The Effects And Mechanisms Of 5-nitro-2-(3-phenylpropylamino) Benzoic Acid(NPPB) On Lps-induced Cytokines Expression In RAW264.7

Objective:5-Nitro-2-(3-phenylpropylamino) benzoic acid(NPPB) is known as one of the chloride channel blockers. At the same time, NPPB is also a GPR35 agonist. While both chloride channel and GPR35 relate to inflammation. Recent reports suggested that TNF-α could activate a Clcurrent in human umbilical vein endothelial cells with an existence of Cl C-3. Besides, Clchannel blockers, Cl C-3 knockdown or knock out remarkably reduced TNF-α-induced HUVECs intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocyte to endothelial cell adhesion, and NF-κB activation. These all indicated that Cl C-3 chloride channel was concerned with the process of vascular inflammation. However, GPR35 could be detected in many immune cells, such as monocytes, neutrophils, and i NKT cells and it was implicated in human IBD. These all indicated that GPR35 might had an effect on promoting inflammation.NPPB is both a chloride channel blocker and a GPR35 agonist. There were many reports about its effects on vascular inflammation. But the effects of NPPB on macrophage inflammatory response are still unknown. So in this research, we investigated the effects and mechanisms of NPPB on LPS-induced cytokines expression in Raw264.7 to reveal the role of NPPB in the inflammatory response of macrophages.Method:1. Western blot and Quantitative PCR techniques were used to detect the effects of NPPB on LPS-induced cytokines expression in RAW264.7 cells.2. Quantitative PCR technique was used to analyze the m RNA expression of Cl C-3 in RAW264.7 cells.3. Immunofluorescence technique was used to observe the distribution of Cl C-3.4. Specific si RNAs were used to down-regulate the expression of Cl C-3 protein.5. Western blot technique was used to detect the effects of DIDS and tamoxifen on LPS-induced TNF-α expression in RAW264.7 cells.6. Western blot technique was used to detect the effects of NPPB on LPS-induced phosphorylation of NF-κB, p38 MAPK, ERK, JNK and c-Jun in RAW264.7 cells.7. Western blot technique was used to detect the effects of GPR35 agonist on LPS-induced cytokines expression in RAW264.7 cells.Results:1. NPPB up-regulated the expression of cytokines in LPS-induced RAW264.7 both at gene and protein levels.2. The expression of Cl C-3 was remarkably higher than the other subtypes of Cl C family.3. Treatment with Cl C-3 si RNA inhibited the protein expression of TNF-α in LPS-induced RAW264.7.4. The effects of DIDS and tamoxifen on LPS-induced TNF-α expression in RAW264.7cells differed with NPPB.5. NPPB down-regulated the phosphorylation of NF-κB in LPS-induced RAW264.7.6. GPR35 agonist up-regulated the expression of TNF-α in LPS-induced RAW264.7 both in gene and protein levels.7. NPPB up-regulated the phosphorylation of p38 MAPK in LPS-induced RAW264.7.Conclusion:1. NPPB up-regulated the expression of cytokines in LPS-induced RAW264.7 both in gene and protein levels.2. Cl C-3 chloride channel might not be the target of up-regulation of TNF-α by NPPB in LPS-induced RAW264.7 cells.3. NPPB might activate GPR35 to promote the phosphorylation of p38 MAPK then up-regulate the expression of TNF-α on LPS-induced RAW264.7 cells.