Study On The Mechanism Of Yifeijianpi Recipe Regulating TLR4/MyD88/NF-κB Signaling Pathway To Inhibit 16HBE Inflammatory Injury And Mucus Hypersecretion

1 Objective:Through the study of the cell level of the medicine-containing serum of the Yifei Jianpi Recipe,it is revealed that the TLR4/NF-κB signaling pathway is the molecular target of the prescription to inhibit airway inflammation and mucus hypersecretion.2 Methods:(1)40 SD rats were randomly divided into two groups: blank serum group and medicated serum group,with 20 rats in each group.Rats in blank serum group and medicated serum group were given stroke-physiological saline solution and Yifei Jianpi Recipe by gavage respectively.After the last gavage,rats were anesthetized and blood was collected from abdominal aorta under aseptic condition;(2)Human Bronchial Epithelial Cells(16HBE)were stimulated by Cigarette Smoke Extract(CSE)prepared by self-made gas suction pump.CSE and serum containing Yifei Jianpi Recipe were screened by CCK8 method to intervene Chronic Obstructive Pulmonary Disease(COPD),the optimal concentration and time of COPD cell model;(3)The effects of TLR4 gene silencing and overexpression on TLR4 mRNA expression in 16 HBE were detected by qRT-PCR;(4)Western Blot(WB)and qRT-PCR were used to detect TLR4,NF-κB,MUC5 AC,MUC7,muc8 mRNA and protein expression after TLR4 silencing and overexpression.3 Results:(1)Compared with 5%,10% and 15% CSE for 6h,there was no significant difference in cell viability after 12 h and 24h;Compared with 15% CSE for 6h,the cell viability decreased significantly after 48 h and 72h(P<0.05);Compared with 20% and25% CSE for 6h and 12 h,the cell viability decreased significantly after 24 h,48h and72h(P<0.05).The best concentration and time of 16 HBE stimulated by 20% CSE for 24 hours were selected.Compared with 5%,10% and 15% medicated serum for 6h,12 h,24h,48 h and 72 h,the cell viability of 20% and 25% medicated serum increased significantly(P<0.01);Compared with 20% medicated serum for 6h and 12 h,the cell viability increased significantly after 24 h,48h and 72h(P<0.01).The optimal concentration and time of Yifei Jianpi formula were selected as 20% serum for 24 hours;(2)Compared with the blank control group,the expression of TLR4 mRNA in each CSE experimental group was significantly increased(P<0.01);Compared with CSE group,TLR4 mRNA in CSE+si RNA TLR4 group was significantly decreased,and TLR4 mRNA expression in CSE+Lv-TLR4 group was significantly increased(P<0.01).There was no significant difference in TLR4 mRNA expression between CSE+si RNA TLR4-NC group and CSE+ Lv-TLR4-NC group(P>0.05);Compared with CSE+si RNA TLR4-NC group,the expression of TLR4 mRNA in CSE+si RNA TLR4 group was significantly decreased(P<0.01);Compared with CSE + Lv-TLR4-NC group,the expression of TLR4 mRNA in CSE+ Lv-TLR4 group was significantly increased(P<0.01);(3)Compared with the blank control group,the expressions of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding proteins in the CSE group were significantly increased(P<0.01);Compared with the CSE group,the TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding protein expressions in the CSE group were significantly increased(P<0.01);The expression of the corresponding proteins of TLR4,NF-κB,MUC5 AC,MUC7,and MUC8 decreased significantly(P<0.01),and the expression of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA decreased(P<0.05),and the difference in MUC7 was not statistically significant(P>0.05);Compared with the CSE+si RNA-TLR4-NC group,the expressions of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding proteins in the CSE+si RNA-TLR4 group were significantly reduced(P<0.01);Compared with the drug-containing serum group,the expressions of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding proteins in the CSE+si RNA-TLR4+ drug-containing serum group were reduced(P<0.05,P<0.01);(4)Compared with the CSE group Compared with the CSE+drug-containing serum group,the expressions of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding proteins were significantly reduced;Compared with the CSE+Lv-TLR4-NC group,the TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and corresponding protein expression of the CSE+Lv-TLR4 group increased significantly;Compared with CSE+Lv-TLR4 group,CSE+Lv-TLR4+ drug-containing serogroup,the expression of TLR4,NF-κB,MUC5 AC,MUC7,MUC8 mRNA and the corresponding protein in the CSE+Lv-TLR4+ drug-containing serogroup was significantly increased compared with the CSE+drug-containing serogroup.The above differences are all There is statistical significance(P<0.05,P<0.01).Conclusion:(1)Intervene 16 HBE cells through CSE,and establish a COPD cell model,the modeling method is reasonable and well-documented;(2)Yifei Jianpi Recipe can interfere with the COPD cell model,reduce the secretion of MUC and inhibit COPD tract inflammation and mucus hypersecretion;(3)TLR4/NF-κB signaling pathway is one of the potential molecular targets of Yifei Jianpi Recipe to inhibit airway inflammation and mucus hypersecretion;(4)Yifei Jianpi Recipe reduces TLR4 by down-regulating TLR4 NF-κB transcription,inhibiting the production or synthesis of MUC,may be its molecular mechanism for inhibiting airway inflammation and mucus hypersecretion.