丹酚酸B对去卵巢骨质疏松小鼠的生物学变化及机制

doi:10.3969/j.issn.1000-8535.2023.09.004

摘要/Abstract

摘要： 目的丹酚酸B是传统中药丹参的重要生物活性组分,临床应用广泛。新近发现,丹酚酸B具有防治骨质疏松的作用。本研究拟在前期工作基础上,系统地研究丹酚酸B对去卵巢骨质疏松小鼠的作用,探讨其对小鼠间充质干细胞（MSCs）成骨分化的影响。方法将18只8周龄SPF级别的C57雌性小鼠分为假手术组、骨质疏松组、丹酚酸B治疗组。骨质疏松组和丹酚酸B治疗组行双侧卵巢摘除术。而假手术组则保持正常的卵巢结构,同时去除局部的脂肪。3组均于术后3日内给予抗生素进行抗感染治疗。4周内,丹酚酸B治疗组给予丹酚酸B 2.5 mg/kg腹腔注射,连续12周,2天1次,其余2组在相同的时间内给予等量生理盐水。16周后,在麻醉状态下将所有小鼠处死。应用MicroCT测量了小鼠右后股骨的骨密度。采用qRT-PCR技术,分析小鼠左后股骨骨髓MSCs中Runx2和Osterix的表达。将小鼠右后股骨进行液氮研磨处理,提取蛋白质,用WB法测定OPG和RANKL的含量。结果骨质疏松组小鼠股骨骨密度比假手术组低（P<0.05）,丹酚酸B治疗组小鼠股骨骨密度比骨质疏松组高,但差异无显著性意义（P<0.05）。骨质疏松组小鼠Runx2和Osterix水平低于假手术组（P<0.05）,丹酚酸B治疗组小鼠Runx2和Osterix水平比骨质疏松组高（P<0.05）。骨质疏松组小鼠OPG和RANKL蛋白水平低于假手术组（P<0.05）,丹酚酸B组小鼠OPG和RANKL蛋白水平比骨质疏松组高（P<0.05）。结论绝经后骨质疏松症早期对小鼠的骨质疏松具有一定的影响,但还需要更多的实验来验证本研究的结论。

关键词: 丹酚酸B, 去卵巢, 骨质疏松, 生物学变化

Abstract: Objective Salvianolic acid B is an important bioactive component of traditional Chinese medicine Salvia miltiorrhiza and is widely used in clinic．Recently,salvianolic acid B has been found to have the effect of preventing osteoporosis．On the basis of previous work,this study intends to systematically explore the effect of salvianolic acid B on ovariectomized mice with osteoporosis,and its effect on the osteogenic differentiation of mouse mesenchymal stem cells（MSCs）．Methods Eighteen eight-week-old SPF C57 female mice were divided into sham operation group,osteoporosis group and salvianolic acid B treatment group（6 mice in each group）．The osteoporosis group and salvianolic acid B group underwent bilateral ovariectomy．The sham group maintained normal ovarian structure while removing local fat tissue．All three groups were given antibiotics for anti-infection treatment within 3 days after operation．Within 4 weeks,salvianolic acid B treatment group was given salvianolic acid B 2.5 mg/kg intraperitoneal injection for 12 weeks,once every 2 days,and the other 2 groups were given the same amount of saline at the same time．After 16 weeks,all mice were sacrificed under anesthesia．The bone density of the mouse right posterior femur was measured by MicroCT．The expressions of Runx2 and Osterix in the bone marrow of mouse left posterior femur were analyzed by qRT-PCR．The right posterior femur of mice was ground with liquid nitrogen to extract protein,and the contents of OPG and RANKL were determined by WB．Results The bone mineral density of the femur in the osteoporosis group was lower than that in the sham operation group（P<0.05）,and the bone mineral density of the femur in the salvianolic acid B treatment group was higher than that in the osteoporosis group,but the difference was not significant（P<0.05）．The levels of Runx2 and Osterix in the osteoporosis group were significantly lower than those in the sham operation group（P<0.05）,and the levels of Runx2 and Osterix in the salvianolic acid B treatment group were higher than those in the osteoporosis group（P<0.05）．The levels of OPG and RANKL protein in the osteoporosis group were significantly lower than those in the sham operation group（P<0.05）,while the levels of OPG and RANKL protein in the salvianolic acid B group were higher than those in the osteoporosis group（P<0.05）．Conclusions The early stage of postmenopausal osteoporosis has certain effects on osteoporosis in mice,but more experiments are needed to verify the conclusions of this study．

Key words: salvianolic acid B, ovariectomy, osteoporosis, biological changes

岑美婷. 丹酚酸B对去卵巢骨质疏松小鼠的生物学变化及机制[J]. 广州医药, 2023, 54(9): 23-27.

CEN Meiting. Biological changes and mechanism of salvianolic acid B in ovariectomized osteoporosis mice[J]. Guangzhou Medical Journal, 2023, 54(9): 23-27.

参考文献

[1] WU D,CLINE-SMITH A,SHASHKOVA E,et al.T-Cell Mediated Inflammation in Postmenopausal Osteoporosis[J].Front Immunol,2021(12):687551.
[2] MOHSIN S,BANIYAS M M,ALDARMAKI R S,et al.An update on therapies for the treatment of diabetes-induced osteoporosis[J].Expert Opin Biol Ther,2019,19(9):937-948.
[3] WANG M,LI J,WANG D,et al.The effects of mesenchymal stem cells on the chemotherapy of colorectal cancer[J].Biomed Pharmacother,2023(160):114373.
[4] RAHMANI-MOGHADAM E,ZARRIN V,MAHMOODZADEH A,et al.Comparison of the characteristics of breast milk-derived stem cells with the stem cells derived from the other sources:A comparative review[J].Curr Stem Cell Res Ther,2022,17(1):71-90.
[5] GUAN M,YAO W,LIU R,et al.Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass[J].Nat Med,2012,18(3):456-462.
[6] JIANG Z,GAO W,HUANG L.Tanshinones,critical pharmacological components in Salvia miltiorrhiza[J].Front Pharmacol,2019(10):202.
[7] LIU Q,LU J,LIN J,et al.Salvianolic acid B attenuates experimental skin fibrosis of systemic sclerosis[J].Biomed Pharmacother,2019(110):546-553.
[8] TAO S,DUAN R,XU T,et al.Salvianolic acid B inhibits the progression of liver fibrosis in rats via modulation of the Hedgehog signaling pathway[J].Exp Ther Med,2022,23(2):116.
[9] GAO X,WU Q,ZHANG X,et al.Salvianolate ameliorates osteopenia and improves bone quality in prednisone-treated rheumatoid arthritis rats by regulating RANKL/RANK/OPG signaling[J].Front Pharmacol,2021(12):710169.
[10] WU Y,ZHANG C,WU J,et al.Angiogenesis and bone regeneration by mesenchymal stem cell transplantation with danshen in a rabbit model of avascular necrotic femoral head[J].Exp Ther Med,2019,18(1):163-171.
[11] KIM R,LEE S,LEE C Y,et al.Salvia miltiorrhiza enhances the survival of mesenchymal stem cells under ischemic conditions[J].J Pharm Pharmacol,2018,70(9):1228-1241.
[12] SOTHORNWIT J,SOMBOONPORN W,SOONTRAPA S,et al.Carotid intima-media thickness and osteoporosis in postmenopausal women:A cross-sectional study[J].Climacteric,2018,21(3):280-285.
[13] LIANG G,KOW A S F,THAM C L,et al.Ameliorative Effect of Tocotrienols on Perimenopausal-Associated Osteoporosis-A Review[J].Antioxidants (Basel),2022,11(11):2179.
[14] DAS A,DESTOUNI A.Novel insights into reproductive ageing and menopause from genomics[J].Hum Reprod,2023,38(2):195-203.
[15] Geng Q,Gao H,Yang R,et al.Pyrroloquinoline quinone prevents estrogen deficiency-induced osteoporosis by inhibiting oxidative stress and osteocyte senescence[J].Int J Biol Sci,2019,15(1):58-68.
[16] MA L,HUA R,TIAN Y,et al.Connexin 43 hemichannels protect bone loss during estrogen deficiency[J].Bone Res,2019(7):11.
[17] BATOON L,MILLARD S M,RAGGATT L J,et al.Osteal macrophages support osteoclast-mediated resorption and contribute to bone pathology in a postmenopausal osteoporosis mouse model[J].J Bone Miner Res,2021,36(11):2214-2228.
[18] JERBAN S,MA Y,WEI Z,et al.Quantitative magnetic resonance imaging of cortical and trabecular bone[J].Semin Musculoskelet Radiol,2020,24(4):386-401.
[19] MONTAÑEZ-RIVERA I,NYAKATURA J A,AMSON E.Bone cortical compactness in 'tree sloths' reflects convergent evolution[J].J Anat,2018,233(5):580-591.
[20] SEEMAN E,MARTIN T J.Antiresorptive and anabolic agents in the prevention and reversal of bone fragility[J].Nat Rev Rheumatol,2019,15(4):225-236.
[21] LANGDAHL B L,STENKJAER L,CARSTENS M,et al.A CAG repeat polymorphism in the androgen receptor gene is associated with reduced bone mass and increased risk of osteoporotic fractures[J].Calcif Tissue Int,2003,73(3):237-43.
[22] QIN X,JIANG Q,NAGANO K,et al.Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts[J].PLoS Genet,2020,16(11):e1009169.
[23] TANG J,XIE J,CHEN W,et al.Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation[J].J Biol Chem,2020,295(33):11669-11681.
[24] CHOI Y H,KIM G S,CHOI J H,et al.Ethanol extract of Lithospermum erythrorhizon Sieb.et Zucc.promotes osteoblastogenesis through the regulation of Runx2 and Osterix[J].Int J Mol Med,2016,38(2):610-618.
[25] SANCISI V,MANZOTTI G,GUGNONI M,et al.RUNX2 expression in thyroid and breast cancer requires the cooperation of three non-redundant enhancers under the control of BRD4 and c-JUN[J].Nucleic Acids Res,2017,45(19):11249-11267.
[26] LIU Q,LI M,WANG S,et al.Recent Advances of Osterix Transcription Factor in Osteoblast Differentiation and Bone Formation[J].Front Cell Dev Biol,2020(8):601224.
[27] CHEN X,WANG Z,DUAN N,et al.Osteoblast-osteoclast interactions[J].Connect Tissue Res,2018,59(2):99-107.
[28] 王诗尧,许少策,师政伟,等.骨重建过程中降钙素基因相关肽与RANK/RANKL/OPG作用机制的研究进展[J].中国修复重建外科杂志,2019,33(4):511-515.
[29] LIN B,KE Q,LEAMAN D W,et al.Regulation of RANKL-induced osteoclastogenesis by RING finger protein RNF114[J].J Orthop Res,2018,36(1):159-166.
[30] ZHANG X,ZOU L,LI J,XU B,et al.Salvianolic acid B and danshensu induce osteogenic differentiation of rat bone marrow stromal stem cells by upregulating the nitric oxide pathway[J].Exp Ther Med,2017,14(4):2779-2788.
[31] YANG K,LUO Y,LU S,et al.Salvianolic acid B and ginsenoside Re synergistically protect against Ox-LDL-induced endothelial apoptosis through the antioxidative and antiinflammatory mechanisms[J].Front Pharmacol,2018(9):662.
[32] XU D,XU L,ZHOU C,et al.Salvianolic acid B promotes osteogenesis of human mesenchymal stem cells through activating ERK signaling pathway[J].Int J Biochem Cell Biol,2014(51):1-9.
[33] BIAN Y,XIANG J.Salvianolic acid B promotes the osteogenic differentiation of human periodontal ligament cells through Wnt/β-catenin signaling pathway[J].Arch Oral Biol,2020 (113):104693.