| 摘要: | 雞鳴散（JMS）為一中藥處方包含七種中藥材，數百年來用於治療風濕; 足痛; 筋骨腫脹; 利尿和不同類型的發炎。我們的研究目的是藉由研究三種不同細胞株 （RAW 264.7巨噬細胞; SW1353 軟骨細胞和初代培養的大鼠軟骨細胞）的抗發炎和關節炎活性。我們使用鹿角菜膠誘導的大鼠急性足掌發炎和單碘乙酸鹽誘導的大鼠退化性關節炎模型這兩種動物模型進行急性和慢性關節炎研究。為了分析草藥處方成分和血清代謝，我們使用了兩種不同的質譜工具：超高效液相色譜-四極桿時間飛行質譜儀 （UPLC-Q-TOF-MS） 和三重四極桿質譜儀 (UPLC- MS/MS)。對於利尿研究，我們使用代謝籠來收集尿液樣本，並使用 UPLC-MS/MS 測定大鼠的血清成分。Venn diagram 和 MetaboAnalyst 用於研究體內代謝與時間藥理學的關聯性。結果顯示雞鳴散具有降低RAW264.7巨噬細胞 COX-2; PGE2; NO 和 iNOS 的表現量，初代培養軟骨細胞iNOS表現降低，SW1353的MMP-13降低。在動物模型中發現，雞鳴散顯著抑制鹿角菜膠誘導大鼠急性足掌發炎的腫脹，及退化性關節炎模型中大鼠雙足承重分佈的平衡，同時顯著降低足掌腫脹和降低血清的發炎因子 PGE2表現量血清藥理學分析血清成分得知 hesperidin 在體內會被代謝成 hesperetin 3'-O-glucuronide, hesperetin 5-glucuronide, hesperetin 7-glucoside 及 hesperetin 7-neohesperidoside 等代謝物，而 narirutin 在體內會被代謝成 2-hydroxyisoflavanone naringenin, naringenin 4'-O-glucuronide, naringenin 5-rhamnoside, naringenin 7-sulfate, naringenin-7-O-glucuronide, (S)-Naringenin 8-C-(2''-rhamnosylglucoside) and (2S)-Naringenin 8-C-alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranoside 等代謝物， 結合能力結果顯示 11種血清成分結合能力高於正對照組 indomethacin。利尿實驗結果顯示，雞鳴散顯著增加給藥後 6 小時的總尿量。及改變400; 800 mg/kg 組的尿鉀（K+）含量。尿生化檢查; pH分析顯示給藥後並無對大鼠產生急性的腎臟毒性，在時間藥理學部分 JMS-95E (800 mg/kg) 的四時段給藥採集(0-6 a.m., 6-12 a.m., 0-6 p.m., 6-12 p.m.) 的結果顯示 JMS-95E 可顯著改變(0-6 a.m., 6-12 a.m.)的尿量。在方劑晝夜節律的時間藥理學研究，我們將每天 24 小時區分為四組給藥時間分別為(0-6 a.m., 6-12 a.m., 0-6 p.m., 6-12 p.m.)小時。結果顯示，口服JMS 6小時後，0-6點; 6-12點組發生顯著變化。 Venn diagram 分析質譜數據顯示 0和6時給藥中重複的 key biomarker為 naringenin-7-O-glucoside。MetaboAnalyst 體內代謝途徑結果顯示六大途徑參與胃腸和肝臟的代謝。我們的研究證明了JMS方劑具有抗發炎; 抗退化性關節炎; 利尿及具有時間藥理學中的服藥時間作用差異。
The prescription of Ji Ming Shan (JMS) contains seven herbal medicines and has been used for hundreds of years to combat rheumatism, foot pain, reduce tendon swelling, induce diuresis, and various types of inflammation. Our research aim is to investigate the anti-inflammatory and osteoarthritis activities through three different cell lines (RAW 264.7 cells, SW1353 chondrocytes, and primary cultured rat chondrocytes). Two animal models, carrageenan-induced paw edema, and monoiodoacetate-induced rat models were used for acute and chronic studies. To analyze the herbal prescription components and serum metabolism, two different mass spectrometric tools were used: Ultra-performance liquid chromatography-tandem quadrupole time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) and triple quadrupole-tandem mass spectrometry (UPLC-MS/MS). For the diuresis study, the metabolize cage was used to collect urine samples and UPLC-MS/MS was used to determine serum components from rat. Venn diagrams and MetaboAnalyst were used to investigate the chronopharmacology of body organ regulation. Results showed decreased expression levels of COX-2, PGE2, NO and iNOS from macrophage cells, decreased iNOS expression from primary cultured chondrocytes and MMP-13 of SW1353 were reduced. Animal studies demonstrated a significant reduction in rat paw volume in both models and the recovery of hind-paw bearing distribution in the monoiodoacetate model. In metabolism, hesperidin underwent oxidation and glucuronidation to form hesperetin 3'-O-glucuronide, hesperetin 5-glucuronide, hesperetin 7-glucoside and hesperetin 7-neohesperidoside. Narirutin was converted to 2-hydroxyisoflavanone naringenin, naringenin 4'-O-glucuronide, naringenin 5-rhamnoside, naringenin 7-sulfate, naringenin-7-O-glucuronide, (S)-Naringenin 8-C-(2''-rhamnosylglucoside) and (2S)-Naringenin 8-C-alpha-L-rhamnopyranosyl-(1->2)-beta-D-glucopyranoside. Eleven compounds showed high binding ability with the object protein COX-2 compared with the positive control (indomethacin) from LibDock results. Diuresis results showed a significant change in urine volume at 6 hours continues collection with no kidney toxicity, and urine pH ranged from 7.0 to 7.5 in all treated groups. Chronopharmacology revealed significant changes in the 0-6 and 6-12 hour groups. A key biomarker from the Venn diagram cross-analysis named "naringenin-7-O-glucoside" was identified. MetaboAnalyst demonstrated that the major metabolism pathways from 0-3, 6-9, 12-15 and 18-21 hours within Butanoate metabolism, D-Glutamine and D-glutamate metabolism, Glycerophospholipid metabolism, Linoleic acid metabolism, Primary bile acid biosynthesis, Riboflavin metabolism, Sphingolipid metabolism. JMS-95E can inhibited inflammatory pathway of NO, iNOS, COX-2, PGE2, MMP-13, reduced the paw volume, recover hind-paw weight bearing. In the serum sample 15 compounds from the narirutin and hesperidin were identified and in silico analysis showed hersperidin-7-glucoside exhibited the highest binding ability with COX-2 enzyme. JMS significantly changed 0-6 and 6-12 groups urine volume within no kidney toxicity. The naringenin-7-O-glucoside was identified as the diuresis key compound and metabolize analysis result with six major pathways are involved in the gastrointestinal system and the liver metabolites. Our study demonstrated the ability of JMS prescription in anti-inflammation, anti-osteoarthrosis, diuresis, and chronopharmacology effect. |
