Fang Li1, Peng Hao1,2,3, Guangjie Liu4, Weiyi Wang5, Ruifang Han1,2,3, Zhixin Jiang1,2,3, Xuan Li6,7,8. 1. Nankai University Eye Hospital, Tianjin, 300020, China. 2. Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, 300020, China. 3. Tianjin Eye Hospital, Tianjin Eye Institute, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin, 300020, China. 4. Department of Respiratory Medicine, Beijing Tongren Hospital, Capital Medical University, Beijing, 100730, China. 5. Department of Ophthalmology, Tianjin Huanhu Hospital, Tianjin, 300350, China. 6. Nankai University Eye Hospital, Tianjin, 300020, China. xuanli08@yahoo.com. 7. Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, 300020, China. xuanli08@yahoo.com. 8. Tianjin Eye Hospital, Tianjin Eye Institute, Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin, 300020, China. xuanli08@yahoo.com.
Abstract
PURPOSE: To investigate the effects of hyaluronic acid (HA) on the inflammation of corneal fibroblasts induced by lipopolysaccharide (LPS). METHODS: Primary rabbit corneal keratocytes were isolated with collagenase. The keratocytes were cultured in a serum-containing medium to induce corneal fibroblasts, which represented the wound repair phenotype of corneal keratocytes. Corneal fibroblasts were treated with LPS with or without 4-methylumbelliferone (4-MU) / high molecular weight hyaluronic acid (HMWHA). The gene expression was evaluated via real-time PCR, immunofluorescence, and western blot. The release of inflammatory cytokines and HA was determined by ELISA. RESULTS: Three types of hyaluronan synthase (HAS) were detected in corneal fibroblasts. LPS stimulation caused the up-regulation of HAS1 and HAS2 expression in corneal fibroblasts. LPS-induced HAS2 expression was significantly inhibited by 4-MU, and accompanied by decreased HA release by the corneal fibroblasts. In the corneal fibroblasts, 4-MU reduced the LPS-stimulated up-regulation of inflammatory cytokines including IL-1, IL-6, IL-8, TNF-α, and also attenuated the LPS-induced up-regulation of inflammatory related receptors including TLR2, TLR4, CD44, and CXCR1. HMWHA treatment resulted in a significant decline in the expression of IL-6, IL-8, TLR4, and CXCR1 responded to LPS stimulation. Consistent with mRNA expression of level, the up-regulation of the release of IL-6 and IL-8 induced by LPS in corneal fibroblasts was significantly attenuated by 4-MU and HMWHA. The LPS-induced expression of IL-8 and its receptor CXCR1 at both the mRNA and protein level were significantly attenuated by 4-MU and HMWHA. CONCLUSION: The inhibitor of HA synthesis 4-MU, and HMWHA successfully reduced LPS-induced inflammation in corneal fibroblasts. The mechanism might be via the inhibition of LPS-induced TLR4 up-regulation.
PURPOSE: To investigate the effects of hyaluronic acid (HA) on the inflammation of corneal fibroblasts induced by lipopolysaccharide (LPS). METHODS: Primary rabbit corneal keratocytes were isolated with collagenase. The keratocytes were cultured in a serum-containing medium to induce corneal fibroblasts, which represented the wound repair phenotype of corneal keratocytes. Corneal fibroblasts were treated with LPS with or without 4-methylumbelliferone (4-MU) / high molecular weight hyaluronic acid (HMWHA). The gene expression was evaluated via real-time PCR, immunofluorescence, and western blot. The release of inflammatory cytokines and HA was determined by ELISA. RESULTS: Three types of hyaluronan synthase (HAS) were detected in corneal fibroblasts. LPS stimulation caused the up-regulation of HAS1 and HAS2 expression in corneal fibroblasts. LPS-induced HAS2 expression was significantly inhibited by 4-MU, and accompanied by decreased HA release by the corneal fibroblasts. In the corneal fibroblasts, 4-MU reduced the LPS-stimulated up-regulation of inflammatory cytokines including IL-1, IL-6, IL-8, TNF-α, and also attenuated the LPS-induced up-regulation of inflammatory related receptors including TLR2, TLR4, CD44, and CXCR1. HMWHA treatment resulted in a significant decline in the expression of IL-6, IL-8, TLR4, and CXCR1 responded to LPS stimulation. Consistent with mRNA expression of level, the up-regulation of the release of IL-6 and IL-8 induced by LPS in corneal fibroblasts was significantly attenuated by 4-MU and HMWHA. The LPS-induced expression of IL-8 and its receptor CXCR1 at both the mRNA and protein level were significantly attenuated by 4-MU and HMWHA. CONCLUSION: The inhibitor of HA synthesis 4-MU, and HMWHA successfully reduced LPS-induced inflammation in corneal fibroblasts. The mechanism might be via the inhibition of LPS-induced TLR4 up-regulation.
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