黄连解毒汤及其主要活性成分抑制LPS所致BV2小胶质细胞M1型极化的机制OA

Objective:To investigate whether Huanglian Jiedutang(HLJD)and its major active constituents(geniposide,baicalin,and berberine)can inhibit the inflammatory response of BV2 cells under lipopolysaccharide(LPS)stimulation via the high-mobility group protein B1(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway,and to explore differences in therapeutic efficacy among the three monomers,their combined formula,and HLJD under equal content ratios.Methods:BV2 microglial cells were used as the primary experimental model.Cell viability was assessed using the cell counting kit-8(CCK-8)method to examine the effects of different concentrations of dimethyl sulfoxide(DMSO,0.8％,0.4％,0.2％,0.1％,and 0.05％)on cell viability.IncuCyte was employed to monitor the growth of cells under different concentrations of HLJD(200,100,50,25,12.5,6.25 mg·L-1).Nitric oxide(NO)assay was used to screen the optimal HLJD concentration.High-performance liquid chromatography(HPLC)determined the content of geniposide,baicalin,and berberine in HLJD,and experimental groups were subsequently established according to the relative proportions of these constituents.CCK-8 assay evaluated cell viability under different treatments.Enzyme-linked immunosorbent assay(ELISA)measured levels of inflammatory factors(TNF-α,IL-1β,IL-6,IL-10)in the supernatant.Flow cytometry assessed the effects of treatments on M1-type polarization of BV2 cells.Western blot determined the expression levels of HMGB1,TLR4,and NF-κB-related proteins.Results:Compared with the blank group,DMSO at concentrations ≤0.2％did not affect cell viability within 48 h.BV2 cell growth plateaued at 24 h after treatment with 200 mg·L-1 HLJD.Under stimulation with 2 mg·L-1 LPS,this concentration of HLJD effectively reduced NO release,and 6 h pre-treatment had a stronger inhibitory effect on NO than direct administration.HPLC results showed that 1 mg of HLJD freeze-dried powder contained approximately 24 μg of geniposide,15 μg of baicalin,and 30 μg of berberine.Based on these ratios,experimental groups were blank,LPS(2 mg·L-1),HLJD(200 mg·L-1),monomer combination,geniposide(4.8 mg·L-1),baicalin(3 mg·L-1),and berberine(6 mg·L-1).The monomer combination group consisted of all three active constituents dissolved together.LPS and HLJD or its active constituents did not affect cell viability compared with the blank group.LPS significantly increased TNF-α,IL-1β,IL-6,and IL-10 in the supernatant(P＜0.01).HLJD and its active constituents significantly reduced pro-inflammatory factors TNF-α,IL-1β,and IL-6(P＜0.05,P＜0.01)while upregulating anti-inflammatory IL-10(P＜0.01),with the monomer combination showing the strongest effect(P＜0.05,P＜0.01).Compared with the blank group,LPS significantly increased the proportion of CD80+CD86+(M1-type)BV2 cells(P＜0.01).HLJD and its constituents partially inhibited M1 polarization(P＜0.05,P＜0.01),with the monomer combination exhibiting the most pronounced effect(P＜0.05,P＜0.01).Compared with the blank group,LPS upregulated HMGB1,TLR4,and NF-κB-related proteins(P＜0.01),whereas HLJD and its active constituents significantly reduced their expression(P＜0.05,P＜0.01),with the monomer combination having the strongest regulatory effect(P＜0.05,P＜0.01).Conclusion:HLJD and its major active constituents(geniposide,baicalin,berberine)can inhibit LPS-induced inflammatory responses in BV2 cells.The combination of the three active constituents demonstrates the most potent anti-inflammatory effect,significantly attenuating M1-type polarization of BV2 cells via the HMGB1/TLR4/NF-κB signaling pathway.