Study On The Effect Of COPD Acupuncture Serum On Alveolar Macrophage Based On The Post Signal Pathway Of Cholinergic Alpha 7nACh Receptor

Background: Chronic obstructive pulmonary disease(COPD)is a common worldwide and major health-care burden disease,cigarette smoking is one of the main risk factors.COPD is associated with chronic inflammation affecting predominantly the lung parenchyma and peripheral airways,this is more responsive to corticosteroids and bronchodilators.Although these drugs can effectively relieve the clinical symptoms and reduce the number of acute attacks,but can not effectively improve lung function.As a part of traditional Chinese medicine,electro-acupuncture can improve pulmonary function and improve the quality of life of COPD patients.Although the mechanism of EA effect on COPD is not clear,but Studies have shown that the immune regulation of acupuncture may be related to the activation of cholinergic anti-inflammatory pathways.Objective: To study the effect of acupuncture serum on AM inflammatory factors and intracellular signaling pathways,we use cigarette smoking extract(CSE)to induce alveolar macrophages(AMs)cell as inflammation models,and use ?-bungarotoxin(?-BGT)to block key protein ?7nAChR of cholinergic anti-inflammatory pathway(CAP)to demonstrate that the CAP can anti-inflammat to AM in the treatment of COPD by acupuncture,to provides an experimental basis for electro-acupuncture treatment.Methods:(1)We used cigarette smoking combind with tracheal instillation of lipopolysaccharide(LPS)to replicate COPD rat models.Used rat lung function and pathological sections to evaluate whether the model was successful.Extracted acupuncture serum after electroacupuncture to treat model rats for one week.(2)Extracted the primary AMs,and the cells were identified by trypan blue exclusion assay,Giemsa staining,and immunofluorescence methods;extracted CSE and examined the cytotoxicity of CSE to AM by CCK-8 method;(3)The cells were divided into 6 groups: control group,cigarette smoke extract group(CSE),cigarette smoke extract + acupuncture serum group(CSE + serum),and ?-BGT+acupuncture serum group(?-BGT+serum),?-BGT+cigarette smoke extract+acupuncture serum group(?-BGT+CSE+serum),acupuncture of serum.(4)Detected the effect of acupuncture serum on IL-1?,IL-6 and TNF-? in AM supernatant by ELISA.(5)Detected the mRNA and protein expression of ?7nAChR,NF-?B,JAK2,and STAT3 in AM by Rea-ltime PCR and Western-Blot.Result:(1)The levels of FEV0.1,FEV0.3,FEV0.1/FVC and FEV0.3/FVC were lower in the COPD model group compared with the normal control group(P<0.05).Airway chronic inflammation,alveolar structure was changed,and secretion was increased observed in the pathological section.(2)The extracted cells were identified as AM,and the concentration of 20% CSE was the best to induce the AM inflammatory cell model;(3)The content of IL-1?,IL-6 and TNF-? in AM supernatant was observed by ELISA.The results showed that the CSE group had higher levels than the control group(all P<0.05);after the acupuncture serum was added,the CSE+serum group content decreased;after blocking the ?7nAChR,the ?-BGT+CSE+serum group content increased(All P<0.05),indicated that acupuncture serum had anti-inflammatory effects on CSE-induced AM,and this effect may be exerted by ?7nAChR;(4)Reatime PCR method was used to observe the mRNA expression of ?7nAChR,NF-?B,JAK2,and STAT3 in AM.The results showed that the CSE group was upregulated compared to the control group(all P<0.05);after the acupuncture serum was added,the CSE+serum group was further elevated(P<0.05);after blocking the?7nAChR,the mRNA of ?7nAChR,NF-?B,JAK2,and STAT3 expression in ?-BGT+CSE+serum group was decreased(P<0.05).In addition,the expression in the serum group was significantly higher than that in the control group(P<0.05).After blocking the ?7nAChR,the ?-BGT+ serum group was down-regulated(P<0.05).Theseresults suggest that acupuncture serum can upregulate the mRNA expression of?7nAChR,NF-?B,JAK2,STAT3,and this effect may be exerted by ?7nAChR.(5)The protein expression of ?7nAChR,NF-?B,JAK2,STAT3 in AM was observed by Western-Blot.The results showed that the expression of CSE group was higher compared to control group(all P<0.05);after acupuncture serum was added,the expression of CSE+serum group was decreased(P<0.05);after blocking ?7nAChR,?-BGT+CSE+serum group was blocked.,all expression was decreased(P<0.05).In addition,the expression of serum in the serum group was significantly higher than that in the control group(P<0.05);the expression in the ?-BGT+ serum group was decreased after the ?7nAChR was blocked(P<0.05).These results suggest that acupuncture serum can down-regulate the expression of ?7nAChR,NF-?B,JAK2,STAT3 in AM,and the effect is related to the activation of ?7nAChR.Conclusion:1)Model rats established by smoking combind with LPS tracheal instillation,satisfied with clinical pathology and lung function of the characteristics of COPD.COPD model rats were successfully prepared in this study;2)CSE successfully induced AM to release the inflammatory factors of IL-1?,TNF-?,and IL-6,and the inflammatory cell model was established.In the inflammatory state of AM,acupuncture serum can reduce the release of inflammatory factors through?7nAChR,which has the effect of inhibiting inflammation.In the normal state of AM,acupuncture serum has pro-inflammatory effects,and this effect is not related to?7nAChR.3)CSE activates NF-?B and JAK2/STAT3 inflammatory signaling pathways of AM;acupuncture serum eventually inhibits NF-?B and JAK2/STAT3 pathway proteins through ?7nAChR.Outlook: Acupuncture serum and ?-BGT as a foreign protein has effect of stimulatory on AM,may also activate other intracellular signaling pathways through other receptors on the cell membrane,Increased the expression of inflammatory factors.Acupuncture serum up-regulated the expression of ?7nAChR,NF-?B,JAK2,STAT3 mRNA,but down-regulated the protein,which may be related to post-transcriptional modification,as the acupuncture serum up-regulated the process of translation of certain modified proteins in AM,which participated in the shear of the protein,leading to a decrease in the amount of protein,we need further research to the specific regulatory factors.