Journal of Traditional Chinese Medicine ›› 2024, Vol. 44 ›› Issue (2): 268-276.DOI: 10.19852/j.cnki.jtcm.20240203.005
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Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway
ZHENG Xueying1, GUO Liang1, LAI Siyi3,4, LI Fengyue1, LIANG Mingli1, LIU Wanting1, MENG Chun2(
), LIU Guanghui3,4()
- 1 Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350104, China
2 Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350104, China; Eye Institute of Integrated Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
3 Department of Ophthalmology, Affiliated People's Hospital (Fujian Provincial People's Hospital), Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
4 Eye Institute of Integrated Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China
-
Received:2022-11-22Accepted:2023-04-27Online:2024-04-15Published:2024-02-03 -
Contact:Prof. MENG Chun, Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou 350108, China; Eye Institute of Integrated Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China.mengchun@fzu.edu.cn Telephone: +86-591-83947028; Dr. LIU Guanghui, Department of Ophthalmology, Affiliated People's Hospital (Fujian Provincial People's Hospital), Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China; Eye Institute of Integrated Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350004, China.latiny@gmail.com -
Supported by:Fujian Major Research Grants for Young and Middle-aged Health Professionals(Research and Development of Anti-Keratitis Protein Drug Sgp130)(2021ZQNZD012);National Natural Science Foundation of China(Study on Mechanism of Yijing Decoction in Preventing Microvascular Damage of Early Diabetic Retinopathy based on MMPs/TIMPs)(81774369)
Cite this article
ZHENG Xueying, GUO Liang, LAI Siyi, LI Fengyue, LIANG Mingli, LIU Wanting, MENG Chun, LIU Guanghui. Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway[J]. Journal of Traditional Chinese Medicine, 2024, 44(2): 268-276.
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Figure 1 Apoptosis and cell viability with exposure to different doses of emodin were identified using flow cytometry and the CCK-8 assay A: HUVEC were incubated with different concentrations (0-20 μM, 24 h) of emodin, CCK-8 assay was applied to determine cells viability; B: the rate of cell apoptosis in each group, the ratio of the second quadrant plus the fourth quadrant represents the ratio of cell apoptosis; C: apoptosis was assessed by flow cytometry assay. C1: 0 μM emodin group; C2: 10 μM emodin group; C3: 20 μM emodin group. CCK8: cell-counting-kit-8; HUVEC: human umbilical vein endothelial cell. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, compared with 0 μM emodin treatment group.
Figure 1 Apoptosis and cell viability with exposure to different doses of emodin were identified using flow cytometry and the CCK-8 assay A: HUVEC were incubated with different concentrations (0-20 μM, 24 h) of emodin, CCK-8 assay was applied to determine cells viability; B: the rate of cell apoptosis in each group, the ratio of the second quadrant plus the fourth quadrant represents the ratio of cell apoptosis; C: apoptosis was assessed by flow cytometry assay. C1: 0 μM emodin group; C2: 10 μM emodin group; C3: 20 μM emodin group. CCK8: cell-counting-kit-8; HUVEC: human umbilical vein endothelial cell. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, compared with 0 μM emodin treatment group.
Figure 2 Emodin inhibited migration, invasion, and tube formation of HUVEC A: HUVEC were treated with various concentrations of (0, 10 and 20 μM) emodin 24 h, and the migration of HUVEC was determined by transwell migration assay; B: HUVEC were treated with various concentrations of emodin (0, 10 and 20 μM), and the migration of HUVEC was determined transwell invasion assay; C: HUVEC were treated with various concentrations of emodin (0, 10 and 20 μM), and the migration of HUVEC was determined by matrigel tube-formation assay; D: results of quantitative analysis of HUVEC transwell migration assay. E: results of quantitative analysis of HUVEC transwell invasion assay. F: results of quantitative analysis of HUVEC matrigel tube-formation assay. A1, B1, C1: cells treated with 0 μM emodin 24 h; A2, B2, C2: cells treated with 10 μM emodin 24 h; A3, B3, C3: cells treated with 20 μM emodin 24 h. HUVEC: human umbilical vein endothelial cells. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.01, bP < 0.05, compared with 0 μM emodin group.
Figure 2 Emodin inhibited migration, invasion, and tube formation of HUVEC A: HUVEC were treated with various concentrations of (0, 10 and 20 μM) emodin 24 h, and the migration of HUVEC was determined by transwell migration assay; B: HUVEC were treated with various concentrations of emodin (0, 10 and 20 μM), and the migration of HUVEC was determined transwell invasion assay; C: HUVEC were treated with various concentrations of emodin (0, 10 and 20 μM), and the migration of HUVEC was determined by matrigel tube-formation assay; D: results of quantitative analysis of HUVEC transwell migration assay. E: results of quantitative analysis of HUVEC transwell invasion assay. F: results of quantitative analysis of HUVEC matrigel tube-formation assay. A1, B1, C1: cells treated with 0 μM emodin 24 h; A2, B2, C2: cells treated with 10 μM emodin 24 h; A3, B3, C3: cells treated with 20 μM emodin 24 h. HUVEC: human umbilical vein endothelial cells. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.01, bP < 0.05, compared with 0 μM emodin group.
Figure 3 In mouse corneas, emodin inhibits alkali burn-induced neovascularization and inflammatory cell infiltration A: slit-lamp microscopy was used to detect CNV on Days 7 and 14; A1: pictures of corneas of mouse 7 d after treatment of corneal alkali burns with PBS eye drops. A2: pictures of corneas of mouse 14 d after treatment of corneal alkali burns with PBS eye drops. A3: pictures of corneas of mouse 7 d after treatment of corneal alkali burns with emodin eye drops. A4: pictures of corneas of mouse 14 d after treatment of corneal alkali burns with emodin eye drops. B: corneal inflammatory cell infiltration in various groups of mouse (hematoxylin-eosin staining, × 100); B1: normal mouse corneas without any treatment; B2: treatment of mouse corneal alkali burns with PBS drops for 14 d; B3: treatment of mouse corneal alkali burns with emodin drops for 14 d; C: statistical analysis of the CNV-covered area at various time points; D: quantification of inflammatory cell infiltration on 14 d. CNV: corneal neovascularization. PBS: phosphate buffered saline. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, bP < 0.01, compared with PBS group.
Figure 3 In mouse corneas, emodin inhibits alkali burn-induced neovascularization and inflammatory cell infiltration A: slit-lamp microscopy was used to detect CNV on Days 7 and 14; A1: pictures of corneas of mouse 7 d after treatment of corneal alkali burns with PBS eye drops. A2: pictures of corneas of mouse 14 d after treatment of corneal alkali burns with PBS eye drops. A3: pictures of corneas of mouse 7 d after treatment of corneal alkali burns with emodin eye drops. A4: pictures of corneas of mouse 14 d after treatment of corneal alkali burns with emodin eye drops. B: corneal inflammatory cell infiltration in various groups of mouse (hematoxylin-eosin staining, × 100); B1: normal mouse corneas without any treatment; B2: treatment of mouse corneal alkali burns with PBS drops for 14 d; B3: treatment of mouse corneal alkali burns with emodin drops for 14 d; C: statistical analysis of the CNV-covered area at various time points; D: quantification of inflammatory cell infiltration on 14 d. CNV: corneal neovascularization. PBS: phosphate buffered saline. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, bP < 0.01, compared with PBS group.
Figure 4 Effect of emodin on the expression of CD31 and VEGFR2 signaling pathway after corneal alkali burns A: expression of CD31 in mouse cornea by immunofluorescence detection; A1: normal mouse corneas without any treatment; A2: treatment of mouse corneal alkali burns with PBS drops for 14 d; A3: treatment of mouse corneal alkali burns with emodin drops for 14 d; B: quantitative analysis of CD31 expression. Control: blank control group mice, without any treatment. PBS: negative control group mice treated with phosphate buffered saline eye drops. Emodin: experimental group mice, treated with emodin eye drops; C: quantitative analysis of VEGFR2, pVEGFR2, pSTAT3, tSTAT3, pPI3K, tPI3K, pAkt, and tAkt; D: representative Western blot of VEGFR2, pVEGFR2, pSTAT3, tSTAT3, pPI3K, tPI3K, pAkt, and tAkt after treatment of VEGF-stimulated HUVEC with PBS and 20 μM emodin for 24 h; E: VEGFR2 expression bands in alkali-burned mouse corneas from the emodin treatment and control groups on 14 d; F: quantitative analysis of VEGFR2 in alkali-burned mouse corneas from the emodin treatment and control groups on 14 d. CD31: platelet endothelial cell adhesion molecule-1; VEGF: vascular endothelial growth factor; VEGFR2: vascular endothelial growth factor receptor 2; p-VEGFR2: phospho-vascular endothelial growth factor receptor 2. STAT3: signal transducer and activator of transcription 3; p-STAT3: phospho-signal transducer and activator of transcription 3; PI3K: phosphoinosmde-3-kinase; p-PI3K: phospho-phosphoinosmde-3-kinase; Akt: total protein kinase B; p-Akt: phospho-protein kinase B. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, bP < 0.01, compared with PBS group.
Figure 4 Effect of emodin on the expression of CD31 and VEGFR2 signaling pathway after corneal alkali burns A: expression of CD31 in mouse cornea by immunofluorescence detection; A1: normal mouse corneas without any treatment; A2: treatment of mouse corneal alkali burns with PBS drops for 14 d; A3: treatment of mouse corneal alkali burns with emodin drops for 14 d; B: quantitative analysis of CD31 expression. Control: blank control group mice, without any treatment. PBS: negative control group mice treated with phosphate buffered saline eye drops. Emodin: experimental group mice, treated with emodin eye drops; C: quantitative analysis of VEGFR2, pVEGFR2, pSTAT3, tSTAT3, pPI3K, tPI3K, pAkt, and tAkt; D: representative Western blot of VEGFR2, pVEGFR2, pSTAT3, tSTAT3, pPI3K, tPI3K, pAkt, and tAkt after treatment of VEGF-stimulated HUVEC with PBS and 20 μM emodin for 24 h; E: VEGFR2 expression bands in alkali-burned mouse corneas from the emodin treatment and control groups on 14 d; F: quantitative analysis of VEGFR2 in alkali-burned mouse corneas from the emodin treatment and control groups on 14 d. CD31: platelet endothelial cell adhesion molecule-1; VEGF: vascular endothelial growth factor; VEGFR2: vascular endothelial growth factor receptor 2; p-VEGFR2: phospho-vascular endothelial growth factor receptor 2. STAT3: signal transducer and activator of transcription 3; p-STAT3: phospho-signal transducer and activator of transcription 3; PI3K: phosphoinosmde-3-kinase; p-PI3K: phospho-phosphoinosmde-3-kinase; Akt: total protein kinase B; p-Akt: phospho-protein kinase B. The mean ± standard deviation is indicated by each value (n = 3). aP < 0.05, bP < 0.01, compared with PBS group.
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