Lu Qingyi, Jiang Haixu, Zhu Qingqing, Xu Jie, Cai Yanan, Huo Guiyang, Yuan Kai, Huang Guangrui, Xu Anlong
School of Life Sciences, Beijing University of Chinese Medicine, Beijing, China.
The Seventh Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Evid Based Complement Alternat Med. 2022 Feb 16;2022:8589121. doi: 10.1155/2022/8589121. eCollection 2022.
Rheumatoid arthritis (RA) is a common autoimmune disease worldwide. Neutrophils play critical roles in the onset and development of RA and are the promising target for RA treatment. Tetrandrine is a bis-benzyl isoquinoline alkaloid derived from the traditional Chinese herbal S. Moore. Tetrandrine is effective in alleviating RA by inhibiting macrophage inflammatory response, fibroblast overproliferation, and pannus formation. However, whether tetrandrine regulates the activities of neutrophils in RA is largely unknown. In this study, we adopted adjuvant-induced arthritis (AA) murine model to explore the effect of tetrandrine on RA and neutrophils. Twenty-eight mice were divided into four groups. The control group was injected with PBS in the limbs and treated with PBS by intraperitoneal injection (i.p.) from Day 10 to Day 37. The arthritis murine model was induced by injecting FCA into the ankle joints of hind limbs. The AA group, the AA + TET group, and the AA + DEX group mice were treated with PBS, tetrandrine (6 mg/kg), or dexamethasone (1 mg/kg) i.p. daily, respectively. Arthritic scores were evaluated, and the joint diameter was measured every three days. A cytometric bead assay was performed to measure the concentrations of IFN-, TNF- and IL-6 in the serum. H&E staining and Safranin O-fast staining were adopted to monitor the tissue changes in the joint. Immunohistochemistry assays were applied to detect the MPO, NE, CitH3, and PAD4 expression levels. To assess the effect of tetrandrine on neutrophil activities , CCK8 tests were applied to determine cell viability. The qPCR and ELISA were performed to determine IL-1 and IL-6 expression levels. Immunofluorescence assays were performed to measure the formation of NETs. The results indicated that tetrandrine significantly alleviated the symptoms of RA in terms of the ankle diameter (from 4.629 ± 2.729 to 3.957 ± 0.257; < 0.01) and ankle score (from 4.000 ± 0.000 to 3.286 ± 0.756; < 0.05). Tetrandrine treatment significantly increased the cartilage areas and decreased serum IL-6 significantly (from 5.954 ± 2.127 to 2.882 ± 2.013; < 0.01). The immunohistochemistry assays also showed decreased expression levels of NE, MPO, PAD4, and CitH3 induced by tetrandrine in comparison with the AA group ( < 0.01). The qPCR assays and ELISAs showed that tetrandrine had an anti-inflammatory effect by significantly inhibiting IL-6 ( < 0.01). The immunofluorescence assays showed that NET formation induced by PMA could be reduced by tetrandrine ( < 0.01). In conclusion, tetrandrine has good efficacy in treating RA by regulating neutrophil-involved inflammation and NET formation.
类风湿关节炎（RA）是一种全球常见的自身免疫性疾病。中性粒细胞在RA的发病和发展中起关键作用，是RA治疗的有前景的靶点。粉防己碱是一种从传统中草药汉防己中提取的双苄基异喹啉生物碱。粉防己碱通过抑制巨噬细胞炎症反应、成纤维细胞过度增殖和血管翳形成，有效缓解RA。然而，粉防己碱是否调节RA中中性粒细胞的活性在很大程度上尚不清楚。在本研究中，我们采用佐剂诱导性关节炎（AA）小鼠模型来探讨粉防己碱对RA和中性粒细胞的影响。28只小鼠分为四组。对照组小鼠在四肢注射PBS，并在第10天至第37天腹腔注射（i.p.）PBS。通过将弗氏完全佐剂（FCA）注射到后肢踝关节来诱导关节炎小鼠模型。AA组、AA + TET组和AA + DEX组小鼠分别每天腹腔注射PBS、粉防己碱（6 mg/kg）或地塞米松（1 mg/kg）。评估关节炎评分，每三天测量关节直径。进行细胞计数珠分析法以测量血清中IFN-、TNF-和IL-6的浓度。采用苏木精-伊红（H&E）染色和番红O-固绿染色来监测关节组织变化。应用免疫组织化学分析法检测髓过氧化物酶（MPO）、中性粒细胞弹性蛋白酶（NE）、瓜氨酸化组蛋白H3（CitH3）和肽酰精氨酸脱亚氨酶4（PAD4）的表达水平。为了评估粉防己碱对中性粒细胞活性的影响，应用CCK8试验来测定细胞活力。进行qPCR和ELISA以测定IL-1和IL-6的表达水平。进行免疫荧光分析以测量中性粒细胞胞外陷阱（NETs）的形成。结果表明，粉防己碱在踝关节直径（从4.629±2.729减小至3.957±0.257；P<0.01）和踝关节评分（从4.000±0.000降至3.286±0.756；P<0.05）方面显著减轻了RA症状。粉防己碱治疗显著增加了软骨面积，并显著降低了血清IL-6水平（从5.954±2.127降至2.882±2.013；P<0.01）。免疫组织化学分析还显示，与AA组相比，粉防己碱诱导的NE、MPO、PAD4和CitH3表达水平降低（P<0.01）。qPCR分析和ELISA显示，粉防己碱通过显著抑制IL-6发挥抗炎作用（P<0.01）。免疫荧光分析显示，粉防己碱可减少佛波酯（PMA）诱导的NET形成（P<0.01）。总之，粉防己碱通过调节中性粒细胞参与的炎症和NET形成，在治疗RA方面具有良好疗效。
