| 谢大伟,张来鑫,李青松,等.吉非替尼通过调控表皮生长因子受体影响兔膝骨关节炎软骨损伤作用的机制[J].安徽医药,2021,25(6):1075-1079. |
| 吉非替尼通过调控表皮生长因子受体影响兔膝骨关节炎软骨损伤作用的机制 |
| Action mechanism of gefitinib in influencing cartilage injury of rabbit knee osteoarthritis by regulating EGFR |
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| DOI：10.3969/j.issn.1009-6469.2021.06.004 |
| 中文关键词: 骨关节炎，膝 吉非替尼 表皮生长因子受体 软骨损伤 兔 蛋白， |
| 英文关键词: Osteoarthritis, knee Knee osteoarthritis Gefitinib Epidermal growth factor receptor Cartilage injury Rabbits |
| 基金项目: |
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| 中文摘要: |
| 目的研究吉非替尼通过调控表皮生长因子受体( EGFR)影响兔膝骨关节炎模型软骨损伤作用机制。方法将 30只新西兰兔随机分为假手术组、模型组、吉非替尼组，模型组与吉非替尼组均采用 Hulth-Telhag方式制成膝骨关节炎模型，假手术组仅剪开关节全不做任何处理，造模成功后吉非替尼组给予吉非替尼灌胃给药 85 mg/kg，1次/天，其他组给予等量的生理盐水，连续给药 4周。停药 1周后苏木精 -伊红( HE)染色观察软骨病理组织情况及 Mankin′s评分，原位末端标记法( TUNEL)染色观察软骨细胞凋亡情况，酶联免疫吸附测定( ELISA)检测兔关节液中白细胞介素 -1β(IL-1β)、肿瘤坏死因子 α(TNF-α)水平。免疫组化法检测软骨组织中骨代谢标志物 Ⅱ型胶原蛋白( Col-Ⅱ)、基质金属蛋白酶 13(MMP-13)阳性表达情况，蛋白质印迹法(Western blotting)检测软骨组织中 EGFR、丝裂素活化蛋白激酶( p38MAPK)蛋白的表达情况。结果与假手术组相比，模型组关节软骨层明显变薄，并且有溃裂和裂隙情况存在，同时软骨柱状排列消失，结构遭到严重损坏，软骨细胞明显减少，与模型组相比，吉非替尼组软骨病理损伤情况更严重，且三组 Mankin′s评分［(0.32±0.13)分、(7.38±0.28)分、(8.65±0.27)分］关系为假手术组 <模型组 <吉非替尼组(均 P<0.05)；三组间软骨凋亡率［( 4.12±0.38)%、(29.83±3.05)%、(37.61±3.72)%］、 IL-1β［( 541.23± 27.28)ng/L、(738.61±68.37)ng/L、(825.42±75.19)ng/L］、 TNF-α［( 49.31±7.28)ng/L、(82.62±25.42)ng/L、(95.05±32.56)ng/L］水平之间的关系均为假手术组 <模型组 <吉非替尼组(均 P<0.05)； COL-Ⅱ阳性表达率间的关系为吉非替尼组 <模型组 <假手术组(均 P<0.05)MMP-13阳性表达率间的关系为假手术组 <模型组 <吉非替尼组(均 P<0.05)；蛋白质印迹法结果显示，吉非替尼组中 EGFR磷酸化水平显著低于模型组，模型组又显著低于假手术组，同时吉非替尼组中 p38MAPK蛋白磷酸化水平显著高于模型组，模型组又显著高于假手术组(均 P<0.05)。结论吉非替尼通过抑制 EGFR磷酸化活化 p38MAPK信号途径，促进 MMP-13蛋白表达，降解 COL-Ⅱ，同时促进软骨处炎症细胞因子水平，从而加重软骨损伤。 |
| 英文摘要: |
| Objective To study the action mechanism of gefitinib in influencing cartilage injury of rabbit knee osteoarthritis (KOA)model by regulating epidermal growth factor receptor (EGFR).Methods Thirty New Zealand rabbits were randomly assigned intosham operation group, model group and gefitinib group. In model group and gefitinib group, Hulth-Telhag method was performed tomake KOA models. In sham operation group, joints were cut off and no treatment was conducted. After successful modeling, gefitinibgroup was given gefitinib gavage of 85 mg/kg, once/d. The other groups were given the same amount of normal saline. Continuous administration lasted for 4 weeks. One week after drug withdrawal, cartilage pathological tissues and Mankin′s score were observed by hematoxylin and eosin (HE) staining. The apoptosis of cartilage cells was observed by TdT-mediated dUTP-biotin nick end labeling assay (TUNEL) staining. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rabbit joint fluid were detected by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect positive expression of bone metabolismmarkers such as type Ⅱ collagen (Col-Ⅱ) and matrix metalloproteinase 13 (MMP-13) in cartilage tissues. Western blotting was performed to detect the expressions of EGFR and p38 mitogen-activated protein kinase (p38MAPK) protein in cartilage tissues.Results Compared with sham operation group, articular cartilage layer was significantly thinner in model group. There were debacle and fissures. At the same time, the columnar arrangement of cartilage disappeared, structure was severely damaged, and cartilage cells weresignificantly reduced. Compared with model group, pathological damage of cartilage was more severe in gefitinib group. ArrangingMankin′s scores [(0.32±0.13) points, (7.38±0.28) points, (8.65±0.27) points] from low to high, the corresponding order was sham operation group, model group and gefitinib group (P<0.05). Arranging cartilage apoptosis rates [(4.12±0.38)%, (29.83±3.05)%, (37.61± 3.72)%], IL-1β [(541.23±27.28) ng/L, (738.61±68.37) ng/L, (825.42±75.19) ng/L] and TNF-α [(49.31±7.28) ng/L, (82.62±25.42) ng/L,(95.05±32.56) ng/L] levels from low to high, the corresponding order was sham operation group, model group and gefitinib group (P< 0.05). Arranging positive expression rates of COL-Ⅱ from low to high, the corresponding order was gefitinib group, model group and sham operation group (P<0.05). Arranging positive expression rates of MMP-13 from low to high, the corresponding order was sham operation group, model group and gefitinib group (P<0.05). Western blotting results showed that phosphorylation level of EGFR protein ingefitinib group was significantly lower than that in model group, and this in model group was significantly lower than that in sham operation group. The phosphorylation level of p38MAPK protein in gefitinib group was significantly higher than that in model group, and thisin model group was significantly higher than that in sham operation group (all P<0.05).Conclusion Gefitinib can promote the expression of MMP-13 protein, degrade COL-Ⅱ and promote inflammatory cytokine levels in cartilage by inhibiting EGFR phosphorylationand activating p38MAPK signaling pathway, thus aggravating cartilage damage. |
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