加味四君子汤对阿霉素肾病大鼠骨代谢的影响

Effect of Modified Sijunzi Decoction on the Bone Metabolism of Adriamycin Induced Nephropathy Rats

目的探讨加味四君子汤对泼尼松干预的阿霉素肾病大鼠骨代谢的影响。方法制作大鼠阿霉素肾病模型,将50只SD大鼠随机分为5组,即模型组、激素组、中药组、中药加激素组及正常组。各组于造模后7、21、35天收集24 h尿标本,以双缩脲比色法测定大鼠24 h尿蛋白,以ELISA法测定各组血清骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator for NF-κB ligand,RANKL)、骨钙素(osteocalcin,BGP)以及抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRACP)水平,采用实时定量PCR和Western blot检测胫骨组织中OPG、RANKL的表达。结果 (1)与正常组比较,模型组第7、21、35天24 h尿蛋白均有不同程度增高(P<0.05,P<0.01);与模型组同期比较,激素组及中药加激素组24 h尿蛋白均有不同程度下降,差异有统计学意义(P<0.05,P<0.01);并随着治疗时间的延长,降低更明显(P<0.05,P<0.01);中药组治疗35天,与模型组比较,差异亦有统计学意义(P<0.05)。(2)与本组21天比较,激素组TRACP、RANKL均明显升高(P<0.05,P<0.01);与模型组比较,第21、35天,激素组TRACP、RANKL均升高(P<0.05,P<0.01),OPG、BGP均降低(P<0.05,P<0.01);与中药组同期比较,激素组、中药加激素组OPG均降低,RANKL均升高(P<0.05,P<0.01);且激素组TRACP升高,BGP降低(P<0.05,P<0.01);与激素组同期比较,中药加激素组OPG、BGP升高(P<0.05,P<0.01),RANKL降低(P<0.01);第35天,TRACP降低(P<0.01)。(3)与正常组比较,模型组第21天OPG、RANKL mRNA升高(P<0.05,P<0.01),第35天OPG、RANKL mRNA降低(P<0.01)。与中药组同期比较,激素组OPG mRNA降低(P<0.01),RANKL mRNA升高(P<0.05);中药加激素组OPG mRNA降低(P<0.05)。(4)与本组21天比较,激素组OPG降低(P<0.05),RANKL升高(P<0.05),中药加激素组RANKL降低(P<0.05)。与模型组同期比较,激素组OPG降低(P<0.01),RANKL升高(P<0.01)。与中药组同期比较,激素组、中药加激素组OPG均降低(P<0.01),RANKL均升高(P<0.01);与激素组同期比较,中药加激素组OPG均升高(P<0.01),RANKL均降低(P<0.01)。结论泼尼松能通过OPG/RANKL/RANK通路诱导骨质疏松。加味四君子汤在降蛋白尿的同时,可通过上述通路促进成骨细胞分化,抑制破骨细胞生成,从而减缓泼尼松诱导的骨质疏松的形成。

Objective To explore the effect of Modified Sijunzi Decoction （MSD） on the bone me- tabolism of prednisone intervened adriamycin-induced nephropathy rats. Methods The adriamycin-induced nephropathy rat model was prepared. Totally 50 SD rats were randomly divide into five groups, i.e.,the model group, the hormone group, the Chinese medicine （CM） group, the CM ＋hormone group, and the normal control group. The 24-h urine samples were collected on the 7th ,21 st, and 35th day after mod- eling. The 24-h urine protein was measured by biuret colorimetry. Serum levels of osteoprotegerin （OPG）, receptor activator of nuclear factor-κB ligand （RANKL）, osteocalcin （BGP）, and tartrate-resistant acid phosphatase （TRACP） were determined by ELISA. Expressions of OPG and RANKL in the tibia tissue were detected using real-time quantitative PCR and Western blot. Results （1） Compared with the normal control group, the 24-h urine protein increased in each group on the 7th, 21 st, and 35th day （P 〈 0.05, P 〈0.01）. Compared with the model group, the 24-h urinary protein decreased in the hormone group and the CM ＋hormone group （P 〈0.05, P 〈0.01 ）. The decrement was more obvious along with the treatment time went by （P 〈0.05, P 〈0.01 ）. There was statistical difference in the reduction of urine protein on the 35th day between the CM group and the model group （P 〈0.05）. （2） Compared with the 21st-day of the same group, the serum levels of TRACP and RANKL increased （P 〈0.05, P 〈0.01 ）. Compared with the model group, the serum levels of the TRACP and RANKL increased （P 〈0.05, P 〈0.01 ）, OPG and BGP de- creased （P〈0.05, P〈0.01） in the hormone group. Compared with the CM group at the same period, serum OPG level decreased and the RANKL level increased in the hormone group and the CM ＋ hormone group （P〈0.05, P 〈0.01 ）. Besides, the serum level of TRACP increased and BGP decreased （P 〈0.05, P 〈 0.01）. Compared with the hormone group at the same period, OPG and BGP increased （P 〈0.05, P 〈 0.01）, RANKL decreased （P 〈0.01） in the CM ＋hormone group. On the 35th day TRACP decreased （P 〈 0.01）. （3） Compared with the normal group, mRNA expressions of OPG and RANKL on the 21 st day in- creased （P 〈0.05, P 〈0.01 ）, mRNA expressions of OPG and RANKL on the 35th day decreased in the model group （P 〈0.01 ）. Compared with the CM group at the same period, OPG mRNA expression de- creased （P 〈0.01 ） and RANKL mRNA expression increased in the hormone group （P 〈0.05）. OPG mRNA expression decreased in the CM ＋hormone group （P 〈0.05）. （4） Compared with the hormone group on the 21 st day, the OPG level decreased and the RANKL protein increased （both P 〈0.05）. RANKL decreased in the CM ＋hormone group （P 〈0.05）. Compared with the model group at the same period, OPG decreased and RANKL increased in the hormone group （P 〈0.01 ）. Compared with the CM group at the same period, OPG decreased （P 〈0.01 ）, RANKL increased （P 〈0.01 ） in the hormone group and the CM ＋ hormone group. Compared with the hormone group at the same period, OPG increased and RANKL decreased in the CM ＋ hormone group （ both P 〈 0.01 ）. Conclusions Prednisone could induce osteoporosis through the OPG/RANKL/RANK pathway. MSZ could slow down the formation of prednisone-induced osteoporosis through promoting osteoblast differentiation, and inhibiting osteoclastogenesis.