| Objective: Calcium oxalate crystal induces renal tubular epithelial cell inflammatory response is one of the basic links of calcium oxalate renal stones.The process is intervened with this process through ginkgo biloba extract EGB761,and can be discussed by inhibiting cytosocial inflammation induced by calcium oxalate crystals,thereby inhibiting kidney Stone formation.Methods: The cell-crystal reaction system was constructed by renal tubular epithelial cells,and CCK-8 method was used to detect the effect of different concentrations of ginkgo biloba extract EGB761 on the activity of renal tubular epithelial cells.The renal tubular epithelial cell is divided into 4 groups: control group(NC;cultured in complete medium),Ca Ox group(Ca Ox;cultured in complete medium containing 2.0 mmol/L Ca Ox crystal),EGB761group(EGB761;culture medium with 0.5 mg / ml ginkgo biloba extract EGB761),Ca Ox+ EGB761 group(cultured in complete medium containing 2.0mmol/L Ca Ox crystal and 0.5 mg / ml ginkgo biloba extract EGB761).After 24 hours,we extracted cells from each group,and then western blot and immunofluorescence was used to detect the expression of the intracellular inflammatory marker protein NF-κB in the cells;Western blot was used to detect the expression of the intracellular inflammatory marker protein TLR4.The crystal adhesion,cell morphology,cell and nucleus morphology were observed under light microscope.NF-κB m RNA expression was detected by RT-q PCR.It is thus to explore the ginkgo biloba extract EGB761 to inhibit cytosocial inflammatory response induced by calcium oxalate crystals.Results: The results showed that Ginkgo biloba extract EGB761 did not change the cell viability when the concentration was as high as 0.5mg/ m L.Therefore,0.5mg/ m L was used in subsequent experiments.The cells of the control group and the EGB761 group were observed to grow adherently under light microscope,the shape is fusiform and close arrangement.In the Ca Ox group,the fusiform form of renal tubular epithelial cells disappeared and some of them became round,the cell space widened and separated from the surrounding cells,and the suspended cells increased.In the EGB761 group,the adhesion of calcium oxalate crystals around the cells was reduced,most of the cells were fusiform,suspension cells were reduced,and most of the cells are in the form of normal epithelial cells.The DAPI staining results suggested that the cellular nucleus were round and the morphology of the cellular nucleus was not significantly changed in the control group and EGB761 group;In the Ca Ox group,the cellular nucleus began to be broken and concentrated,and the staining was deepened;However,in the EGB761 group,cellular nucleus fragmentation was reduced and most of the nucleus is mostly normal renal tubular epithelial cell nucleus.Western blot results showed that the expression of TLR4 and NF-κB in EGB761 group were lower than those in the control group,but the difference was not statistically significant(P>0.05),the expression amount of TLR4 and NF-κB in Ca Ox group were observably increased,and the difference was statistically significant(P<0.05),the expression levels of TLR4 and NF-κB in EGB761 group were significantly lower than those in the Ca Ox group,and the difference was statistically significant(P<0.05).The expression of NF-κB in cells detected by immunofluorescence assay was similar to that by Western blot.RT-q PCR results showed that compared with the control group,the Ca Ox group NF-κB m RNA expression was significantly increased,the difference was statistically significant(P<0.05),while the EGB761 group was not significantly different from the control group(P>0.05).Conclusion: 1.The optimal concentration of ginkgo biloba extract EGB761 on the activity of renal tubular epithelial cells was determined.2.Verified that renal tubular epithelial cells could develop inflammatory response under the stimulation of calcium oxalate crystals.3.Ginkgo biloba extract EGB761 can inhibit the inflammatory response of renal tubular epithelial cells induced by calcium oxalate crystals through TLR4/NF-κB signaling pathway. |
