补肾壮骨方对骨关节炎大鼠软骨细胞外基质稳态的影响

张辽; 傅科上; 邓颖萍; 金甬; 吴权; 史燕红

doi:10.16766/j.cnki.issn.1674-4152.003995

补肾壮骨方对骨关节炎大鼠软骨细胞外基质稳态的影响

doi: 10.16766/j.cnki.issn.1674-4152.003995

张辽^1, ,,
傅科上¹,
邓颖萍²,
金甬¹,
吴权¹,
史燕红¹

1.
浙江中医药大学附属宁波市中医院中医骨伤科，浙江宁波 315000
2.
浙江中医药大学附属宁波市中医院肾病科

基金项目:

国家中医药管理局科技司-浙江省中医药管理局共建科技计划项目重点项目 GZY-ZJ-KJ-23037

浙江省中医药科技计划项目 2024ZL144

宁波市自然科学基金项目 2021J300

详细信息

通讯作者:
张辽，E-mail：drzhangliao@163.com

中图分类号: R684.3 R-332
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- 被引次数: 0
出版历程
- 收稿日期: 2024-05-15
- 网络出版日期: 2025-08-14

The effect of Bushen Zhuanggu Decoction on the extracellular matrix homeostasis of chondrocytes in osteoarthritis rats

1.
Department of Orthopaedics, Ningbo Hospital of Traditional Chinese Medicine Affiliated to Zhejiang Chinese Medical University, Ningbo, Zhejiang 315000, China

摘要

摘要: 目的探讨补肾壮骨方对大鼠软骨细胞外基质稳态失衡的影响及作用机制。方法将36只大鼠采用随机数字表法分为6组，即假手术组、模型组、补肾壮骨方低浓度组、中浓度组、高浓度组和阳性对照组，每组6只。采用HE染色和番红O染色检测软骨组织病理学变化；检测Wnt/β-catenin信号通路相关蛋白及软骨退化相关蛋白(蛋白质印迹法)和mRNA(RT-PCR法)表达。结果模型组软骨表层明显纤维化，软骨细胞减少，低、中、高浓度组和阳性对照组软骨细胞排列整齐，且高剂量组软骨病理学变化最显著。与模型组[(12.49±1.34)分]相比，低、中、高浓度组和阳性对照组大鼠改良Mankin ' s评分[分别为(10.17±1.19)分、(7.50±0.89)分、(3.99±0.45)分、(6.51±0.73)分]下降，软骨组织中IL-1β、Wnt5α、β-catenin、GSK-3β、MMP-13、ADAMTS5蛋白及mRNA表达明显降低，Axin、Collagen Ⅱ、Aggrecan蛋白和mRNA表达均明显升高(P＜0.05)。结论补肾壮骨方能抑制关节炎大鼠软骨细胞外基质降解，发挥治疗作用，其作用机制可能和抑制Wnt/β-catenin信号通路相关。
- 骨关节炎 /
- 补肾壮骨方 /
- Wnt/β-catenin信号通路 /
- 软骨细胞外基质
Abstract: Objective To investigate the effect and mechanism of Bushen Zhuanggu Decoction on the imbalance of extracellular matrix homeostasis in chondrocytes of osteoarthritis (OA) rats. Methods Thirty-six rats were randomly divided into six groups using a random number table: sham group, model group, Bushen Zhuanggu Decoction low-dose, medium-dose, high-dose group, and positive control group, with six in each group. Histological changes in cartilage tissue were evaluated using HE and Safranin O staining. The expression of proteins and mRNA related to the Wnt/β-catenin signaling pathway and cartilage degradation was assessed using Western blotting and RT-PCR, respectively. Results The model group exhibited significant cartilage surface fibrosis and reduced chondrocyte numbers. In contrast, the low-, medium-, and high-dose Bushen Zhuanggu Decoction groups, as well as the positive control group, showed an orderly arrangement of chondrocytes, with the most pronounced pathological improvement observed in the high-dose group. Compared to the model group (12.49±1.34), the Mankin ' s scores significantly decreased in the low-dose (10.17±1.19), medium-dose (7.50±0.89), high-dose (3.99±0.45), and positive control groups (6.51±0.73). The expression levels of IL-1β, Wnt5α, β-catenin, GSK-3β, MMP-13, and ADAMTS5 proteins and mRNA in cartilage tissue were significantly reduced, while the expression of Axin, Collagen Ⅱ, and Aggrecan proteins and mRNA were significantly increased (P < 0.05). Conclusion Bushen Zhuanggu Decoction inhibits the degradation of the extracellular matrix in chondrocytes of OA rats and exerts therapeutic effects. Its mechanism may be related to the inhibition of the Wnt/β-catenin signaling pathway.
- Osteoarthritis /
- Bushen Zhuanggu Decoction /
- Wnt/β-catenin signaling pathway /
- Cartilage extracellular matrix

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图 1 各组大鼠软骨组织HE染色(×100)

Figure 1. HE staining of rat cartilage tissue in each group(×100)

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图 2 各组大鼠软骨组织番红O染色(×40)

Figure 2. Safranin O staining results of rat cartilage tissue in each group(×40)

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图 3 各组大鼠软骨组织中蛋白表达结果

Figure 3. Protein expression results in cartilage tissue of rats in different groups

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表 1 RT-PCR引物序列

Table 1. The primer sequences for RT-PCR

蛋白名称	引物序列(5'→3')
Wnt5a	F：AGCTACCCGATCTGGTGGTC
	R：CAAACTCGATGTCCTCGCTAC
β-catenin	F：CTTCCAGACACGCCATCATG
	R：CACACAGAGTACTTGCGCTC
MMP-13	F：CATGAGCTTGGCCACTCCCT
	R：TGAACGTCATCATCTGGGAGCA
ADAMTS-5	F：AGGGCACTGGCTATTACG
	R：GTTCTCACGCACCTTCCT
Collagen Ⅱ	F：GGCTTCCAAGTGCCAGTAGG
	R：TGGAGAGGAGAGTGCAGTCA
Aggrecan	F：AGGTGTCGCTCCCCAACTAT
	R：CTTCACAGCGGTAGATCCCAG
Axin	F：GTAGACGGTACAACGAAGGCA
	R：AGCCAGAGTTGACGTAGTAGG
GSK-3β	F：GTCCGACTGCGGTATTTCTTC
	R：CTCGATGGCAGATTCCAAAGG
IL-1β	F：TTCAGGCAGGCAGTATCACTC
	R：GAAGGTCCACGGGAAAGACAC
β-actin	F：TCTTCCAGCCTTCCTTCCTG
	R：CACACGAGTACTTGCGCTC

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表 2 各组大鼠软骨组织中蛋白表达水平比较(x±s)

Table 2. Comparison of protein expression levels in cartilage tissue of rats among different groups(x±s)

组别	n	IL-1β	Wnt5α	β-catenin	Axin	GSK-3β	MMP13	ADAMTS5	Collagen Ⅱ	Aggrecan
假手术组	6	0.52±0.11	0.61±0.13	0.46±0.10	1.03±1.08	0.73±0.10	0.36±0.11	0.54±0.11	1.02±0.14	1.83±0.27
模型组	6	2.04±0.29^a	1.83±0.27^a	1.63±0.20^a	0.53±0.15^a	1.20±0.17^a	0.91±0.19^a	1.55±0.21^a	0.46±0.12^a	0.63±0.13^a
低浓度组	6	1.53±0.18^b	1.42±0.21^b	1.24±0.19^b	0.68±0.14^b	1.06±0.18^b	0.73±0.13^b	0.84±0.13^b	0.53±0.15^b	0.70±0.14^b
中浓度组	6	1.31±0.17^b	1.35±0.22^b	1.13±0.14^b	0.71±0.13^b	0.96±0.13^b	0.64±0.15^b	0.74±0.12^b	0.64±0.12^b	0.87±0.14^b
高浓度组	6	1.01±0.14^b	1.03±0.14^b	0.92±0.13^b	0.84±0.16^b	0.74±0.12^b	0.44±0.14^b	0.62±0.14^b	0.83±0.14^b	1.06±0.21^b
阳性对照组	6	1.24±0.19^b	1.13±0.16^b	1.04±0.15^b	0.73±0.12^b	0.83±0.14^b	0.55±0.13^b	0.63±0.13^b	0.77±0.11^b	0.73±0.15^b
F值		217.963	203.254	143.452	24.610	39.364	31.662	101.624	31.620	148.476
P值		< 0.001	< 0.001	< 0.001	< 0.001	<0.001	< 0.001	< 0.001	< 0.001	< 0.001
注：与假手术组比较, ^aP < 0.05；与模型组比较，^bP < 0.05。

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表 3 各组大鼠软骨组织中相关基因的相对表达量比较(x±s)

Table 3. Comparison of relative expression levels of related genes in cartilage tissue of rats among different groups(x±s)

组别	n	IL-1β	Wnt5α	β-catenin	Axin	GSK-3β	MMP13	ADAMTS5	Collagen Ⅱ	Aggrecan
假手术组	6	0.65±0.12	0.80±0.15	0.50±0.10	1.30±0.15	1.50±0.12	0.45±0.10	1.70±0.13	2.45±0.20	2.20±0.18
模型组	6	3.05±0.25^a	2.85±0.22^a	2.35±0.20^a	0.55±0.08^a	2.10±0.18^a	3.25±0.28^a	2.50±0.22^a	1.65±0.12^a	1.50±0.11^a
低浓度组	6	2.45±0.20^b	2.25±0.18^b	1.90±0.16^b	0.64±0.09^b	1.85±0.15^b	2.60±0.20^b	2.20±0.18^b	1.75±0.17^b	1.60±0.12^b
中浓度组	6	2.12±0.18^b	2.05±0.16^b	1.70±0.14^b	0.72±0.11^b	1.70±0.13^b	2.40±0.18^b	2.00±0.16^b	1.80±0.13^b	1.60±0.16^b
高浓度组	6	1.65±0.15^b	1.72±0.13^b	1.40±0.12^b	0.80±0.12^b	1.58±0.12^b	1.85±0.14^b	1.85±0.18^b	1.95±0.14^b	1.95±0.15^b
阳性对照组	6	2.01±0.17^b	1.92±0.15^b	1.58±0.14^b	0.68±0.10^b	1.60±0.20^b	2.10±0.15^b	1.90±0.14^b	2.15±0.16^b	1.75±0.13^b
F值		728.024	462.733	348.972	88.124	47.682	1 341.523	60.192	85.564	77.823
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
注：与假手术组比较, ^aP < 0.05；与模型组比较，^bP < 0.05。

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参考文献(20)

[1]	ZHU J, YANG S, QI Y, et al. Stem cell-homing hydrogel-based miR-29b-5p delivery promotes cartilage regeneration by suppressing senescence in an osteoarthritis rat model[J]. Sci Adv, 2022, 8(13): eabk0011. DOI: 10.1126/sciadv.abk0011.
[2]	LIU Y, ZENG Y, SI H B, et al. Exosomes derived from human urine-derived stem cells overexpressing miR-140-5p alleviate knee osteoarthritis through downregulation of VEGFA in a rat model[J]. Am J Sports Med, 2022, 50(4): 1088-1105.
[3]	ZHUANG H, REN X, JIANG F, et al. Indole-3-propionic acid alleviates chondrocytes inflammation and osteoarthritis via the AhR/NF-κB axis[J]. Mol Med, 2023, 29(1): 17. DOI: 10.1186/s10020-023-00614-9.
[4]	吴志明, 袁振兴, 勒春, 等. 补肾壮骨方通过EphB4/Ephrin-B2轴缓解绝经后骨质疏松[J]. 江西中医药, 2023, 54(11): 68-72. WU Z M, YUAN Z X, LE C, et al. Bushen Zhuanggu prescription alleviated postmenopausal osteoporosis through EphB4/Ephrin-B2 axis[J]. Jiangxi Journal of Traditional Chinese Medicine, 2023, 54(11): 68-72.
[5]	张辽, 韩晶晶, 邓颖萍, 等. 叶海教授基于"经纬辨证"理论治疗膝骨关节炎的临证经验[J]. 中国中医骨伤科杂志, 2019, 27(4): 69-70, 73. ZHANG L, HAN J J, DENG Y P, et al. Professor Ye Hai' s clinical experience in the treatment of knee osteoarthritis based on the theory of "meridian and latitude differentiation"[J]. Chinese Journal of Traditional Medical Traumatology & Orthopedics, 2019, 27(4): 69-70, 73.
[6]	张辽, 叶有骏, 邓颖萍, 等. 叶海从肝脾肾论治绝经后骨质疏松性骨关节炎经验撷菁[J]. 浙江中医杂志, 2023, 58(11): 803-804. ZHANG L, YE Y J, DENG Y P, et al. Ye Hai picks from the experience of treating postmenopausal osteoporotic osteoarthritis from liver, spleen and kidney[J]. Zhejiang Journal of Traditional Chinese Medicine, 2023, 58(11): 803-804.
[7]	周雪来, 邓敦, 沈斌, 等. 揿针联合耳穴压豆在气血虚弱证膝骨关节炎患者中的应用效果分析[J]. 中华全科医学, 2022, 20(1): 121-124. doi: 10.16766/j.cnki.issn.1674-4152.002292 ZHOU X L, DENG D, SHEN B, et al. Application of combing thumbtack needle with ear point pressing beans in knee osteoarthritis patients with syndrome of qi and blood weakness[J]. Chinese Journal of General Practice, 2022, 20(1): 121-124. doi: 10.16766/j.cnki.issn.1674-4152.002292
[8]	JI X, DU W, CHE W, et al. Apigenin inhibits the progression of osteoarthritis by mediating macrophage polarization[J]. Molecules, 2023, 28(7): 2915. DOI: 10.3390/molecules28072915.
[9]	ZHOU Y, ZHAO Y, WU Y, et al. Human umbilical cord mesenchymal stem cells alleviate rat knee osteoarthritis via activating Wnt/β-catenin signaling pathway[J]. Curr Stem Cell Res Ther, 2024, 19(2): 234-244. http://www.nstl.gov.cn/paper_detail.html?id=20ab92270f49aa091ac6b7e83ad55827
[10]	SONG X Y, ZHAO M, ZHANG P, et al. Cangxitongbi capsules protect the articular cartilage in the rat knee through the long non-coding RNA HOTAIR/p38MAPK pathway[J]. Ann Transl Med, 2022, 10(1): 23. DOI: 10.21037/atm-21-6539.
[11]	ZHANG R, HAN L, LIN W, et al. Mechanisms of NLRP3 inflammasome in rheumatoid arthritis and osteoarthritis and the effects of traditional Chinese medicine[J]. J Ethnopharmacol, 2024, 321: 117432. DOI: 10.1016/j.jep.2023.117432.
[12]	赵东方, 尉志强, 邢姝琴, 等. 巴戟天对骨关节炎小鼠软骨细胞自噬、凋亡的影响及机制[J]. 新乡医学院学报, 2023, 40(11): 1001-1007. ZHAO D F, WEI Z Q, XING S Q, et al. Effect and mechanism of morinda root on autophagy and apoptosis of chondrocytes in osteoarthritis mice[J]. Journal of Xinxiang Medical University, 2023, 40(11): 1001-1007.
[13]	DU X, XIN R, CHEN X, et al. TAF15 regulates the BRD4/GREM1 axis and activates the gremlin-1-NF-κB pathway to promote OA progression[J]. Regen Ther, 2023, 24: 227-236.
[14]	ZHAO S S, KARHUNEN V, MORRIS A P, et al. ADAMTS5 as a therapeutic target for osteoarthritis: Mendelian randomisation study[J]. Ann Rheum Dis, 2022, 81(6): 903-904. http://ard.bmj.com/content/81/6/903.full
[15]	HAN H, CHEN M, LI Z, et al. Corosolic acid protects rat chondrocytes against IL-1β-induced ECM degradation by activating autophagy via PI3K/AKT/mTOR pathway and ameliorates rat osteoarthritis[J]. Drug Des Devel Ther, 2022, 16: 2627-2637.
[16]	LI F, XU Z, XIE Z, et al. Adipose mesenchymal stem cells-derived exosomes alleviate osteoarthritis by transporting microRNA-376c-3p and targeting the WNT-beta-catenin signaling axis[J]. Apoptosis, 2023, 28(3-4): 362-378.
[17]	LV R, DU L, BAI L. RNF125, transcriptionally regulated by NFATC2, alleviates osteoarthritis via inhibiting the Wnt/β-catenin signaling pathway through degrading TRIM14[J]. Int Immunopharmacol, 2023, 125(Pt B): 111191. DOI: 10.1016/j.intimp.2023.111191.
[18]	CHENG L, HUANG C, LI M, et al. Chonggu granules improve cartilage matrix metabolism in knee osteoarthritis via the miR-148a-3p/Wnt/β-Catenin pathway[J]. J Inflamm Res, 2023, 16: 4751-4762. doi: 10.2147/JIR.S428582
[19]	MA T W, RUAN H R, LV L Y, et al. Oleanolic acid, a small-molecule natural product, inhibits ECM degeneration in osteoarthritis by regulating the Hippo/YAP and Wnt/β-catenin pathways[J]. Food Funct, 2023, 14(22): 9999-10013. http://www.keyanzhidian.com/doc/detail?id=2073857734
[20]	肖强, 郭子龙, 杨晓宏. 基于Wnt/β-catenin信号通路探讨川芎嗪延缓膝骨关节炎软骨退变的机制[J]. 中医药导报, 2022, 28(2): 37-42, 52. XIAO Q, GUO Z L, YANG X H. Study of the mechanism of ligustrazine in delaying cartilage degeneration in knee osteoarthritis based on Wnt/β-catenin signaling pathway[J]. Guiding Journal of Traditional Chinese Medicine and Pharmacology, 2022, 28(2): 37-42, 52.