化脂复肝颗粒通过抑制TLR4/NF-κB/NLRP3减轻Kupffer细胞炎症改善非酒精性脂肪性肝病机制研究

Huazhi Fugan granule alleviates Kupffer cell inflammation by inhibiting TLR4/NF-κB/NLRP3 and improves nonalcoholic fatty liver disease

目的:探讨化脂复肝颗粒在非酒精性脂肪性肝病(NAFLD)中对Kupffer细胞(KCs)TLR4/MyD88/NF-κB/NLRP3信号通路的影响。方法:将分离培养的KCs随机分为空白组、LPS干预组、LPS+空白血清组、LPS+化脂复肝组、高糖+对照组、高糖+LPS干预组、高糖+LPS+空白血清组、高糖+LPS+化脂复肝组,LPS干预浓度为100 ng/ml,高糖干预为于KCs培养基中另加入葡萄糖30 mmol/L。用CCK-8法检测各组KCs增殖情况;酶联免疫吸附法(ELISA)测定各组KCs培养基上清液中促炎因子TNF-α、IL-1β的含量;反转录实时荧光定量PCR(RT-qPCR)法检测各组KCs中TLR4、MyD88、NLRP3、Caspase-1、NF-κB p65、TNF-α、IL-1β的基因表达情况。结果:CCK-8检测显示,空白及高糖培养对照组KCs细胞不断增殖,长势良好;LPS+化脂复肝组与高糖+LPS+化脂复肝组KCs缓慢增殖,非高糖组KCs均较含高糖组KCs增殖速率更快;而LPS或高糖+LPS诱导模型组及各加空白血清组KCs于第6小时开始出现凋亡,含高糖组KCs均较非高糖组KCs凋亡速率更快。非高糖各组与含高糖各组中促炎因子TNF-α、IL-1β含量以及炎症相关基因(TLR4、MyD88、NLRP3、Caspase-1、NF-κB/p-65、TNF-α、IL-1β、IL-18)表达水平均呈相同变化趋势,且含高糖各组上述促炎因子及炎症相关基因表达水平均较非高糖组高。以非高糖组为例,LPS诱导模型组和LPS+空白血清组的促炎因子TNF-α与IL-1β含量均显著高于对照组(P<0.05),炎症相关基因TLR4、MyD88等的mRNA表达水平也均显著高于对照组(P<0.05),而LPS诱导模型组与LPS+空白血清组间上述各指标比较差异均无统计学意义(P>0.05);与LPS诱导模型组或LPS+空白血清组比较,LPS+化脂复肝组促炎因子TNF-α与IL-1β含量均显著降低(P<0.05),炎症相关基因TLR4、MyD88等的mRNA表达水平也均显著降低(P<0.05)。结论:化脂复肝颗粒含药血清可改善KCs活力,有效减轻KCs炎症反应,减少KCs细胞焦亡的发生,具有显著的抗炎、抗氧化损伤及保肝作用,其作用机制可能与抑制TLR4/MyD88/NF-κB p65/NLRP3信号通路的激活相关。

Objective:To investigate the effect of Huazhi Fugan granules on TLR4/MyD88/NF-κB/NLRP3 signaling pathway in KCs in nonalcoholic fatty liver disease(NAFLD).Methods:The isolated and cultured Kupffer cells(KCs)were randomly divided into blank control group,LPS intervention group,LPS+blank serum group,LPS+Huazhi Fugan group,high glucose+control group,high glucose+LPS intervention group,high glucose+LPS+blank serum group,high glucose+LPS+Huazhi Fugan group,LPS intervention concentration was 100 ng/ml,and high glucose intervention was adding glucose 30 mmol/L to KCs medium.The proliferation of KCs in each group was detected by CCK-8 method;the content of pro-inflammatory factors TNF-αand IL-1βin the supernatant of KCs medium in each group was determined by enzyme-linked immunosorbent assay(ELISA);RT-qPCR method to detect the gene expression of TLR4,MyD88,NLRP3,Caspase-1,NF-κB p65,TNF-α,IL-1βin KCs of each group.Results:The detection of CCK-8 showed that KCs cells in the blank and high glucose culture control groups proliferated continuously and grew well;KCs in the LPS+HZFGXQ group and the high glucose+LPS+Huazhi Fugan group proliferated slowly,and the KCs in the non-high glucose group were higher than those in the high glucose group.The proliferation rate of KCs was faster;while the KCs in the LPS or high glucose+LPS-induced model group and the blank serum groups began to appear apoptosis at 6 h,and the apoptosis rate of KCs in the high glucose group was faster than that in the non-high glucose group.The contents of pro-inflammatory factors TNF-α,IL-1βand inflammation-related genes(TLR4,MyD88,NLRP3,Caspase-1,NF-κB p65,TNF-α,IL-1β,IL-18)showed the same trend of change,and the expression levels of the above pro-inflammatory factors and inflammation-related genes in each group containing high glucose were higher than those in the non-high glucose group.Taking the non-high glucose group as an example,the contents of pro-inflammatory factors TNF-αand IL-1βin the LPS-induced model group and the LPS+blank serum group were significantly higher than those in the control group(P<0.05),and the mRNAs of inflammation-related genes TLR4 and MyD88 were significantly higher than those in the control group(P<0.05).The expression levels were also significantly higher than those in the control group(P<0.05),while there was no significant difference in the above indexes between the LPS-induced model group and the LPS+blank serum group(P>0.05);Compared with the LPS-induced model group or the LPS+blank serum group Compared with the LPS+Huazhi Fugan group,the contents of pro-inflammatory factors TNF-αand IL-1βwere significantly decreased(P<0.05),and the mRNA expression levels of inflammation-related genes TLR4 and MyD88 were also significantly decreased(P<0.05).Conclusion:Huazhi Fugan granule-containing serum can improve the viability of KCs,effectively reduce the inflammatory response of KCs,and reduce the occurrence of KCs cell pyroptosis.It has significant anti-inflammatory,anti-oxidative damage and hepatoprotective effects.Inhibited activation of TLR4/MyD88/NF-κB p65/NLRP3 signaling pathway.