蛴螬提取物对兔光损伤视网膜变性Caspase 8、Bax、Bid及Caspase 3表达的影响

doi:10.3877/cma.j.issn.2095-2007.2019.02.004

中华眼科医学杂志(电子版) ›› 2019, Vol. 09 ›› Issue (02) : 83 -89. doi: 10.3877/cma.j.issn.2095-2007.2019.02.004

论著

蛴螬提取物对兔光损伤视网膜变性Caspase 8、Bax、Bid及Caspase 3表达的影响

蒋鹏飞¹, 彭俊², 吴大力³, 彭清华⁴^,^†(

)

^1. 410208　长沙，湖南中医药大学中医学院2017级硕士研究生
^2. 410007　长沙，湖南中医药大学第一附属医院眼科
^3. 530023　南宁，广西中医药大学第一附属医院眼科
^4. 410208　长沙，湖南中医药大学中医学院

收稿日期:2019-03-03 出版日期:2019-04-28
通信作者: 彭清华
基金资助:
湖南省中央财政支持地方高校改革发展资金建设项目(2018年); 湖南省中医药科研计划项目(2010012); 中医药防治眼耳鼻咽喉疾病湖南省重点实验室建设项目(2017TP1018); 国家中医药管理局中医眼科学重点学科建设项目(ZK1801YK015); 湖南省研究生科研创新项目(CX2018B497)

Effects of grub extract on the expressions of Caspase 8, Bax, Bid, and Caspase 3 in rabbits with photorefractive retinal degeneration

Pengfei Jiang¹, Jun Peng², Dali Wu³, Qinghua Peng⁴^,^†()

^1. Master′s Degree 2017, Chinese Medicine College, Hunan University of Chinese Medicine, Changsha 410208, China
^2. Department of Ophthalmology, The First Hospital of Hunan University of Chinese Medicine, Changsha 410007, China
^3. Department of Ophthalmology, The First Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China
^4. Chinese Medicine College, Hunan University of Chinese Medicine, Changsha 410208, China

Received:2019-03-03 Published:2019-04-28
Corresponding author: Qinghua Peng

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引用本文：

蒋鹏飞, 彭俊, 吴大力, 彭清华. 蛴螬提取物对兔光损伤视网膜变性Caspase 8、Bax、Bid及Caspase 3表达的影响[J]. 中华眼科医学杂志(电子版), 2019, 09(02): 83-89.

Pengfei Jiang, Jun Peng, Dali Wu, Qinghua Peng. Effects of grub extract on the expressions of Caspase 8, Bax, Bid, and Caspase 3 in rabbits with photorefractive retinal degeneration[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(02): 83-89.

目的

探讨蛴螬提取物对兔光损伤视网膜变性半胱氨酸天冬氨酸蛋白酶8(Caspase 8)、B细胞淋巴瘤-2相关的x蛋白(Bax)、B细胞淋巴瘤-2同源区域3凋亡诱导蛋白(Bid)及半胱氨酸天冬氨酸蛋白酶3(Caspase 3)表达的影响。

方法

将30只实验性兔随机分成空白组、模型组、蛴螬低剂量组、蛴螬中剂量组及蛴螬高剂量组。每组6只兔(12只眼)。除空白组外，其余四组均建立光损伤视网膜变性模型，在光照架的6个面分别放置1根40瓦的白色荧光灯管，调节光照强度，并用ZDS-10型数字式照度计测定兔活动水平。多点照度平均为(2000±200)Lx，在12 h明及12 h暗环境下循环光环境适应7 d，自由摄食饮水。7 d后光照前先暗适应24 h，然后光照12 h，再进行暗适应12 h，连续3个循环，光照时间总计为36 h。成模后，给予空白组、模型组生理盐水5 ml/kg灌胃，蛴螬低剂量组以0.45 g/kg(中剂量组以0.9 g/kg、高剂量组以1.8 g/kg)蛴螬提取物配成5 ml蛴螬提取物混悬液灌胃。在给药14 d处死全部兔后取材做苏木精-伊红染色，观察视网膜细胞结构及免疫组化检测Caspase 8、Bax、Bid及Caspase 3的表达。Caspase 8、Bax、Bid及Caspase 3表达的积分光密度值，以均数±标准差表示。采用单因素方差分析比较不同组别Caspase 8、Bax、Bid及Caspase 3表达水平的差异，当差异有统计学意义时，进一步采用SNK法两两比较。

结果

视网膜显微形态学观察结果显示，空白组兔视网膜结构层次分明，各层细胞排列整齐规则，细胞染色均匀且形态规整；模型组兔视网膜层次结构模糊，分界不清，出现不规则淡染，细胞排列紊乱，细胞数目减少；蛴螬低剂量组兔视网膜外核层胞核排列较疏松；蛴螬中剂量组兔视网膜外核层增厚，视网膜外核层胞核排列较整齐密集；蛴螬高剂量组兔视网膜外核层胞核排列较整齐，厚度有所增加。空白组视网膜外核层细胞浆中Caspase 8、Bax、Bid及Caspase 3阳性物质分布稀疏，含量较少；模型组视网膜外核层细胞浆中Caspase 8、Bax、Bid及Caspase 3阳性物质弥漫分布，含量较多。空白组和模型组的Caspase 8、Bax、Bid及Caspase 3表达与蛴螬各组相比差异均有统计学意义(P<0.05)。蛴螬中剂量组Caspase 8、Caspase 3表达与蛴螬低剂量组相比，差异有统计学意义(F＝7.30，5.43；P<0.05)；蛴螬中剂量组Bid、Bax表达与蛴螬低剂量组相比，差异无统计学意义(F＝2.47，5.79；P>0.05)。蛴螬高剂量组Caspase 8、Bax、Bid及Caspase 3表达与蛴螬低剂量组相比，差异均无统计学意义(F＝0.11，0.11，0.95，0.01；P>0.05)。蛴螬中剂量组Caspase 8表达与蛴螬高剂量组相比，差异有统计学意义(F＝6.14，P<0.05)；蛴螬中剂量组Bax、Bid及Caspase 3表达与蛴螬高剂量组相比，差异均无统计学意义(F＝0.11，0.79，1.71；P>0.05)。

结论

蛴螬提取物能够抑制Caspase 8、Bax、Bid及Caspase 3的表达，不同剂量效果有差异，蛴螬中剂量组效果较好。

关键词: 蛴螬提取物, 光损伤, 半胱氨酸天冬氨酸蛋白酶8, 半胱氨酸天冬氨酸蛋白酶3, B细胞淋巴瘤-2相关的x蛋白, B细胞淋巴瘤-2同源区域3凋亡诱导蛋白

Objective

The aim of this study was to investigate the effects of grub extract on the expression of cysteine aspartic protease 8 (Caspase 8), B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 homology 3 interacting domain death agonist (Bid), and caspase aspartic protease 3 (Caspase 3) in rabbits with photorefractive retinal degeneration.

Methods

Thirty experimental rabbits were randomly divided into 5 groups: blank group, model group, low-dose group, medium-dose group and high-dose group. Each group contained 6 rabbits (12 eyes). Except for the blank group, the other four groups established a model of photodamage retinal degeneration. One 40 W white fluorescent tube was placed on each of the six sides of the light frame to adjust light intensity, and the rabbit activity level was measured by ZDS-10 digital illuminometer. Multi-point illumination, with an average of (2000±200) Lx, was adapted to cyclic light for 7 days in 12 h bright and 12 h dark environment, and the rabbits were free for food and water. After 7 days, the light was darkly adapted for 24 h, then illuminated for 12 h, and then dark-adapted for 12 h for 3 consecutive cycles. The total illumination time was 36 h. After the model was established, the blank group and the model group were intragastrically administered with 5 ml/kg of normal saline, and the low-dose group was intragastrically administered with 5 ml of grub extract suspension by 0.45 g/kg grub extract (the medium-dose group by 0.9 g/kg and the high-dose group by 1.8 g/kg). After 14 days all the rabbits were sacrificed, HE staining was performed to observe the structure of retinal cells and immunohistochemistry to detect the expression of Caspase 8, Bax, Bid, and Caspase 3. The integrated optical density values of Caspase 8, Bax, Bid, and Caspase 3 expression were expressed as mean ± standard deviation. One-way ANOVA was used to compare the expression levels of Caspase 8, Bax, Bid, and Caspase 3 in different groups. When the difference was statistically significant, SNK method was used for further comparisons.

Results

The micromorphological observation of the retina showed that the retinal structure of the blank group was well-defined, cells in each layer were arranged neatly, uniformly stained, and the morphology was regular. The retina of the model group was fuzzy, boundary was unclear, and irregular staining occurred. The arrangement was disordered and the number of cells decreased. In the low-dose group, the nucleus of the outer nuclear layer of the retina was looser. In the middle-dose group, the outer nuclear layer of the retina became thick, and the nucleus of the outer nuclear layer of the retina was arranged neatly. The nucleus of the outer nuclear layer of the retina was arranged neatly and showed increased thickness. The distribution of Caspase 8, Bax, Bid, and Caspase 3 positive substances in the cytoplasm of the outer nuclear layer of the blank group was sparse and less, while they were diffusely distributed and contained more in the model group. The expressions of Caspase 8, Bax, Bid, and Caspase 3 in the blank group and the model group were significantly different from those in the other three groups (P<0.05). There were statistically significant differences between the medium-dose group and the low-dose group for the expressions of Caspase 8, and Caspase 3 (F=7.30, 5.43; P<0.05), while no difference for the expression of Bid and Bax (F=2.47, 5.79; P>0.05). There were no significant differences between the high-dose group and the low-dose group for the expression of Caspase 8, Bax, Bid, and Caspase 3 (F=0.11, 0.11, 0.95, 0.01; P>0.05). There was statistically significant difference between the medium-dose group and the high-dose group for the expression of Caspase 8 (F=6.14, P<0.05), while no differences for the expressions of Bax, Bid, and Caspase 3 (F=0.11, 0.79, 1.71; P>0.05).

Conclusions

The grub extract could inhibit the expressions of Caspase 8, Bax, Bid, and Caspase 3. Different dose leads to different effect. For all dosages, medium dose shows better.

Key words: Grub extract, Photodamage, Cysteine aspartic protease 8, Cysteine aspartic protease 3, B-cell lymphoma-2 associated X protein, B-cell lymphoma-2 homology 3 interacting domain death agonist

图1 高倍光学显微镜观察各组实验性兔视网膜组织的显微结构图像　A图示空白组兔的视网膜组织结构层次分明；B图示模型组兔的视网膜组织结构模糊，分界不清；C图示蛴螬低剂量组兔的视网膜组织排列较疏松；D图示蛴螬中剂量组兔的视网膜组织排列整齐；E图示蛴螬高剂量组兔的视网膜组织排列较整齐（苏木精-伊红染色染色　×400）

表1 各组兔视网膜Caspase 8、Bax、Bid及Caspase 3表达的比较[±s ，积分光密度(×10⁶)]

组别	眼数(只)	Caspase 8	Bax	Bid	Caspase 3
空白组	12	2.72±0.17	2.39±0.12	2.34±0.27	2.54±0.27
模型组	12	5.24±0.26^a	6.30±0.11^a	6.12±0.36^a	5.96±0.29^a
蛴螬低剂量组	12	3.88±0.43^ab	4.23±1.31^ab	4.08±0.41^ab	4.08±0.39^ab
蛴螬中剂量组	12	3.32±0.27^abc	3.86±0.48ab	3.68±0.47^ab	3.60±0.32^abc
蛴螬高剂量组	12	3.80±0.39^abd	4.01±1.00^ab	3.88±0.29^ab	4.11±0.90^ab
F值		51.48	19.75	81.83	37.67
P值		<0.05	<0.05	<0.05	<0.05

表1 各组兔视网膜Caspase 8、Bax、Bid及Caspase 3表达的比较[±s ，积分光密度(×10⁶)]

组别	眼数(只)	Caspase 8	Bax	Bid	Caspase 3
空白组	12	2.72±0.17	2.39±0.12	2.34±0.27	2.54±0.27
模型组	12	5.24±0.26^a	6.30±0.11^a	6.12±0.36^a	5.96±0.29^a
蛴螬低剂量组	12	3.88±0.43^ab	4.23±1.31^ab	4.08±0.41^ab	4.08±0.39^ab
蛴螬中剂量组	12	3.32±0.27^abc	3.86±0.48ab	3.68±0.47^ab	3.60±0.32^abc
蛴螬高剂量组	12	3.80±0.39^abd	4.01±1.00^ab	3.88±0.29^ab	4.11±0.90^ab
F值		51.48	19.75	81.83	37.67
P值		<0.05	<0.05	<0.05	<0.05

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Effects of grub extract on the expressions of Caspase 8, Bax, Bid, and Caspase 3 in rabbits with photorefractive retinal degeneration

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Effects of grub extract on the expressions of Caspase 8, Bax, Bid, and Caspase 3 in rabbits with photorefractive retinal degeneration