Search

Additional file 1. Additional figures.

Additional file 1: Supplementary Figure 1. CCL2 expression at different time points after SCI. The volcano map of differentially expressed genes in GEO: GSE42828. Supplementary Figure 2. Expression and localization of CCR2 after spinal cord injury. (A, B) Western blotting detection of CCR2 expression in spinal cord tissue. (C) qRT-PCR detection of CCR2 expression in spinal cord tissue. (D) FISH detects the location and expression of CCR2 in spinal cord injury tissue. Supplementary Figure 3. Identification of primary neurons, microglia and astrocytes. (A) NeuN identifies neuron nuclei, and MAP-2 identifies neuron axons. (B) CD11b identifies microglia cells. (C) GFAP identifies astrocytes. Supplementary Figure 4. Inhibiting the release of sEVs from activated astrocytes can reduce microglia activation and neuronal apoptosis. (A) Western blotting detection of Rab27a expression in astrocytes. (B) Rab27a siRNA can reduce the release of NO from microglia after inhibiting the release of sEVs. (C, D) TUNEL staining to detect neuronal apoptosis. Supplementary Figure 5. The effects of sEVs containing CCL2 on microglial activation and neuron apoptosis after inhibition of CCR2. (A-C) ELISA method to detect TNF-α, IL-1β and IL-6. (D) Detection of changes in NO content. (E-H) qRT-PCR and western blotting to detect the expression of TNF-α, IL-1β and IL-6. (I, J) Transwell assessment of the migration ability of BV2 microglia. (K-N) TUNEL staining and western blotting to detect neuronal apoptosis. Supplementary Figure 6. Further verification in vitro that CCL2-induced activation of microglia aggravates neuronal apoptosis. (A, B) Western blotting detection of IL-1Ra protein expression in neurons. (C) Immunofluorescence detection of neurons IL-1Ra protein expression. (D, E) TUNEL staining to detect neuronal apoptosis. (F, G) Western blotting was used to detect the expression of apoptosis related proteins in neurons.

Hao Wang,1,2,* Xuhui Ge,1,3,* Huiheng Qu,1,2,* Ning Wang,1,2 Jiawen Zhou,4 Wenjing Xu,1,2 Jingjing Xie,1,2 Yongping Zhou,1,2 Liqing Shi,1 Zhongke Qin,1 Zhuang Jiang,1,2 Wenjie Yin,1 Jiazeng Xia1,2 1Department of General Surgery and Translational Medicine Center, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, Wuxi 214002, Jiangsu, People’s Republic of China; 2Department of General Surgery, Wuxi Clinical College Affiliated to Nantong University, Wuxi 214002, Jiangsu, People’s Republic of China; 3Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, People’s Republic of China; 4The State Key Laboratory of Reproductive Medicine; Key Laboratory for Aging & Disease, Research Centre for Bone and Stem Cells, Department of Human Anatomy, Nanjing Medical University, Nanjing 211166, Jiangsu, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jiazeng XiaDepartment of General Surgery and Translational Medicine Center, The Affiliated Wuxi No.2 People’s Hospital of Nanjing Medical University, 68, Zhong Shan Road, Wuxi 214002, Jiangsu, People’s Republic of ChinaEmail jiazengxia@yahoo.comPurpose: Glycyrrhizic acid (GA) is the main active ingredient extracted from Chinese herb licorice root, and it shows anti-tumor effects in many cancer types, while its role in gastric cancer (GC) is still unknown. In this study, we evaluated the effects of GA on GC cells and explored the underlying mechanisms.Methods: The anti-proliferation effect of GA on GC cells was assessed by CCK-8, colony formation, and EdU assay. The effects of GA on cell cycle and apoptosis were detected by flow cytometer. Western blotting was performed to explore the underlying mechanisms.Results: Our results showed thatGA had a time- and dose-dependent inhibitory effect on proliferation of GC cells. Flow cytometer analysis demonstrated thatGA would lead to G1/S-phase arrest and apoptosis. GA treatment down-regulated the levels of G1 phase-related proteins, including cyclin D1, D2, D3, E1, and E2. In terms of apoptosis, GA treatment up-regulated thelevels of Bax, cleaved PARP, and pro-caspase-3, -8, -9, but did not influence their cleavage patterns. The expression of Bcl-2, survivin and p65 was attenuated after treatment. Besides, GA would down-regulate the phosphorylation of PI3K/AKT pathway.Conclusion: This study focused on inhibitory effect of GA on GC cells by inducing cell cycle arrest and apoptosis. Several important cyclins- and apoptosis-related proteins were involved in the regulation of GA to GC cells, and phosphorylated PI3K and AKT were attenuated. The results of this study indicated that GA is a potential and promising anti-cancer drug for GC.Keywords: glycyrrhizic acid, gastric cancer, cell cycle, apoptosis, PI3K/AKT pathway