Cinnamomi ramulus exhibits anti-proliferative and anti-migration effects on MH7A rheumatoid arthritis-derived fibroblast-like synoviocytes through induction of apoptosis &amp; cell arrest and suppression of matrix metalloproteinase
Authors: Liu Jia , Zhang Qing , Li Ruo-Lan , Wei Shu-Jun , Gao Yong-Xiang , Jin Wei , Wu Chun-Jie , Pu Xufeng 
Published: February 2020

Abstract
Background: Rheumatoid arthritis (RA) is a complex chronic inflammatory disease that is associated with the aberrant activation of fibroblast-like synoviocytes (FLS). The extract of Cinnamomi ramulus has been reported to exert alleviates pain, anti-tumor and anti-inflammatory effects. The present study was designed to investigate the effects of Cinnamomi ramulus on RA and explore the underlying mechanisms. Material/methods: TNF-α induced human synoviocyte MH7A cells was performed to evaluate the anti-proliferative and anti-migration effect of Cinnamomi ramulus. The anti-proliferative effect of Cinnamomi ramulus was determined by CCK-8 assay and colony formation assay. Apoptosis was measured by AnnexinV FITC/PI staining and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis and cell cycle-related molecules, including Bcl-2, Bax, C-Caspase-3, CDC2 and Cycylin B1 were determined by Western blotting. Furthermore, the migration and invasion abilities of MH7A cells were determined using scratch wound healing assay and transwell assay. mRNA expressions of (MMP)-1, -2, & -3, P53, P21 and Cyclin D were determined using qRT-PCR analysis. For qualitative analysis on its chemical components, an ultra-high performance liquid chromatography (UPLC) coupled with Q-Exactive MS (QE-MS) was established for rapid separation and structural identification of the constituents in Cinnamomi ramulus. The further computationally study on the relationships between the 9 compounds and the potential target proteins of RA were carried out with molecular docking strategy. Results: Our data demonstrate that Cinnamomi ramulus inhibited proliferation of MH7A cells, induced cell apoptosis, blocked the cell cycle in the G2/M phase and regulated the protein expression of Bcl-2, Bax, C-Caspase-3, CDCD2 and Cyclin B1. Moreover, Cinnamomi ramulus was proven to significantly inhabited migration and invasion of MH7A cells through inhibition of levels of matrix metalloproteinase (MMP)-1, -2, & -3.Cinnamomi ramulus reduced mRNA levels of CDK4 whereas increased the expression of P53, P21 and CyclinD, implying its regulation effects on apoptosis and cell cycle distribution in MH7A cells. The chromatographic profiling of the extract by UPLC-QE-MS/MS analysis showed 9 compounds are the main components. And the molecular docking strategy results showed that the compounds in Cinnamomi ramulus have good affinity with protein crystal, and benzyl cinnamate may be the main active component of Cinnamomi ramulus to induce cell apoptosis and cycle resistance. Conclusions: Cinnamomi ramulus exhibits anti-proliferative and anti-migration effects on MH7A rheumatoid arthritis-derived fibroblast-like synoviocytes through induction of apoptosis & cell arrest and suppression of matrix metalloproteinase.