The Regulation And Mechanism Of Tea Polyphenols On Inflammation Of Orbital Fibroblasts In Thyroid-associated Ophthalmopathy

Objective: To investigate the effect and the mechanism of tea polyphenols(TP)on the inflammation of thyroid-associated ophthalmopathy(TAO)orbital fibroblasts mediated by lipopolysaccharide,which may provide new ideas for the treatment of thyroid-associated ophthalmopathy.Methods: TAO group: Patients with thyroid-associated ophthalmopathy undergoing orbital decompression due to oppressive optic neuropathy were enrolled(n = 6).Orbital connective tissue specimens were taken after surgery.Control group: The patients who were matched with the age and gender of the experimental group for eyeball removal due to old eye trauma or non-inflammatory corneal scleral staphyloma were selected(n = 4),and the orbital connective tissue specimens were taken after surgery.None of the included patients had active inflammation.Immunohistochemistry was used to determine that the primary cells we extracted were orbital fibroblasts.The expression of IL-1β,IL-6 and MCP-1 in orbital fibroblasts of thyroid-associated ophthalmopathy and control group was detected by RT-q PCR.Passage at a ratio of 1: 3,plant the same amounts of cells in a petri dish.After the orbital fibroblasts adhere to the wall,the CCK-8 method was used to detect the effect of different concentrations of tea polyphenols on the proliferation of orbital fibroblasts.The cells were divided into three groups: TAO group,TAO + LPS(concentration: 10 μg / m L)group,TAO + LPS + TP(concentration: 100 μmol /L)group;after 24 hours of low serum intervention,RT-q PCR and ELISA were used to investigate the effect of tea polyphenols on the LPS-mediated inflammation in TAO orbital fibroblasts.We also used western blot to detect the expression of NF-κB and NLRP3 among the groups.The expression of NLRP3 was detected by immunofluorescence.Results: We cultured primary cells of orbital fibroblasts from orbital tissues,and observed the growth rules,cell morphology of our primary cells.The results of immunohistochemical analysis shown that vimentin expression was positive,indicating that the extracted cells were of mesenchymal origin;desmin,myoglobin,cytokeratin 17,and S100 B were negative,excluding the possibility of other cells.In sum,the primary cells were orbital fibroblasts.RT-q PCR results showed that the expression of IL-1βm RNA in orbital fibroblasts derived from patients with thyroid-related eye disease was 50 times that of the control group(P <0.05),and the expression of IL-6 mRNA was 4.7 times that of the control group(P <0.05),MCP-1 m RNA expression was 18 times that of the control group(P <0.05).We used different concentrations of tea polyphenols to intervene in orbital fibroblasts.CCK-8 was used to detect the absorbance of orbital fibroblasts at various concentrations,and to calculate the survival rate of cells at each concentration.The results showed that when the tea polyphenol concentration was above 100 μmol / L,the cell viability decreased and the difference was statistically significant(P <0.05).This shows that,to a certain extent,the higher the concentration of tea polyphenols,the lower the cell viability of TAO orbital fibroblasts.We set the intervention concentration of tea polyphenols to 100 μmol / L.Under the stimulation of lipopolysaccharide,the inflammation level of orbital fibroblasts increased significantly: The m RNA expression of IL-1β in TAO +LPS group was about 3 times in the TAO group(P <0.001),IL-6 m RNA was about 10 times(P <0.001),and the MCP-1 m RNA expression was about 4 times(P <0.001);tea polyphenol intervention could significantly reduce the IL-1β,IL-6 and MCP-1 m RNA expression in orbital fibroblast(P <0.001).Taken the supernatant of the cell culture medium after intervention,the results of ELISA and RT-q PCR showed a consistent trend: lipopolysaccharide stimulation could induce the expression of IL-1β,IL-6 and MCP-1 in orbital fibroblasts to increase significantly.Treatment with tea polyphenols could effectively inhibited the lipopolysaccharide-induced cellular inflammation and reduced the overexpression of IL-1β,IL-6 and MCP-1.In order to explore the mechanism of the tea polyphenols on inflammatory factors in orbital fibroblasts of thyroid-associated ophthalmopathy,we used western blot to detect the protein expression of NLRP3 and NF-κB,and detected the expression of NLRP3 by immunofluorescence.Western blot analysis showed that the expression of NF-κB and NLRP3 showed the same trend with the expression of inflammatory factors: the expression of NF-κB and NLRP3 in the TAO + LPS group was increased compared with the TAO group;in TAO+LPS+TP group,the expression of NF-κB and NLRP3 decreased.In addition,the NLRP3 immunofluorescence results was consistent with western blot results.Conclusion: The expression of inflammatory factors in orbital fibroblasts of patients with thyroid-associated ophthalmopathy is higher than that of orbital fibroblasts in patients with control group;LPS can stimulate the inflammatory of orbital fibroblasts in thyroid-associated ophthalmopathy;TP can attenuates inflammatory response which caused by LPS stimulation in thyroid-associated ophthalmopathy orbital fibroblasts via NF-κB / NLRP3 pathway;in-depth studies on the effects of TP on expression of inflammatory factors in orbital fibroblasts of thyroid-associated ophthalmopathy may provide new ideas for the treatment or adjuvant treatment of thyroid-associated ophthalmopathy.