| OBJECTIVE:We established such a lung and colon injury mice model infected with influenza virus.Further,we tested the hypothesis that the well-known anti-virus monomer of Chinese traditional medicine-berberine(BBR)might inhibit the influenza virus infection and improve the physiological condition both in lung and colon by detecting the transcription and/or expression of cytokines IL-1β,TNF-α,IL-6,IL-10,INF-y,CCL25 and IL-17A;Detect the effect of BBR on the intestinal flora in mice model by 16S rRNA sequencing to explain from the perspective of intestinal flora of BBR’s protective effect on lung and intestinal injury.METHODS:1.BALB/c mice were randomly divided into normal group,model group,ribavirin group,BBR high-dose group(10mg/kg)and BBR low-dose group(5mg/kg).The mice model of lung and colon injury was established by nasal infected with A/PR/8/34(PR8)influenza virus strain and treated with intraperitoneal injection of BBR or ribavirin.Record the general condition and body weight then calculate and measure the index of each organ and the length and weight of colon of mice model.The lung,small intestine and colon of mice were collected for pathological analysis by hematoxylin-eosin staining(HE).Evaluate comprehensively the success of the model and the intervention effect of BBR on the mice model.2.Expression of the serum cytokines like IL-6,IL-17A and IFN-γ were quantitatively detected by enzyme-linked immunosorbent assay(ELISA),and the mRNA transcription of cytokines like IL-1β,IL-6,IL-10,TNF-α,CCL25 and IL-17A were analyzed by the Real time PCR(RT-PCR).3.The effects of influenza virus infection on intestinal microflora of mice and the intervention effect of BBR on intestinal micro flora of mice were analyzed by 16S rRNA sequencing and the information was organized through the Trimmomatic、FLASH、Pear and usearch.RESULT:1.Compared with the normal group,after influenza virus infection,the general condition of the model group is poor activity,rough hair,and some mice had loose stools and even died,the mortality rate of the model group was 25%,and their body weight continued to decrease,with lung organ coefficient was significantly increased(P<0.0001),colon weight(P<0.01)and length(P<0.001)were significantly reduced,however,Small intestine index did not see significant change(P>0.05).Lung HE staining of the model group showed that lung bronchial epithelial cell falls off,alveolar structure fuzzy,pulmonary interstitial thickening,a large number of lymphocytes and mononuclear cell infiltration.Colon HE staining of the model group showed that mucosa lamina propria and muscle layer thinning with a large number of inflammatory cells infiltration,glandular structures disappear or fall off.Small intestine HE staining of the model group showed that small intestinal glands disorder arrangement and partial glands shedding.Compared with the model group,low-dose BBR group could effectively reduce the mortality of mice,maintain the body weight,reduce the ecchymosis of the lung,reduce the lung index(P<0.05),and maintain the colon weight(P<0.05)and length(P<0.05).In the HE staining,the low-dose BBR group can alleviate the pulmonary interstitial edema,alleviate the infiltration of lymphocytes and monocytes,and improve the disappearance and exfoliation of the small intestine and colon glandular structure;However,there was no significant difference in lung and intestinal inflammation of mice in the high-dose BBR group.2.Compared with the normal group,the levels of serum IL-6(P<0.001),IL-17A(P<0.05)and IFN-γ(P<0.05)in model group were significantly increased after influenza virus infection,the mRNA expression levels of IFN-y(P<0.001)and IL-10(P<0.001)in lung were significantly increased,IL-17A mRNA expression level was significantly decreased(P<0.05).Compared with model group,low-dose BBR group significantly decreased the expression of serum IFN-y(P<0.05),decreased the mRNA expression levels of IFN-y(P<0.001)and IL-10(P<0.001)in lung and increased the transcription of IL-17A(P<0.05);Meanwhile,low-dose BBR group significantly decreased the expression of IL-6 mRNA in colon(P<0.05).3.The 16s rRNA intestinal flora sequencing results showed that after influenza virus infection,the abundance of dominant bacteria decreased and the proportion of opportunistic bacteria increased in model group.BBR could increase the abundance of dominant bacteria and reduce the proportion of opportunistic bacteria.The Ruminococcaceae had a significant increase after BBR treatment.CONCLUSION:1.BBR had obvious protective effect on lung and colon of BALB/c mice infected with influenza virus.Compared with the model group,low dose BBR reduced the mortality of the mice,reduced the inflammatory changes of the lung and intestinal tract,and improved the inflammatory infiltration of the lung,small intestine and colon;2.BBR reduced the expression of IFN-y and IL-10 mRNA in lung,increasing the expression of IL-17A mRNA,reducing the level of serum IFN-y,and inhibiting the expression of IL-6 mRNA in colon which might interprete the BBR protected the lung and colon in mice.3.BBR changed the abundance of dominant microflora and the overall structure of intestinal microflora,and significantly increased the proportion of Ruminococcaceae,thus played a protective role against colon injury of mice infected with influenza virus. |
