Study On The Therapeutic Mechanism Of Qingfei Tongluo Formula By Regulating NLRP3 Inflammatory On Mycoplasma Pneumoniae Pneumonia

Part One-Study on Mechanism of MP-induced NLRP3 Inflammatory Pathway in A549 Cells and Intervention of Qingfei Tongluo formula on Inflammatory of NLRP3 in MP Infected A549 Cells Objective To investigate the immune in flammation induced by Mycoplasma pneumoniae(MP)after infection with NLRP3 inflammation of A549 cells in vitro,and the regulation of NLRP3 inflammatory by Qingfei Tongluo formula(QTF)and the control of its downstream cytokines IL-18,IL-1?.Methods(1)The Qingfei Tongluo formula was extracted by water extraction and added to MP-infected A549 cells.The optimal concentration of the drug was calculated by CCK-8 test to detect the QTF of different concentrations(0.1mg/L,0.5mg/L,1mg/L,2mg/L,4mg/L)and different time points(24h,48 h,72h).(2)Inhibition/over-expression of the NLRP3 plasmid was prepared and the experiments were grouped according to the optimal drug concentration measured by the CCK-8 assay.(3)Western blot was used to detect the expression of ASC,Caspase-1 and NLRP3 in NLRP3 inflammatory vesicles;The ASC,Caspase-1 and NLRP3 genes were detected by Real-time PCR;The inflammatory cytokines IL-18 and IL-1? were detected by ELISA;apoptosis rate was detected by flow cytometry.To observe the activation of NLRP3 inflammatory by MP and the effect of QTF on NLRP3 inflammatory.Results(1)The results of CCK-8 showed that the IC50 of Qingfei Tongluo formula was 52.856%,which corresponds to the survival rate of A549 cells at 48 h,and the increase of drug concentration enhanced the protection of cells and concentration-dependent.(2)Western blot and Real-time PCR showed that the expression of ASC,Caspase-1 and NLRP3 in NLRP3 inflammatory on A549 cells increased after A549 cells were infected by MP.The ELISA showed that the downstream inflammatory cytokine IL-18,IL-1? was significantly increased,indicating that NLRP3 inflammatory corpuscles can be activated by MP and produce a series of inflammatory reactions.Flow cytometry analysis showed that the apoptosis rate in creased after MP infection of A549 cells.(3)After using Qingfei Tongluo formula,Western blot and Real-time PCR showed that the expression levels of ASC,Caspase-1 and NLRP3 in NLRP3 inflammatory decreased,and ELISA showed that the downstream related in flammatory cytokines IL-18,IL-1? expression level was significantly decreased,indicating that Qingfei Tongluo formula can inhibit the activated NLRP3 inflammatory corpuscle and restore the downstream in flammatory cytokines IL-18 and IL-1? to normal levels,and flow cytometry found that Qingfei Tongluo formula was used.After the complex,the apoptotic rate decreased.Conclusions(1)After infection of A549 cells with MP,it can activate the NLRP3 inflammatory corpuscle in the inhibited state,and produce a series of immune inflammatory reactions,increase the release of downstream in flammatory factors,increase the apoptosis rate,indicating that NLRP3 inflammatory bodies were induced by MP.(2)In vitro experiments have shown that Qingfei Tongluo formula can regulate NLRP3 inflammatory,reduce inflammation and inhibit cell apoptosis,and Qingfei Tongluo formula has a concentration-dependent regulation of NLRP3 inflammatory bodies.The greater the concentration,the protective effect more obvious.Part Two-Study on Expression of NLRP3 Inflammatory pathway in MPP Mice and intervention of Qingfei Tongluo formula Objective To investigate the effects of Qingfei Tongluo formula on NLRP3 inflammatory corpuscles Caspase-1,NLRP3 and downstream in flammatory cytokines in MPP mice,and further verify the inflammatory effects of Qingfei Tongluo formula on NLRP3 Inflammatory pathway in vivo.Methods(1)100 BALB/C mice were randomly divided into 5 groups,20 in each group-A group:control;B group:model;C group:Qingfei Tongluo formula;D group:azithromycin;E group:combination of Qingfei Tongluo formula and azithromycin.(2)Mice were modeled by MP nasal dropping on the 1st to 3th days and the 8th to 10 th days of the experiment with administered by intragastricad ministration on the first day.A and B group were both treated with normal saline for 10 days;C group were treated with Qingfei Tongluo formula for 10 days;D group were treated with azithromycin.Liquid for the 1st to 3th days and the 8th to 10 th days;E group were used azithromycin solution plus Qingfei Tongluo formula for 10 days.(3)Mice were sacrificed on the 6th and 11 th days,ten in each group.Western blot was used to detect the expression of Caspase-1 and NLRP3 in NLRP3 inflammatory of MPP mice.The Caspase-1 and NLRP3 genes were detected by Real-time PCR.The inflammatory cytokines IL-18 and IL-1? were detected by ELISA;HE staining was used to observe the pathophysiological morphology of lung tissue in MPP mice.Results(1)On the 6th and 11 th days,the expression levels of Caspase-1 and NLRP3 in the NLRP3 inflammatory corpuscles of MPP mice were increased.MP-DNA gene number in control group showed no MP infection,while MP-DNA of model and other treatment group were positive.ELISA test showed that the expression levels of downstream in flammatory factors IL-18 and IL-1? were elevated.The results of the two samples were relatively high.On the 11 th day,the expression levels of Caspase-1 and NLRP3,IL-18 and IL-1? were significantly higher than those on the 6th day.(2)Comparison of each treatment groups and model group,the expression levels of Caspase-1 and NLRP3 in the NLRP3 inflammatory corpuscles of MPP mice were decreased on the 6th and 11 th day,and the MP-DNA gene copy number of each treatment group were decreased.The decrease was obvious,and the expression levels of downstream inflammatory factors IL-18 and IL-1? were significantly decreased.On the 11 th day,the expression levels of Caspase-1 and NLRP3,IL-18 and IL-1? were significantly lower than those on the 6th day.(3)Compared with azithromycin group,the expression levels of caspase-1,NLRP3 and inflammatory factors IL-18,IL-1? in lung tissue of MPP mice in Qingfei Tongluo Formula group were not significantly decreased.Compared with Qingfei Tongluo Formula group and azithromycin group,the expression levels of caspase-1,NLRP3 and inflammatory factors IL-18,IL-1? in lung tissue of MPP mice in integrated traditional Chinese and Western medicine group was significantly different.(4)HE results showed that the lung tissue of the control group was intact.The bronchial and alveolar hyperemia in the model group was obvious,and it was infiltrated by a large number of inflammatory cells.After using Qingfei Tongluo formula,each treatment group had different degrees of improvement.The bronchial and alveolar congestion and inflammatory cell in filtration were most significantly improved in the integrated Chinese and Western medicine group.Conclusions(1)Experiments in vivo show that Qingfei Tongluo formula can alleviate inflammation by regulating NLRP3 inflammation corpuscles and alleviate lung tissue damage.(2)From the perspective of repeated Mycoplasma pneumoniae infection,Qingfei Tongluo formula combined with azithromycin has a better advantage in the treatment of MPP.