The Research Of The Action And Mechanism Of Triptolide In Lipopolysaccharide Inducing The Corneal Stromal Fibroblasts

Corneal stromal fibroblasts are the major cellular components, play a key role in keeping transparency of the cornea. Lipopolysaccharide is an integral part of the Gram-negative bacteria cell membrane,could promoting cytokines secretion in inflammatory cells. Human corneal stromal fibroblasts with LPS receptor can identify LPS through CD14, MD-2 and LPS-binding protein (LBP) receptor complex , and release the pro-inflammatory factors, such as IL- 6,8 and ICAM-1, etc. These come into leading the infiltration of leukocytes and the formation of corneal ulcers.Triptolide has a strong action of anti-inflammatory and immunosuppressive, it is an inhibitor of NF-кB. Triptolide not only effects on T cells, B cells, monocytes and other immune cells, and also effects on fibroblasts, epithelium and other non-immune cells.We study the role of triptolide in corneal inflammation in order to guide the clinical application. Method:1. Cultured primary and passaged human corneal stromal fibroblasts in vitro, cryopres- ervation and recovery of cells.2. Observed different concentrations of LPS, triptolide and dexamethasone act on human corneal stromal fibroblasts, and whether there is cytotoxicity and proliferation.3. Detected the changes of IL-1?, IL-6, IL-4, IL-8, TNF-α, ICAM-1, VCAM-1, IκBα, NF-κB in lipopolysaccharide inducing the human corneal stromal fibroblasts.4. Triptolide and/or Dexamethasone play a role on the above-mentioned factors in lipopolysaccharide inducing the human corneal stromal fibroblasts.Results:1. Human corneal stromal fibroblasts of primary may get 80% confluence after cultured 10-14 day , subcultured cells grow well ,and every 3-5 days into next generation, the 7th generation cells remain in good condition; cells of cryopreservation and recovery is also in good condition. Purity of the corneal fibroblast cultures was judged with antibodies to vimentin and to cytokeratin.2. There were no effect on proliferatio and survival of cell with low concentrations of lipopolysaccharide (≤100ng/ml ) in 48 hours by human corneal stromal fibroblasts;High concentrations of lipopolysaccharide (≥1000 ng/ml) effect on survival of cell after 24 hours;There were no effect on proliferatio and survival of cell with triptolide (1nM-100nM) and dexamethasone (0.1nM-1000nM) in 48 hours.3. IL-6, IL-8, ICAM, VCAM-1, NF-κB were increased with LPS (100ng/ml) stimulated after 24 hours by human corneal stromal fibroblasts,and IκBαwere decreased.4. Triptolide inhibited the LPS-induced release of IL-6,IL-8,ICAM,VCAM-1,NF-κB by human corneal stromal fibroblasts.5. Dexamethasone inhibited the LPS-induced release of IL-6,IL-8,ICAM by human corneal stromal fibroblasts6. Triptolide(10nM) and dexamethasone(10nM) inhibited the LPS-induced release of IL-6,IL-8 ,ICAM by human corneal stromal fibroblasts,which is stronger than any one.Conclusion:Human corneal stromal fibroblasts cultured in vitro grew well, can be used for further experimental study after subculture, cryopreserved and resuscitation. LPS of low concentration can promote the proliferation of human corneal stromal fibroblasts, and promote the expression of inflammatory cytokines, adhesion molecules, chemokines through signal transduction pathway of NF-κB in cultured human corneal stromal fibroblasts. Triptolide or dexamethasone inhibit the LPS-induced expression of the inflammatory cytokines, chemokines expression. Through this research, we suppose triptolide may become the substitute or complementary medicine of dexathamesone in treating corneal inflammation.