密蒙花颗粒剂对去势雄兔泪腺细胞凋亡因子Bax、Bcl-2、Fas和FasL的影响

Effect of Buddleia Granule on Bax,Bcl-2,Fas,and FasL of Lacrimal Gland Cells of Castrated Male Rabbits

目的观察密蒙花颗粒剂对去势雄兔泪腺细胞凋亡因子Bax、Bcl-2、Fas和Fas L的影响,探讨密蒙花颗粒剂对去势雄兔干眼症模型的疗效。方法将密蒙花原药材制备成颗粒剂。将30只健康成年新西兰长耳白兔(雄性),随机分为空白组(A组),模型组(B组),密蒙花颗粒剂组(C组),安慰剂组(D组),睾酮组(E组)5组,每组6只。除A组外其余4组实验用兔行双侧睾丸及附睾切除术。除A、B组外,从术后第3日开始C组予密蒙花颗粒剂,按100 mg/kg灌胃,3次/日;D组予生理盐水20 m L灌胃,3次/日;E组按2 mg/kg在大腿肌肉处注射丙酸睾酮注射液,每3日注射1次。各组实验用兔,分别于术前和术后第4周各进行1次SchirmerⅠ试验(SchirmerⅠtest,SⅠT)和泪膜破裂时间(tear break-up time,BUT)测定。用药观察4周后采用空气栓塞法处死各组兔子,摘取双眼泪腺组织,采用免疫组化法对泪腺细胞凋亡因子Bax、Bcl-2、Fas和Fas L的表达进行检测,并观察泪腺组织结构形态图。结果 (1)SⅠT及BUT结果:与本组术前及术后A组比较,B、D组SⅠT及BUT均明显下降(P<0.01);与B、D组术后比较,C、E组SⅠT及BUT升高,且差异均有统计学意义(P<0.01)。(2)Bax、Bcl-2、Fas、Fas L表达:与A组比较,B、D组Bax、Fas、Fas L表达均明显增多,Bcl-2表达明显减少(P<0.01);与B、D组比较,C、E组Bax、Fas、Fas L表达均明显减少,Bcl-2表达明显增多(P<0.01)。(3)Bax、Bcl-2、Fas、Fas L表达观察结果:A组:泪腺结构清晰,未见Bax、Bcl-2、Fas、Fas L表达;B组:泪腺结构模糊,Bax、Fas、Fas L均大量表达于细胞膜和细胞浆中,呈棕黄色颗粒,Bcl-2仅少量表达;C组:泪腺结构清晰,散见Bax、Fas、Fas L表达,Bcl-2大量表达于细胞膜和细胞浆中,呈棕黄色颗粒;D组:泪腺结构模糊,Bax、Fas、Fas L均大量表达于细胞膜和细胞浆中,呈棕黄色颗粒,Bcl-2仅少量表达;E组:泪腺结构清晰,散见Bax、Fas、Fas L表达,Bcl-2未见表达。结论密蒙花颗粒剂具有与雄激素相似的抑制泪腺细胞凋亡因子Bax、Fas和Fas L表达,同时上调Bcl-2的作用,能抑制雄激素缺乏所致兔泪腺细胞的凋亡,维持泪腺基础分泌量,但其作用弱于雄激素。

Objective To observe the effect of Buddleia Granule （BG） on apoptosis factors （Bax, Bcl-2, Fas, and FasL） of lacrimal gland cells of castrated male rabbits, and to study its effect on dry eye model of castrated male rabbits. Methods Buddleia raw materials were made into granules. Totally 30 healthy adult New Zealand rabbits were randomly divided into blank group （Group A）, model group （Group B）, BG group （Group C）, the placebo group （Group D）, testosterone group （Group E）, 6 rabbits in each group. Bilateral testis and epididymis were cut off to rabbits of Group B to E. The rabbits of Group C were given BG according to 100 mg/kg by gastrogavage,3 times per day. Those in Group D were given normal saline, 20 mL each time, 3 times per day. Those in Group E were injected with Testosterone Propionate Injection （2 mg/kg） in thigh muscle, once injection per every 3 days. All rabbits were tested Schirmer [ test （S I T） and tear break-up time （BUT） before operation and 4 weeks after operation. Af- ter 4 weeks, all rabbits were scarified by air embolism and bilateral lacrimal glands clipped. Expressions of inflammatory factors （Bax, Bcl-2, Fas, and FasL） from lacrimal gland cells were checked by immunohistochemical staining method. The morphology of lacrimal gland was also observed. Results （1） Results of S I T and BUT. Compared with before operation and Group A after operation, S I T and BUT were significantly decreased in Group B and D after opeIration （P 〈0.01 ）. After operation, as compared with Group B and D, S I T and BUT were significantly increased in Group C and E, with statistical difference （P 〈0.01 ）. （2） Expressions of Bax, Bcl-2, Fas, and FasL： Compared with Group A, the expression of Bax, Fas, and FasL were obviously increased, but Bcl-2 was decreased obviously in Group B and D （P 〈 0.01 ）. Compared with Group B and D, the expressions of Bax, Fas, and FasL were decreased, but Bcl- 2 was increased obviously in Group C and E, with statistical difference （P 〈0.01 ）. （3） The results of Bax, Bcl-2, Fas, FasL expressions ： Group A ; The structure of lacrimal gland was clear. The expressions of Bax, Bcl-2, Fas, FasL were not found. Group B and D： The structure of lacrimal gland was indistinct. Bax, Fas, and FasL were expressed in cell membrane and cytoplasm heavily, showing brownish granules with yellow. The expression of Bcl-2 was only expressed in a large amount. Group C： The structure of lacrimal gland was clear. The expression of Bax, Bcl-2, Fas, and FasL were expressed scarcely. Bcl- 2 was expressed in cell membrane and cytoplasm heavily, showing brownish granules with yellow. Group E. The structure of lacrimal gland was clear. The expression of Bax, Bcl-2, Fas, and FasL were expressed in a small amount. Bcl-2 was not found. Conclusions Compared with androgen, BG had a similar effect of depressing the expression of Bax, Fas, FasL and increasing the expressions of Bcl-2. It could inhibit the apoptosis of lacrimal gland cells in castrated male rabbits, maintain the basic secretion. But its effect was slightly weaker than that of the androgen.