龟鹿二仙膏对糖皮质激素性骨质疏松大鼠骨细胞凋亡影响的实验研究

Experimental Study on Effect of Guilu Erxian Ointment(龟鹿二仙膏) on Bone Cell Apoptosis in Rats with Glucocorticoid-Induced Osteoporosis

目的 通过建立糖皮质激素性骨质疏松(glucocorticoid-induced osteoporosis, GIOP)大鼠模型,探讨龟鹿二仙膏是否通过调控凋亡相关蛋白Cyt-c、Caspase-9、Bcl-2、Bax,抑制骨细胞凋亡,发挥防治大鼠GIOP的作用。方法 60只3月龄Wistar大鼠(雌雄各半)按照随机数字表法将大鼠分为5组,每组12只。分别为:空白组(CON)、模型组(DEX)、龟鹿二仙膏高剂量组(HD)、龟鹿二仙膏中剂量组(MD)、龟鹿二仙膏低剂量组(LD),除CON组外,其余各组采用地塞米松肌肉注射(2.5 mg/kg, 2次/周)复制GIOP大鼠模型,正常组给予等量生理盐水注射,连续注射9周。在建立GIOP模型的同时,开始灌胃给药,连续灌胃9周。灌胃结束后,各组大鼠腹腔麻醉,腹主动脉采血处死,双能X线检测腰椎骨密度(BMD);HE染色观察腰椎骨小梁形态;ELISA法测定血清骨碱性磷酸酶(BALP)含量;蛋白质印迹法(Western blot)检测Cyt-c、Caspase-9、Bcl-2和Bax等4个目标蛋白表达情况;Tunel法检测腰椎骨细胞凋亡情况,实验结果进行统计学分析。结果 9周后,DEX组大鼠BMD、骨微结构状况、血清BALP水平均显著降低(P<0.05),GLEXG各组上述指标均有所增加(P<0.05)。DEX组Caspase-9、Bax、Cyt-c蛋白表达增加,Bcl-2蛋白表达下降(P<0.05),而GLEXG组Caspase-9、Bax、Cyt-c表达蛋白下降,Bcl-2蛋白表达增加(P<0.05)。DEX组TUNEL染色显示腰椎骨细胞凋亡数量增加(P<0.05),而GLEXG组较DEX组相比骨细胞凋亡数量有所减少(P<0.05)。结论 龟鹿二仙膏可以提高GIOP大鼠腰椎BMD,改善骨小梁结构,增加血清BALP含量,减少地塞米松引起的骨细胞凋亡数量,从而防治GIOP,其作用机制可能与提高抗凋亡蛋白Bcl-2的表达,抑制促凋亡蛋白Cyt-c、Caspase-9、Bax表达相关。

Objective To establish a rat model of glucocorticoid-induced osteoporosis(GIOP)to explore whether Guilu Erxian Ointment(龟鹿二仙膏)can inhibit bone cell apoptosis by regulating apoptosis-related proteins Cyt-c,Caspase-9,Bcl-2 and Bax,playing a role in preventing and treating GIOP in rats.Methods Sixty 3-month-old Wistar rats(half male and half female)were divided into 5 groups according to the random number table method,with 12 rats in each group:blank group(CON),model group(DEX),Guilu Erxian Ointment high-dose group(HD),Guilu Erxian Ointment medium-dose group(MD),Guilu Erxian Ointment low-dose group(LD).Except the CON group,the other groups were given intramuscular injection of dexamethasone(2.5 mg/kg,2 times/week)to replicate the GIOP rat model.The normal group was given the same amount of normal saline injection for 9 weeks.At the same time as the GIOP model was established,intragastric administration was started for 9 consecutive weeks.After the gavage,rats in each group were anesthetized by abdominal cavity.The rats were sacrificed and the blood was taken out from the abdominal aorta.Dual-energy X-ray was used to detect the lumbar bone mineral density(BMD).The morphology of lumbar trabecular bone was observed by HE staining.The serum bone alkaline phosphatase(BALP)was determined by ELISA.Western blot method was used to detect the expressions of Cyt-c,Caspase-9,Bcl-2 and Bax and other four target proteins.Tunel method was used to detect lumbar bone cell apoptosis and the experimental results were statistically analyzed.Results After 9 weeks,the BMD,bone microstructure status,and serum BALP levels of rats in the DEX group were significantly reduced(P<0.05),and the above indicators in each Guilu Erxian Ointment group were increased(P<0.05).The expressions of Caspase-9,Bax and Cyt-c protein in DEX group increased,and the expression of Bcl-2 protein decreased(P<0.05),while the expressions of Caspase9,Bax and Cyt-c protein in Guilu Erxian Ointment groups decreased,and the expression of Bcl-2 protein increased(P<0.05).TUNEL staining in the DEX group showed an increase in the number of bone cell apoptosis in the lumbar spine(P<0.05),while the number of bone cell apoptosis in the Guilu Erxian Ointment groups was less than that in the DEX group(P<0.05).Conclusion Guilu Erxian Ointment can increase lumbar spine BMD in GIOP rats,improve bone trabecular structure,increase serum BALP content,reduce the number of bone cell apoptosis caused by dexamethasone,and prevent GIOP.Its mechanism may be related to improving anti-apoptosis.The expression of apoptosis protein Bcl-2 inhibits the expressions of pro-apoptotic proteins Cyt-c,Caspase-9 and Bax.