基于p38 MAPK信号通路探讨隐丹参酮对膝骨关节炎模型大鼠滑膜纤维化的干预作用及机制

Investigating the role of Cryptotanshinone in alleviating synovial fibrosis in knee osteoarthritic rats via the p38 MAPK signaling pathway.

关键词:  膝骨关节炎  滑膜纤维化  隐丹参酮  丝裂原活化蛋白激酶

Key words:  Knee osteoarthritis  Synovial fibrosis  Cryptotanshinone  Mitogen-activa ed protein kinase.

摘要:

探讨隐丹参酮（Cryptotanshinone）通过调控p38 MAPK信号通路改善膝骨关节炎（KOA）大鼠滑膜纤维化的作用机制。方法：运用网络药理学筛选隐丹参酮对KOA的关键靶点和信号通路，将隐丹参酮与关键靶点进行分子对接；采用前交叉韧带离断法构建KOA大鼠模型，低、高浓度组给予隐丹参酮（0.2、0.6mg/kg/d）灌胃，激动剂组给予p38 MAPK?激动剂Anisomycin（5.0 mg/kg）腹腔注射联合高浓度隐丹参酮处理14天后，采用组织病理学方法（HE染色和Masson染色）观察滑膜组织的形态学变化及纤维化程度；通过免疫组织化学技术检测滑膜组织中TGF-β、α-SMA和Ⅰ型胶原蛋白（Collagen I）的表达定位与水平；应用ELISA法定量分析大鼠血清中炎性因子IL-1β和TNF-α含量；结合实时荧光定量PCR和Western blot技术，系统检测滑膜组织内TGF-β、α-SMA、Collagen I等纤维化相关指标及p38 MAPK信号通路关键分子的mRNA水平与蛋白表达量。结果：通过网络药理学分析，获得250个隐丹参酮靶点、4368个KOA靶点，61个药物－疾病共同靶点，经过PPI网络分析得到核心靶点为丝裂原活化蛋白激酶1（MAPK1）、丝裂原活化蛋白激酶14（MAPK14）、胰岛素（?INS?）等，GO功能富集条目1768条，其中生物过程（BP）1568条、细胞组分（CC）53条、分子功能（MF）147条，KEGG通路富集显示交集靶点共涉及221条通路，依据P值排序前6条为上皮细胞信号传导（Epithelial cell signaling），癌症中的信号通路（Pathways in cancer），催乳素信号通路（Prolactin signaling pathway），胰岛素抵抗（Insulin resistance），MAPK信号通路?（MAPK signaling pathway），分子对接结果显示隐丹参酮与核心通路靶点的结合能均小于﹣7.5 kcal/mol，具有较好结合活性；动物实验显示：与假手术组比较，滑膜衬里层显著增厚，细胞排列紊乱；滑膜下层可见大量炎症细胞浸润，并伴有弥漫性胶原纤维沉积；TGF-β、α-SMA、Collagen I 蛋白阳性区域比例明显增加；血清中IL-1β、TNF-α含量，滑膜组织中纤维化相关蛋白（TGF-β、α-SMA、Collagen I）及mRNA的表达，以及p38MAPK蛋白及mRNA，p-p38MAPK蛋白的表达均显著升高。与模型组比较，隐丹参酮组大鼠滑膜组织的病理改变明显改善，上述定量指标均显著逆转，且高浓度组较低浓度组效果更明显。与高浓度组相比，激动剂组大鼠炎症细胞浸润与胶原沉积更加明显，免疫组化中TGF-β、α-SMA、Collagen I表达增加，滑膜组织中p38MAPK、p-p38MAPK、TGF-β、α-SMA、Collagen I 蛋白表达，及TGF-β、α-SMA、Collagen I、p38MAPK的mRNA表达均增高。结论：隐丹参酮能够改善KOA大鼠滑膜组织纤维化，且高浓度隐丹参酮改善效果更佳；其作用机制可能与抑制p38 MAPK信号通路有关。

Abstract:

Objective: To explore the mechanism by which cryptotanshinone regulates the p38 MAPK signaling pathway to improve synovial fibrosis in a rat model of knee osteoarthritis (KOA). Methods: To identify the key targets and signaling pathways of Cryptotanshinone in the treatment of KOA using network pharmacology, and to perform molecular docking of Cryptotanshinone with these key targets. A KOA rat model was established using the anterior cruciate ligament transection (ACLT) method. Rats were treated with low and high doses of cryptotanshinone (0.2, 0.6 mg·kg^-1·d^-1) via oral gavage, while the agonist group received Anisomycin (5.0 mg·kg^-1) intraperitoneally along with high-dose cryptotanshinone for 14 days. Histopathological alterations in the synovium were assessed by HE and Masson staining. Immunohistochemistry was used to detect the expression of TGF-β, α-SMA, and Collagen I in the tissues. Serum levels of IL-1β and TNF-α were measured by ELISA. mRNA and protein expression of TGF-β, α-SMA, Collagen I, and p38MAPK in synovial tissues were assessed by quantitative real-time PCR and Western blot. Results: Network pharmacology identified 250 cryptotanshinone targets and 4,368 KOA targets, yielding 61 common targets. Subsequent PPI analysis revealed core targets, including MAPK1, MAPK14, and INS. GO enrichment analysis returned 1,768 terms, while KEGG analysis implicated 221 pathways. The most significantly enriched pathways included the MAPK signaling pathway, insulin resistance, and pathways in cancer. Molecular docking confirmed strong binding activity between cryptotanshinone and the core targets, with binding energies all below -7.5 kcal/mol. Compared with the sham-operated group, the model group exhibited significant proliferation and disorganization of synovial lining cells, inflammatory cell infiltration, and collagen deposition. The positive staining areas for TGF-β, α-SMA, and Collagen I increased significantly, and levels of IL-1β and TNF-α in the serum, as well as the expression of fibrosis-related proteins (TGF-β, α-SMA, Collagen I) and mRNA in synovial tissues, were elevated. Protein and mRNA levels of p38MAPK, and p-p38MAPK were also significantly higher. Compared to the model group, rats in the cryptotanshinone-treated group showed significantly improved synovial pathology, with a marked reversal of the above quantitative indicators. The high-dose group exhibited a more pronounced effect than the low-dose group. In contrast, the agonist group showed more severe inflammatory cell infiltration and collagen deposition compared to the high-dose group, with increased expression of TGF-β, α-SMA, Collagen I, p38MAPK, p-p38MAPK proteins, and mRNA. Conclusion: Cryptotanshinone can improve synovial fibrosis in KOA rats, with a more pronounced effect at higher concentrations. The mechanism may involve the suppression of the p38 MAPK signaling pathway.