基于“肾主骨”理论分析左归丸对大鼠破骨细胞凋亡的影响
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摘要
目的基于"肾主骨"理论分析左归丸对大鼠破骨细胞(OC)凋亡的影响。方法选取15只雌性健康Wistar大鼠,按照随机数字表法随机将其分为卵巢切除(OVX)加左归丸组、OVX组及对照组各5只,给予相关处理后,制备相应血清。取1日龄Wistar乳鼠制备大鼠OC及成骨细胞(OB)体外分离和培养,并分别分为6组和3组与上述血清共同培养,检测比较前6组OC所引起骨吸收陷窝数目和面积,以及后3组OB骨保护素(OPG)及破骨细胞核因子k B受体活化因子配体(RANKL)蛋白表达。结果随着培养时间延长,各组骨吸收陷窝数目及面积随之增大。培养第7天时,各组骨吸收陷窝数目及面积总体比较差异均有统计学意义(P <0. 05)。培养第7天时,OC+OVX血清组骨吸收陷窝数目及面积均高于OC+OVX含药血清组及OC+正常血清组,OC+OVX含药血清组骨吸收陷窝数目及面积均高于OC+正常血清组; OC+OB+OVX血清组骨吸收陷窝数目及面积均高于OC+OB+OVX含药血清组及OC+OB+正常血清组; OC+OB+OVX血清组骨吸收陷窝数目及面积均高于OC+OVX血清组,OC+OB+OVX含药血清组骨吸收陷窝数目及面积均低于OC+OVX含药血清组,差异有统计学意义(P <0. 05)。各组OB OPG及RANKL蛋白表达总体比较差异有统计学意义(P <0. 05)。OB+OVX血清组OB OPG蛋白表达明显低于OB+正常血清组,OB RANKL蛋白表达明显高于OB+正常血清组; OB+OVX含药血清组OB OPG蛋白表达高于OB+OVX血清组,OB RANKL蛋白表达低于OB+OVX血清组,差异有统计学意义(P <0. 05)。结论左归丸含药血清可直接抑制OC,还可通过OB间接抑制OC,作用机制可能与调节OB OPG、RANKL蛋白表达相关,提示基于"肾主骨"理论的左归丸可治疗骨质疏松症。
Objective To investigate the effect of Zuogui Pill on apoptosis of osteoclasts( OC) in rats based on the Theory of Kidney Dominating the Bone. Methods Fifteen healthy female Wistar rats were selected and randomly divided into three groups: ovariectomy( OVX) + Zuogui Pill group( n = 5),OVX group( n = 5) and the control group( n = 5) according to random number table method. After relevant treatment,the corresponding serum was prepared. OC and osteoblasts( OB) of rats were isolated from tibia,tibia,femur and skull of 1 day-old Wistar rats and cultured. They were divided into 6 groups and another 3 groups and co-cultured with the above serum. The number and area of bone resorption cavities caused by OC were compared in the first six groups. In addition,expression of OB osteoprotegerin( OPG) and receptor activator of nuclear factor kappa-Β ligand( RANKL) of OC was detected and compared in another 3 groups. Results With the extension of culture time,the number and area of bone resorption cavities were increased. At the 7thday after culture,there were significant differences in the number and area of bone resorption cavities( P < 0. 05). At the 7thday after culture,the number and area of bone resorption cavities in OC + OVX serum group were larger than those in OC + OVX drug-containing serum group and OC + normal serum group( P <0. 05),and those in OC + OVX drug-containing serum group were larger than those in OC + normal serum group. The number and area of bone resorption cavities in OC + OB + OVX serum group were larger than those in OC + OB + normal serum group and OC + OB + OVX drug-containing serum group,those indicators were larger in OC + OB + OVX serum group than in OC + OVX serum group,while those were smaller in OC + OB + OVX drug-containing serum group than in OC + OVX drug-containing serum group( P < 0. 05),there were significant differences in expressions of OB OPG and RANKL protein( P < 0. 05). The expression of OB OPG protein was lower in OB + OVX serum group than in OB + normal serum group,while expression of OB RANKL protein was significantly higher than OB + normal serum group( P < 0. 05). The epxression of OB OPG protein was higher but that of OB RANKL protein was lower in OB + OVX drug-containing serum group than in OB + OVX serum group,suggesting significant differences( P <0. 05). Conclusion The drug-containing serum of Zuogui Pill can directly inhibit OC and indirectly inhibit OC via OB. The mechanism of action may be related to the regulation of protein expression of OC OPG and RANKL,sugggesting that Zuogui Pill can treat osteoporosis based on the Theory of Kidney Dominating the Bone.
Objective To investigate the effect of Zuogui Pill on apoptosis of osteoclasts( OC) in rats based on the Theory of Kidney Dominating the Bone. Methods Fifteen healthy female Wistar rats were selected and randomly divided into three groups: ovariectomy( OVX) + Zuogui Pill group( n = 5),OVX group( n = 5) and the control group( n = 5) according to random number table method. After relevant treatment,the corresponding serum was prepared. OC and osteoblasts( OB) of rats were isolated from tibia,tibia,femur and skull of 1 day-old Wistar rats and cultured. They were divided into 6 groups and another 3 groups and co-cultured with the above serum. The number and area of bone resorption cavities caused by OC were compared in the first six groups. In addition,expression of OB osteoprotegerin( OPG) and receptor activator of nuclear factor kappa-Β ligand( RANKL) of OC was detected and compared in another 3 groups. Results With the extension of culture time,the number and area of bone resorption cavities were increased. At the 7thday after culture,there were significant differences in the number and area of bone resorption cavities( P < 0. 05). At the 7thday after culture,the number and area of bone resorption cavities in OC + OVX serum group were larger than those in OC + OVX drug-containing serum group and OC + normal serum group( P <0. 05),and those in OC + OVX drug-containing serum group were larger than those in OC + normal serum group. The number and area of bone resorption cavities in OC + OB + OVX serum group were larger than those in OC + OB + normal serum group and OC + OB + OVX drug-containing serum group,those indicators were larger in OC + OB + OVX serum group than in OC + OVX serum group,while those were smaller in OC + OB + OVX drug-containing serum group than in OC + OVX drug-containing serum group( P < 0. 05),there were significant differences in expressions of OB OPG and RANKL protein( P < 0. 05). The expression of OB OPG protein was lower in OB + OVX serum group than in OB + normal serum group,while expression of OB RANKL protein was significantly higher than OB + normal serum group( P < 0. 05). The epxression of OB OPG protein was higher but that of OB RANKL protein was lower in OB + OVX drug-containing serum group than in OB + OVX serum group,suggesting significant differences( P <0. 05). Conclusion The drug-containing serum of Zuogui Pill can directly inhibit OC and indirectly inhibit OC via OB. The mechanism of action may be related to the regulation of protein expression of OC OPG and RANKL,sugggesting that Zuogui Pill can treat osteoporosis based on the Theory of Kidney Dominating the Bone.
引文
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[12]王彬,李霄,马凰富,等.左归丸对少弱精子症模型大鼠睾丸组织干细胞因子及其mRNA表达的影响研究[J].中国全科医学,2018,21(3):334-337.
[13]梁超,金国琴,蒋嘉烨,等.左归丸及其拆方对大鼠骨髓间充质干细胞向神经样细胞分化的影响[J].中华中医药杂志,2018,32(8):225-227.
[14]安方玉,颜春鲁,刘永琦,等.左归丸对去势骨质疏松大鼠钙磷代谢和细胞核因子κB受体活化因子配体/骨保护素的影响[J].中华骨质疏松和骨矿盐疾病杂志,2018,11(3):261-266.
[15]吴倩,杨静,梁嘉丽,等.左归丸联合干细胞治疗对化疗后卵巢早衰大鼠的影响[J].中国中西医结合杂志,2017,37(11):1361-1366.
[16]孙千惠,吴琼,任艳玲,等.左、右归丸对去卵巢大鼠骨髓间充质干细胞凋亡的研究[J].辽宁中医杂志,2016,58(12):2648-2651.
[17]阳松威,孙晓峰,贺又舜,等.左归丸对化疗致卵巢早衰小鼠卵巢功能的影响[J].中成药,2016,38(4):717-722.
[18]杨锋,潘乐,马秋涛,等.左归丸对骨髓间充质干细胞成骨分化相关基因甲基化的影响[J].中医杂志,2015,56(15):1326-1329.
[19]张添昊,任艳玲,艾思羽,等.左归丸和右归丸对去卵巢大鼠股骨骨髓PPARγ、C/EBPβ、C/EBPα蛋白表达的影响[J].中医杂志,2017,58(6):511-515.
[20]杨锋,杨利学,李小群,等.左归丸、右归丸对去卵巢骨质疏松大鼠神经肽CGRP、SP、VIP、NPY的影响[J].中国骨质疏松杂志,2016,22(6):761-765.
[21]陈东阳,林庶茹,尚德阳,等.左归丸含药血清对骨髓间充质干细胞GPR48、BSP表达影响的实验研究[J].辽宁中医杂志,2017,59(1):178-180.
[2]吴佳莹,李岳泽,刘红,等.“肾主骨”机理研究——左归丸对Hepcidin、Fpn1及OPG/RANKL mRNA表达的影响[J].中国中医基础医学杂志,2017,22(11):1548-1551.
[3]刘立萍,李雪峰,姜波,等.补肾填精方对去卵巢大鼠炎症因子与破骨细胞活性的影响[J].辽宁中医杂志,2017,59(9):1982-1984.
[4]付剑江,刘婷,墨啸筝,等.左归丸抗乳腺癌破骨性骨转移机制探讨[J].中国实验方剂学杂志,2016,22(12):123-127.
[5]胡咏新,孙利,褚晓秋,等.滋阴补肾对甲强龙脉冲治疗Graves眼病骨代谢水平的影响[J].现代中西医结合杂志,2017,26(9):925-927.
[6]孙千惠,任艳玲,吴琼,等.左归丸及右归丸对骨髓间充质干细胞成骨及成脂分化后凋亡的影响[J].中华中医药杂志,2017,31(12):5331-5334.
[7]赵旭,郝传铮,杨仁旭,等.左归丸对去卵巢大鼠骨髓间充质干细胞基因表达谱时间序列的影响[J].中医杂志,2015,56(16):1414-1419.
[8] Marzban M,Gu Q P,Jia X. New analysis and computational study for the planar connected dominating set problem[J]. J Combinatorial Optimization,2015,32(1):1-28.
[9]祝雨田,王彬,马凰富,等.左归丸对生精功能障碍大鼠睾丸Bcl-2、Bax蛋白及mRNA表达的影响[J].中华中医药杂志,2017,31(12):5327-5330.
[10]詹菲,徐志伟,黄进,等.左归丸对衰老大鼠骨髓间充质干细胞增殖的影响[J].中华中医药杂志,2015,30(7):2518-2521.
[11]黄进,徐志伟,詹菲,等.左归丸对衰老大鼠骨髓间充质干细胞成骨和成脂分化的影响[J].中药新药与临床药理,2015,26(1):5-8.
[12]王彬,李霄,马凰富,等.左归丸对少弱精子症模型大鼠睾丸组织干细胞因子及其mRNA表达的影响研究[J].中国全科医学,2018,21(3):334-337.
[13]梁超,金国琴,蒋嘉烨,等.左归丸及其拆方对大鼠骨髓间充质干细胞向神经样细胞分化的影响[J].中华中医药杂志,2018,32(8):225-227.
[14]安方玉,颜春鲁,刘永琦,等.左归丸对去势骨质疏松大鼠钙磷代谢和细胞核因子κB受体活化因子配体/骨保护素的影响[J].中华骨质疏松和骨矿盐疾病杂志,2018,11(3):261-266.
[15]吴倩,杨静,梁嘉丽,等.左归丸联合干细胞治疗对化疗后卵巢早衰大鼠的影响[J].中国中西医结合杂志,2017,37(11):1361-1366.
[16]孙千惠,吴琼,任艳玲,等.左、右归丸对去卵巢大鼠骨髓间充质干细胞凋亡的研究[J].辽宁中医杂志,2016,58(12):2648-2651.
[17]阳松威,孙晓峰,贺又舜,等.左归丸对化疗致卵巢早衰小鼠卵巢功能的影响[J].中成药,2016,38(4):717-722.
[18]杨锋,潘乐,马秋涛,等.左归丸对骨髓间充质干细胞成骨分化相关基因甲基化的影响[J].中医杂志,2015,56(15):1326-1329.
[19]张添昊,任艳玲,艾思羽,等.左归丸和右归丸对去卵巢大鼠股骨骨髓PPARγ、C/EBPβ、C/EBPα蛋白表达的影响[J].中医杂志,2017,58(6):511-515.
[20]杨锋,杨利学,李小群,等.左归丸、右归丸对去卵巢骨质疏松大鼠神经肽CGRP、SP、VIP、NPY的影响[J].中国骨质疏松杂志,2016,22(6):761-765.
[21]陈东阳,林庶茹,尚德阳,等.左归丸含药血清对骨髓间充质干细胞GPR48、BSP表达影响的实验研究[J].辽宁中医杂志,2017,59(1):178-180.