| Objective: Diabetic nephropathy (DN) is one of the most seriouscomplications of diabetes and also the leading cause of chronic renal failure.The mechanism of chronic inflammatory reaction in DN is concerned greatlythese years because of its complexity. Toll-like receptor4(TLR4) plays animportant role in the progress of cell signal activation and inflammatoryreaction. The role of TLR4in the pathogenesis of DM and DN is arousingincreasing interest. TLR4is confirmed to be highly expressed in many kindsof cells, but the detailed mechanism of the up-regulation of it in DM and DNhas not been fully elucidated. Therefore, in order to discuss how the TLR4activation affects the development of DN and the regulation of themacrophage recruitment and renal interstitial fibrosis，we studied the effect ofthe highly expressed TLR4in vitro and vivo in high glucose surrounding tothe generation of inflammatory factors in renal tubular epithelial cells andmacrophages stimulated with different ligands of TLR4based on the literatureand preliminary experimental results,. The purpose of this study is to provide atreatment strategies and experimental basis for DN.Methods:1Observing the influence of high glucose to the expression of TLR4andinflammatory factors in HK-2and THP-1cells: HK-2and THP-1cells werecultured in1640medium containing different concentrations of glucose. Theexpression of TLR4on the surface of HK-2and THP-1cells was examined byimmunofluorescence and flow cytometer. Cells were stimulated with LPS andHSP60in1640medium containing different concentrations of glucose.Real-time PCR was used to examine the mRNA (TNF-α, MCP-1, IL-6, IL-8)expression at different time (2,4,6,8h).2Observing the effect of supernatant of macrophages activated by LPS to HK-2cells: We observed the generation of the inflammation factors of HK-2cells after treated by the supernatant. THP-1cells were cultured in1640medium containing different concentrations of glucose and stimulated withLPS for24hours. Then, we collected the supernatant of THP-1and put it intoHK-2cells. Real-time PCR was used to examine the mRNA expression,Western blot was used to examine the NF-κBp65expression, andimmunohistochemistry was used to the FN expression.3Inducing the experimental diabetic rat model: We used streptozotocin(STZ)(45mg/kg) to induce experimental diabetic rat model and then testedblood glucose of tail vein in72h. The model with blood glucose more than16.7mmol/L twice in a row was considered to be qualified.4Observing the inflammatory response mediated by peritonealmacrophage activation in DM rats dynamically: Peritoneal macrophages werecollected at different phases (2,4,8and12w) after the onset of diabetes, andthen stimulated with LPS at different time (2,4and6h). Real-time PCR wasused to examine the TNF-α and IL-1β mRNA expression in the cells. At thesame time, peritoneal macrophages were stimulated with LPS and HSP60for48h for ELISA test.5Observing the inflammatory response of renal tissue dynamically:Renal tissue was separated at different phases (2,4,8and12w) after the onsetof diabetes. Real-time PCR was used to examine the TLR4, TNF-α and IL-1βmRNA expression in the renal tissue. Renal lesions were observed after HEstaining and FN expression was detected with immunohistochemistry.Results:1High glucose surrounding can promote the expression of the TLR4onthe surface of THP-1and HK-2cells.2Compared with the control group，stimulation with LPS and HSP60canup-regulate the TNF-α, MCP-1, IL-6, IL-8mRNA expression in THP-1andHK-2cells in high glucose surrounding.3Compared with the control group and the high glucose group，theTNF-α, MCP-1, IL-6, IL-8mRNA expression in the HK-2cells were up-regulate in the supernatant stimulating group. Western Blot results showedthat the expression of NF-κBp65increased significantly in the HK-2cellstreated with the supernatant in72h. Immunohistochemistry results showed thatFN expression increased significantly when the supernatant made up40%ofthe medium.4The model of type2diabetic rat was induced successfully.5Real-time PCR was used to examine the TNF-α and IL-1β mRNAexpression in the rat peritoneal macrophages stimulated with LPS. The resultsshowed that TNF-α and IL-1β mRNA expression reached the peak at2hoursafter LPS stimulation, and the peak value increased gradually with thedevelopment of DM.6ELISA test results showed that TNF-α and IL-1β mRNA rose graduallyand reached the peak at8w and12w with the values of600pg/ml and700pg/ml respectively.7Renal tissue was separated at different phases (2w,4w,8w,12w) afterthe onset of diabetes. Real-time PCR was used to examine the TLR4, TNF-αand IL-1β mRNA expression in the renal tissue. The results showed that theTLR4expression at12w in DM group was markedly increased compared withthe control group and other groups at the same time. The expression of TNF-αincreased obviously at different periods in DM group compared with thecontrol group, with the peak reached at12w. Similar results appeared in theexpression of IL-1β mRNA, which reached the peak at8w and12w.8Renal tissue HE staining results showed that macrophages began toinfiltrate into the tissue at4w after the onset of DM, and the number ofmacrophages was significantly increased at12w. At the same time, theexpression of FN appeared at4w and aggravated step by step from4w to12wafter the DM onset.9Renal tissue immunohistochemistry revealed that FN expressionappeared at4w and enhanced gradually from4w to12w after the DM onset.Conclusions：1High glucose surrounding can promote the expression of the TLR4on THP-1and HK-2cells. THP-1and HK-2cells stimulated with LPS andHSP60can heighten inflammatory factors m RNA expression in high glucosesurrounding.2Treating the HK-2cells with the supernatant of THP-1can lead to moreintense inflammatory reaction and higher expression of the FN.3Inflammatory factors in the peritoneal macrophages after treated withLPS and HSP60, and the TLR4and inflammatory factor mRNA (TNF-α andIL-1β) can be increased gradually with the development of DM.4Renal fibrosis and inflammation infiltration aggravate gradually alongwith the development of DM. |
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