| Objective: To observe the effects of nourishing yin and clearing lung soup plus flavor on the expression of lung surface active substances SP-A,SP-D,DPPC and specific transcription factors RORγt and Foxp3 of immune pathway Th17/Treg,as well as T-bet and VDR in a mouse model of severe Mycoplasma pneumoniae pneumoniae(SMPP)induced by high load MP nasal drip,and to elucidate the mechanism of action and targets of nourishing yin and clearing lung soup plus flavor intervention in SMPP.The mechanism of action and the target of SMPP.Materials and methods: 120 BALB/c mice were randomly divided into 5 groups,including normal group,model group,nourishing yin group,aci group and nourishing yin + aci group,with 24 mice in each group,and each mouse was treated with 0.1 m L of 1×1010 CCU/m L MP bacterial solution for 3 consecutive days by nasal drops according to the modeling method of the previous study.Six mice were randomly executed on the 3rd,7th,10 th and14th days after modeling,and peripheral blood,alveolar lavage fluid and lung tissue specimens were collected from the mice.The expression of SP-A and SP-D in alveolar lavage fluid was measured by ELISA,and the content of DPPC in alveolar lavage fluid was measured by HPLC.The protein levels of Foxp3 in mouse lung tissues were detected by IHC method;the protein levels of Foxp3 in mouse lung tissues were detected by Western blot;the gene levels of RORγt,Foxp3,T-bet and VDR in mouse lung tissues were determined by q PCR method.Results:1.The pathological scores of mice in the model group were higher than those of each drugadministered group at all time points,and the differences were statistically significant(P <0.05),and the pathological scores of mice in all groups peaked at day 7.The pathological scores of the aci group were slightly lower than those of the nourishing yin group at all time points,but the difference was not statistically significant(P > 0.05),and the pathological scores of the nourishing yin + aci group were lower than those of the nourishing yin group at all time points except day 3(P < 0.05),and the pathological scores of the nourishing yin + aci group were lower than those of the Chinese medicine or western medicine administration groups alone on day 14,and the difference was statistically significant(P < 0.05).2.The SP-A level in the alveolar lavage fluid of mice decreased significantly(P < 0.05)after modeling compared with the normal group,and the SP-A level measured on day 3 was the lowest;compared with the model group,the SP-A level in the alveolar lavage fluid of mice in each administration group increased,and the effect was obvious from day 7(P < 0.05)in the aci and nourishing yin + aci groups,and from day 10 in the nourishing yin group(P < On day10 and 14,the efficacy of nourishing yin + aci group was better than that of Chinese medicine or western medicine alone(P < 0.05),indicating that nourishing yin and clearing lung soup plus flavor combined with azithromycin was more effective.3.After modeling,compared with the normal group,the SP-D level in the alveolar lavage fluid of mice generally decreased and reached the lowest level on day 7;compared with the model group,there was a significant difference in the SP-D level of mice in each administration group(P < 0.05),showing the effect of increasing the SP-D level in the alveolar lavage fluid of mice;on days 7,10 and 14 after administration,the efficacy of the nourishing yin + aci group was better than that of the nourishing yin group(P < 0.05),but compared with the aci group,the efficacy of the nourishing yin + aci group was better than that of the nourishing yin group(P < 0.05).),but the advantage was not significant when compared with the Aci group(P > 0.05).4.After modeling,compared with the normal group,the concentration of DPPC in the alveolar lavage fluid of mice decreased at all time points and reached the peak again on day 14,but never exceeded that of the normal group.Compared with the model group,the DPPC concentration in the alveolar lavage fluid of mice increased at the same time point(P < 0.05),but the upregulation rate of the Yang Yin group was slower than that of the Aci group and the Yang Yin + Aci group,indicating that the expression of DPPC in the alveolar lavage fluid of mice could be increased in all the administered groups;on days 7 and 10,the efficacy of the Yang Yin + Aci group was better than that of the Aci group and the Yang Yin group(P <0.05);on day 14,the efficacy of the Yang Yin + Aci group was higher than that of the Aci group and the Yang Yin group.+On day 14,the difference was not significant(P>0.05)between the Nourishing Yin + Aci group and the Aci group,but was significant(P<0.05)compared with the Nourishing Yin group.5.The lung tissues of mice in the normal group showed staining expression of Foxp3 in the cytoplasm.In the model group,the expression intensity of Foxp3 brownish-yellow positive sites was reduced and discontinuously distributed in the cytoplasm.In the nourishing Yin and Aci groups,Foxp3 brown-yellow positive expression was found around the pulmonary vasculature and interstitium,with a tendency of concentrated distribution.The positive staining of the expression products in the Nourishing Yin + Archie group was higher than that of the drug-alone group.6.The expression level of RORγt m RNA in mice after modeling showed an increasing trend,which was significantly different from that of the normal group(P < 0.05).Compared with the model group,the effect of Aci group started from day 3(P<0.05);the effect of nourishing Yin + Aci group started on day 7(P<0.05);the effect of all dosing groups started on day 10(P<0.05)and the effect of nourishing Yin + Aci group was better than that of nourishing Yin group(P<0.05),but the effect was not significant compared with Aci group(P>0.05).7.After modeling,the Foxp3 m RNA expression level was down-regulated in the model group mice.The difference was statistically significant(P < 0.05)compared with the normal group.Compared with the model group,mice in the nourishing yin + aci group started to take effect on day 10 after transcutaneous administration(P < 0.05);the group administered alone started to take effect on day 14(P < 0.05),but there was no significant difference between the groups administered(P > 0.05).8.T-bet m RNA expression levels were downregulated in mice at all time points after modeling,and the differences were statistically significant compared with the normal group(P < 0.05).After the drug intervention,compared with the model group,the nourishing Yin +Aci group started to take effect from day 3(P < 0.05);all drug groups started to take effect from day 7(P < 0.05),among which the Aci and nourishing Yin + Aci groups were more effective than the nourishing Yin group(P < 0.05),but the difference between the Aci and nourishing Yin + Aci groups was not statistically significant(P > 0.05).9.The VDR m RNA expression levels of mice increased at all time points after modeling,and the difference was statistically significant compared with the normal group(P < 0.05).After the pharmacological intervention,the effect started between the administration groups on day3(P < 0.05);from day 10 onwards,the effect was more pronounced in the Aci group and the nourishing Yin + Aci group than the nourishing Yin group(P < 0.05),but the difference was not significant between the Aci group and the nourishing Yin + Aci group(P > 0.05).10.After MP infection,the level of Foxp3 in the lung tissue of mice decreased compared with the normal group(P < 0.05)and reached the lowest point on day 7;after drug intervention,compared with the model group,the level of Foxp3 in the lung tissue of mice started to increase in the Aqi group and the nourishing Yin + Aqi group at day 7(P < 0.05),and the efficacy of the Aqi group was better than that of the nourishing Yin group at day 7(P < 0.05),and compared with the nourishing Yin +The efficacy of the Aci group was better than that of the nourishing Yin group on day 7(P < 0.05),but was not significant compared with that of the nourishing Yin + Aci group(P > 0.05);the efficacy of the nourishing Yin + Aci and Aci groups was better than that of the nourishing Yin group on days 10 and 14,with statistically significant differences(P < 0.05).Conclusion:1.Nourishing Yin and clearing lung soup plus flavor can repair alveolar air-fluid plane damage in SMPP by regulating the lung surface active substances SP-A,SP-D and DPPC,which may be the target of Nourishing Yin and clearing lung soup plus flavor for the treatment of SMPP.2.Nourishing Yin and clearing lung soup plus flavor can effectively improve the immune imbalance in SMPP mice,which may be related to its regulation of the specific transcription factors RORγt and Foxp3 of the immune pathway Th17/Treg,as well as T-bet and VDR. |
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