| Objective1 To evaluate the efficacy and safety of Qushi Tongluo Decoction combined with azithromycin in the treatment of Mycoplasma pneumoniae pneumonia(MPP)in children,and to explore its regμLatory role in the microcircuLation disorder‘puLmonary blood stasis’ caused by MPP;2 To study the up-reguLation of inflammatory factors in the supernatant of human respiratory epithelial cells(Beas-2b)infected by Mycoplasma pneumoniae(MP)and the down-reguLation of Qushi Tongluo Decoction through the PI3K/AKT signaling pathway;3 Preliminary exploration of Qushi Tongluo Decoction to treat MP-infected mice by reguLating PI3K/AKT signaling pathway,activating its downstream factors vascuLar endothelial growth factor(VEGFA),inhibiting Autophagy-related protein(LC3B),which plays a role in reguLating immunity.Method1 Clinical evaluation of Qushi Tongluo Decoction combined with azithromycin in the treatment of MPP.Clinical trial:A total of 96 diagnosed children from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine and the First Affiliated Hospital of Henan University of Traditional Chinese Medicine were collected from February 2021 to December 2022.According to the ratio of 2:1,they were randomLy divided into groups(64 cases in the treatment group and 32 cases in the control group).The treatment group was given oral Qushi Tongluo formuLa granuLes combined with intravenous infusion of azithromycin,and the control group was given oral administration of azithromycin on the basis of intravenous infusion of azithromycin.Qushi Tongluo formuLa analog agent,the course of treatment is 5 days.After the treatment,the main curative effect index(the total effective rate of the disease,the curative effect of TCM syndrome),the secondary curative effect index(complete antipyretic time,antipyretic onset time,cough,sputum,asthma onset time)and blood coaguLation four items were analyzed.,and evaluate the security of the party.2 The effect of Qushi Tongluo Decoction on MP-induced Beas-2b cells.Cell experiments:In the early stage of the experiment,the identification of active ingredients of Qushi Tongluo Decoction,the concentration screening of Qushi Tongluo Decoction and azithromycin intervention were carried out.At the end of the preliminary work,the Beas-2b cells were divided into 7 groups,namely blank group,model group,3 groups of traditional Chinese medicine(low,medium and high doses),azithromycin group,and combined drug group(medium dose of traditional Chinese medicine+azithromycin).After the intervention,the changes in cell morphology were observed,and the cell supernatant and cells were collected.The cell supernatant was used to detect the levels of inflammatory factors IL-6,TNF-α,and IL-1 β by ELISA,and the cells were collected to extract RNA for real-time fluorescence quantification.The expression of IL-6 and IL-1β genes was detected by PCR method.3 Mechanism of Qushi Tongluo Recipe on MP-infected BALB/c mouse model3.1 Effect of Qushi Tongluo Recipe on MP-infected BALB/c mouse model 72 mice were randomLy divided into 9 groups,8 in each group,respectively blank group(3rd day,referred to as K3;7th day,referred to as K7;14th day,referred to as K14),model group(3rd day,referred to as K14)M3;the 7th day,referred to as M7;the 14th day,referred to as M14),the combined drug group(Azithromycin+Qushi Tongluo Fang,the 3rd day,referred to as L3;the 7th day,referred to as L7;the 14th day,referred to as L14).Intervention method:After 3 days of MP modeling,the blank group and the model group were given continuous gastric administration of normal saline for 3 days,7 days,and 14 days respectively,200 μL/day/animal;9.01mg/mL azithromycin for 3 days,7 days,14 days,200 μL/day/cause.Observation indicators:During the intervention period,the body weight,food intake and behavioral changes of the mice were recorded.If the mice appeared listless,less active,duLI hair erection,less food and water intake,faster breathing rate,weight loss,etc.,it was considered as model establishment success.After the intervention,lung tissues were collected for HE staining,immunohistochemistry,and Western blot experiments.3.2 Study on the Mechanism of Qushi Tongluo Recipe on BALB/c Mouse Model in the Acute Stage of MP InfectionGrouping method:72 male BALB/c mice were randomLy divided into 8 groups,9 in each group,respectively blank group,model group,traditional Chinese medicine group(low,medium and high doses),azithromycin group,combined drug group(traditional medicine Medium dose+azithromycin),combination drug,inhibitor group.Intervention method:After 3 days of MP modeling,all groups were given intragastric administration for 3 consecutive days(blank and model groups:normal saline,traditional Chinese medicine group:Qushi Tongluo prescription at concentrations of 270mg/mL,540mg/mL,and 1080mg/mL,and azithromycin group:Concentration 9.0lmg/mL,combined drug group:traditional Chinese medicine dose+azithromycin,inhibitor group:continuous intravenous injection of LY294002 pathway inhibitor for 3 days,and gavage traditional Chinese medicine dose+azithromycin),200 μL/time/cause.Observation indicators:During the intervention period,the body weight,food intake,and behavioral changes of the mice were recorded.After the intervention,the lung tissues were collected for HE staining,immunohistochemical detection,and Western blot experiments.The plasma was collected for prothrombin time(PT)and activated partial coaguLation.Active Enzyme Time(APTT).ResuLts1 Clinical research1.1 Main curative effect index The total effective rate of the two groups was statistically compared,and the difference was statistically significant(P=0.041<0.05).The total effective rate of the treatment group was better than that of the control group.The curative effect of TCM syndromes in the two groups was statistically compared,and the difference was statistically significant(P=0.039<0.05).The curative effect of TCM syndromes in the treatment group was better than that in the control group.1.2 Secondary curative effect index Comparing the complete antipyretic time,antipyretic,cough,sputum,and asthma onset time of the two groups,complete antipyretic time(P=0.041<0.05);antipyretic onset time(P=0.035<0.05);cough onset time(P=0.030<0.05);sputum onset time(P=0.048<0.05);asthma onset time(P=0.041<0.05),the differences were statistically significant,the treatment group The complete antipyretic time,antipyretic,cough,sputum,and asthma onset time were shorter than those in the control group.1.3 Comparison of four coaguLation items.The levels of PT,APTT,FIB,and TT in the two groups were compared before and after treatment,and there was no significant difference in the levels of PT,APTT,FIB,and TT between the treatment group and the control group before treatment(P>0.05),which was comparable;There was no significant difference in the levels of PT,APTT,FIB,and TT between the latter two groups(P>0.05),indicating that there were no significant differences in the levels of PT,APTT,FIB,and TT between the two groups after treatment.There were statistically significant differences in PT,APTT,FIB,and TT levels between the treatment group and the control group before and after treatment(P=0.039<0.05,t=4.925,P=0.008<0.05),indicating that after treatment,the two groups of children in PT,APTT levels were significantly increased,and FIB and TT levels were significantly decreased after treatment.Comparing the elevated levels of PT and APTT and the decreased levels of FIB and TT in the two groups,P>0.05,suggesting that the treatment group and the control group had no statistical significance in increasing the levels of PT and APTT and decreasing the levels of FIB and TT,and there was no significant difference.It shows that Qushi Tongluo Recipe combined with azithromycin and azithromycin alone can increase the levels of PT and APTT,and reduce the levels of FIB and TT,and there is no difference between the degree of increase and decrease.2 Cell experiment research2.1 Identification of components of Qushi Tongluo prescription found that the effective active ingredients mainly include Formononetin,Resveratrol,Quercetin,Chrysin,Scutellarin,Vanillin,Isorhamnetin,Rutin,Naringin,Kaempferol-7-0-glucoside,Luteolin,Wogonin,etc.2.2 Drug Intervention Concentration Screening Concentration screening of Qushi Tongluo Decoction and Azithromycin were carried out,and finally Qushi Tongluo Decoction 600ug/mL,300ug/mL,150ug/mL and Azithromycin 7.8ug/mL were selected as the drug intervention concentrations in this experiment.2.3 Observation of cell morphology Comparing the administration group with the model group,a large number of floating dead cells appeared in the model group,about 30%of the adherent cells,cell deformation,contracture,cell nucleus rupture,and a large number of cell fragments appeared.The cell morphology of the administration group was basically normal,and the cells More than 50%adherence,scattered cell debris.It shows that Qushi Tongluo prescription,azithromycin and the combined drug group can protect Beas-2b cells at the level of cell morphology.2.4 Detection of IL-6,TNF-α,and IL-1β levels in cell supernatants by Elisa method Except for low doses of traditional Chinese medicine(IL-6:P=0.7879,TNF-α:P=0.9886),middle and high doses of Chinese medicine groups,Both the azithromycin group and the combined drug group couLd reduce the levels of IL-6 and TNF-α in the cell supernatant(P<0.05);The level of IL-1β in both groups decreased,and there were statistical differences(P<0.05).In addition,it was also found that Qushi Tongluo Decoction combined with azithromycin had a better effect on reducing the levels of IL-6,TNF-α,and IL-1β in cell supernatant than single medication(P<0.05).2.5 The expression of IL-6 and IL-1β genes in cells detected by fluorescent quantitative PCR.The low,medium and high dose groups of traditional Chinese medicine,the azithromycin group and the combined drug group had a significant down-reguLation effect on the expression of IL-6 and IL-1 β genes(P<0.0001),indicating that each group of drugs had anti-inflammatory effects on MP-infected Beas-2b cells;the difference between the combined drug group and the single drug group was significant(P<0.05),indicating that the combination of Chinese and Western medicine had a synergistic effect.3 Animal Experimental Research3.1 Effect of Qushi Tongluo Recipe on MP-infected BALB/c mouse model3.1.1 Body weight and food intake(1)The body weight of the blank group gradually increased and stabilized at around 16-17g,the other six groups The mice lost weight on the second day after modeling,and the weight loss in the model group was faster than that in the combination drug group.(2)The body weight of the M14 and L14 groups recovered at 8 to 10 days(5 to 7 days after administration),but the weight recovery time of the model group was later than that of the combination drug group.The model group was slower in ascending speed than the combination drug group,and the weight loss was easy to repeat.(3)For mice in the three blank groups,the food intake was stable and fluctuated between 14 and 16 g.The food intake of the model group and the combination drug group suddenly decreased on the second day of modeling,and the food intake began to increase on the eighth day.Compared with the L14 group,the food intake tended to be the same after 12 days.3.1.2 Behavioral changes(1)The three groups of mice in the blank group were in good mental state,with normal and shiny hair,good activities,no shortness of breath,no secretion around the mouth and nose,and normal stool.(2)The three groups of the model group showed poor mental state and drowsiness on the second day of modeling,and then gradually became sluggish,unresponsive,decreased activity,hair piloerection and duLlness,shivering,rapid breathing rate,and secretions around the nose and mouth.Matter,the stool was sticky,and the smell was worse than that of the blank group.(3)On the second day after modeling,the combined drug group experienced poor spirits,drowsiness,dμL1 hair erection,chills,rapid breathing rate,and secretions around the mouth and nose.The above symptoms began to improve on the second day of administration.However,it was worse than that of the blank group.On the 7th day of administration,the mental state of the L14 group improved,without drowsiness,with normal hair,no vertical hair,and no chills.On the 13th day of administration,the above symptoms were completely improved and the same as the blank group,but the body weight was lower than that of the blank group.3.1.3 Pathological observation of lung histopathology(1)In K3 and K7 groups,the alveolar structure was clear,the epithelial cells were arranged neatly,the thickness of the bronchi at all levels was consistent,there was no obvious secretion in the alveoli,and there were no obvious macrophages and eosinophils around the trachea.Infiltration of granμ Locytes,slight thickening of alveolar septa and mild hyperplasia of epithelial cells in the K14 group,no obvious secretions in the lumen,and a small amount of blood vessel bleeding;(2)Compared with the blank group,the three groups of the model group showed lung consolidation,The alveoli disappeared,a large number of inflammatory cells and eosinophils infiltrated,and the epithelial cells hyperplasia was severe.The inflammatory exudation around the alveoli of M3 was more severe than that of the M7 group,and a large number of blood vessels appeared in the M3 group.Compared with M3 and M7,the inflammatory cells of the M14 group Reduced,but the airway epithelium is still hyperplastic.The degree of tissue lesions was ranked M3>M7>M14.(3)Compared with the model group,there was no obvious large lung consolidation,airway inflammatory cells and eosinophils were significantly reduced,and epithelial cell hyperplasia was alleviated.Compared with the three groups of the combined drug group,L7 group and L3 group In contrast,the degree of alveolar cavity fusion was reduced,and the alveolar structure of L14 group was clearer than that of L3 and L7 groups.3.1.4 Immunohistochemical detection of IL-6 and TNF-α(1)Compared with the model group,the difference was statistically significant(P<0.0001).IL-6 in the lung tissue of the mice in the model group was significantly increased.,the IL-6 level was significantly lower than that of the model group(M3 vs L3 P=0.001<0.05,M7 vs L7 P=0.0287<0.05,M14 vs L14 P=0.0215<0.05).(2)TNF-α in the lung tissue of mice after MP infection was significantly increased,and the difference was statistically significant(P<0.0001).After drug intervention,TNF-α in the model group was significantly lower than that in the combined drug group(M3 vs L3 P=0.001<0.05,M7 vs L7 P=0.0018<0.05,M14 vs L14 P=0.0085<0.05).3.1.5 Western blot detection of PI3K/AKT pathway proteinPI3K/AKT pathway protein expression in each group Compared with the blank group,the p-PI3K/PI3K protein expression level decreased in the model group after modeling,the difference was statistically significant(P<0.0001),and the p-AKT/AKT protein expression level decreased,the difference was statistically significant(K3vs M3 P=0.0021<0.05,K7 vs M7 P<0.0001,K14vs M14 P=0.0063<0.05).After combined drug intervention,the PI3K/AKT pathway protein was significantly increased,and the difference in sex ratio between the combined group and the model group was statistically significant(M3 vs L3 P=0.0002<0.05,M7 vs L7 P=0.0002<0.05,M14 vs L14 P=0.0037<0.05),suggesting that MP infection can inhibit the expression of PI3K/AKT pathway.The PI3K/AKT pathway is activated to achieve therapeutic effects.3.2 Study on the mechanism of action of Qushi Tongluo Decoction on the BALB/c mouse model of MP infection in the acute phase3.2.1 Body weight and food intake(1)The body weight of the blank group increased steadily and remained stable when it reached about 19g;the body weight of the other groups increased slowly after modeling,and the model group increased at the lowest speed,and all of them lost weight after the second day of modeling.(2)The food intake of the blank group remained stable,all around 30g.After modeling,the food intake of the other groups except the blank group decreased.The food intake of the treatment group recovered on the third day,and the average food intake of the model group was lower than other groups.3.2.2 Behavioral changes(1)There was no obvious abnormality in the mental state of the mice in the blank group,smooth and shiny hair,flexible activities,no shortness of breath,cough,no abnormal secretions around the mouth and nose,and normal stool.(2)Compared with the blank group,the model group began to experience poor mental state,drowsiness and malaise,rapid respiratory rate,coughing,dμL1 hair erection,chills,secretions around the nose and mouth,and sticky stools on the second day of modeling.(3)The above-mentioned symptoms of the model group appeared on the second day of modeling in the low,medium and high doses of traditional Chinese medicine,azithromycin,and combined medication groups,but the degree was between the model group and the blank group.3.2.3 Lung histopathological observation(1)Blank group and model group were similar to the corresponding lung tissue structures in the first section of blank group and model group(2)Lung tissue consolidation was seen in the low-dose Chinese medicine group,and alveoli fused into buLlae.Inflammatory cells and eosinophils exuded around the alveoli,but the degree of infiltration was lower than that of the model group;large-scale lung tissue consolidation,massive proliferation of epithelial cells,and lower infiltration of neutrophils were seen in the middle-dose group of traditional Chinese medicine than that of the middle-dose group;In the high-dose Chinese medicine group,the azithromycin group,and the combined drug group,the degree of lung tissue consolidation was low,with mild epithelial cell hyperplasia and a small amount of intravascuLar hemorrhage;(3)In the inhibitor group,the lung tissue was slightly consolidated,with epithelial cell hyperplasia and around the alveoli Inflammatory cells and eosinophils infiltrated,massive intravascuLar hemorrhage.3.2.4 Immunohistochemistry and Western blot detection(1)The levels of VEGFA in the model group were significantly lower than those in the blank group(P<0.05);after treatment,the differences in VEGFA levels in the drug intervention groups were statistically significant compared with the model group(all P<0.05),the levels of VEGFA in the low-dose group,middle-dose group,high-dose group,azithromycin group,and combination group increased significantly;after the application of PI3K pathway inhibitors,the level of VEGFA decreased compared with the combination group(P All<0.05).(2)The level of LC3B in the model group was significantly higher than that in the blank group(P<0.0001).After treatment,the level of LC3B in each group of drug intervention was significantly different from that in the model group(P<0.0001),and the level of LC3B in each group of medication decreased significantly;Compared with the combination group(P<0.05),the level of LC3B was significantly increased in the inhibitor group.(3)There was no significant difference in Beclinl between the blank group and the model group(P=0.8093>0.05),and there was no significant difference in Beclinl between each drug group and the model group(P>0.05),suggesting that Beclinl was not significantly different in this experiment.There was no significant difference between MP-infected mice and after drug treatment.It is specuLated that it is related to the insensitivity of the lung tissue to Beclinl factor in this experiment,which needs to be verified by follow-up experiments.(4)The PI3K/AKT pathway was found to have significant changes in the first section of the second part of this experiment.In this section,the PI3K/AKT pathway inhibitor recovery experiment was carried out in the acute phase of MPP.Diseases are associated with this pathway.Compared with the model group,the expression levels of p-PI3K/PI3K protein and p-AKT/AKT protein decreased in the blank group,and the difference was statistically significant(P<0.0001);p-AKT/AKT protein compared with the model group,the difference was statistically significant(all P<0.05),suggesting that the PI3K/AKT pathway was activated after drug intervention;the application of PI3K inhibitors to block the PI3K/AKT pathway,found that p-PI3K/AKT pathway in the inhibitor group PI3K protein and p-AKT/AKT protein decreased significantly,and the difference was statistically significant(all P<0.0001).3.2.5 Comparison of PT and APTTThe PT and APTT in the model group decreased significantly(P<0.0001),and the PT and APTT in the drug treatment group increased compared with the model group(P<0.0001),indicating that the decline in PT and APTT in mice after MP infection was high CoaguLation state,when PT and APTT increase after administration,the hypercoaguLability state is relieved.Conclusion1 Compared with azithromycin alone in the treatment of MPP,Qushi Tongluo Decoction combined with azithromycin also has a higher clinical cure rate in Liaoning and Henan,which can shorten the time of antipyretic,reduce the onset time of antipyretic,cough,sputum,and asthma,reduce the damage of MP to the lung tissue of children;relieve the hyper coaguLable state caused by MP infection,and reduce the occurrence of ’pμ Lmonary blood stasis’ in microcircuLation disorders;2 Qushi Tongluo formuLa can reguLate PI3K/AKT signaling pathway,activating VEGFA proteins,thereby inhibiting autophagy,reducing IL-6,TNFα,IL-1β and other inflammatory factors,alleviating mi croc ircuLation disorders,and protecting lung tissue. |
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