Propofol Modulates the miR-142-3p/RAC1/NF-κB Axis to Influence Knee Osteoarthritis in Rats
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摘要:
目的 探讨丙泊酚对膝骨关节炎（knee osteoarthritis，KOA）大鼠疼痛的改善作用及其对微小RNA-142-3p（miR-142-3p）/Ras相关C3肉毒素底物1（ras-related C3 botulinum toxin substrate 1，RAC1）/核因子-κB （nuclear factor-κB，NF-κB）轴的调控机制。 方法 采用手术离断前交叉韧带、内侧副韧带及内侧半月板的方法构建大鼠KOA模型，用miR-142-3p antagomir或丙泊酚干预。干预结束后，检测大鼠机械刺激缩足阈值；酶联免疫吸附法（enzyme-linked immunosorbent assay，ELISA）检测大鼠血清中P物质（substance P，SP）、降钙素基因相关肽（calcitonin gene-related peptide，CGRP）和肿瘤坏死因子-α（tumour necrosis factor-α，TNF-α）、白细胞介素（interleukin，IL）-1β、IL-4、IL-10水平；改良Lequesne MG评分评估膝关节功能；免疫荧光检测脊髓组织中小胶质细胞活化标志物离子钙结合衔接分子1（ionized calcium-binding adaptor molecule 1，IBA-1）表达；定量实时逆转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction，qRT-PCR）测定脊髓组织中诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、精氨酸酶1（Arginase 1，Arg-1）、miR-142-3p相对表达水平；Western blot检测脊髓组织中RAC1、p-NF-κB p65和NF-κB p65蛋白表达水平。 结果 与KOA组相比，miR-142-3p antagomir组大鼠机械疼痛阈值降低（P < 0.05），Lequesne MG评分及血清SP、CGRP、TNF-α、IL-1β水平增加（P < 0.05），血清IL-4、IL-10水平和脊髓组织Arg-1、miR-142-3p表达水平降低（P < 0.05），脊髓组织IBA-1荧光强度、iNOS mRNA表达水平及RAC1、p-NF-κB p65蛋白表达水平增加（P < 0.05）；丙泊酚组大鼠机械疼痛阈值增加（P < 0.05），Lequesne MG评分及血清SP、CGRP、TNF-α、IL-1β水平降低（P < 0.05），血清IL-4、IL-10水平和脊髓组织Arg-1、miR-142-3p表达水平增加（P < 0.05），脊髓组织IBA-1荧光强度、iNOS mRNA表达水平及RAC1、p-NF-κB p65蛋白表达水平降低（P < 0.05）。与miR-142-3p antagomir组相比，丙泊酚组和丙泊酚+miR-142-3p antagomir组大鼠的上述指标结果有明显改善（P < 0.05）；miR-142-3p antagomir则部分逆转了丙泊酚对KOA大鼠的改善作用（P < 0.05）。 结论 丙泊酚可能通过调控miR-142-3p/RAC1/NF-κB轴，抑制小胶质细胞M1极化、促进M2型极化来纠正炎症因子失衡，从而缓解KOA大鼠疼痛症状。 -
关键词:
- 丙泊酚 /
- 微小RNA-142-3p /
- RAC1/NF-κB通路 /
- 膝骨关节炎 /
- 小胶质细胞极化
Abstract:Objective To explore the effect of propofol on pain relief in rats with knee osteoarthritis （KOA） and its regulatory mechanism on the microRNA-142-3p （miR-142-3p）/Ras-related C3 botulinum toxin substrate 1 （RAC1）/nuclear factor-κB （NF-κB） axis. Methods A rat knee osteoarthritis model was established by surgically transecting the anterior cruciate ligament, medial collateral ligament, and medial meniscus, with intervention by miR-142-3p antagomir or propofol. Following the conclusion of the intervention, the mechanical pain withdrawal threshold in rats was assessed. Enzyme-linked immunosorbent assay （ELISA） was employed to detect levels of substance P （SP）, calcitonin gene-related peptide （CGRP）, tumour necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-4 and IL-10 levels in rat serum. Modified Lequesne MG score for assessing knee joint function. Immunofluorescence detection of ionized calcium-binding adaptor molecule 1 （IBA-1） , an activational marker of microglia, in spinal cord tissue. Quantitative real-time reverse transcription polymerase chain reaction （qRT-PCR） was employed to determine the relative expression levels of inducible nitric oxide synthase （iNOS）, arginase 1 （Arg-1）, and miR-142-3p in spinal cord tissue. Western blot analysis of RAC1, p-NF-κB p65 and NF-κB p65 protein expression levels in spinal cord tissue. Results Compared with the KOA group, rats in the miR-142-3p antagomir group exhibited a reduced mechanical pain threshold （P < 0.05）, increased Lequesne MG scores and serum levels of SP, CGRP, TNF-α, and IL-1β （P < 0.05）, serum IL-4 and IL-10 levels, and spinal cord tissue Arg-1 and miR-142-3p expression levels decreased （P < 0.05）. Spinal cord tissue IBA-1 fluorescence intensity, iNOS mRNA expression levels, and RAC1 and p-NF-κB p65 protein expression levels increased （P < 0.05）. The propofol group exhibited increased mechanical pain thresholds in rats （P < 0.05）, reduced Lequesne MG scores and serum levels of SP, CGRP, TNF-α, and IL-1β （P < 0.05）, serum IL-4 and IL-10 levels, and spinal cord tissue Arg-1 and miR-142-3p expression levels increased （P < 0.05）. Spinal cord tissue IBA-1 fluorescence intensity, iNOS mRNA expression levels, and RAC1 and p-NF-κB p65 protein expression levels decreased （P < 0.05）. Compared with the miR-142-3p antagomir group, the propofol group and the propofol+miR-142-3p antagomir group demonstrated significant improvement in the aforementioned indicators in rats （P < 0.05）. The miR-142-3p antagomir partially reversed the ameliorative effects of propofol in KOA rats （P < 0.05）. Conclusion Propofol may inhibit the M1 polarization of microglia, promote the M2 polarization, exert anti-inflammatory effect and inhibit the pain response of KOA rats by regulating the miR-142-3p/RAC1/NF-κB axis. -
Key words:
- Propofol /
- MicroRNA-142-3p /
- RAC1/NF-κB pathway /
- Knee osteoarthritis /
- Microglial polarization
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图 1 各组大鼠机械疼痛阈值结果（$\bar x \pm s $，n = 15）
与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 1. Mechanical pain threshold results for each group of rats （$\bar x \pm s $，n = 15）
图 2 各组大鼠血清疼痛相关指标水平（$\bar x \pm s $，n = 15）
A：各组大鼠血清CGRP水平；B：各组大鼠血清SP水平。与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 2. Serum levels of pain-related markers in each group of rats （$\bar x \pm s $，n = 15）
图 3 各组大鼠Lequesne MG评分（$\bar x \pm s $，n = 15）
与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 3. Lequesne MG scores for rats in each group （$\bar x \pm s $，n = 15）
图 4 各组大鼠关节软骨组织HE染色（×400）
Figure 4. Hematoxylin and eosin staining of articular cartilage tissue from each group of rats （×400）
图 5 各组大鼠血清中炎症因子水平（$\bar x \pm s $，n = 15）
A：各组大鼠血清中TNF-α水平；B：各组大鼠血清中IL-1β水平；C：各组大鼠血清中IL-4水平；D：各组大鼠血清中IL-10水平。与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 5. Levels of inflammatory cytokines in the serum of rats from each group （$\bar x \pm s $，n = 15）
图 6 免疫荧光染色检测大鼠脊髓组织中IBA-1表达（$\bar x \pm s $，n = 5）
A：各组大鼠脊髓组织中小胶质细胞免疫荧光染色结果；B：各组大鼠脊髓组织中IBA-1荧光强度。与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 6. Immunofluorescence staining for IBA-1 expression in rat spinal cord tissue （$\bar x \pm s $，n = 5）
图 7 各组大鼠脊髓组织小胶质细胞极化标志物水平（$\bar x \pm s $，n = 10）
A：各组大鼠脊髓组织iNOS mRNA水平；B：各组大鼠脊髓组织Arg-1 mRNA水平。与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 7. Levels of glial cell polarisation markers in spinal cord tissue from rats in each group （$\bar x \pm s $，n = 10）
图 8 各组大鼠脊髓组织miR-142-3p、RAC1和p-NF-κB p65的表达（$\bar x \pm s $，n = 10）
A：miR-142-3p和RAC1的结合位点预测；B：双荧光素酶报告基因测定验证miR-142-3p与RAC1的靶向关系；C：各组大鼠脊髓组织miR-142-3p相对表达水平；D：各组大鼠脊髓组织RAC1 mRNA相对表达水平；E：各组大鼠脊髓组织RAC1、p-NF-κB p65、NF-κB p65蛋白表达。与miR-NC相比，▲P < 0.05；与Sham组相比，*P < 0.05；与KOA组相比，#P < 0.05；与miR-142-3p antagomir组相比，△P < 0.05；与丙泊酚组相比，$P < 0.05。
Figure 8. Expression of miR-142-3p，RAC1 and p-NF-κB p65 in spinal cord tissue from each group of rats （$\bar x \pm s $，n = 10）
表 1 引物序列
Table 1. Primer sequences
基因 引物序列（5'-3'） 引物长度（bp） miR-142-3p F：TGCTGCTGTGTAGTGTTTCCTACT 83 R：TATGGTTGTTCACGACTCCTTCAC U6 F：CTCGCTTCGGCAGCACA 171 R：GCGAGCACAGAATTAATACGAC iNOS F：CGGAGAACAGCAGAGTTGG 122 R：GGAATAGCACCTGGGGTTT Arg-1 F：CAGTATTCACCCCGGCTA 198 R：CCTCTGGTGTCTTCCCAA GAPDH F：GTTACCAGGGCTGCCTTCTC 134 R：GGGTTTCCCGTTGATGACC 
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