秦皮素通过Nrf2/HO-1信号通路抑制铁死亡减轻骨关节炎大鼠软骨损伤

doi:10.12300/j.issn.1674-5817.2026.033

摘要/Abstract

摘要：

目的采用前交叉韧带横断术（anterior cruciate ligment transection，ACLT）诱导大鼠骨关节炎，探索秦皮素能否减轻软骨损伤及其潜在作用机制。方法将18只SD大鼠随机分为3组：假手术（Sham）组、模型（ACLT）组、秦皮素干预（ACLT+Fra）组。除假手术组外均通过ACLT法建立OA大鼠模型，手术1周后，ACLT+Fra组膝关节注射秦皮素（5 mg?kg^-1?d^-1），每周2次，连续4周；使用H&E染色和番红-固绿染色观察各组大鼠膝关节软骨损伤情况；采用Mankin评分法、OARSI评分法评估膝关节破坏程度；使用免疫组织化学染色法检测各组膝关节软骨基质金属蛋白酶13（matrix metallopeptidase 13，MMP13）、II型胶原（collagen type II，Col2）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、血红素氧合酶1（heme oxygenase，HO-1）；采用WB检测大鼠软骨MMP13、Col2、核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）、HO-1、溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）、GPX4的蛋白表达水平；采用qRT-PCR检测MMP13、Col2、Nrf2、HO-1、SLC7A11、GPX4的mRNA表达水平；采用试剂盒检测各组大鼠膝关节软骨谷胱甘肽谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）、Fe²⁺含量的差异。结果与Sham组相比，ACLT组大鼠关节面软骨磨损严重，软骨基质降解，Mankin评分与OARSI评分显著降低（P＜0.01）；免疫组化结果显示，MMP13阳性表达显著升高（P＜0.01），Col2、GPX4、HO-1阳性表达显著降低（P＜0.01）；MDA、Fe2?含量显著升高（P＜0.01），而GSH含量显著降低（P＜0.01）；MMP13的蛋白及mRNA表达显著升高（P＜0.01），Col2、Nrf2、HO-1、SLC7A11、GPX4的蛋白及mRNA表达显著降低（P＜0.01）；与ACLT组相比，ACLT+Fra组大鼠软骨表面损伤程度减轻，Mankin评分与OARSI评分显著升高（P＜0.01）；免疫组织化学结果显示，MMP13阳性表达显著降低（P＜0.05），Col2、GPX4、HO-1阳性表达显著升高（P＜0.01）；MDA、Fe2?含量显著降低（P＜0.01），GSH含量显著升高（P＜0.05）；MMP13的蛋白及mRNA表达显著降低（P＜0.01），Col2、Nrf2、HO-1、SLC7A11、GPX4的蛋白及mRNA表达显著升高（P＜0.05）。结论秦皮素可能通过激活Nrf2/HO-1信号通路抑制软骨细胞铁死亡，从而发挥软骨保护作用。

关键词: 秦皮素, 骨关节炎, 软骨细胞, 铁死亡, 大鼠

Abstract:

Objective To explore whether fraxetin can alleviate cartilage injury and its potential mechanism in rats with ACLT-induced osteoarthritis. Methods Eighteen SD rats were randomly divided into three groups: the sham operation (Sham) group, the model (ACLT) group, and the fraxetin intervention (ACLT+Fra) group; Except for the Sham group, OA rat models were established by ACLT method in the other two groups; One week after surgery, the ACLT+Fra group was injected with fraxetin (5 mg/kg/day) into the knee joint, twice a week for 4 consecutive weeks; H&E staining and safranin O-fast green staining were used to observe the knee joint cartilage injury in each group; The Mankin scoring system and the OARSI scoring system were adopted to evaluate the degree of knee joint damage; Immunohistochemical staining was used to detect the expression of MMP13, Col2, GPX4, and HO-1 in the knee joint cartilage of each group; The WB was used to detect the protein expression levels of MMP13, Col2, Nrf2, HO-1, SLC7A11, and GPX4 in rat cartilage; qRT-PCR was used to detect the mRNA expression levels of MMP13, Col2, Nrf2, HO-1, SLC7A11, and GPX4; Kits were used to detect the differences in the contents of GSH, MDA, and Fe2? in the knee joint cartilage of rats in each group. Results Compared with the Sham group, the ACLT group rats showed severe cartilage surface damage, degradation of the cartilage matrix, and significantly reduced Mankin and OARSI scores(P<0.01); immunohistochemistry results showed a significant increase in MMP13 positive expression (P<0.01), and a significant decrease in Col2, GPX4, and HO-1 positive expression(P<0.01); MDA and Fe2? contents were significantly increased(P<0.01), while GSH levels were significantly decreased(P<0.01); MMP13 protein and mRNA expressions were significantly elevated(P<0.01), and Col2, Nrf2, HO-1, SLC7A11, and GPX4 protein and mRNA expressions were significantly decreased(P<0.01).Compared with the ACLT group, the ACLT+Fra group rats showed reduced cartilage surface damage, with significantly increased Mankin and OARSI scores(P<0.01); immunohistochemical results showed a significant decrease in MMP13 positive expression(P<0.05), and a significant increase in Col2, GPX4, and HO-1 positive expression(P<0.01); MDA and Fe2? contents were significantly reduced(P<0.01), and GSH levels were significantly increased(P<0.05); MMP13 protein and mRNA expressions were significantly decreased(P<0.01), and Col2, Nrf2, HO-1, SLC7A11, and GPX4 protein and mRNA expressions were significantly increased(P<0.05). Conclusion Fraxetin may exert a cartilage protective effect by activating the Nrf2/HO-1 signaling pathway to inhibit chondrocyte ferroptosis.

Key words: Fraxetin, Osteoarthritis, Cartilage, Ferroptosis, Rats

中图分类号:

Q95-33

杨然,冯戟玮,马飞,等.秦皮素通过Nrf2/HO-1信号通路抑制铁死亡减轻骨关节炎大鼠软骨损伤[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2026.033.

YANG Ran,FENG Jiwei,MA Fei,et al. Fraxetin Inhibits Ferroptosis and Alleviates Cartilage Damage in Osteoarthritis Rat Through Nrf2/HO-1 Signaling Pathway[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2026.033.

图/表 7

表1 引物序列

Table1 Sequence of primer

基因 Gene	正向引物 Forward primer	反向引物 Reverse primer
MMP13	CTTCTTCTTGTTGAGCTGGACTC	CTGTGGAGGTCACTGTAGACT
Col2	GGGAATGTCCTCTGCGATGAC	GAAGGGGATCTCGGGGTTG
Nrf2	TCTTGGAGTAAGTCGAGAAGTGT	GTTGAAACTGAGCGAAAAAGGC
HO-1	AAGCCGAGAATGCTGAGTTCA	GCCGTGTAGATATGGTACAAGGA
SLC7A11	GGCACCGTCATCGGATCAG	CTCCACAGGCAGACCAGAAAA
GPX4	GATGGAGCCCATTCCTGAACC	CCCTGTACTTATCCAGGCAGA
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

注：A，HE染色和番红固绿染色，比例尺为200 μm；B，Mankin评分；C，国际骨关节炎研究学会评分（OARSI）。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。
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图1 大鼠膝关节软骨HE染色、番红固绿染色和Mankin、OARSI评分结果
注：A，HE染色和番红固绿染色，比例尺为200 μm；B，Mankin评分；C，国际骨关节炎研究学会评分（OARSI）。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。

Figure 1 HE staining， safranin O-fast green staining of rat knee joint cartilage， and Mankin's and OARSI scores
Note： A， HE staining and safranin O-fast green staining， scale bar=200 μm； B， Mankin score； C， Osteoarthritis Research Society International （OARSI） international score. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6. ^*P＜0.05，^**P＜0.01.

注：A，各组大鼠MMP13和Col2的免疫组化染色（棕黄色为阳性染色），比例尺为200 μm；B，MMP13阳性区域定量分析；C，Col2阳性区域定量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。
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图2 秦皮素对大鼠膝关节软骨MMP13和Col2含量的影响
注：A，各组大鼠MMP13和Col2的免疫组化染色（棕黄色为阳性染色），比例尺为200 μm；B，MMP13阳性区域定量分析；C，Col2阳性区域定量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。

Figure 2 The effect of sinapine on the content of MMP13 and Col2 in rat knee joint cartilage
Note： A. Immunohistochemical staining of MMP13 and Col2 in each group of rats（Positive staining is shown in brown-yellow.）， scale bar=200 μm； B. Quantitative analysis of MMP13 positive areas； C. Quantitative analysis of Col2 positive areas. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6. ^*P＜0.05，^**P＜0.01.

注：A，各组大鼠MMP13、Col2蛋白和Tubulin的蛋白质印迹检测结果；B，MMP13的相对表达量分析；C，Col2的蛋白相对表达量分析；D，MMP13的mRNA表达及其相对量分析；E，Col2的mRNA表达及其相对量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带，1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。 A， Protein bands of MMP13， Col2 and Tubulin in each group of 蛋白质印迹； B， Analysis of relative protein expression of MMP13； C， Analysis of relative protein expression of Col2； D， Analysis of mRNA expression and relative quantity of MMP13； E， Analysis of mRNA expression and relative quantity of Col2. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6.^*P＜0.05，^**P＜0.01.
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图3 大鼠膝关节软骨MMP13、Col2蛋白和mRNA表达
注：A，各组大鼠MMP13、Col2蛋白和Tubulin的蛋白质印迹检测结果；B，MMP13的相对表达量分析；C，Col2的蛋白相对表达量分析；D，MMP13的mRNA表达及其相对量分析；E，Col2的mRNA表达及其相对量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带，1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。 A， Protein bands of MMP13， Col2 and Tubulin in each group of 蛋白质印迹； B， Analysis of relative protein expression of MMP13； C， Analysis of relative protein expression of Col2； D， Analysis of mRNA expression and relative quantity of MMP13； E， Analysis of mRNA expression and relative quantity of Col2. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6.^*P＜0.05，^**P＜0.01.

Figure 3 Expression of MMP13， Col2 proteins and mRNA in rat knee joint cartilage

注：A，MDA含量；B，GSH含量；C，Fe2?含量。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。
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图4 大鼠膝关节软骨MDA、GSH、Fe²⁺含量
注：A，MDA含量；B，GSH含量；C，Fe2?含量。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。

Figure 4 MDA， GSH， and Fe2? contents in rat knee cartilage
Note： A， MDA content； B， GSH content； C， Fe2? content. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n = 6.^*P＜0.05，^**P＜0.01.

注：A，各组大鼠GPX4和HO-1的免疫组化染色（棕黄色为阳性染色），比例尺为200μm；B，GPX4阳性区域定量分析；C，HO-1阳性区域定量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。
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图5 大鼠膝关节软骨GPX4和HO-1含量
注：A，各组大鼠GPX4和HO-1的免疫组化染色（棕黄色为阳性染色），比例尺为200μm；B，GPX4阳性区域定量分析；C，HO-1阳性区域定量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。

Figure 5 The content of GPX4 and HO-1 in the knee joint cartilage of rats
Note： A. Immunohistochemical staining of GPX4 and HO-1 in each group of rats（Positive staining is shown in brown-yellow.）， scale bar=200 μm； B. Quantitative analysis of GPX4 positive areas； C. Quantitative analysis of HO-1 positive areas. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6. ^*P＜0.05，^**P＜0.01.

注：A，各组大鼠Nrf2、HO-1、SLC7A11、GPX4蛋白和Tubulin的蛋白质印迹检测结果；B，Nrf2的蛋白相对表达量分析；C，HO-1的蛋白相对表达量分析；D，SLC7A11的蛋白相对表达量分析；E，GPX4的蛋白相对表达量分析；F，Nrf2的mRNA表达及其相对量分析；G，HO-1的mRNA表达及其相对量分析；H，SLC7A11的mRNA表达及其相对量分析；I，GPX4的mRNA表达及其相对量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。
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图6 大鼠膝关节软骨Nrf2、HO-1、SLC7A11、GPX4 蛋白和mRNA水平
注：A，各组大鼠Nrf2、HO-1、SLC7A11、GPX4蛋白和Tubulin的蛋白质印迹检测结果；B，Nrf2的蛋白相对表达量分析；C，HO-1的蛋白相对表达量分析；D，SLC7A11的蛋白相对表达量分析；E，GPX4的蛋白相对表达量分析；F，Nrf2的mRNA表达及其相对量分析；G，HO-1的mRNA表达及其相对量分析；H，SLC7A11的mRNA表达及其相对量分析；I，GPX4的mRNA表达及其相对量分析。Sham组，假手术组，仅打开关节囊，不切断前交叉韧带， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT组，切断前交叉韧带，构建ACLT骨关节炎模型， 1周后膝关节注射50 μL生理盐水，每周2次，持续4周；ACLT+Fra组，切断前交叉韧带，构建ACLT骨关节炎模型，1周后膝关节注射50 μL秦皮素，每周2次，持续4周。n=6。^*P＜0.05，^**P＜0.01。

Figure 6 Protein and mRNA levels of Nrf2， HO-1， SLC7A11， and GPX4 in rat knee
Note： A， Western Blot of Nrf2， HO-1， SLC7A11， GPX4 proteins and Tubulin bands in each group of mice； B， Analysis of relative protein expression of Nrf2； C， Analysis of relative protein expression of HO-1； D， Analysis of relative protein expression of SLC7A11； E， Analysis of relative protein expression of GPX4； F， Analysis of mRNA expression of Nrf2 and its relative quantity； G， Analysis of mRNA expression of HO-1 and its relative quantity； H， Analysis of mRNA expression of SLC7A11 and its relative quantity； I， Analysis of mRNA expression of GPX4 and its relative quantity. Animals in the Sham group received sham operation， in which the joint capsule was exposed without transection of the anterior cruciate ligament. One week after surgery， 50 μL normal saline was injected into the knee joint cavity twice weekly for 4 consecutive weeks. In the ACLT group， the anterior cruciate ligament was transected to establish the ACLT-induced osteoarthritis model. One week postoperatively， 50 μL normal saline was intra-articularly injected twice weekly for 4 consecutive weeks. For the ACLT+Fra group， the anterior cruciate ligament was transected to construct the ACLT osteoarthritis model. One week after surgery， 50 μL fraxetin solution was administered via intra-articular injection twice weekly for 4 consecutive weeks.n=6.^*P＜0.05，^**P＜0.01.joint cartilage

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