Anti-inflammatory Effect Of Berberine Hydrochloride On LPS Induced Inflammatory Reaction

Berberine hydrochloride is an isoquinoline alkaloid extracted from berberidaceae,rutaceae,and other plants.Previous reports have shown that berberine hydrochloride has anti-inflammatory properties,however,the underlying molecular mechanisms remain unclear.In this experiment,firstly,the anti-inflammatory mechanism of berberine hydrochloride were detected in LPS-stimulated RAW264.7 cells.The potential cytotoxicity of berberine hydrochloride was evaluated by the CCK-8 assay,then an in vitro model of inflammation was induced by LPS in mouse RAW264.7cells before the cells were treated with various concentrations of berberine hydrochloride(12.5,25,and 50 ?g/mL).The concentration and expression level of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay(ELISA)and quantitative real-time polymerase chain reaction(qRT-PCR),respectively.The nuclear expression level of NF-?B was determined by an electrophoretic mobility shift assay(EMSA).The expression levels of TLR4 and NF-?B were determined by Western blot.Secondly,the lipopolysaccharide(LPS)-induced mouse model of mastitis was established to explore the anti-inflammatory action of berberine hydrochloride.Sixty mice that had been lactating for 5-7 days were randomly divided into six groups,including a control,LPS,three berberine hydrochloride treatment groups(5,10,and 20 mg/kg),and a dexamethasone(DEX)(5 mg/kg)group.Berberine hydrochloride was administered intraperitoneally 1 h before and 12 h after LPS induction,and all the mice were sacrificed 24 h after LPS induction.The pathological and histopathological changes of the mammary glands were observed,and the concentrations and mRNA expression of TNF-?,IL-1?,and IL-6 were measured by ELISA and qRT-PCR.The activation of TLR4 and NF-?B signaling pathways were analyzed by western blot.The experimental results in vitro showed that cell viabilities were not affected by the berberine hydrochloride at concentrations of 12.5,25,and 50 ?g/mL.Berberine hydrochloride dose-dependently inhibited the expression of TNF-?,IL-6,and IL-1? in LPS-stimulated RAW264.7 cells.The EMSA analysis showed that berberine hydrochloride significantly inhibited the nuclear expression of p65 protein,and the western blot analysis showed that berberine hydrochloride significantly suppressed LPS-induced TLR4 and NF-?B activation,and the phosphorylation of I-?B.The experimental results in vivo indicated that berberine hydrochloride(5,10,and 20mg/kg)significantly attenuated neutrophil infiltration,dose-dependently decreased the secretion and mRNA expression of TNF-?,IL-1?,and IL-6 within a certain range.Furthermore,berberine hydrochloride suppressed LPS-induced TLR4 and NF-?B P65 activation,and the phosphorylation of I-?B.In conclusion,berberine hydrochloride exerted an anti-inflammatory effect by inhibiting the activation of TLR4/NF-?B signaling pathway and down-regulating the level of pro-inflammatory cytokines,including TNF-?,IL-1? and IL-6.