| Part one: preparation of rat model of acute lung injury and its changes in different periods Objective:The(ALI)rat model of acute lung injury was established by intratracheal instillation of lipopolysaccharide(LPS).The degree of lung injury was observed at 3 h,6 h and 12 h.Methods:1.Rat grouping and modeling: 32 healthy Sprague-Dawley rats were randomly divided into normal group and model group.According to the duration of LPS infusion,the model group was divided into 3 h group,6 h group and 12 h group with 8 rats in each group.The rat model of ALI was established by instilling LPS(2mg/kg)into the trachea of rats.2.Index detection: observe the general condition of rats;detect the lung function of rats in each group;calculate the lung wet / dry weight ratio(W/D)from the left lung;detect the contents of cytokines tumor necrosis factor-α(TNF-α),interleukin-1 β(IL-1 β)and IL-8 in(BALF)of bronchoalveolar lavage fluid(BALF)of rats in each group by enzyme-linked immunosorbent assay(ELISA).The contents of malondialdehyde(MDA)and superoxide dismutase(SOD)in lung tissue were detected by ELISA method,and the pathological changes of lung tissue were observed by HE staining.Results:1.General condition of rats: compared with the normal group,after 3 hours of LPS induction,the spirit of rats was in a state of indifference,food intake decreased,activity decreased significantly,mucus secretion in nasal cavity increased,respiratory frequency increased,and wheezing could be heard.2.Pulmonary function: forced expiratory volume in 0.1 second(FEV0.1),forced expiratory volume in 0.3 second(FEV0.3)and ratio of forced vital capacity to(FVC)(FEV0.1/FVC;FEV0.3/FVC)decreased significantly in rats induced by LPS for 3hours(P < 0.05,P < 0.01).3.Lung pathology: after 3 hours of LPS induction,the alveolar septum thickened,pulmonary interstitial edema and erythrocyte exudation in rats.The ratio of wet to dry weight(W / D)increased significantly(P < 0.01).4.Cytokines and oxidation-antioxidation indexes: after 3 hours of LPS induction,the contents of IL-1 β,IL-8 and TNF-α in BALF were significantly increased(P <0.01),and the content of MDA in lung tissue was significantly increased(P < 0.01),while the content of SOD was significantly decreased(P < 0.01).Conclusions:The ALI rat model was successfully prepared by tracheal instillation of LPS.By comparing the different periods of 3 h,6 h and 12 h,it was found that the characteristics of pulmonary pathological changes,inflammatory factors and oxidation indexes of rats at 3 h after modeling were more consistent with the characteristics of ALI patients,so the ALI model induced by LPS for 3 h was the best.Part two: effect of electroacupuncture preconditioning on M1 polarization of alveolar macrophages in ALI rats Objective:To study whether electroacupuncture at acupoints LU 5 and ST 36 can inhibit the phenotypic polarization to M1 in alveolar macrophages of ALI rats,exert the protective effect of lung,and study its molecular mechanism.Methods:1.Grouping and modeling of rats: 40 healthy Sprague-Dawley rats were randomly divided into normal group,model group,electroacupuncture(EA)pretreatment group and EA group include LU5 group ST36 group and LU5 + ST36 group,with 8 rats in each group.The best time point of LPS induction in the first step was selected to make the model,and the method was the same as above.2.Electroacupuncture pretreatment: Six days before the preparation of the model,each electroacupuncture pretreatment group was pretreated with electroacupuncture at the corresponding bilateral acupoints,and the needle handle was connected with the electroacupuncture therapeutic apparatus,and the stimulation intensity was 1 to 2 Ma at a frequency of 2 Hz,and the intensity gradually increased to a slight tremor at the acupuncture site of rats.The duration of each intervention was 30 minutes for 6consecutive days.3.Index detection: the lung function of rats in each group was observed,and the lung tissue was taken to observe the pathomorphology of lung tissue and the score of lung injury.The contents of cytokines TNF-α,IL-1 β and IL-8 secreted by M1 phenotype in BALF of rats in each group were detected by ELISA,and the contents of MDA,SOD and myeloperoxidase(MPO)in lung tissue of rats in each group were detected.Real-time quantitative polymerase chain reverse transcription(QT-PCR)was used to determine the levels of M1 phenotypic markers(CD86,iNOS)and their key signaling molecules Toll-like receptor 4(TLR4),myeloid differentiation factor 88(My D88)and nuclear factor-κB p65(NF-κB p65)mRNA in AM of rats in each group.The protein expression of CD86 and iNOS and the protein expression of TLR4,My D88 and NF-κB p65 of M1 signal pathway in AM of rats in each group were detected by Western blot method.Results:1.Lung function and pathology: compared with the normal group,the lung function of the model group showed that FEV0.1,FEV0.3,FEV0.1/ FVC and FEV0.3/FVC were significantly decreased(P<0.01),The wet/dry ratio of lung tissue was increased(P<0.01);HE staining showed obvious inflammatory cell infiltration,a large number of erythrocyte exudation,alveolar septum thickening,and the lung injury score was significantly increased(P<0.01).After electroacupuncture preconditioning,FEV0.1,FEV0.3,FEV0.1/FVC and FEV0.3/FVC increased significantly(P<0.05,P<0.01),pathological sections showed that inflammatory cell infiltration decreased,erythrocyte exudation decreased,alveolar septum narrowed,and lung injury score decreased.2.The content of cytokines: compared with the normal group,the contents of M1 cytokines TNF-α,IL-1 β and IL-8 in the BALF of the model group were significantly higher than those in the normal group(P<0.01).Compared with the model group,the contents of cytokines TNF-α,IL-1 β and IL-8 in BALF of rats in electroacupuncture pretreatment group decreased significantly(P<0.01).3.Oxidation-antioxidation index: compared with the normal group,the contents of MDA and MPO in the lung tissue of the model group were significantly higher,and the content of SOD was significantly lower than that in the normal group(P<0.01).Compared with the model group,the contents of MDA and MPO in electroacupuncture pretreatment group decreased significantly,while the content of SOD increased significantly(P<0.05,P<0.01).4.M1 polarization markers: compared with the normal group,the relative expression of CD86,iNOS mRNA and protein in the AM of the model group was significantly higher than that of the normal group(P<0.01).After acupuncture pretreatment,the mRNA expression of CD86 and iNOS in AM was lower than that in the model group(P<0.05,P<0.01),and the detection effect of the above indexes in the LU 5 + ST 36 group was better than that in the LU 5 group and ST 36 group.5.M1 polarization signal pathway molecules: compared with the normal group,the relative expressions of mRNA and protein of TLR4,My D88 and NF-κB p65 in the AM of the model group were significantly higher than those in the normal group(P<0.01).After acupuncture pretreatment,the mRNA expression of TLR4,My D88 and NF-κB p65 in AM was lower than that in the model group(P<0.05,P<0.01),and the detection effect of the above indexes in the LU 5 + ST 36 group was better than that in the LU 5 group and ST 36 group.Conclusions:1.The phenotype of M1 in the lungs of ALI rats was significantly increased.2.Electroacupuncture preconditioning can inhibit the polarization of AM to M1,and its effect may be related to the down-regulation of TLR4/My D88/ NF-κB p65 signal pathway.3.Electroacupuncture at LU 5 and ST 36 can improve the pulmonary function and pathology of ALI rats,and the therapeutic effect of the combination of two acupoints is better than that of single acupoint. |
