独一味通过调控RAGE-PI3K-Akt改善膝骨关节炎大鼠滑膜纤维化的机制研究

Mechanism of Duyiwei in Ameliorating Synovial Fibrosis in Knee Osteoarthritis Rats via Regulation of RAGE-Mediated PI3K-Akt Signaling

关键词:  膝骨关节炎  独一味  滑膜纤维化  网络药理学  外周痛敏

Key words:  knee osteoarthritis  Duyiwei  synovial fibrosis  

摘要:

目的 探讨独一味是否通过调控晚期糖基化终末产物受体-磷脂酰肌醇3-激酶-蛋白激酶B(RAGE-PI3K-Akt，Receptor for Advanced Glycation End-products-Phosphatidylinositol 3-kinase-Protein kinase B)改善膝骨关节炎(KOA，knee osteoarthritis)大鼠滑膜纤维化作用机制。方法 利用网络药理学预测独一味治疗KOA的活性成分及其作用靶点；将SPF级雄性SD大鼠(n=10/组)随机分为对照组、模型组及独一味低、高剂量组(1.0、3.0 g·kg?¹·d?¹)、独一味高剂量+oe-NC组、独一味高剂量+oe-RAGE组；除对照组外，其余行前交叉韧带横断术(ACLT，anterior cruciate ligament transection)建立KOA模型，造模后膝关节注射腺相关病毒，连续灌胃给药4周。分别于造模前及造模后各时间点对各组大鼠进行热痛阈潜伏期与冷痛阈潜伏期检测，以评估外周痛敏变化；HE和Masson染色评估滑膜炎症及纤维化程度；ELISA检测滑膜组织神经生长因子(NGF，Nerve Growth Factor)、白细胞介素-6(IL-6，Interleukin-6)、肿瘤坏死因子-α(TNF-α，Tumor Necrosis Factor-alpha)含量水平；WB检测滑膜组织中RAGE、P-PI3K、PI3K、p-Akt、Akt、p-p65、转录因子p65(P65，Transcription factor p65)、I型胶原蛋白(Collagen I，Collagen Type I)、α-平滑肌肌动蛋白(α-SMA，Alpha-Smooth Muscle Actin)、基质金属蛋白酶组织抑制剂-1(TIMP-1，Tissue Inhibitor of Metalloproteinases-1)、波形蛋白(Vimentin)的蛋白表达；qPCR检测Col1a1、Acta2、Timp1及Vim的mRNA表达；IHC观察纤维化蛋白的组织分布；结果 网络药理学结果显示，独一味包含有效成分97个，对应靶点97个，膝骨关节炎相关基因4525个，得到药物疾病共同靶点85个；药物疾病共同靶点与AGE-RAGE信号通路下游靶基因交集靶点共25个。热痛阈及冷痛阈潜伏期结果显示独一味可显著延长潜伏期时间，而在给予oe-RAGE腺相关病毒后，独一味高剂量组的镇痛效应被显著逆转；ELISA 结果显示独一味显著降低滑膜组织中NGF、IL-6及TNF-α的含量；WB结果显示独一味能够显著下调RAGE-PI3K-Akt通路相关蛋白表达及磷酸化水平。qPCR结果显示独一味显著下调滑膜组织中Col1a1、Acta2、Timp1及Vim的mRNA表达；而oe-RAGE组上述指标均显著回升。结论 独一味能够通过调控RAGE-PI3K-Akt通路表达改善KOA大鼠的滑膜纤维化情况。

Abstract:

OBJECTIVE To investigate whether Duyiwei (Lamiophlomis rotata) improves synovial fibrosis in rats with knee osteoarthritis (KOA) by regulating the Receptor for Advanced Glycation End-products–Phosphatidylinositol 3-kinase–Protein kinase B (RAGE-PI3K-Akt) signaling pathway. METHODS Network pharmacology was employed to predict the active components and potential targets of Duyiwei in the treatment of KOA. SPF-grade male SD rats (n = 10/group) were randomly assigned to the following groups: control group, model group, low-dose Duyiwei group(1.0 g·kg?¹·d?¹), high-dose Duyiwei group (3.0 g·kg?¹·d?¹), high-dose Duyiwei + oe-NC group, and high-dose Duyiwei + oe-RAGE group. Except for the control group, all other groups underwent anterior cruciate ligament transection (ACLT) to establish the KOA model. Following modeling, adeno-associated virus (AAV) was injected into the knee joints, and the rats were continuously administered Duyiwei via intragastric gavage for 4 weeks. Thermal withdrawal latency and cold withdrawal latency were measured before modeling and at various time points post-modeling to assess changes in peripheral pain sensitivity. Hematoxylin-eosin (HE) and Masson''s trichrome staining were performed to evaluate synovial inflammation and the degree of fibrosis. The levels of nerve growth factor (NGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in synovial tissues were measured by enzyme-linked immunosorbent assay (ELISA). Protein expressions of RAGE, p-PI3K, PI3K, p-Akt, Akt, p-p65, Transcription factor p65 (P65), Collagen Type I (Collagen I), Alpha-Smooth Muscle Actin (α-SMA), Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), and Vimentin in synovial tissues were detected by Western blotting (WB). The mRNA expressions of Col1a1, Acta2, Timp1, and Vim were determined using quantitative real-time PCR (qPCR). The tissue distribution of fibrosis-related proteins was observed via immunohistochemistry (IHC). RESULTS Network pharmacology analysis showed that Duyiwei contained 97 active components corresponding to 97 targets, with 4,525 genes related to knee osteoarthritis, yielding 85 common drug-disease targets. There were 25 overlapping targets between the drug-disease common targets and the downstream target genes of the AGE-RAGE signaling pathway. Thermal and cold withdrawal latency tests demonstrated that Duyiwei significantly prolonged the latency periods, whereas this analgesic effect in the high-dose Duyiwei group was markedly reversed after the administration of oe-RAGE adeno-associated virus. ELISA results showed that Duyiwei significantly reduced the contents of NGF, IL-6, and TNF-α in synovial tissues. WB results indicated that Duyiwei significantly downregulated the expression and phosphorylation levels of key proteins in the RAGE-PI3K-Akt pathway. qPCR results revealed that Duyiwei significantly downregulated the mRNA expressions of Col1a1, Acta2, Timp1, and Vim in synovial tissues. whereas these indices were significantly elevated in the oe-RAGE group.CONCLUSION Duyiwei can ameliorate synovial fibrosis in knee osteoarthritis rats by regulating the expression of the RAGE-PI3K-Akt pathway.