| Objective:Pulmonary fibrosis is a progressive fatal lung disease,and there is a lack of effective treatment at present.In our previous studies,we found that the citrus alkaline extract(CAE)has the effect of anti-pulmonary fibrosis,but it is not clear whether the gut microbiota plays a role in bleomycin-induced pulmonary fibrosis and whether the citrus alkaline extract plays an anti-pulmonary fibrosis effect through gut microbiota.The purpose of this study was to explore whether gut microbiota plays a role in bleomycin-induced pulmonary fibrosis in mice;to further evaluate whether CAE can play a role in anti-pulmonary fibrosis by regulating gut microbiota;and to explore the mechanism of CAE against bleomycin-induced pulmonary fibrosis by regulating gut microbiota and promoting Treg cell differentiation.Methods:1.The mouse model of pulmonary fibrosis induced by bleomycin was used to verify the effect of CAE on pulmonary fibrosis.40 C57BL/6J mice were randomly divided into blank group,model group,low dose treatment group(64 mg/kg/d)and high dose treatment group(96 mg/kg/d).Except for the blank group,all the other groups were made by tracheal instillation.In the CAE treatment group,the drug was given on the first day after the establishment of the model,and on the 28th day,the mice were dissected,and the serum,bronchoalveolar lavage fluid and lung tissue were obtained.The changes of body weight,hair and activity of mice were observed and recorded.The inflammation was evaluated by measuring the contents of IL-6,IL-17,TGF-? and IL-10 in bronchoalveolar lavage fluid and serum by ELISA method,the pathological changes of lung tissue were evaluated by HE staining and Masson staining,and the expression of ?-SMA and Collagen I gene in lung tissue of mice in each group was determined by qPCR method.2.Mice were pretreated with antibiotics and then bleomycin was used to induce pulmonary fibrosis in mice to observe the effect of CAE on pulmonary fibrosis in mice.40 C57BL/6J mice were randomly divided into model group,antibiotic group,low dose treatment group(64 mg/kg/d)and high dose treatment group(96 mg/kg/d).Except for the model group,the other three groups were pretreated with antibiotics before endotracheal instillation of bleomycin.After endotracheal instillation of bleomycin,low-dose treatment group and high-dose group were given intragastric administration of CAE 64 mg/kg/d and 96 mg/kg/d,respectively.On the 28th day after intragastric administration,the mice were dissected,and the serum and lung tissue of mice were obtained.The changes of body weight,hair and activity of mice were observed and recorded.The inflammation was evaluated by measuring the contents of IL-6,IL-17,TGF-? and IL-10 in bronchoalveolar lavage fluid and serum by ELISA method,the pathological changes of lung tissue were evaluated by HE staining and Masson staining,and the expression of ?-SMA and Collagen I gene in lung tissue of mice in each group was determined by qPCR method.3.The co-feeding method was used to observe whether CAE played a role through gut microbiota.Forty-eight C57BL/6J mice were randomly divided into three groups,16 mice in each group,and fed in a rat cage,and were divided into three groups:group A,group B and group C.Group An included 8 mice in each of the model group(CHAM)and blank group(CHAB),which were fed in the same cage.The model group(CHAM)was given bleomycin and was fed with normal saline,and the blank group(CHAB)was fed with normal saline.And intragastric administration of normal saline.A total of 8(CHBTA)mice in group B,including model group(CHBM)and CAE low dose group,were raised in the same cage,in which the model group(CHBM)was made by bleomycin and fed with normal saline,and the model was made by(CHBTA)in CAE low dose group.CAE 64 mg/kg was given to the stomach from the first day after the establishment of the model and lasted for 28 days.A total of 8(CHCTB)mice in the model group(CHCM)and CAE high dose group were raised in the same cage,in which the model group(CHCM)was made with bleomycin and fed with normal saline,and the high dose CAE group(CHCTB)was given bleomycin to make the model,and CAE 96 mg/kg was given to the stomach from the first day after the establishment of the model for 28 days.On the 28th day after intragastric administration,the mice were dissected,and the serum and lung tissue of mice were obtained.The changes of body weight,hair and activity of mice were observed and recorded.The inflammation was evaluated by measuring the contents of IL-6,IL-17,TGF-? and IL-10 in bronchoalveolar lavage fluid and serum by ELISA method,the pathological changes of lung tissue were evaluated by HE staining and Masson staining,and the expression of?-SMA and CollagenI gene in lung tissue of mice in each group was determined by qPCR method.4.The effect of CAE on gut microbiota of mice in each group was observed by detecting the gut microbiota in the feces of mice.The feces of mice in each group were collected,and the gut microbiota was detected and analyzed by 16s rDNA,and the changes of gut microbiota in each groups were observed.5.By detecting the content of short-chain fatty acids in feces,FoxP3 mRNA in lung tissue,IL-10 expression in serum and bronchoalveolar lavage fluid(BALF),to explore the mechanism of CAE anti-pulmonary fibrosis through gut microbiota.The contents of short-chain fatty acids in feces,IL-10 in bronchoalveolar lavage fluid and serum were determined by gas chromatography-mass spectrometry(GC-MS),and the expression of FoxP3 gene in lung tissue of mice in each group was determined by qPCR.Result:1.The citrus alkaline extract could delay the weight loss of pulmonary fibrosis mice,improve lung histopathology,reduce the score of pulmonary fibrosis,decrease the expression of ?-SMA and CollagenI mRNA in lung tissue,and reduce the contents of IL-6,IL-17 and TGF-? in bronchoalveolar lavage fluid and serum.2.The mice treated with antibiotic mixture(ABX)in advance could resist the weight loss caused by bleomycin,improve the lung histopathology,reduce the pulmonary fibrosis score,decrease the expression of ?-SMA and CollagenI mRNA in lung tissue,and decrease the contents of IL-6,IL-17 and TGF-? in serum.3.Co-feeding with mice treated with citrus alkaline extract could improve lung histopathology,reduce the score of pulmonary fibrosis,decrease the expression of?-SMA and CollagenI mRNA in lung tissue and decrease the content of IL-6,IL-17 and TGF-? in serum.4.The citrus alkaline extract can increase the species richness of gut microbiota and the relative abundance of butyric acid-producing bacteria such as actinobacteria and firmicutes.5.The citrus alkaline extract could increase the content of butyric acid in intestinal contents,the expression of FoxP3 mRNA in lung tissue,the expression of ZO-1 mRNA in colon tissue and the content of IL-10 in bronchoalveolar lavage fluid and serum.Conclusion:1.Gut microbiota plays an important role in the occurrence and development of bleomycin-induced pulmonary fibrosis;2.The citrus alkaline extract may increase the content of butyric acid in the intestine by increasing the abundance of butyric acid-producing bacteria in the intestine,and butyric acid may play an anti-pulmonary fibrosis effect by promoting the secretion of IL-10 by Treg cells in lung tissue. |
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