Study On TLR4 Functions Of Glycometabolism And Relevant Mechanism In Liver Cells

Purpose:An elevated rate of glucose metabolism and the dependency on aerobic glycolysis for ATP generation have long been observed in liver cells. The altered TLR4 express in liver cells provides an attractive opportunity for developing novel energy metabolism syndrome therapeutic strategies. It has been reported that the level of PFKFB3 expression was decreased in cells and was associated with TLR4. TLR4, which catalyzes the regulation of glucose metabolism, plays a vital role in the process of Glycolysis. However, the effect of insulin inhibits the regulation of TLR4 in glucose metabolism, In this study, the effect of TLR4 in glucose metabolism was explored and the involving mechanism was also studied.Methods:1. Human liver cell lines:QSG-7701, HL-7702, and THLE-2, Rat liver cell line:BRL-3A, Human liver cancer cell lines:LM3 and HepG2 were preserved in our research.2. The liver cell lines expression of TLR4 and PFKFB3 mRNA and protein were monitored by RT-PCR and Western blot assay, respectively.3. The siRNA/TLR4 was transfected into the HL-7702 cell lines,, via Lipofectamine transfection assay. G418 resistant clones were developed and collected by cultured in G418-containing medium.4. Western blot assay was used to measure change of proteins related to TLR4 and PFKFB3.5. qRT-PCR was used to quantitative mRNA level changes.6. Wound healing assay and Transwell chamber assay were performed to assess cell growth and migration.7. Flow cytometric analysis was used to detect the alteration of cell cycle distribution and apoptosis; Apoptosis was also detected by assay of phosphatidylserine valgus.8. JC-1 staining was used to test the change of mitochondrial membrane potential. ATP content was detected by related kits.Results:1. The inflammatory cytokines of TLR4, the expression of normal liver cells, is higher than that of liver tumor cells. PFKFB3, this energy metabolism related gene, the distribution of normal liver cells is lower than the liver cancer cells.2. siRNA TLR4 could knocked TLR4 down in the mRNA level and in protein level. Following TLR4 expression decreased, the expression of PFKFB3 sequential down-regulated.3. FAA can induce TLR4 expression in liver cells, and also cause PFKFB3 expression a rise.4. Insulin increase TLR4 and PFKFB3 expression, blocked by siRNA Which target TLR4 in liver cell lines HL-77025. Compared with parental cells, a significant increase of cell proliferation was observed in siRNA TLR4 transfected cells, accompanied with a GO/Gl phase arrest. siRNA TLR4 induced G0/G1 cell cycle arrest via down-regulation of pi3k/AKT pathway.6. In the siRNA TLR4 transfected cells, decreased TLR4 expression promoted apoptosis through enhancing mitochondrial energy metabolism,7. In the siRNA TLR4 transfected cells, JC-1 decreased linearly with the expression of TLR4, meanwhile, ATP was also decreased.Conclusion:In conclusion, this study provides evidence for the TLR4 could regulate glucose metabolism on the liver cells, and indicates that targeting TLR4 may be an effective strategy for diabetes therapy. Further studies are required to explore whether the TLR4 regulation of glucose metabolism in the glycolytic pathway have similar effects as the Stimulating factor of insulin and still more efforts are needed to develop novel high effective regulation of TLR4 in the future.