Exploring the Effect of Warming Meridian and Invigorating Blood External Treatment on Pain in Knee Osteoarthritis Rats Based on Netrin-1 Mediated Neural Sprouting
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摘要:
目的 研究Netrin-1介导感觉神经萌发对膝骨关节炎(Knee osteoarthritis, KOA)大鼠疼痛行为的影响及温经活血外治法的干预作用。 方法 随机将30只大鼠平均分为空白组、KOA组及外治法组。KOA模型通过前交叉韧带横断术构建, 造模成功后, 外治法组使用温经活血外治法代表方——“易层”贴敷28 d。给药完成后各大鼠腹主动脉采血并提取各组滑膜组织和背根神经节(Dorasal root ganglion, DRG)组织, 苏木精-伊红(Hematoxylin-eosin staining, HE)染色观察滑膜炎症, 神经镀银染色滑膜观察神经萌发; ELISA检测血清中促炎因子白介素-6(Interleukin 6, IL-6)和疼痛介质P物质(Substance p, SP)、降钙素基因相关肽(Calcitonin gene-related peptide, CGRP)含量; Western blot和qPCR检测滑膜组织Netrin-1、CGRP和DRG组织神经元表面受体结直肠癌缺失基因(Deleted in colorectal cancer, DCC)、非协调同源-5(Uncoordinated-5, UNC5)蛋白和基因表达量。 结果 HE染色显示外治法组炎性细胞浸润较KOA组明显减少, 神经镀银显示外治法组较KOA组神经纤维数量明显减少; KOA组较空白组IL-6和SP含量明显升高, 外治法组较KOA组IL-6和SP含量明显减少; KOA组较空白组Netrin-1、CGRP、DCC蛋白和基因表达明显升高, UNC5则明显降低, 外治法组较KOA组Netrin-1、CGRP、DCC蛋白和基因表达明显降低, UNC5则明显升高。 结论 温经活血外治法能够缓解KOA疼痛, 其作用机制可能与抑制Netrin-1、影响神经萌发和抑制疼痛因子有关。 Abstract:OBJECTIVE To study the effect of Netrin-1 mediated sensory neural sprouting on pain behavior in knee osteoarthritis (KOA) rats and the intervention effect of warming meridians and invigorating blood external treatment. METHODS 30 rats were randomly divided into the blank group, KOA group, and external treatment group. The KOA model was constructed by the anterior cruciate ligament transaction (ACLT). After successful modeling, the external treatment group was applied with the representative formula of warming meridians and invigorating blood external treatment, Easy Layer, for 28 days. After administration, blood was collected from the abdominal aorta of each rat; synovial tissue and dorsal root ganglia (DRG) tissue were extracted from each group. Hematoxylin eosin staining (HE) was used to observe synovial inflammation; nerve silver staining was used to observe nerve sprouting in synovium; ELISA was used to detect the levels of inflammatory factor interleukin 6 (IL-6), pain factors substance p (SP) and Calcitonin gene related peptide (CGRP) in serum; western blot and qPCR were used to detect protein and gene levels of Netrin-1 and CGRP in synovial tissue and deleted in colorectal cancer (DCC), uncoordinated homology-5 (UNC5) of neuronal surface receptors in DRG tissue. RESULTS HE staining showed a significant decrease in inflammatory cell infiltration in the external treatment group compared to the KOA group; nerve silver plating showed a significant decrease in the number of nerve fibers in the external treatment group compared to the KOA group. The content of IL-6 and SP in the KOA group was significantly increased compared to the blank group, while the content of IL-6 and SP in the external treatment group was significantly reduced compared to the KOA group. The protein and gene expression of Netrin-1, CGRP, DCC was significantly increased and UNC5 was reduced in the KOA group compared to the blank group; the protein and gene expression of Netrin-1, CGRP, DCC was significantly reduced and UNC5 was increased in the external treatment group compared to the KOA group. CONCLUSION The external therapy of warming meridians and invigorating blood can alleviate KOA pain, and the mechanism may be related to the inhibition of Netrin-1-mediated sensory nerve sprouting and the reduction of pain mediator secretion. -
图 2 滑膜组织神经镀银染色切片(×200)
Figure 2. Nerve silver staining sections of synovial tissue(× 200)
图 3 各组大鼠血清中促炎因子IL-6和疼痛因子SP、CGRP含量
注: 与空白组比较, **P＜0.01;与KOA组比较, ##P＜0.01。x±s，n=10。
Figure 3. Levels of pro-inflammatory factor IL-6 and pain mediator SP and CGRP in the serum of rats in each group
图 4 各组大鼠不同时间冷板抬爪时间(x±s，n=10)
Figure 4. Cold plate paw lifting time for each group of rats at different time
图 5 各组大鼠滑膜组织Netrin-1、CGRP相对蛋白和基因表达比较
注: 与空白组比较, *P＜0.05, **P＜0.01;与KOA组比较, ##P＜0.01。x±s，n=10。
Figure 5. Comparison of relative protein and gene expression of Netrin-1 and CGRP in synovial tissue of rats in each group
图 6 各组大鼠滑DRG组织DCC、UNC5相对蛋白和基因表达比较
注: 与空白组比较, *P＜0.05, **P＜0.01;与KOA组比较, #P＜0.05, ##P＜0.01。x±s，n=10。
Figure 6. Comparison of relative protein and gene expression of DCC and UNC5 in the smooth DRG tissue of rats in each group
表 1 引物序列
Table 1. Primer sequence
基因名称 序列 Netrin-1 F: 5′-AAGCCCTTCCACTACGAT-3′ R: 5′-GTTGAGACAGACACCCCC-3′ DCC F: 5′-ATCCTCCCGTCTCCCACA-3′ R: 5′-TTCCTGCTCCGAAACCTC-3′ UNC5 F: 5′-GAGGGCAGTTGGGAGGAT-3′ R: 5′-ACGGGAGCAAACAGAAGG-3′ CGRP F: 5′-AAGTTCTCCCCTTTCCTG-3′ R: 5′-GTTCCTCCTCCTGCTCCA-3′ β-actin F: 5′-GAGAGGGAAATCGTGCGT-3′ R: 5′-GGAGGAAGAGGATGCGG-3′ 
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