3.1. Changes of lung inflammation in IAV-infected mice
Most of the infected mice developed the disease three days after infection, with the symptoms of bristling, curling, tarnished hair, reduced activity, food intake, weight loss, rapid breathing, loose stool and other symptoms. In the NC group, the lung tissue structure was normal and no inflammatory lesions were found (Fig.1A,D-E). The lung tissue of IAV-C group showed different degrees of inflammatory lesions, such as inflammatory cell infiltration, bronchiolitis with edema, and perivascular interstitial edema. The lung index in IAV-C group was significantly higher than that in NC group (p<0.01) (Fig.1B,D-E). Compared to the IAV-C group, puerarin can significantly improve the inflammatory pathological degree of the lung and lessening the lung index(p<0.01) (Fig.1C-E).
3.2. Pathological changes of intestinal tract in IAV-infected mice
HE staining showed that inflammatory lesions were found in the small and large intestines of IAV-infected mice, especially in the ileum and colon (Fig. 2-IAV-C). Compared with the normal control group, there were different degrees of inflammatory lesions in the duodenum, jejunum, ileum, colon and rectum in the virus-infected group, especially in the jejunum, ileum and colon (Fig. 2-NC and IAV-C). The local mucosal epithelial cells of the duodenum were necrotic and exfoliated, intestinal villi were destroyed, inflammatory cell infiltration was seen in the mucosal layer, no obvious atrophy was found in the glands in the lamina propria, inflammatory cell infiltration was seen, and some edema was found in the submucosa (Fig. 2-IAV-C-duodenum). The local mucosal epithelial cells of the jejunum were necrotic, and the glands in the lamina propria disappeared and were replaced by new connective tissue. Inflammatory cell infiltration was obvious, and the submucosal structure was loose and edema was present (Fig. 2-IAV-C-jejunum). The mucosal epithelial cells of the ileum were necrotic and exfoliated, with local mucosal defects, and the glands in the lamina propria disappeared and were replaced by connective tissue (Fig. 2-IAV-C-ileum). Inflammatory cell infiltration was seen in the submucosa. Some of the colonic mucosal epithelial cells were necrotic and exfoliated, with local mucosal epithelial layer defect, lamina propria glands disappeared, the missing mucosal epithelial cells were replaced by new connective tissue, and inflammatory cell infiltration was found in the new tissue(Fig. 2-IAV-C-colon). Local villous epithelial cells of the rectal mucosa were necrotic and exfoliated, mucosal structure was destroyed, inflammatory cell infiltration was visible in the mucosal layer, the gland structure of the lamina propria was damaged, submucosal structure was loose and there was some edema (Fig. 2-IAV-C-rectum). Influenza virus infection can lead to inflammation of mesenteric adipose tissue in mice. The mesenteric lymph nodes were significantly enlarged, and the germinal center was obvious (Fig. 2-IAV-C-mesenteric lymph nodes). In the germinal center, there was proliferation of lymphoblasts and some thickening of the cap-like area. Some atrophy of adipose tissue was observed in mesenteric fat, the number of adipocytes decreased, and a large number of inflammatory cells infiltrated around the fat (Fig. 2-IAV-C-mesenteric adipose tissue). The inflammatory lesions in the puerarin group were lighter than those in IAV-C group, especially in the ileum and colon (Fig. 2-IAV-C-puerarin).
3.3. Changes of viral load in lung and intestine of IAV-infected mice
Five days after the mice were infected with influenza virus, viral RNA was found in lung, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph nodes and mesenteric adipose tissue in IAV-C group. The content of viral RNA was significantly higher than that of NC group(Fig.3A-H). Compared with the IAV-C group, the level of pulmonary enterovirus titer in puerarin group was significantly lower (P < 0.01) (Fig. 3A-H).
3.4. Changes of inflammatory cytokines in lung and intestine of IAV-infected mice
On the 5th day of influenza virus infection, the levels of inflammatory cytokines TNF-a, IL-6, IL-17 in lung, duodenum, jejunum, ileum and rectum in IAV-C group were significantly increased(Fig.4B-D), and the levels of TNF-a and IL-6 in colon were significantly higher compared to NC group(Fig.4B,D). Puerarin could significantly reduce the above-mentioned tissue cytokine levels(Fig.4B-D). The level of IFN-g was not significantly increased in the lung and intestine tissues, and there was no significant difference between IAV-C group and puerarin group(Fig.4A).
3.5. Changes of adipokines in lung and intestine of IAV-infected mice
The level of leptin, visfatin and chemokine in lung, mesenteric lymph node and mesenteric adipose tissue in IAV-C group were significantly higher than those in NC group (P < 0.05-0.001) (Fig.5-7). Puerarin could significantly reduce the levels of leptin, lactone and chemokin, and increase the level of adiponectin (P < 0.01) (Fig. 5-7). The analysis showed that the adiponectin levels in lung, mesenteric lymph node and mesenteric adipose tissue had a significant decrease after 5 days of infection than that in NC group (Fig. 8). Puerarin (100 mg / kg) could inhibit the increase of adiponectin in lung, mesenteric lymph node and mesenteric adipose tissue (Fig. 8A-C, p < 0.05–0.01).
3.6. Changes of SIgA level in lung and intestine of mice after infection
Mucosal immune response was evaluated to determine the effects of puerarin. IgA levels in broncho-alveolar lavage and intestinal lavage were detected on the 5th day post-infection. The sIgA levels in bronchoalveolar lavage fluid and small intestinal lavage fluid in IAV-C group were significantly lower than those in NC group(P<0.01~0.001) (Fig. 9). The level of sIgA in bronchoalveolar lavage fluid and small intestinal lavage fluid of puerarin group was significantly higher than that of IAV-C group (Fig. 9A-B).
3.7. Changes of mucosal immune function in IAV-infected mice
On the 5th day after influenza virus infection, the molecular levels of toll receptor (TLR)3, 4, 9 and integrin (aVb3, a 4) in lung, duodenum, jejunum, ileum, colon and rectum in IAV-C group were significantly higher than that in NC group(P<0.05~0.001, Fig. 10A-E). Puerarin could significantly improve the molecular levels of toll receptor and integrin in the above tissues ((P<0.05~0.001, Fig. 10 A-E).