Zhang Zong-Xing, Jiang Lu, Liu Dao-Zhong, Tao Bo-Nan, Hou Zi-Ming, Tian Meng-Jie, Feng Jia, Yuan Lin
Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases, Hubei Minzu University Enshi 445000, China Medical School, Hubei Minzu University Enshi 445000, China.
Zhongguo Zhong Yao Za Zhi. 2023 Apr;48(8):2203-2211. doi: 10.19540/j.cnki.cjcmm.20230103.706.
This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
本研究旨在通过磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）信号通路，探讨飞龙掌血根及根皮乙醇提取物（TAAE）对大鼠胶原诱导性关节炎（CIA）的治疗作用。具体而言，先诱导大鼠发生CIA，然后分别用TAAE和雷公藤多苷片（TGT）对大鼠进行治疗（口服，每日一次）。每周对大鼠后肢关节的肿胀程度进行评分。给药35天后，基于苏木精-伊红（HE）染色观察组织病理学变化。采用酶联免疫吸附测定（ELISA）检测细胞因子[肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6]水平。进行末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色以检测大鼠滑膜细胞的凋亡情况。使用蛋白质免疫印迹法检测凋亡相关蛋白B细胞淋巴瘤-2（Bcl-2）相关X蛋白（Bax）、Bcl-2和半胱天冬酶-3以及通路相关蛋白磷酸肌醇3激酶（PI3K）、磷酸化（p）-PI3K、蛋白激酶B（Akt）和p-Akt的表达水平。进行逆转录-定量聚合酶链反应（RT-qPCR）检测Bax、Bcl-2、半胱天冬酶-3、TNF-α、IL-6和IL-1β的mRNA水平以及通路相关蛋白PI3K、p-PI3K、Akt和p-Akt的mRNA水平。TAAE可减轻CIA大鼠的关节肿胀，降低血清炎症细胞因子水平，改善滑膜组织病理学变化，促进滑膜细胞凋亡，并抑制滑膜炎症。此外，RT-qPCR和蛋白质免疫印迹结果表明，TAAE上调Bax水平，下调Bcl-2水平，并激活半胱天冬酶-3以促进滑膜细胞凋亡。TAAE有效下调p-PI3K和p-Akt的蛋白水平。在本研究中，TAAE对大鼠CIA具有治疗作用并减轻炎症。其机制是抑制PI3K/Akt信号通路并促进滑膜细胞凋亡。总体而言，本研究为TAAE抗炎机制的研究提供了新线索，为TAAE在炎症性及自身免疫性疾病治疗中的更好临床应用奠定了理论基础。
