补肺汤对IPF相关细胞活性及TLR2、4信号通路的影响

Effects of Bufei Decoction(补肺汤) on the Activity and TLR2、4 Signal pathway of IPF Cells

目的探究补肺汤对高迁移率族蛋白B1(HMGB1)诱导的人非小细胞肺癌细胞(A549)和人肺成纤维细胞(HFL1)活性及TLR2、4/NF-κB信号通路的影响。方法使用冷冻技术制备补肺汤冻干粉,SPF级大鼠正常饲养3周,补肺汤灌胃4周后处死,取含药血清。将A549和HFL1细胞正常培养、传代、冻存后,随机分为空白组、HMGB1诱导组、补肺汤冻干粉预培养+HMGB1诱导组(冻干粉浓度分别为0.1%、0.15%、0.2%、0.25%、0.3%)、补肺汤含药血清预培养+HMGB1诱导组(含药血清浓度分别为5%、10%、15%、20%、25%)、阳性药物(甲泼尼松龙)+HMGB1诱导组及空白血清预培养+HMGB1诱导组(空白血清浓度为10%、20%),按照分组处理细胞。采用MTT法检测细胞活性,免疫组化S-P法检测TLR2、TLR4、MyD88、NF-κB蛋白的阳性表达。结果MTT试验:与HMGB1诱导组相比,补肺汤冻干粉组和含药血清组A549和HFL1细胞的OD值均显著下降(P<0.01);免疫组化S-P:与HMGB1诱导组相比,补肺汤冻干粉组和含药血清组A549和HFL1细胞TLR2、TLR4、MyD88、NF-κB的平均吸光度均呈下降趋势(P<0.05,P<0.01),且其下降程度均与药物剂量呈正相关。结论补肺汤冻干粉及含药血清对A549和HFL1细胞活性有抑制作用,其机制可能与下调TLR2、4/MyD88/NF-κB信号通路有关。

Objective To investigate the effects of Bufei Decoction(补肺汤)on the activity and TLR2,4/NF-κB signal pathway in human lung adenocarcinoma cell(A549)and human lung fibroblasts(HFL1)induced by high mobility family protein B1(HMGB1).Methods Bufei Decoction lyophilized powder was prepared by freezing technique,and the SPF rats were killed after gavage of Bufei Decoction for 4 weeks after 3 weeks of normal feeding.A549 and HFL-1 cells were cultured,subcultured and cryopreserved,then randomly divided into:blank group,HMGB1-induction group,Bufei Decoction lyophilized powder pre-culture plus HMGB1-induction group(the concentration of lyophilized powder was 0.1%,0.15%,0.2%,0.25%and 0.3%),Bufei Decoction medicated serum pre-culture plus HMGB1-induction group(the concentration of medicated serum was 5%,10%,15%,20%and 25%),positive drug group(Methylprednisolone)and blank serum group,these cells were treated in their respective groups.The cells activity was detected by MTT,and TLR2,4,MyD88,and NF-κB positive expression of protein genes were detected by immunohistochemical S-P method.Results MTT:Compared with HMGB1-induction group,the OD values of A549 and HFL1 cells in Bufei Decoction lyophilized powder and medicated serum groups were significantly decreased(P<0.01);Immunohistochemical S-P:Compared with HMGB1-induction group,the average optical densities of TLR2,4,MyD88,NF-κB of A549 and HFL1 cells in Bufei Decoction lyophilized powder and medicated serum groups were significantly decreased(P<0.05,P<0.01),and both the degrees of decrease were positively correlated with drug dose.Conclusion The lyophilized powder and medicated serum of Bufei Decoction can restrain activity of A549 and HFL1 cells,and the mechanism may be related to the down-regulation of TLR2,4/MyD88/NF-κB signaling pathway.