Effect of Berberine on Activation of TLR4-NFB Signaling Pathway and NLRP3 Inflammasome in Patients with Goat

Objective: To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)kappa B (NF-kappa B) signaling and NLRP3 inflammasome in patients with gout. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-kappa B (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1 beta (IL-1 beta) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-kappa B (p50/65), inhibitor of kappa B kinase alpha/beta (IKK alpha/beta), NF-kappa B inhibitor alpha (IKB alpha), phospho-IKK alpha/beta (p-IKK alpha/beta), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1 beta protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 mu g/mL), 0.1% dimethyl sulfoxide, 25 mu mol/L BBR, and 10, 25, and 50 mu mol/L BBR+100 mu g/mL MSU for different time periods. The protein levels of IL-1 beta and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-kappa B (p50/p65), IKK alpha/beta, I kappa B beta, p-IKK alpha/beta, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting. Results: (1) TLR4, NF-kappa B (p65), PYCARD, CASP1, and IL-1 beta mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P<0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P<0.05, P<0.01). (2) The protein levels of TLR4, IKK beta, MyD88, NF-kappa B, p-IKK alpha/beta, PYCARD, and CASP1 in PBMCs were significantly higher, and those of I kappa B alpha, IKK alpha, and NLRP3 were found to be significantly lower in the AP group than in the HC group (PP<0.01). (3) The serum IL-1 beta protein levels were significantly higher in the AP and NAP groups than in the HC group (P<0.01). (4) The IL-1 beta protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 mu moL/L BBR groups compared with that in the MSU group (P<0.01). (5) The protein levels of IL-1 beta and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P<0.01). (6) The protein levels of TLR4, MyD88, NF-kappa B (p50, p65, p105), IKK alpha/beta, p-I kappa B alpha, p-IKK alpha/beta, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (PP<0.01). Conclusions: Activation of TLR4-NF kappa B signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-kappa B signaling and NLRP3 inflammasome expression.