Study On The Regulatory Mechanism Of Yangyin Qingfei Decoction On Oxidative Damage In SMPP Mic

Purpose:To investigate the damage of oxidative stress in SMPP mice by observing the changes of SOD,MDA and ROS levels in serum and lung tissues of mice with severe Mycoplasma pneumoniae pneumoniae（SMPP）;to investigate the intervention of different doses of Yangyin Qingfei Decoction Plus on the factors related to ROS-Nrf2-ARE signaling pathway and SOD and MDA,and to investigate the efficacy mechanism of SMPP.Material and method:48 BALB/c mice were randomly divided into 2 groups:normal group and model group.The model group mice were anesthetized and nasal drops of MP bacterial solution at 10¹⁰CCU/ml were used to construct the SMPP model.After the model was successfully established,the samples were taken on the 3rd,7th,10th and 14th days.The general conditions of the mice were observed;the contents of SOD and MDA in the serum and lung tissues of the mice were measured by UV spectrophotometry.the contents of ROS in the serum and lung tissues of the mice were measured by ELISA.36 BALB/c mice were randomly divided into six groups:normal group,model group,azithromycin group,Yangyin low-dose group,Yangyin medium-dose group and Yangyin high-dose group.Except for the normal group,the mice in other groups were constructed as the SMPP model by the same method as in Experiment 1,and all groups were sampled on day 7 after the intervention after successful modeling.The general conditions of the mice were observed;the lung index of the mice was calculated;the levels of MDA and SOD in the serum of the mice were detected by UV spectrophotometry,and the levels of ROS in the serum of the mice were detected by ELISA.The gene contents of Keap1,Nrf2 and HO-1 in lung tissues were determined by qPCR,and the protein expression of Keap1,Nrf2 and HO-1 in mouse lung tissues were detected by Western blot.Results:Experiment 11.The mice in the normal group were in good general condition,with normal activity,bright hair,even breathing,no abnormal nasal secretions,normal diet and water intake,and normal defecation.From the 3rd day after modeling,some mice in the model group were mentally tired,less active and lost weight.On the 14th day of modeling,the mental condition of mice slightly improved,but the overall condition was still poor.2.Morphology of mouse lung tissues under light microscopy:the lung tissues of mice in the normal group had normal morphological structure and did not show the condition of inflammatory cell infiltration.The lung tissue structure of the mice in the model group was obviously damaged,and it was difficult to see intact alveoli,and a significant widening of the alveolar septum could be observed,with a large amount of inflammatory cell exudation.The pathological score of mice showed that the pathological score of mice in the model group reached a peak at day 7 and then gradually decreased.3.Comparing the levels of SOD,MDA and ROS in the serum of each group of mice:after MP infection,the overall levels of serum SOD decreased and the overall levels of MDA and ROS increased in the model group compared with the normal group,with the peak and trough values occurring on day 7;compared with the normal group,the levels of SOD in the serum of the model group decreased significantly on days 7 and 10,and the levels of MDA and ROS increased significantly with statistically significant（P<0.05）.Comparing the levels of SOD,MDA and ROS in the lung tissues of mice in each group:compared with the normal group,the overall lung tissue levels of SOD were lower and the levels of MDA and ROS were higher in the model group.Compared with the normal group,the levels of SOD in lung tissues on day 7 and day 10 in the model group were significantly lower and the levels of MDA and ROS were significantly higher,which were statistically significant（P<0.05）;compared with day 3 in the model group,the levels of SOD in lung tissues on day 7 and day 10 in the model group were lower and the levels of MDA and ROS were higher,which were significantly different and statistically significant（P<0.05）;The levels of SOD in lung tissue decreased and the levels of MDA and ROS increased on day 7 of the model group compared with day14 of the model group,and were statistically significant（P<0.05）.There was no statistical significance in the model group on day 10 compared with day 14,although the SOD content was lower and the MDA and ROS content was higher on day 10（P>0.05）.Experiment 21.Mice general status observation:mice in the normal group were in good general status with normal mental and activity level.Mice in other groups showed different degrees of changes starting from day 3 after modeling,and mice in the model group were in the worst condition.On the 7th day after modeling,almost all mice in the model group were in poor mental condition and showed vertical hair,anorexia and reduced water intake.Except for the normal group,the mice in the nourishing Yin high dose group and the azithromycin group were in better condition.On the 7th day after administration,the condition of all treatment groups improved,and the improvement was more obvious in the mice in the nourishing Yin high-dose group and the azithromycin group.2.Morphology of mouse lung tissues under light microscopy:the lung tissue structure of mice in the model group was obviously damaged,only individual intact alveoli were present,alveolar interval appeared significantly widened,and a large number of inflammatory cell exudation could be observed.In the low dose group,partial inflammatory cell infiltration and widening of alveolar septum could be observed,and more lymphocyte infiltration could be observed near the fine bronchi.In the medium dose group,there was a partial inflammatory infiltration with mild widening of the alveolar septum and moderate lymphocyte infiltration near the fine bronchi.The Yangyin high-dose group had a small amount of inflammatory cell infiltration and mildly impaired lung tissue morphology and structure,which was overall better than the Yin group in the other two dose groups.The Western medicine group had some inflammatory cell infiltration near the bronchi and mild morphological structural damage of lung tissue.3.Pulmonary index and pathology scores in mice:The lung index of the mice was observed to increase in all other groups compared with the normal group,with the highest in the model group and the lowest in the azithromycin group.Compared with the model group,the lung index of each treatment group decreased and was statistically significant（P<0.05）.Compared with the Yangyin low-dose group,the lung index decreased in the Yangyin medium-dose group,the Yangyin high-dose group and the azithromycin group with statistical significance（P<0.05）.There was also statistical significance（P<0.05）in the Yangyin medium-dose group compared with the Yangyin high-dose group and the azithromycin group,but there was no statistical significance（P>0.05）in the azithromycin group compared with the Yangyin high-dose group.The pathological scores of mice were observed.The highest pathological score was in the model group and the lowest in the azithromycin group.Compared with the Yangyin low-dose group,the Yangyin medium-dose group,Yangyin high-dose group and azithromycin group had lower pathological scores,with statistically significant differences（P<0.05）.The pathology scores in the Yangyin high-dose group and the azithromycin group were statistically significantly lower compared with the Yangyin medium-dose group（P<0.05）.The pathology score of the azithromycin group was slightly lower than that of the Yangyin high-dose group,but it was not statistically significant（P>0.05）.4.Observation of ROS levels in mouse serum:The ROS content in the serum of mice in the model group was found to be the most elevated,and its difference was statistically significant（P<0.05）when compared with the normal group,and the content of ROS in the remaining treatment groups decreased compared with the model group,which was statistically significant（P<0.05）.Compared with the Yangyin low-dose group,the ROS content decreased in the Yangyin high-dose group and the azithromycin group,which was statistically significant（P<0.05）.The ROS in the Yangyin high-dose group was slightly lower than that in the azithromycin group,but there was no significant difference that was statistically significant（P>0.05）.5.Determination of SOD and MDA in mice serum by UV spectrophotometric method:Compared with the normal group,the serum SOD levels were decreased and the values of MDA were increased in all other groups.Compared with the model group,the serum SOD values were increased and the values of MDA were decreased in each treatment group group.There was a statistically significant difference in the content of SOD in the model group compared with the nourishing Yin medium-dose group and the azithromycin group（P<0.05）.Although the content of SOD in the nourishing Yin high-dose group was higher than that in the model group,it was not statistically significant（P>0.05）.The content of SOD was significantly higher in the azithromycin group compared with the nourishing Yin high-dose group and was statistically significant（P<0.05）.The MDA content was significantly decreased in the Yangyin medium dose group,Yangyin high dose group and azithromycin group compared to the Yangyin low dose group with statistical significance（P<0.05）.It was also statistically significant（P<0.05）in the Yangyin medium dose group compared with the Yangyin high dose group and the azithromycin group.The azithromycin group was slightly lower than the Yangyin high-dose group,but it was not statistically significant（P>0.05）.6.qPCR assay for mRNA content of Keap1,Nrf2 and HO-1 in lung tissue:The gene expression of Keap1,Nrf2 and HO-1 in lung tissue was detected,and the lowest gene expression levels were found in the model group.The levels of Keap1,Nrf2 and HO-1 genes were significantly higher in the other groups compared with the normal group,and the differences were statistically significant（P<0.05）.The gene levels of Keap1,Nrf2 and HO-1were significantly increased and statistically significant in each treatment group compared with the model group（P<0.05）.The gene levels of Keap1 and Nrf2 were higher in the medium-dose group compared with those in the low-dose group,and the difference was statistically significant（P<0.05）,but the gene levels were only slightly higher in the medium-dose group compared with HO-1 in the low-dose group,and the difference was not statistically significant（P>0.05）.Compared with the Nrf2 and HO-1 gene levels in the medium-dose and low-dose groups,the levels in the Yangyin high-dose group were significantly higher and statistically significant（P<0.05）.The azithromycin group had the highest levels of Nrf2 and HO-1 genes and the difference was statistically significant（P<0.05）compared with the Yangyin high-dose group.7.WB measurement of protein expression of Keap1,Nrf2 and HO-1 in lung tissue:After MP infection,the protein expression of Keap1,Nrf2 and HO-1 was decreased in lung tissues of all other groups compared with the normal group.The protein expressions of Keap1,Nrf2 and HO-1 were significantly increased in each treatment group compared with the model group,and the difference was statistically significant（P<0.05）.The elevation of Keap1,Nrf2 and HO-1 protein expression in the Yangyin high-dose group was statistically significant compared with the medium-dose and low-dose groups（P<0.05）.The differences in Keap1,Nrf2 and HO-1 protein expression in the azithromycin group were statistically significant compared with the Yangyin high-dose group（P<0.05）.Conclusion:1.SMPP can lead to an increase in the content of ROS and MDA and a decrease in the content of SOD,further confirming that the pathogenesis of SMPP is closely related to oxidative damage,and that changes in its indicators can predict the occurrence of SMPP and determine the severity of the disease.2.Yangyin Qingfei Decoction Plus can increase the expression of Keap1,Nrf2 and HO-1 and decrease the content of ROS,increase the content of antioxidant SOD and decrease the content of oxidative index MDA,suggesting that Yangyin Qingfei Decoction Plus can regulate the oxidative stress damage of SMPP,and there is a dose-dependent relationship,and the ROS-Nrf2-ARE signaling pathway may be one of its action mechanisms.