Gu-Ben-Fang-Xiao Decoction Ameliorated Murine Asthma In Remission Stage By Modulating Microbiota-acetate-Tregs Axis

Objective:Gu-Ben-Fang-Xiao decoction(GBFXD),derived from traditional Chinese medicine,is an effective and safe therapeutic formula for asthma in the remission stage(ARS).Manipulating gut microbiota is a promising therapeutic intervention in asthma and is being extensively studied.This study aims to clarify the mechanisms of GBFXD in treating ARS by investigating the role of gut microbiota in ARS and the the effects of GBFXD on microbiotaMethods:The ARS mouse model induced by a combination of ovalbumin and respiratory syncytial virus(RSV).Enhanced pause(Penh)in awake state and resistance of airway(Rrs)in anesthetic state were detected respectively to evaluate the respiratory function of mice.Flow cytometer(FCM)was used to detect the total number of inflammatory cells in the bronchoalveolar lavage fluid(BALF).Hematoxylin-eosin staining was used to evaluate the lung histopathology.16S rDNA sequencing was used to detect and analyze the diversity and composition of bacteria in colonic contents.The contents of short-chain fat acid(SCFAs)in feces and serum were determined by Gas chromatography-mass spectrometry(GC-MS)or enzyme-linked immunosorbent assay.The relative proportion of regulatory T cells(Treg)in peripheral blood mononuclear cells(PBMC)was detected by FCM.The co-housing experiment and the validation experiment using broad-spectrum antibiotics and complementary acetate were conducted to investigate the role of gut microbiota and its product,acetate,in the therapeutic mechanism of GBFXD in preventing and treating ARS.Results:(1)Results of airway function,lung histopathology and cells count of BALF:Compared with the CTRL group,after the stimulations by methacholine,Penh and Rrs in ARS model mice increased more significantly,and GBFXD treatment reversed the abnormal Penh and Rrs in ARS mice;inflammation around the bronchi and blood vessels and the total number of inflammatory cells in BALF increased significantly in the ARS model mice.GBFXD treatment effectively reduced the pathological state of lung tissue in mice and the total number of cells in BALF.(2)Results of the colonic microbiota composition and the levels of SCFAs in feces and serum:The dominant community composition of colonic contents exhibited significant changes between the CTRL mice and the ARS mice,which was mainly reflected by a significant increase in the abundance of Bacteroidetes and a decrease in the proportion of Firmicutes.At the level of phylum,family,and genus,GBFXD intervention improved the bacterial disorders in ARS mice to a certain extent by increasing the ratio of Firmicutes to Bacteroidetes,the relative abundance of SCFAs-producing bacteria such as Firmicutes,Lachnospiraceae,Bifidobacteriaceae,and the SCFAs levels(especially acetic acid)in feces and total SCFAs in serum Level(3)Results of the co-housing experiment:Total cells in BALF of the Co-Model group were markedly lower than that of the Model mice,while the total cell counts in B ALF from Co-GBFXD tended to increase compared to the GBFXD group.Consistently,the inflammatory infiltrations surrounding bronchi and vessels in the Co-Model group were milder than those in the Model mice.However,mice in the Co-GBFXD group showed more severe peribranchial and perivascular inflammations when compared to the GBFXD group.The level of acetate in the Co-GBFXD group was significantly lower than that in the GBFXD group,while the level of acetate showed a tendency to increase in the Co-Model group,compared to that in Model mice(P=0.117).(4)Results of the acetate replenishment experiment on ABX treated ARS mice:The fecal contents of acetate in the G-ABX group was significantly lower than that of the G-ABX group The protective effects of GBFXD were attenuated after ABX treatment,manifested as the augmented histopathological score in the lung sections and a massive amount of total cells in the BALF,while acetate supplemented restored the therapeutic effects of GBFXD on ARS mice(5)Results of the proportion of Treg in PBMCs:The percentage of Tregs was prominently reduced in the Model group when compared with the CTRL group,and GBFXD treatment markedly enhanced the proportion of Tregs.The ARS mice co-housed with GBFXD-treated mice showed an increased percentage of Tregs in PBMCs,demonstrating that GBFXD could increase the percentage of Tregs through gut microbiota.Moreover,the effect of GBFXD on Tregs was abolished after ABX treatment,while acetate supplement restored this effect of GBFXDConclusion:(1)Airway inflammation and lung function dysfunction existed in the OVA-RSV-induced murine model of ARS.GBFXD ameliorated respiratory function and lung inflammation,with clear pharmacodynamic effects(2)GBFXD treatment partly reversed the gut dysbiosis in asthmatic mice at the phylum,family,and genus levels,increased the abundance of SCFAs-producing bacteria,and elevated the local colonic and systemic levels of SCFAs,particularly the level of acetate,suggesting that gut microbiota and acetate might participate in the efficacy mechanism of GBFXD in the treatment of ARS(3)The co-housing experiment and the acetate replenishment experiment on ABX treated mice suggest that the transmission and depletion of gut bacteria and the supplementation of acetate directly affect the effects of GBFXD on Tregs differentiation in PBMC and the improvement of pulmonary inflammation,suggesting that GBFXD might ameliorate lung inflammation in ARS mice through the microbiota-acetate-Tregs axis.(4)The dysbiosis of colonic microbiota existed in ARS model mice and might play a critical role in the pathogenesis of ARS.To manipulate gut microbiota-acetate-Tregs axis might be a promising therapeutic intervention in asthma.