| Influenza is an acute respiratory infection caused by the influenza virus.The "Spanish flu" of 1918-1919 and the "Asian flu" of 1957,as well as H1N1 and H7N9 which have had huge impact on global scale in recent years,have caused a large number of death and social panic,and have become one of the major public health problems faced by human[1,2]Influenza virus can infect respiratory epithelial cells and is an important pathogen causing acute respiratory infection.It stimulates the host’s innate immune response and adaptive immune response,causing interstitial pneumonia,diffuse alveolar damage,pulmonary congestion,edema,and hemorrhage,which cause the occurrence of influenza virus pneumonia and seriously threaten human health[3,4].Influenza virus pneumonia is classified exogenous warm disease category in Chinese medicine.The main pathogenesis is poisonous injury and blood stasis.Therefore,the treatment should focus on heat-clearing,detoxifying,and activating blood circulation.Xijiao Dihuang decoction combined with Yinqiao powder(XDY),created by Wu Jutong,is the classic combination from "Jiedu Liangxue Sanyu" in the Qing Dynasty.Patients with influenza A,infectious atypical pneumonia,or common cold combined with hemorrhagic diseases(anemia of recurrent disorders,thrombocytopenic purpura,etc.)are treated by XDY in clinic.A large number of clinical studies have proved that the combination achieves the purpose of heat-clearing,detoxifying,and activating blood circulation,and it has good effect on viral pneumonia with poisonous injury and blood stasis.According to the research,XDY significantly reduced the mortality rate of mice with influenza virus pneumonia.Through suppressing the release of the prostaglandin 2(PGE2),phospholipase S2(PLA2),and alkene B4(LT-B4),XDY can increases the expression of aquaporin(AQP-1),inhibits the production of inflammatory cytokines and oxygen free radical,reduce pulmonary vascular permeability,and reduce pulmonary edema and pulmonary tissue hemorrhage.In short,XDY can relieve inflammation in the lungs,and have a good effect on influenza virus pneumonia.In the vitro experiments,the drug-containing serum of XDY significantly inhibited the production of inflammatory cytokines,inflammatory mediators and free radicals by alveolar macrophage,and have anti-inflammatory effects.It can reduce the cellular permeability and the reconstruction of F-actin through the inhibition of phosphorylated ERM expression and protect the microvascular endothelium.ObjectiveIn the previous studies,we investigated the effects of XDY on the body from different perspectives such as cytoskeleton,cytokines,and inflammatory mediators,but lacked a comprehensive demonstration of its therapeutic mechanism.Micro RNA,which is involved in the regulation of almost all known signal pathways and pathologic and physiological processes in body,is the core component of gene regulation network.In the related studies of viral disease infection,miRNA was involved in the interaction between host and pathogen.During the development of influenza viral pneumonia,miRNA is an important regulatory factor for influenza viral pneumonia caused by influenza virus.XDY has a clear effect on the influenza viral pneumonia mice.However,the therapeutic effect of XDY on the level of miRNA was not reported,neither was the effect of mRNA level fully studied.Therefore,this topic is planned to be based on the previous research,with influenza viral pneumonia mice as the model,and through high-throughput sequencing analysis,comprehensively display the regulation of XDY in the level of miRNA and mRNA.The relationship between miRNA-mRNA interactions focuses on its effects on vascular permeability changes,apoptosis,adhesion molecules,TLR and other related biological processes and signaling pathways,in order to clarify the effect of XDY on innate immune response and adaptive immune response in vivo experiments,and comprehensive analysis of the mechanism of drug action.It provides experimental basis and theoretical basis for the clinical use of XDY,the classic combination of "Jiedu Liangxue Sanyu".Methods1.Therapeutic effect of XDY on influenza viral pneumonia mousePreliminary study on the treatment of influenza virus pneumonia by XDY was collated,and the therapeutic effect of the combination was verified again.A mouse model of influenza virus pneumonia was established,and the normal group,virus group,and XDY group were set up to observe the life status of the mice such as body weight,hair,activity,reaction sensitivity,eyesight,and mental status,and to observe the lung tissue of mice pathological changes by HE-staining,and the therapeutic effect of XDY on influenza viral pneumonia was demonstrated from the overall level.2.Screening and functional analysis of miRNA and mRNA in lung tissue of influenza virus pneumonia mouse with XDY treatmentA mouse model of influenza virus pneumonia was established with normal,virus,and XDY groups.Lung tissues were collected on the 4 dpi and 7 dpi mice for high-throughput sequencing.The expression profiles of miRNA and mRNA in the lung tissues of influenza viral pneumonia mouse were comprehensively presented at the genetic level,and the regulation of the immune response of the body was explored.The miRNA and mRNA associated with such signaling pathways as TLRs,MAPK,Jak-STAT,TNF were selected for verification.3.Effect of XDY on miRNA and mRNA in lung tissue of influenza virus pneumonia mouseA mouse model of influenza virus pneumonia was established with normal,virus,and XDY groups.Lung tissues were collected on the 4 dpi and 7 dpi mice.QRT-PCR was used to detect the expression levels of 11 miRNAs and 5 mRNAs in the lung tissues of each group.Results1.Therapeutic effect of XDY on influenza viral pneumonia miceObservation of the comprehensive vital signs of influenza virus pneumonia mice revealed that mice in the virus group had sparse hair,dim eyes,unresponsiveness,and continued weight loss,whereas the normal group and XDY group were have smooth hair,bright eyes,rapid response,relatively flat body weight,and various vital signs are significantly stronger than the virus group mice.The results of HE show that,the lung tissue of the virus group mice showed severe interstitial inflammation.The extent of lung lesions tissue in the XDY group were significantly improved compared with the virus group,and were basically similar to the normal group.From the overall perspective,it is proved that the combination of XDY has a good therapeutic effect on influenza virus pneumonia.2.Screening and functional analysis of miRNA and mRNA in lung tissue of influenza virus pneumonia mice with XDY treatment2.1.With the increasing degree of inflammation,the expression of miRNA and mRNA in the lung of the virus group and the normal group showed a significant difference.After the XDY group mice treated with medicine,the degree of inflammation had a significant improvement.Differentially expressed miRNA may be involved in the immune inflammatory response to influenza viruses.2.2.According to the many-to-many correspondences and multiples of changes between miRNA and mRNA expressed differentially in high-throughput sequencing,11 miRNAs(miR-7b-5p,miR-146b-3p,miR-146b-5p,miR-147-3p,miR-155-5p,miR-223-3p,miR-34c-5p,miR-200b-3p,miR-503-5p,miR-669a-3p,miR-351-5p),and 5 mRNAs(Mapk3,Mapk10,Fos,Statl,Ifnb1)were selected for the next step primary research.3.Effect of XDY on miRNA and mRNA in lung tissue of influenza virus pneumonia mice3.1.Expression of miRNA in lung of influenza virus pneumonia mice:Based on the results of miRNA high-throughput sequencing and prediction of their target genes,we selected 11 miRNAs which are related,with significant difference,to immune inflammatory response,apoptosis,cell permeability,antivirus pathways from differentially expressed mi RNA for qRT-PCR validation.The experimental results showed that,qRT-PCR verification was consistent with the results of high-throughput sequencing.In the virus group,6 mi RNA were down-regulated within overall trend expression,and 5 miRNAs were up-regulated.There was no statistical difference between the XDY group and the normal group.At 4 dpi,in the virus group,comparing with the normal group,the expression of miR-146b-3p,miR-146b-5p,miR-147-3p,miR-155-5p,miR-223-3p were up-regulated(p<0.001,p<0.01,p<0.001,p<0.001,p<0.001),the expression of miR-351-5p were down-regulated(p<0.01),and the expression of miR-7b-5p,miR-34c-5p,miR-200b-3p,miR-503-5p,miR-669a-3p expressed no significant difference(p>0.05,p>0.05,p>0.05.p>0.05,p>0.05).At 4 dpi,in the XDY group,comparing with the normal group,the expression of miR-146b-3p,miR-146b-5p,miR-147-3p,miR-155-5p,miR-223-3p,miR-200b-3p were up-regulated(p<0.001,p<0.01,p<0.001,p<0.001,p<0.001,p<0.05),but had no significant difference to the virus group(p>0.05,p>0.05,p>0.05,p>0.05,p>0.05,p>0.05).Comparing with the virus group,the expression of miR-351-5p was up-regulated(p<0.05),but had no significant difference to the normal group(p>0.05).The expression of miR-7b-5p,miR-34c-5p,miR-503-5p,miR-669a-3p have no difference neither from the normal group nor from the virus group(p>0.05,p>0.05,p>0.05,p>0.05).At 7 dpi,in the virus group,the expression of miR-7b-5p,miR-146b-3p,miR-146b-5p.miR-147-3p,miR-155-5p,miR-223-3p were up-regulated,when comparing with the normal group(p<0.001,p<0.001,p<0.01,p<0.001,p<0.001),and the expression of miR-34c-5p,miR-200b-3p,miR-351-5p,miR-503-5p,miR-669a-3p were down-regulated(p<0.001,p<0.001,P<0.001,p<0.001,p<0.01).At 7 dpi,in the XDY group,the expression of miR-7b-5p,miR-146b-3p,miR-146b-5p were down-regulated when comparing with the virus group(p<0.01,p<0.01,p<0.05),but they have no significant difference to the normal group(p>0.05,p>0.05,p>0.05).The expression of miR-34c-5p,miR-200b-3p,miR-351-5p,miR-503-5p were up-regulated,comparing with the virus group(p<0.001,p<0.05,p<0.001,p<0.001)but had no significant difference to the normal group(p>0.05,p>0.05,p>0.05,p>0.05).The expression of miR-147-3p,miR-155-5p were down-regulated comparing with the virus group(p<0.01,p<0.01),but up-regulated comparing with the normal group(p<0.001,p<0.05).The expression of miR-223-3p was up-regulated comparing with the normal group(p<0.001),and had no significant difference but a downward trend,comparing with the virus group(p>0.05).The expression of mir-669a-3p had no significant difference neither form the normal group nor from the virus group(p>0.05).3.2.Expression of mRNA in lung of influenza virus pneumonia mice:There was no significant difference in Mapk3 mRNA expression at 4 dpi in the three groups(p>0.05),and it was significantly down-regulated in virus group compared with the normal group and the XDY group(p<0.001、p<0.001)at 7 dpi;there was no difference between normal and XDY groups(p>0.05)at 4 dpi and 7 dpi.The expression of Mapk10 mRNA was significantly down-regulated in virus group compared with the normal group and the XDY groupi(p<0.01,p<0.05)at 4 dp,and the down-regulated was further increased(p<0.001,p<0.001)at 7 dpi;there was no difference between normal and XDY groups(p>0.05)at 4 dpi and 7 dpi.There was no significant difference with an upward trend in the expression of Fos mRNA at 4 dpi in the virus group(p>0.05),which was significantly up-regulated at 7 dpi in the virus group(p<0.001,p<0.001).No difference was found between the normal and XDY group(p>0.05)at 4 dpi and 7 dpi.The expression of Statl mRNA was significantly up-regulated in virus group and the XDY group at 4 dpi,compared with the normal group(p<0.001,p<0.001),but the ratio of XDY group to virus group was lower than that of the normal group,and there was a significant difference(p<0.001).The expression of Stat1 mRNA at 7 dpi in the virus and XDY group was significantly down-regulated compared with the normal group(p<0.001,p<0.001).Ifnb1 mRNA at 4 dpi was significantly up-regulated in both virus and XDY group compared with the normal group(p<0.001,p<0.001);the ratio of the XDY group was lower than the virus group but the difference was not significant(p>0.05).Ifnb1 mRNA was significantly down-regulated in the virus group at 7 dpi compared with the normal group and the XDY group(p<0.001,p<0.01),and there was no difference in the XDY or normal group(p>0.05).3.3.Expression of JNK,ERK and AP-1 proteins in lung of influenza virus pneumonia mice:at 4 dpi,the expression of JNK and ERK in the virus group were up-regulated compared with the normal group,while AP-1 was down-regulated;the expression of the XDY group was close to the normal group.At 7 dpi,compared with the normal group,the expressions of JNK,ERK in the virus group was significantly up-regulated,while AP-1 was significant down-regulated;Compared with the virus group,the expression of all protein in the XDY group was close to the normal group,and AP-1 was no difference from the normal group.Conclusions1.After influenza virus infection and treatment with XDY,the expression of miRNA and mRNA in lung of influenza virus pneumonia mice changed tremendously,and their changes were mostly distributed in a number of signaling pathways,such as TLRs,MAPK,Jak-STAT,TNF etc.2.According to the many-to-many correspondences of miRNA and mRNA,miR-7b-5p,miR-146b-3p,miR-146b-5p,miR-147-3p,miR-155-5p,miR-223-3p,miR-34c-5p,miR-200b-3p,miR-503-5p,miR-669a-3p,miR-351-5p were selected for qRT-PCR differential expression verification,which were consistent with the results of high-throughput sequencing.We presume that XDY may regulate immune inflammation,apoptosis,cell permeability,antiviral,anti fibrosis and other pathways in the development of influenza viral pneumonia by regulating the expression of the 11 miRNAs,in order to treat influenza viral pneumonia.3.According to the differentially expressed mRNA verification,in the virus group,comparing with the normal group,the expression of Mapk3 and Mapk 10 were down-regulated,and that of Fos was up-regulated.In the XDY group,when comparing with the normal group,the expression of Mapk3 and Mapk 10 was up-regulated,and Fos was down-regulated.And there was no statistical difference between the normal group and the virus group.It is suggested that,during the development of influenza viral pneumonia and the treatment of XDY,XDY probably regulates the expression of inflammatory responses associated with influenza viral pneumonia through the ERK/JNK-AP-1 pathway.In the virus group,the expression of Ifnb1and Statl was significantly up-regulated and then significantly down-regulated.In the XDY group,comparing with the virus group,the expression of Ifnb1 was significantly down-regulated and then significantly up-regulated,and had no difference to the normal group,while the expression of Stat1 was lower than that of the virus group.It is suggested that,there is a certain correlation between STAT and IFN-β,but the treatment of XDY may play a role in different pathways,which will be confirmed in the further studies in future.4.The therapeutic mechanism of XDY in the treatment of influenza viral pneumonia may be related to the regulation of 11 miRNAs(miR-7b-5p,miR-146b-3p,miR-146b-5p,miR-147-3p,miR-155-5p,miR-223-3p,miR-34c-5p,miR-200b-3p,miR-503-5p,miR-669a-3p,miR-351-5p),and ERK/JNK-AP-1,STAT and IFN-β pathways. |
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