮中储存,用于实时荧光定量PCR检测Ku70、Ku80mRNA.采用实时荧光定量PCR的方法,检测放射后第30天各组Ku70、Ku80mRNA的表达量。
33.统计学处理
使用SPSS16.0软件进行统计学分析,多组间比较用方差分析；两组比较采用q检验。检验水准α=0.05。
结果：
1.放射后,与放射模型组相对而言,木豆叶组出现创面的时间较为延缓;同时期皮肤损伤和创面溃疡的程度较轻,愈合率较大；愈合时间缩小；创面愈合过程中,大鼠的活动情况比较正常。与放射模型组相比较,木豆叶组在放射后第30、45、60天的愈合率均有统计学意义(P＜0.05)；第45、60天的创面外观评分均有统计学意义(P＜0.05)；愈合时间亦具有统计学意义(P＜0.05)。
2.放射后各时期,在光镜和透射电镜下,观察到木豆提取物组的成纤维细胞、土皮细胞、血管内皮细胞、真皮结缔组织及胶原纤维数量明显多于放射组,细胞的超微结构的损伤较放射组轻；且木豆提取物组第30天大鼠创面组织中Ku70、 Ku80mRNA(?)的表达均高于放射组,结果有统计学意义(P＜0.05)
结论：木豆提取物能够通过改善放射损伤皮肤组织的作用,延缓细胞凋亡,减轻放射损伤产生创面的严重程度,同时还促进毛细血管的新生和组织的自我修复,促进皮肤创面的愈合,缩短愈合时间;而检测到创面皮肤中Ku70、Ku80mRNA的高表达,与放射损伤引起DNA断裂的修复有关,木豆叶提取物可能通过提高DNA修复蛋白的表达,促进胶原蛋白的合成而促进创面的愈合。
The studies about radioresistance are increasing as the universal attention of radialization endangering the normal life of people.Nuclearleak of Janpan has aroused our regard again.This study was researching about the acute radiodermatitis chiefly.
Radiodermatitis is the common side reaction of actinotheraphy to malignancy. Basal layer cell should be injured by free radicals and active oxygen which are produced by ionizing radiation.The biological effect is expressing as the DNA damnification.The mechanism of damnification is complicated and the damage of organism is serious.The foreign medicine used for treatment can't fulfil the patients' requirements.Nowadays, Many researchers attach importance to study of Chinese Pharmacy. The active constituents of Chinese Pharmacy are abundance,so there are many acting targets, The multiway methods of administration and the less adverse reaction are also the advantages of Chinese Pharmacy. Our university have researched Cajanus cajan leaf for many years, Hao Yuan,Yongqi
Fang and Yong Tang had discovered that it was effective for Cajanus cajan leaf improving the healing of skin wound and geting rid of the skin inflammation.In this study,we using the extraction of Cajanus cajan leaf for experimentation, make sure the effect of the extraction to prevent and therapy acute radiodermatitis;and research the function and relativity with the DNA double strand break(DSB) repair protein Ku70/80,to explore the mechanism of Cajanus cajan leaf influencing radiodermatitis.So Cajanus cajan leaf is expected to be a effective developed medicine to prevent and cure acute radiodermatitis.
Purpose:From observating the occurrence and healing of wound and wound tissue pathological changes of Acute radiation dermatitis pharmacological rat models,which was injured by beta ray radiation and daubed with the cajan leaf extract,we would determine the pharmacodynamics efficacy of the cajan leaf extract;And from detecting Ku70, Ku80mRNA,we would explore the mechanism of cajanus cajan effecting of radioactive dermatitis.
Method:
1. Pharmacodynamic study of Cajanus cajan leaf extract to acute radiation dermatitis
1.1Grouped and treated the rats Divided40SD female rats into4groups randomly, each group of10, as blank control group, radiation pattern group, Cajanus cajan leaf group and positive control(biafine)group,respectively.Blank control group without radiation treatment; The rats of radiation pattern group, Cajanus cajan leaf group and positive control group should be gave radiation injury on the buttock skin.The second day after the radiation, The rats of Cajanus cajan leaf group and positive control group should be daubed with the corresponding drug1G/kg in the irradiated site alter saline debridement, two times a day, keeping the medicine60days after radiation.The radiation pattern group was given saline debridement only. The blank control group was not given any medicine treatment.
1.2Built the cute radiation dermatitis model
Removed the hair of irradiated site of rats with paste consisted of saturated sodium sulfide and equivalent amount of talcum powder, Abdominal anaesthesia with0.3%pentobarbital sodium at the dose of10mL/kg,and doing a single irradiation6MeV bata ray fired by linear accelerator on the gluteal skin. Irradiation distance of1m, the dose rate of200cGy/min, the irradiation time for15min, the dose of30Gy, irradiated area of2cm X4cm, around the site shielded with leading of10cm.
1.3Observing the activity and the appearance of the wound of each groups rats; In the15,30,45,60days after irradiation, measured healing area of each rat's wound, and converted to the healing rate. Rated the wound appearance and diagnostic classification; recorded the healing time.
2. Mechanism research of Cajanus cajan leaf extract effecting on acute radiation dermatitis
2.1Grouped and treated the rats Divided10from100SD female rats randomly, as blank control group; Divided the other90rats into3groups randomly,each group of30, as radiation pattern group, Cajanus cajan leaf group and positive control group, respectively. The method of radiation model and administration was as1.1.Sacrificed6rats of each groups in2,8,15,30,45clays after radiation, except the blank control group should be sacrificed2rats.
2.2Sampled and detected Cut3pieces of buttock skin wound tissues from each rats of the radiation field,the first for light microscopic observation;the second stored in low temperature for transmission electron microscopic observation;and the third for detecting the content of Ku70, Ku80mRNA using real time fluorescence quantitative PCR method.
3. Statistical process Used SPSS16.0software to conduct statistical analysis, using analysis of variance for multiple groups comparison;Used Q test for comparison of the two groups. Inspection standard of a=0.05.
Result:
1. Compared with the rats of radiation pattern group, of the rats of Cajanus cajan leaf group,the time of emergence of wound was delayed, the skin injury wound and ulcer was a lesser degree, the wound healing rate was larger and healing time was reduced; In the process of wound healing, the activity of rat is more normal. Compared with radiation pattern group, The wound healing rate of Cajanus cajan leaf group in the30,45,60days were statistically significant(P<0.05);the score of wound appearance of Cajanus cajan leaf group in the45,60days were statistically significant(P<0.05);. The healing time was also statistically significant(P<0.05).
2. Each period after the radiation,Used light microscopy and transmission electron microscopy,more fibroblasts, epithelial cells, endothelial cells, dermal connective tissue and collagen fiber can be observed during the skin wound tissue of rats of Cajanus cajan leaf extract group than the rats of radiation group. And cells ultrastructure was damaged lighter than the radiation group. And the Ku70, Ku80mRNA expression in wound tissue of Cajanus cajan leaf extract group were higher than the radiation group's, the results were statistically significant(P<0.05).
Conclusion:Cajanus cajan leaf extract can improve the radiation injured epidermal tissue,delay the apoptosis of cell, relieve the severity of radiation injured wound. At the same time promote capillary angiogenesis and repair tissues of itself. The high expression of Ku80mRNA in Ku70, may be associated with radiation injury caused by DNA fracture repair. Cajanus cajan leaf extract promote the synthesis of collagen and promote wound healing possibly by increasing DNA repair protein expression.
引文
[1]朱茂祥.核辐射损伤效应及其医学防护[J].人民军医,2011,54(4)：263-266.
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