Regulation Of Chaipu Decoction On HMGB1,TLR4 And NF-kB In Airway Epithelial Cells Of Asthmatic Mice

Objective:To explore the regulatory effect of Chaipu Decoction on HMGB1,TLR4 and NF-κB in airway epithelial cells of asthmatic mice.Methods:Prepare 24 Balb/c mice,4-6 weeks old,by normal feeding for 7 days,and then equally divide them into group A（normal group）,group B（hepatic depression type asthma group）,and group C（Chaiputang group）,each group has 8 animals.Among the 3 groups,group A continues to take normal feeding measures,while groups B and C undergo chronic and unpredictable stimulation such as fasting,water deprivation,and tail clamping within 1-28 days.The liver depression model was established by using every 7 days as the measurement time to determine the weight change of the mice in each group.Before modeling and one week before the completion of the modeling,the immobility time of the mice in each group was measured to jointly evaluate whether this model is successful.After the liver depression model was made,the asthma model was made as follows:A mixture of 10μg ovalbumin（OVA）and 400μg aluminum hydroxide（Al（OH）₃）was prepared for intraperitoneal treatment of groups B and C on the 1st and 7th days for injection,each group was nebulized on the 14th day.The C group was given Chaipu Decoction（1.5g/kg）30minutes before nebulization,and the A and B groups were injected with the same amount of 0.9%normal saline into the stomach.Each group of mice was individually placed in a semi-closed nebulization box to receive nebulization intervention.The solution used for nebulization in group B and C was OVA solution（1%）,and group A was nebulized with 0.9%saline,each time for 30 minutes,after 7 consecutive days,the mouse showing irritability and nodding breathing represents the establishment of the asthma model successfully.On the 7th day,1 hour after the last administration,the lung tissues of each group were taken under aseptic conditions for HE staining.To observe the inflammatory infiltration,blood was collected from the orbit in A,B,and C groups,and the serum was separated.The serum of A,B,and C groups were added to normal mouse airway epithelial cells and cultured for 24 hours.Fluorescence real-time quantitative polymerase chain reaction（RT-q PCR）was used to detect extracellular high mobility group protein B1（HMGB1）,TOLL-like receptor 4（TLR4）and nuclear transcription factor-κB（NF-κB）in mouse airway epithelial cells,interleukin 6（IL-6）,monocyte chemoattractant protein 1（MCP-1）m RNA expression.Western-Blot was used to detect the protein expression of HMGB1,TLR4,NF-κB,IL-6,and MCP-1 in mouse airway epithelial cells;The concentration of IL-6 and MCP-1 in the supernatant of mouse airway epithelial cells was determined by enzyme-linked immunosorbent assay（ELISA）technology.;Results:1.Modeling of liver depression model:There is no difference in weight and forced swimming immobility time between the normal group,liver depression asthma group,and Chai Pu Tang group（P>0.05）.After modeling The weight of mice in the liver depression asthma group and Chai Pu Tang group was generally lighter,and the weight growth rate of each mouse in the two groups was slower than that of the normal group（P<0.01）,forced swimming immobility time,it was also significantly longer than the normal group of mice（P<0.05）,and the comparison of body weight changes and forced swimming immobility time between the liver-stagnation asthma group and the Chai Pu Tang group was not statistically significant.（P>0.05）2.HE staining results:the alveoli in the lung tissue of the mice in the normal group were intact,the airway wall was not thickened,and the airway mucosa was intact,and there was no inflammatory cell distribution around it.In the liver-stagnation asthma group,there were extensive alveolar destruction and fusion in the lung tissue,bronchial wall edema,and a large number of inflammatory cell infiltrations around it.Compared with the asthma group,the airway lumen of the Chai pu tang group was enlarged,but still narrower than the normal group.Compared with the liver-stagnation asthma group,the degree of inflammatory infiltration around the tube wall was reduced.3.ELISA detected the concentration of IL-6 and MCP-1 in the supernatant of airway epithelial cells in mice:Compared with the normal group,the IL-6 concentration in the liver-stagnation asthma group was significantly increased（P<0.01）,In Chai Pu Tang group,the expression of IL-6 was significantly lower than that in the liver-stagnation asthma group（P<0.01）,and the expression of MCP-1 in the liver-stagnation asthma group was higher than that in the normal group（P<0.05）.Compare to the Chai Pu Tang group,the expression of MCP-1 was decreased（P<0.05）.4.WB detection of HMGB1,TLR4,NF-κB,IL-6,and MCP-1 protein expression in mouse airway epithelial cells:The expression of HMGB1,TLR4,and MCP-1 protein in the liver-stagnation asthma group was significantly higher than that in the normal group（P<0.01）,the expression of NF-κB and IL-6 increased in liver depression type asthma（P<0.05）.After the intervention of Chai Pu Tang,the expression of HMGB1,TLR4,MCP-1 was higher than that in the liver-stagnation asthma group（P<0.01）,and the expression of NF-κB protein was lower than that in the liver-stagnation asthma group（P>0.05）.The expression of IL-6 was lower than that in the liver-stagnation asthma group（P<0.05）.5.RT-q PCR detection of HMGB1,TLR4 and NF-κB,IL-6,MCP-1m RNA expression in mouse airway epithelial cells:HMGB1,TLR4,NF-κB,MCP-1 expression in the liver-stagnation asthma group was significantly increased compare with the normal Group（P<0.01）,the expression of IL-6in liver-stagnation asthma was increased compared with the normal group（P<0.05）.After the intervention of Chai Pu Tang,compared with the liver-stagnation asthma group,The expression levels of HMGB1,TLR4,NF-κB,MCP-1 and IL-6 were all significantly decreased（P<0.01）.Conclusion:1.The serum of mice with liver-stagnation asthma can induce the expression of HMGB1,TLR4,NF-κB,MCP-1 and IL-6 in airway epithelial cells.2.The serum containing Chaipu Decoction can inhibit the expression of HMGB1,TLR4,NF-κB,MCP-1,IL-6 in the airway epithelial cells of mice with liver depression type asthma.