肾衰营养胶囊对肾性骨病模型大鼠肾功能和骨代谢的改善作用及其对BMP-7/Smads信号通路的影响

doi:10.13481/j.1671-587X.20240309

摘要/Abstract

摘要：

目的探讨肾衰营养胶囊对肾性骨病模型大鼠的改善作用及其对骨形态发生蛋白（BMP）-7/Smads信号通路的影响，阐明肾性骨病模型大鼠肾功能和骨代谢与肾性骨病的关系。方法选取50只SPF级SD大鼠，随机选取10只大鼠作为对照组，另外40只大鼠建立肾性骨病模型。将30只造模成功大鼠分为模型组、阳性对照组和肾衰营养胶囊组，每组各10只。对照组和模型组大鼠采用生理盐水灌胃，阳性对照组大鼠给予0.01 mg·kg^－1骨化三醇灌服，肾衰营养胶囊组大鼠给予1.2 g·kg^－1肾衰营养胶囊灌服，均灌胃12周。采用HE染色观察各组大鼠股骨组织病理形态表现，全自动生化分析仪和化学发光法检测各组大鼠血清中肾功能和钙磷代谢指标，实时荧光定量PCR（RT-qPCR）法检测各组大鼠股骨组织中BMP-7和Smad1/5/8 mRNA表达水平，Western blotting法检测各组大鼠股骨组织中BMP-7、磷酸化BMP-7（p-BMP-7）、Smad1/5/8和磷酸化Smad1/5/8（p-Smad1/5/8）蛋白表达水平。结果 HE染色，对照组大鼠骨小梁排列正常，成骨细胞和类骨质面积未发生改变；模型组大鼠骨小梁宽度和平均类骨质面积增加，成骨细胞数量减少；阳性对照组和肾衰营养胶囊组大鼠骨小梁宽度及平均类骨质面积减小，成骨细胞数量增加。与对照组比较，模型组大鼠血尿素氮（BUN）和血肌酐（Scr）水平均升高（P<0.05）；与模型组比较，阳性对照组和肾衰营养胶囊组大鼠BUN及Scr水平均降低（P<0.05）；与阳性对照组比较，肾衰营养胶囊组大鼠BUN和Scr水平均降低（P<0.05）。与对照组比较，模型组大鼠血清中钙离子（Ca²⁺）水平降低（P<0.05），磷离子（P^3－）、甲状旁腺激素（PTH）和碱性磷酸酶（ALP）水平均升高（P<0.05）；与模型组比较，阳性对照组和肾衰营养胶囊组大鼠血清中Ca²⁺水平升高（P<0.05），P^3－、PTH和ALP水平均降低（P<0.05）；与阳性对照组比较，肾衰营养胶囊组大鼠血清中Ca²⁺水平升高（P<0.05），P^3－、PTH和ALP水平均降低（P<0.05）。RT-qPCR法，与对照组比较，模型组大鼠股骨组织中BMP-7和Smad1/5/8 mRNA表达水平降低（P<0.05）；与模型组比较，阳性对照药组和肾衰营养胶囊组大鼠股骨组织中BMP-7及Smad1/5/8 mRNA表达水平升高（P<0.05）；与阳性对照组比较，肾衰营养胶囊组大鼠股骨组织中BMP-7和Smad1/5/8 mRNA表达水平升高（P<0.05）。Western blotting法，与对照组比较，模型组大鼠股骨组织中BMP-7、p-BMP-7、Smad1/5/8和p-Smad1/5/8蛋白表达水平均降低（P<0.05）；与模型组比较，阳性对照组和肾衰营养胶囊组大鼠股骨组织中BMP-7、p-BMP-7、Smad1/5/8和p-Smad1/5/8蛋白表达水平均升高（P<0.05）；与阳性对照组比较，肾衰营养胶囊组大鼠股骨组织中BMP-7、p-BMP-7、Smad1/5/8和p-Smad1/5/8蛋白表达水平均升高（P<0.05）。结论肾衰营养胶囊可改善肾性骨病模型大鼠的肾功能和钙磷代谢紊乱，促进骨髓基质细胞增殖，改善骨代谢情况，其机制可能与激活BMP-7/Smads信号通路有关。

关键词: 肾性骨病, 肾衰营养胶囊, 骨形态发生蛋白7, Smad同源物重组蛋白, 骨密度

Abstract:

Objective To discuss the improvement effect of Shenshuai Yingyang Capsule in the renal osteodystrophy model rats and its effect on the bone morphogenetic protein （BMP）-7/Smads signaling pathway，and to clarify the relationship between renal function and bone metabolism in the renal osteodystrophy model rats. Methods Fifty SPF SD rats were selected， and 10 of which were randomly chosen as control group， and the remaining 40 rats were used to establish the renal osteodystrophy model. Thirty successful modeling rats were divided into model group， positive control group， and Shenshuai Yingyang Capsule group， and there were 10 rats in various groups. The rats in control and model groups were given saline， the rats in positive control group were given 0.01 mg·kg^-1 calcitriol， and the rats in Shenshuai Yingyang Capsule group were given 1.2 g·kg^-1 Shenshuai Yingyang Capsule， all the rats were treated for 12 weeks. HE staining was used to observe the pathomorphology of femur tissue of the rats in various groups； automatic biochemical analyzer and chemiluminescence were used to detect the kidney function and calcium-phosphorus metabolism indicators in serum of the rats in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of BMP-7 and Smad1/5/8 mRNA in femur tissue of the rats in various groups； Western blotting method was used to detect the expression levels of BMP-7， phosphorylated BMP-7（p-BMP-7）， Smad1/5/8， and phosphorylated-Smad1/5/8 （p-Smad1/5/8） proteins in femur tissue of the rats in various groups. Results The HE staining results showed that there was normal trabecular arrangement in control group with unchanged osteoblasts and osteoid area. In model group， the trabecular width and average osteoid area were increased， while the number of osteoblasts was decreased. In positive control and Shenshuai Yingyang Capsule groups， the trabecular width and average osteoid area were decreased， and the number of osteoblasts was increased. Compared with control group，the levels of blood urea nitrogen （BUN） and serum creatinine （Scr） of the rats in model group were increased （P<0.05）； compared with model group， the levels of BUN and Scr of the rats in positive control and Shenshuai Yingyang Capsule groups were decreased （P<0.05）； compared with positive control group， the levels of BUN and Scr of the rats in Shenshuai Yingyang Capsule group were decreased （P<0.05）. Compared with control group， the level of calcium ion （Ca²⁺） in serum of the rats in model group was decreased（P<0.05）， and the levels of phosphorus ion （P^3-）， parathyroid hormone （PTH）， and alkaline phosphatase （ALP） were increased （P<0.05）； compared with model group， the levels of Ca²⁺ in serum of the rats in positive control and Shenshuai Yingyang Capsule groups were increased（P<0.05）， and the levels of P^3-， PTH， and ALP were decreased （P<0.05）； compared with positive control group， the level of Ca²⁺ in serum of the rats in Shenshuai Yingyang Capsule group was increased， and the levels of P^3-， PTH， and ALP were decreased （P<0.05）. The RT-qPCR results showed that compared with control group，the expression levels of BMP-7 and Smad1/5/8 mRNA in femur tissue of the rats in model group were decreased （P<0.05）； compared with model group， the expression levels of BMP-7 and Smad1/5/8 mRNA of the rats in positive control drug group and Shenshuai Yingyang Capsule group were increased （P<0.05）； compared with positive control group， the expression levels of BMP-7 and Smad1/5/8 mRNA in femur tissue of the rats in Shenshuai Yingyang Capsule group were increased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of BMP-7， p-BMP-7， Smad1/5/8， and p-Smad1/5/8 proteins in femur tissue of the rats in model group were decreased （P<0.05）； compared with model group， the expression levels of BMP-7， p-BMP-7， Smad1/5/8， and p-Smad1/5/8 proteins in femur tissue of the rats in positive control group and Shenshuai Yingyang Capsule group were increased （P<0.05）； compared with positive control group， the expression levels of BMP-7， p-BMP-7， Smad1/5/8， and p-Smad1/5/8 proteins in femur tissue of the rats in Shenshuai Yingyang Capsule group were increased （P<0.05）. Conclusion Shenshuai Yingyang Capsule can improve the kidney function and calcium-phosphorus metabolism disorders in the renal osteodystrophy model rats， promote the proliferation of the bone marrow stromal cells， and enhances the bone metabolism； its mechanism may be related to the activation of BMP-7/Smads signaling pathway.

Key words: Renal osteodystrophy, Shenshuai Yingyang Capsule, Bone morphogenetic protein-7, Recombinant mothers against decapentaplegic homolog, Bone density

中图分类号:

R692

陈杰彬,胡蓉,吕佩佳,李成杰,魏连波. 肾衰营养胶囊对肾性骨病模型大鼠肾功能和骨代谢的改善作用及其对BMP-7/Smads信号通路的影响[J]. 吉林大学学报(医学版), 2024, 50(3): 658-665.

Jiebin CHEN,Rong HU,Peijia LYU,Chengjie LI,Lianbo WEI. Improvement effect of Shenshuai Yingyang Capsule on renal function and bone metabolism in renal osteodystrophy model rats and its effect on BMP-7/Smads signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2024, 50(3): 658-665.

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参考文献 21

1	PAZIANAS M， MILLER P D. Osteoporosis and chronic kidney disease-mineral and bone disorder （CKD-MBD）： back to basics［J］. Am J Kidney Dis， 2021， 78（4）： 582-589.
2	CANNATA-ANDA J B， MARTN-CARRO B， MARTN-VRGALA J， et al. Chronic kidney disease-mineral and bone disorders： pathogenesis and management［J］. Calcif Tissue Int， 2021， 108（4）： 410-422.
3	GINSBERG C， IX J H. Diagnosis and management of osteoporosis in advanced kidney disease： a review［J］. Am J Kidney Dis， 2022， 79（3）： 427-436.
4	伍晓辉，尤海岩，胡蓉，等. 肾衰营养胶囊对肾性骨病大鼠骨代谢及股骨组织BMP-2表达的影响［J］. 天津医药， 2020， 48（2）： 91-95.
5	张晶晶，武垣伶，黄丹娜，等. BMP-7/Smads通路参与EndoMT在大鼠HHPH中的作用［J］. 中国病理生理杂志， 2020， 36（2）： 316-322.
6	郭华慧，梁群卿，李贺生，等. 加味六味地黄汤对CKD-MBD模型大鼠肾/骨组织损伤的影响［J］. 中国药房， 2021， 32（6）： 690-696.
7	PEREIRA L， MAGALHÃES J， MENDONÇA L， et al. Evaluation of renal osteodystrophy and serum bone-related biomarkers in a peritoneal dialysis population［J］. J Bone Miner Res， 2022， 37（9）： 1689-1699.
8	EVENEPOEL P， CUNNINGHAM J， FERRARI S， et al. European Consensus Statement on the diagnosis and management of osteoporosis in chronic kidney disease stages G4-G5D［J］. Nephrol Dial Transplant， 2021， 36（1）： 42-59.
9	JØRGENSEN H S， BEHETS G， BAMMENS B， et al. Patterns of renal osteodystrophy 1 year after kidney transplantation［J］. Nephrol Dial Transplant， 2021， 36（11）： 2130-2139.
10	ABDALBARY M， SOBH M， ELNAGAR S， et al. Management of osteoporosis in patients with chronic kidney disease［J］. Osteoporos Int， 2022， 33（11）： 2259-2274.
11	郑华，孙艺，纪镇华. 肾衰营养胶囊改善CRF大鼠骨骼肌萎缩中Wnt7a/PCP信号通路的作用探析［J］. 航空航天医学杂志， 2020， 31（11）： 1324-1325.
12	杨勇，王霄一，丁敏，等. 肾衰宁胶囊对慢性肾功能衰竭患者营养状况及血液流变学的影响［J］. 中国基层医药， 2017， 24（10）： 1450-1454.
13	赵继荣，杨涛，赵宁，等. 杜仲诱导骨髓间充质干细胞成骨分化防治骨质疏松症相关信号通路研究进展［J］. 中国骨质疏松杂志， 2020， 26（12）： 1868-1872.
14	SHATAGOPAM K， BAHLER C D， SUNDARAM C P. Renorrhaphy techniques and effect on renal function with robotic partial nephrectomy［J］. World J Urol， 2020， 38（5）： 1109-1112.
15	SÁNCHEZ ARCO A M， SEGURA JIMÉNEZ I， PLATA ILLESCAS C， et al. Renal function in receptors with simultaneous liver-kidney transplant from the same donor［J］. Transplant Proc， 2022， 54（1）： 45-47.
16	张媛，赵瑾，朱小静. 血液透析方式的选择对肾性骨病患者骨代谢、血清生化指标及生活质量的影响［J］. 兰州大学学报（医学版）， 2020， 46（4）： 73-77.
17	刘丽巧，余建华，米弘瑛，等. 每天予1200 IU维生素D滴剂早产儿血清25-（OH）D水平、钙磷代谢及代谢性骨病发生情况观察［J］. 山东医药， 2022， 62（30）： 50-53.
18	陈勇，黄乔木，葛亮，等. 十味芪黄益肾方对腺嘌呤所致肾纤维化大鼠模型TGF-β1/Smads/BMP-7信号通路的影响［J］. 西部医学， 2019， 31（8）： 1178-1184.
19	傅亮，李玉杰，来媛媛，等. 黄芪注射液联合葛根素注射液对HK-2细胞TGF-β1/Smads及BMP-7/Smad5信号通路的影响［J］. 环球中医药， 2019， 12（3）： 343-347.
20	吴伟东，方红槟，吴大华，等. 髋关节置换辅助维生素D治疗老年骨质疏松股骨颈骨折的远期疗效及对BMP-7/25-（OH）-D-3水平的影响［J］. 中华内分泌外科杂志， 2021， 15（6）： 641-645.
21	张舒，李然，徐先知，等. BMP-7/Smad1/5/8信号通路在肾缺血后处理减轻大鼠肾缺血再灌注损伤中的作用［J］. 药学与临床研究， 2021， 29（1）： 7-11.

Group	BUN[c_B/(mmol·L^－1)]	Scr[c_B/(μmol·L^－1)]
Control	5.36±0.64	27.95±3.88
Model	17.28±2.53^*	171.25±25.03^*
Positive control	14.32±2.34^△	123.18±21.14^△
Shenshuai Yingyang Capsule	11.36±2.10^△#	68.36±7.54^△#

Group	Ca²⁺[c_B/(mmol·L^－1)]	P^3－[c_B/(mmol·L^－1)]	PTH[ρ_B/(ng·L^－1)]	ALP[λ_B/(U·L^－1)]
Control	2.35±0.35	1.15±0.20	1.42±0.23	36.52±4.33
Model	1.78±0.25^*	3.18±0.41^*	3.52±0.45^*	79.55±8.74^*
Positive control	1.94±0.29^△	2.61±0.34^△	2.44±0.34^△	61.02±7.18^△
Shenshuai Yingyang Capsule	2.28±0.33^△#	2.25±0.31^△#	1.85±0.32^△#	48.39±5.47^△#

Group	BMD（g·cm^－2）	Bone volume fraction（η/%）
Control	0.18±0.04	0.57±0.06
Model	0.11±0.01^*	0.31±0.03^*
Positive control	0.13±0.02^△	0.39±0.04^△
Shenshuai Yingyang Capsule	0.16±0.03^△#	0.48±0.05^△#

Group	BMP-7 mRNA	Smad1/5/8 mRNA
Control	1.00±0.01	1.00±0.01
Model	0.32±0.04^*	0.33±0.04^*
Positive control	0.51±0.06^△	0.68±0.07^△
Shenshuai Yingyang Capsule	0.84±0.09^△#	0.82±0.09^△#

Group	BMP-7 protein	p-BMP-7 protein	Smad1/5/8 protein	p-Smad1/5/8 protein
Control	1.00±0.01	1.00±0.01	1.00±0.01	1.00±0.01
Model	0.34±0.04^*	0.21±0.03^*	0.41±0.05^*	0.34±0.05^*
Positive control	0.55±0.06^△	0.45±0.05^△	0.63±0.07^△	0.65±0.07^△
Shenshuai Yingyang Capsule	0.79±0.08^△#	0.68±0.07^△#	0.85±0.09^△#	0.77±0.08^△#