OBJECTIVETo observe the effects of berberine hydrochloride on the secretion of monocyte chemoattractant protein-1 (MCP-1) from human periodontal ligament cells (PDLC) in vitro culture.

METHODSPeriodontal ligament was isolated from extracted human premolars, and PDLC were cultured in vitro. PDLC were divided into two groups, lipopolysaccharide (LPS) group and non-lipopolysaccharide (NLPS) group. Then in accordance with the final concentrations of berberine hydrochloride in cells culture medium (0, 0.01, 0.02, 0.03 g/L), the groups were subdivided into LPS and NLPS control group, LPS1 and NLPS1 group, LPS2 and NLPS2 group, LPS3 and NLPS3 group. Cellular concentration of MCP-1 of each group was determined by enzyme-linked immunosorbent assay (ELISA). The data were statistically analyzed.

RESULTSThe MCP-1 contents were not significantly different between the groups of NLPS1, NLPS2 and NLPS3 [(11.33 ± 0.16), (11.45 ± 0.53), (11.25 ± 0.14) ng/L, respectively] and the NLPS control group [(11.32 ± 0.35) ng/L] (P = 0.692, 0.568, 0.524). MCP-1 contents in the groups of LPS1, LPS2 and LPS3 [respectively (38.14 ± 5.34), (34.15 ± 3.36), (26.13 ± 2.12) ng/L] were significantly lower than in LPS control group [(58.42 ± 1.52) ng/L], P = 0.000, 0.000, P = 0.000.

CONCLUSIONSThe inhibitory effect of berberine hydrochloride on the activities of MCP-1 from PDLC was more significant when PDLC were stimulated with LPS and in a concentration-dependent manner.

Adolescent ; Anti-Inflammatory Agents ; administration & dosage ; pharmacology ; Berberine ; administration & dosage ; pharmacology ; Bicuspid ; cytology ; Cells, Cultured ; Chemokine CCL2 ; secretion ; Child ; Dose-Response Relationship, Drug ; Humans ; Lipopolysaccharides ; pharmacology ; Periodontal Ligament ; cytology ; metabolism

BACKGROUNDAdipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms.

METHODSFully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber.

RESULTSOxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes.

CONCLUSIONSOxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

3T3-L1 Cells ; Animals ; CCAAT-Enhancer-Binding Protein-beta ; analysis ; physiology ; Chemokine CCL2 ; genetics ; secretion ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Humans ; Lipoproteins, LDL ; antagonists & inhibitors ; pharmacology ; Mice ; Peptides ; pharmacology ; Signal Transduction ; physiology

PURPOSE: Deposition of polymeric IgA1 in the kidney mesangium is the hallmark of IgA nephropathy, but the molecular mechanisms of IgA-mediated mesangial responses and inflammatory injuries remain poorly understood. We hypothesize that Toll-like receptor 4 (TLR4) is involved in IgA-induced mesangial cell activation. MATERIALS AND METHODS: Mouse mesangial cells were stimulated with lipopolysaccharide (LPS) (1 microg/mL), IgA (20 microg/mL), or both, and TLR4 expression was measured by real time RT-PCR and Western blot. Intracellular responses to LPS or IgA were assessed by Western blot for ERK1/2, JNK, p38 MAP kinases (MAPKs), Ikappa-Balpha degradation and fibronectin secretion. MCP-1 secretion was assessed by ELISA. Small interfering RNA (siRNA) of TLR4 was used to confirm that the effects were caused by TLR4 activity. RESULTS: LPS- or IgA-treatment upregulated the levels of TLR4 mRNA and protein in cultured MMC at 24 h. LPS and IgA induced rapid phosphorylation of MAPKs, but degradation of Ikappa-Balpha was observed only in LPS-treated MMC. LPS, but not IgA, induced increased secretion of MCP-1 and fibronectin at 24 h or 48 h. Combined LPS and IgA treatment did not cause additional increases in TLR4 mRNA and protein levels or Ikappa-Balpha degradation, and MCP-1 and fibronectin secretions were less than with LPS alone. LPS- or IgA-induced TLR4 protein levels and MAPK activation were inhibited by transfection with TLR4 siRNA. CONCLUSION: These results indicate that the activation of MAPKs and MCP-1 secretion are mediated by TLR4, at least in part, in IgA-treated mesangial cells. TLR4 is involved in mesangial cell injury by induction of pro-inflammatory cytokines in IgA nephropathy.
Animals ; Chemokine CCL2/secretion ; Enzyme-Linked Immunosorbent Assay ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Fibronectins/secretion ; Glomerulonephritis, IGA/*metabolism ; I-kappa B Proteins/metabolism ; Mesangial Cells/*metabolism/secretion ; Mice ; Mice, Transgenic ; Phosphorylation ; RNA Interference ; RNA, Messenger/metabolism ; *Signal Transduction ; Toll-Like Receptor 4/antagonists & inhibitors/genetics/*metabolism