基于中医脏腑理论的桂枝芍药知母汤对SD雌鼠骨质疏松症模型骨代谢指标和骨组织蛋白表达水平的影响

Effect of Guizhi Shaoyao Zhimu Decoction on levels of bone metabolism and bone tissue protein expression in osteoporosis model SD female rats based on the viscera theories of traditional Chinese medicine

目的 分析桂枝芍药知母汤对SD雌鼠骨质疏松症(OP)模型骨代谢指标和骨组织蛋白表达水平的影响,并基于中医脏腑理论探讨桂枝芍药知母汤治疗OP的药理机制。方法 将40只SD雌鼠按照随机数字表法分为空白对照组、模型组、骨化醇组和汤剂组,每组10只。模型组、骨化醇组和汤剂组手术摘除双侧卵巢,空白对照组开腹手术只摘取部分卵巢周围脂肪组织。术后1周,模型组、骨化醇组和汤剂组给予地塞米松磷酸钠注射液肌内注射,1 mg/kg, 2次/周,空白对照组肌内注射等量0.9%氯化钠溶液,连续6周。骨化醇组以0.1 mg/kg阿法骨化醇+羧甲基纤维素钠研磨混悬液灌胃,汤剂组以1.6 mg/kg桂枝芍药知母汤冻干粉+羧甲基纤维素钠研磨混悬液灌胃,空白对照组和模型组以羧甲基纤维素钠10 mL/kg灌胃,1次/d, 6次/周,连续8周。采集相关标本检测骨代谢指标[骨体积(BV)、组织体积(TV)、骨矿密度(BMD)、骨小梁数目(Tb.N)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp)、骨小梁连接密度(Tb.CD)及结构模式指数(SMI)]和骨组织蛋白[基质金属蛋白酶-9(MMP-9)、组织蛋白酶K(CTSK)、抗酒石酸酸性磷酸酶5b(TRAP5b)、核转录因子-κB受体活化因子配体(RANKL)、骨保护素(OPG)及碱性磷酸酶(ALP)]水平,然后进行统计学分析。结果 与空白对照组相比,模型组、骨化醇组和汤剂组BV/TV、BMD、Tb.N、Tb.Th、Tb.Sp及Tb.CD明显降低,SMI明显升高,差异均有统计意义(P<0.05);与模型组相比,骨化醇组和汤剂组BV/TV、BMD、Tb.N、Tb.Th及Tb.CD明显升高,Tb.Sp、SMI明显降低,差异均有统计意义(P<0.05);与骨化醇组相比,汤剂组BV/TV、BMD、Tb.N、Tb.Th及Tb.CD明显降低,Tb.Sp、SMI明显升高,差异均有统计意义(P<0.05)。与空白对照组相比,模型组、骨化醇组和汤剂组MMP-9、CTSK、TRAP5b及RANKL明显升高,ALP、OPG明显降低,差异均有统计意义(P<0.05);与模型组相比,骨化醇组和汤剂组MMP-9、CTSK、TRAP5b及RANKL明显降低,ALP、OPG明显升高,差异均有统计意义(P<0.05);与骨化醇组相比,汤剂组MMP-9、CTSK、TRAP5b及RANKL明显升高,ALP、OPG明显降低,差异均有统计意义(P<0.05)。结论 桂枝芍药知母汤能够抑制SD雌鼠OP模型RANKL表达,提高OPG表达,濡养骨骼以促进骨代谢平衡,起到治疗或改善骨质疏松症的作用。

Objective To analyze the effect of Guizhi Shaoyao Zhimu Decoction on bone metabolism, bone tissue protein in osteoporosis(OP) model SD female rats, and to explore the pharmacological mechanism of Guizhi Shaoyao Zhimu decoction in the treatment of OP based on the viscera theory of traditional Chinese medicine. Methods Forty female SD rats were divided into blank control group, model group, calcitol group and decoction group according to random number table method, with 10 rats in each group. The model group, the calciferol group and the decoction group were operated on to remove the bilateral ovaries, while the blank control group was operated on to remove only part of the adipose tissue around the ovaries. One week after operation, the model group, calciferol group and decoction group were intramuscularly injected with dexamethasone sodium phosphate injection, 1 mg/kg, twice a week, and the blank control group was intramuscularly injected with 0.9% sodium chloride solution of the same amount for 6 weeks. In the calciferol group, 0.1 mg/kg alfacalciferol+sodium carboxymethyl cellulose grinding suspension was administered by gavage;in the decoction group, 1.6 mg/kg Guizhi Shaoyao Zhimu Decoction freeze-dried powder+sodium carboxymethyl cellulose grinding suspension was administered by gavage;in the blank control group and model group, 10 mL/kg sodium carboxymethyl cellulose was administered by gavage once a day, 6 times a week, for 8 consecutive weeks. The levels of bone metabolism indexes [bone volume(BV), tissue volume(TV), bone mineral density(BMD), trabecular bone number(Tb.N), trabecular bone thickness(Tb.Th), trabecular bone separation(Tb.Sp), trabecular bone connection density(Tb.CD) and structural pattern index(SMI) ] and bone tissue protein [matrix metalloproteinase-9(MMP-9), cathepsin K(CTSK), tartrate resistant acid phosphatase 5b(TRAP5b),nuclear transcription factor-κB receptor activator ligand(RANKL), osteoprotegerin(OPG) and alkaline phosphatase(ALP)] were collected and statistically analyzed. Results Compared with the blank control group, BV/TV, BMD, Tb.N, Tb.Th, Tb.Sp and Tb.CD of the model group, calciferol group and decoction group were significantly decreased, and SMI was significantly increased, the differences were statistically significant(P<0.05). Compared with the model group, BV/TV, BMD, Tb.N, Tb.Th and Tb.CD in the calcidol group and decoction group were significantly increased, while Tb.Sp and SMI were significantly decreased, the differences were statistically significant(P<0.05). Compared with the calcidol group, BV/TV, BMD, Tb.N, Tb.Th and Tb.CD in the decoction group were significantly decreased, while Tb.Sp and SMI were significantly increased, the differences were statistically significant(P<0.05). Compared with the blank control group, MMP-9, CTSK, TRAP5b and RANKL in the model group, calciferol group and decoction group were significantly increased, while ALP and OPG were significantly decreased, the differences were statistically significant(P<0.05). Compared with the model group, MMP-9, CTSK, TRAP5b and RANKL in the calcidol group and decoction group were significantly decreased, while ALP and OPG were significantly increased, the differences were statistically significant. Compared with the calcidol group, MMP-9, CTSK, TRAP5b and RANKL in the decoction group were significantly increased, while ALP and OPG were significantly decreased, the differences were statistically significant(P<0.05). Conclusion Guizhi Shaoyao Zhimu Decoction can inhibit the expression of RANKL in SD female rats of OP model, increase the expression of OPG, nourish the bones to promote the balance of bone metabolism, and play a role in the treatment or improvement of osteoporosis.