Gut-Lung Dysbiosis Accompanied by Diabetes Mellitus Leads to Pulmonary Fibrotic Change through the NF-κB Signaling Pathway

Growing evidence shows that the lungs are an unavoidable target organ of diabetic complications. However, the pathologic mechanisms of diabetic lung injury are still controversial. This study demonstrated the dysbiosis of the gut and lung microbiome, pulmonary alveolar wall thickening, and fibrotic change in streptozotocin-induced diabetic mice and antibiotic-induced gut dysbiosis mice compared with controls. In both animal models, the NF-κB signaling pathway was activated in the lungs. Enhanced pulmonary alveolar well thickening and fibrotic change appeared in the lungs of transgenic mice expressing a constitutively active NF-κB mutant compared with wild type. When lincomycin hydrochloride–induced gut dysbiosis was ameliorated by fecal microbiota transplant, enhanced inflammatory response in the intestine and pulmonary fibrotic change in the lungs were significantly decreased compared with lincomycin hydrochloride–treated mice. Furthermore, the application of fecal microbiota transplant and baicalin could also redress the microbial dysbiosis of the gut and lungs in streptozotocin-induced diabetic mice. Taken together, these data suggest that multiple as yet undefined factors related to microbial dysbiosis of gut and lungs cause pulmonary fibrogenesis associated with diabetes mellitus through an NF-κB signaling pathway. Growing evidence shows that the lungs are an unavoidable target organ of diabetic complications. However, the pathologic mechanisms of diabetic lung injury are still controversial. This study demonstrated the dysbiosis of the gut and lung microbiome, pulmonary alveolar wall thickening, and fibrotic change in streptozotocin-induced diabetic mice and antibiotic-induced gut dysbiosis mice compared with controls. In both animal models, the NF-κB signaling pathway was activated in the lungs. Enhanced pulmonary alveolar well thickening and fibrotic change appeared in the lungs of transgenic mice expressing a constitutively active NF-κB mutant compared with wild type. When lincomycin hydrochloride–induced gut dysbiosis was ameliorated by fecal microbiota transplant, enhanced inflammatory response in the intestine and pulmonary fibrotic change in the lungs were significantly decreased compared with lincomycin hydrochloride–treated mice. Furthermore, the application of fecal microbiota transplant and baicalin could also redress the microbial dysbiosis of the gut and lungs in streptozotocin-induced diabetic mice. Taken together, these data suggest that multiple as yet undefined factors related to microbial dysbiosis of gut and lungs cause pulmonary fibrogenesis associated with diabetes mellitus through an NF-κB signaling pathway. Diabetes mellitus (DM), also called diabetes, has become a major global public health problem with a considerable increase in the prevalence of DM over the past few decades.1Bhutani J. Bhutani S. Worldwide burden of diabetes.Indian J Endocrinol Metab. 2014; 18: 868Crossref PubMed Scopus (43) Google Scholar As a metabolic disorder characterized by persistent hyperglycemia, DM consists of autoimmune type 1 diabetes and insulin-resistant type 2 diabetes.2American Diabetes AssociationDiagnosis and classification of diabetes mellitus.Diabetes Care. 2010; 33: S62-S69Crossref PubMed Scopus (4319) Google Scholar The pathogenesis of DM is deemed to involve a combination of genetic and environmental factors.3Altobelli E. Petrocelli R. Verrotti A. Chiarelli F. Marziliano C. Genetic and environmental factors affect the onset of type 1 diabetes mellitus.Pediatr Diabetes. 2016; 17: 559-566Crossref PubMed Scopus (15) Google Scholar,4Murea M. Ma L. Freedman B.I. 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Epithelial-cell-intrinsic IKK-β expression regulates intestinal immune homeostasis.Nature. 2007; 446: 552-556Crossref PubMed Scopus (413) Google Scholar The activation of NF-κB signaling depends on the initiation of the signaling cascade via stimulation of cell surface receptors (toll-like receptors) by cytokines/pathogens and the phosphorylation-induced proteasomal degradation of NF-κB proteins (IκBs), which is required for signal transduction to NF-κB.20Oeckinghaus A. Ghosh S. The NF-κB family of transcription factors and its regulation.Cold Spring Harb Perspect Biol. 2009; 1: a000034Crossref PubMed Scopus (1301) Google Scholar The initiation, propagation, and development of metabolic diseases, including type 2 diabetes, are largely derived from chronic low-level inflammation in which NF-κB signaling plays crucial roles in coordinating inflammatory responses and subsequent disease development.21Baker R.G. Hayden M.S. Ghosh S. NF-κB, inflammation, and metabolic disease.Cell Metab. 2011; 13: 11-22Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar Hence, this study investigated the roles of NF-κB signaling in mediating the gut-lung microbiota dysbiosis-induced pulmonary fibrotic changes in the context of DM. The C57BL/6 mice used in this study were obtained from the Institute of Laboratory Animal Science, Jinan University (Guangzhou, China). NF-κB1(Cys-Ser) knock-in mice were obtained from the Modern Animal Research Center of Nanjing University (Nanjing, China). In the knock-in mice, the usually conservative Cys was replaced by Ser, such that the p50 with this substitution retained a maximum DNA-binding activity,22Toledano M. Ghosh D. Trinh F. Leonard W. N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.Mol Cell Biol. 1993; 13: 852-860Crossref PubMed Google Scholar as shown before.23Xu J.J. Wang G. Luo X. Wang L.J. Bao Y. Yang X. Role of nuclear factorins contribute to differential DNA-binding specificities.J Cell Physiol. 2019; 234: 22565-22580Crossref PubMed Scopus (0) Google Scholar Multiplex PCR genotyping used four primers to detect the knock-in alleles (primer 1, primer 2, primer 3, and primer 4). The following PCR conditions were used to detect wild-type and NF-κB1C59S alleles: 94°C for 5 minutes; 41 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds; and 72°C for 5 minutes. Primers were obtained from Sangon Biotech (Shanghai, China), and the sequences are listed in Table 1. The mice were housed in the animal facility under constant room temperature, 50% to 60% humidity, and a 12:12-hour light-dark cycle, and were supplied with standard food pellets and water.Table 1Primer of Mouse GenotypingGenotypingPrimerSequenceNF-κB1C598ForwardReverse5′-CCTGCTTACAACCTGTTTATTC-3′5′-GCATCGCATTGTCTGAGTAGGTG-3′Wild typeForwardReverse5′-CCTGCTTACAACCTGTTATTC-3′5′-GTGAGATTGGTTCCAACATCAAC-3′ Open table in a new tab Type 1 DM mouse model was established using streptozotocin (STZ) injection.24Kumar S.D. Dheen S.T. Tay S.S.W. Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.Cardiovasc Diabetol. 2007; 6: 34Crossref PubMed Scopus (82) Google Scholar, 25Wang L.M. Zhong N.Z. Liu S.J. Zhu X.Y. Liu Y.J. Hypoxia-induced acute lung injury is aggravated in streptozotocin diabetic mice.Exp Lung Res. 2015; 41: 146-154Crossref PubMed Scopus (9) Google Scholar, 26Yuan D. Xu Y. Hang H. Liu X. Chen X. Xie P. Yuan S. Zhang W. Lin X. Liu Q. Edaravone protect against retinal damage in streptozotocin-induced diabetic mice.PLoS One. 2014; 9: e99219Crossref PubMed Scopus (17) Google Scholar Briefly, 8-week–old mice were injected with STZ (Sigma, St. Louis, MO; dissolved in 0.01 mol/L citrate buffer, pH 4.5) at 75 mg/kg body weight for 3 consecutive days, and diabetes mellitus was defined as a fasting blood glucose level >288 mg/dL (>16 mmol/L).27Jin Y. Wang G. Han S.S. He M.Y. Cheng X. Ma Z.L. Wu X. Yang X. Liu G.S. Effects of oxidative stress on hyperglycaemia-induced brain malformations in a diabetes mouse model.Exp Cell Res. 2016; 347: 201-211Crossref PubMed Scopus (10) Google Scholar, 28He M.Y. Wang G. Han S.S. Jin Y. Li H. Wu X. Ma Z.L. Cheng X. Tang X. Yang X. Liu G.S. Nrf2 signalling and autophagy are involved in diabetes mellitus-induced defects in the development of mouse placenta.Open Biol. 2016; 6: 160064Crossref PubMed Scopus (21) Google Scholar, 29Han S.S. Wang G. Jin Y. Ma Z.L. 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Antibiotic-induced dysbiosis of gut microbiota impairs corneal nerve regeneration by affecting CCR2-negative macrophage distribution.Am J Pathol. 2018; 188: 2786-2799Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar and lincomycin hydrochloride (LH) treatment mouse model, were established, as described before.33Song J. Wang C. Long D. Li Z. You L. Brand-Saberi B. Wang G. Yang X. Dysbacteriosis-induced LPS elevation disturbs the development of muscle progenitor cells by interfering with retinoic acid signaling.FASEB J. 2020; 34: 6837-6853Crossref PubMed Scopus (6) Google Scholar The mice in control group (n ≥ 10) were treated with physiological saline solution (containing 0.1% dimethyl sulfoxide). Mice with DM were randomly divided into three groups: i) physiological saline treatment group (n ≥ 12), ii) fecal microbiota transplant (FMT) treatment group (n ≥ 6), and iii) baicalin treatment group (n ≥ 6), from at least three different batches. In control and DM groups, the mice were administered with physiological saline solution via an intragastric gavage for 1 week. For the FMT, 200 mg of stool (from normal mouse pellets) was resuspended in 5 mL of phosphate-buffered saline under anaerobic conditions, vortexed for 3 minutes, and allowed to settle by gravity for 2 minutes.34Suez J. Korem T. Zeevi D. Zilberman-Schapira G. Thaiss C.A. Maza O. Israeli D. Zmora N. Gilad S. Weinberger A. Kuperman Y. Harmelin A. Kolodkin-Gal I. Shapiro H. Halpern Z. Segal E. Elinav E. Artificial sweeteners induce glucose intolerance by altering the gut microbiota.Nature. 2014; 514: 181-186Crossref PubMed Scopus (974) Google Scholar Each DM mouse in the FMT group was given 200 μL of the supernatant by intragastric method every 3 days.32Liu J. Wu M. He J. Xiao C. Xue Y. Fu T. Lin C. Dong D. Li Z. Antibiotic-induced dysbiosis of gut microbiota impairs corneal nerve regeneration by affecting CCR2-negative macrophage distribution.Am J Pathol. 2018; 188: 2786-2799Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar The mice in baicalin group were administered 1 mL/day of baicalin (40 mg/kg body weight) by intragastric gavage for 1 week. Mice with LH-induced microbiota dysbiosis were randomly divided into two groups: FMT treatment group (n ≥ 6) and physiological saline treatment group (n ≥ 6). All research involving the use of animals in this study was performed in accordance with the procedures of the Ethical Committee for Animal Experimentation, Jinan University. Hematoxylin and eosin (H&E), Masson, and Sirius red staining were used to observe histologic pulmonary structures and fibrous tissue of 13-week–old mouse lungs from control, STZ-induced DM, and STZ-induced DM with baicalin treatment groups. Following photography, the lungs, spleens, and large intestines were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin wax, and serially divided into sections (5 mm thick). For histologic analysis, the sections were dewaxed in xylene, rehydrated, and stained with H&E, Masson, or Sirius red,35Lattouf R. Younes R. Lutomski D. Naaman N. Godeau G. Senni K. Changotade S. Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.J Histochem Cytochem. 2014; 62: 751-758Crossref PubMed Scopus (215) Google Scholar,36Wang G. Yeung C.K. Wong W.Y. Zhang N. Wei Y.F. Zhang J.L. Yan Y. Wong C.Y. Tang J.J. Chuai M.L. Lee K.K.H. Wang L.J. Yang X.S. 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They were incubated with collagen I (Col1; 1:200; ab34710; Abcam, New Territories, Hong Kong), pro-surfactant protein C (SPC) (1:200; ab90716; Abcam), aquaporin-5 (AQP5; 1:200; ab72876; Abcam), α-smooth muscle actin (α-SMA; 1:200; ab66050; Abcam), Ki-67 (1:200; AB9260; Merck KGaA, Darmstadt, Germany), proliferating cell nuclear antigen (1:1000; ab29; Abcam), CD3 (1:200; ab5690; Abcam), p65 (1:200; 6956s; Cell Signaling Technology, Danvers, MA), and caveolin-1 (1:200; PA5-17447; Thermo Scientific, Shanghai, China) overnight at 4°C. For immunohistochemistry, following extensive washing, the sections were incubated in horseradish peroxidase goat anti-rabbit IgG secondary antibody (1:400; 7074S; EarthOx, Millbrae, CA) for 2 hours at room temperature in a dark box and were conjugated to diaminobenzidine (Maixin, Fuzhou, China). After immunostaining, the sections were counterstained with hematoxylin. For immunofluorescence staining, the sections were incubated with the corresponding Alexa Fluor 555 or 488 secondary antibody (1:1000; Invitrogen, Waltham, MA) at room temperature for 2 hours in a dark box. All sections were subsequently counterstained with DAPI (1:1000; Invitrogen) at room temperature for 30 minutes. The sections were photographed using a fluorescence microscope (Olympus BX53) linked to the cellSens Standard 1.9 software (Olympus Corporation, Tokyo, Japan). A minimum of five random images from three samples were assayed per group. The percentages of cells with positive fluorescence from the immunostaining assay were quantified by measuring the area of immunofluorescence signals with Image Pro Plus 7.0 software. Immunofluorescence staining for cells was performed 48 hours later using antibodies against pro-SPC (1:100; ab90716; Abcam). A minimum of eight images per treatment group were assayed. The cells were photographed using an inverted fluorescence microscope (Ti-U; Nikon, Tokyo, Japan) linked to the NIS-Elements F3.2 software. MLE-12, a mouse lung epithelial cell line, was obtained from the ATCC (Manassas, VA; CRL-2110). All of the cells were passaged fewer than 6 months after resuscitation and were cultured in compliance with the protocol provided by the ATCC. Sera and media were purchased from Gibco (Gaithersburg, MD). The cells were cultured in a humidified incubator with 5% CO2 at 37°C in 6-well plates (1 × 106 cells/mL) containing Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Cells were then exposed to 10 μg/mL lipopolysaccharide (LPS)39Zhang J. Wang G. Liu J. Gao L.R. Liu M. Wang C.J. Chuai M. Bao Y. Li G. Li R.M. Zhang Y. Yang X. Gut microbiota-derived endotoxin enhanced the incidence of cardia bifida during cardiogenesis.J Cell Physiol. 2018; 233: 9271-9283Crossref PubMed Scopus (6) Google Scholar,40Xu J.J. Wang G. 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Baicalin reversal of DNA hypermethylation-associated Klotho suppression ameliorates renal injury in type 1 diabetic mouse model.Cell Cycle. 2020; 19: 3329-3347Crossref PubMed Scopus (4) Google Scholar or 2.5 to 10 μmol/L baicalin30Wang G. Liang J. Gao L.R. Si Z.P. Zhang X.T. Liang G. Yan Y. Li K. Cheng X. Bao Y. Chuai M. Chen L.G. Lu D.X. Yang X. Baicalin administration attenuates hyperglycemia-induced malformation of cardiovascular system.Cell Death Dis. 2018; 9: 234Crossref PubMed Scopus (26) Google Scholar,42Zhang X.T. Wang G. Ye L.F. Pu Y. Li R.T. Liang J. Wang L. Lee K.K.H. Yang X. Baicalin reversal of DNA hypermethylation-associated Klotho suppression ameliorates renal injury in type 1 diabetic mouse model.Cell Cycle. 2020; 19: 3329-3347Crossref PubMed Scopus (4) Google Scholar, 43Paudel K.R. Kim D.W. Microparticles-mediated vascular inflammation and its amelioration by antioxidant activity of baicalin.Antioxidants (Basel). 2020; 9: 890Crossref Scopus (8) Google Scholar, 44Wang G. Lu J.Q. Ding Y. Zhang T. Song J.H. Long D. Liang J. Cheng X. Si Z. Qi G. Jiang X. Yang X. Baicalin rescues hyperglycemia-induced neural tube defects via targeting on retinoic acid signaling.Am J Transl Res. 2020; 12: 3311-3328PubMed Google Scholar was added to the medium. Cells were harvested 48 hours later for further analysis. Cell viability was assessed using CCK8 assay (cholecystokinin 8). Briefly, 10 μL of CCK8 reagent (Dojindo, Kumamoto, Japan) was added to the 96-well plates for 48 hours at 37°C. The absorbance values were measured at 450 nm using a Bio-Rad model 450 microplate reader (Bio-Rad, Hercules, CA). The cell viability was indirectly determined by examining the ratio of the absorbance value of baicalin or/and LPS or glucose-treated cells relative to the control cells. Proteins from the mouse lung or MLE-12 cells were isolated from tissue homogenates using a radioimmunoprecipitation assay (Sigma) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified with the bicinchoninic acid assay. The extracted proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% nonfat milk and then incubated with Col1 (1:1000; ab34710; Abcam), pro-SPC (1:1000; ab90716; Abcam), AQP5 (1:1000; ab72876; Abcam), α-SMA (1:1000; ab66050; Abcam), p65 (1:1000; 6956s; Cell Signaling Technology), IκBα (1:1000; ab109509; Abcam), IL-6 (1:1000; ab233706; Abcam), zonula occludens protein 1 (1:1000; 14667-1-AP; Proteintech, Wuhan, Hubei, China), engagement of the cell death surface receptor (1:500; 8023s; Cell Signaling), p53 (1:1000; 2527S; Cell Signaling), P50 (1:1000; bs-1194R; Bioss, Beijing, China), β-actin (1:3000; 60008-1-1g; Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (1:3000; 60004-1-Ig; Proteintech) in a tris-buffered saline with Tween 20 buffer at 4°C overnight. After incubation with the secondary antibody, either horseradish peroxidase goat anti-rabbit IgG (1:3000; 7074S; EarthOx) or horseradish peroxidase goat anti-mouse IgG (1:3000; 7076S; EarthOx), the samples were developed with SuperSignal West Femto Chemiluminescent Substrate (ThermoFisher, Shanghai, China) and the Gel Doc XR+ System (Bio-Rad). Quantity One software version 4.6.2 (Bio-Rad) was used to capture the chemiluminescent signals and analyze the data. The diversity and abundance of the microbiota were analyzed with 16S rRNA gene sequencing. The DNA of the gut microbiota from the stool or lung tissue of the mouse was obtained by the E.Z.N.A. DNA Kit (Omega Bio-tek, Norcross, GA). A DNA sample was sent to the Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for 16S rRNA gene sequencing analysis. 16S rRNA sequence data have been deposited in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra; accession numbers SRP301296, SRP254066, and SRP275674). Total RNA was extracted from mouse lung using the TRIzol kit (Invitrogen). First-strand cDNA was synthesized to a final volume of 20 μL using a SuperScript RIII first-strand kit (Invitrogen). Following reverse transcription, PCR amplification of the cDNA was performed using chicken-specific primers. Primer sequences are provided in Table 2. The PCRs were performed in a Bio-Rad S1000 Thermal cycler (Bio-Rad), as previously described.33Song J. Wang C. Long D. Li Z. You L. Brand-Saberi B. Wang G. Yang X. Dysbacteriosis-induced LPS elevation disturbs the development of muscle progenitor cells by interfering with retinoic acid signaling.FASEB J. 2020; 34: 6837-6853Crossref PubMed Scopus (6) Google ScholarTable 2PCR PrimerGenePrimerSequenceCd19ForwardReverse5′-TGGTGGAGGTAGAAGAGGG-3′5′-GGAAGGGTGTTGACTGGTTA-3′Cd20ForwardReverse5′-TATTCAAACTTCCAAGCCGTAT-3′5′-ATCAGCATCGCCGACAGA-3′Cd14ForwardReverse5′-TCTTGAACCTCCGCAACG-3′5′-TGGGCAATACTCAGTACCTTGA-3′Cd45ForwardReverse5′-CCCGCACTGAGCTGGAAT-3′5′-TGACACCGCTGCTCCTCTT-3′Cd68ForwardReverse5′-ATCTTGCTAGGACCGCTTAT-3′5′-GTGGCTGTAGGTGTCATCGT-3′Tgfb1ForwardReverse5′-ACCGCAACAACGCCATCT-3′5′-GGGCACTGCTTCCCGAAT-3′Il6ForwardReverse5′-TACCACTCCCAACAGACC-3′5′-TTTCCACGATTTCCCAGA-3′Casp8ForwardReverse5′-CCCAGATTTCTCCCTACA-3′5′-ATCAGCATCGCCGACAG