Cigarette smoke-induced dysbiosis: comparative analysis of lung and intestinal microbiomes in COPD mice and patients.

BACKGROUND: The impact of cigarette smoke (CS) on lung diseases and the role of microbiome dysbiosis in chronic obstructive pulmonary disease (COPD) have been previously reported; however, the relationships remain unclear. METHODS: Our research examined the effects of 20-week cigarette smoke (CS) exposure on the lung and intestinal microbiomes in C57BL/6JNarl mice, alongside a comparison with COPD patients' intestinal microbiome data from a public dataset. RESULTS: The study found that CS exposure significantly decreased forced vital capacity (FVC), thickened airway walls, and induced emphysema. Increased lung damage was observed along with higher lung keratinocyte chemoattractant (KC) levels by CS exposure. Lung microbiome analysis revealed a rise in Actinobacteriota, while intestinal microbiome showed significant diversity changes, indicating dysbiosis. Principal coordinate analysis highlighted distinct intestinal microbiome compositions between control and CS-exposed groups. In the intestinal microbiome, notable decreases in Patescibacteria, Campilobacterota, Defferibacterota, Actinobacteriota, and Desulfobacterota were observed. We also identified correlations between lung function and dysbiosis in both lung and intestinal microbiomes. Lung interleukins, interferon-É£, KC, and 8-isoprostane levels were linked to lung microbiome dysbiosis. Notably, dysbiosis patterns in CS-exposed mice were similar to those in COPD patients, particularly of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 4 patients. This suggests a systemic impact of CS exposure. CONCLUSION: In summary, CS exposure induces significant dysbiosis in lung and intestinal microbiomes, correlating with lung function decline and injury. These results align with changes in COPD patients, underscoring the important role of microbiome in smoke-related lung diseases.

Neutralization of CX3CL1 Attenuates TGF-ß-Induced Fibroblast Differentiation Through NF-κB Activation and Mitochondrial Dysfunction in Airway Fibrosis.

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-ß. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis. However, it is currently unknown whether neutralization of CX3CL1 decreases TGF-ß-induced fibroblast differentiation and mitochondrial dysfunction in normal human lung fibroblasts (NHLFs). METHODS: CX3CL1/C-X3-C motif chemokine receptor 1 (CX3CR1), CX3CL1 was analyzed by immunofluorescence (IF) or immunohistochemical (IHC) staining of ovalbumin-challenged mice. CX3CL1 release was detected by ELISA. TGF-ß-induced CTGF, fibronectin, and α-SMA expression were evaluated in NHLFs following neutralization of CX3CL1 (TP213) treatment for the indicated times by Western blotting or IF staining. Mitochondrion function was detected by a JC-1 assay and seahorse assay. Cell apoptosis was observed by a terminal uridine nick-end labeling (TUNEL) assay. RESULTS: An increase in CX3CL1 expression was observed in lung tissues from mice with ovalbumin-induced asthma by IF staining. CX3CR1 was increased in the subepithelial layer of the airway by IHC staining. Moreover, CX3CR1 small interfering (si)RNA downregulated TGF-ß-induced CTGF and fibronectin expression in NHLFs. CX3CL1 induced CTGF and fibronectin expression in NHLFs. TGF-ß-induced CX3CL1 secretion from NHLFs. Furthermore, TP213 decreased TGF-ß-induced CTGF, fibronectin, and α-SMA expression in NHLFs. Mitochondrion-related differentially expressed genes (DEGs) were examined after CX3CL1 neutralization in TGF-ß-treated NHLFs. TP213 alleviated TGF-ß-induced mitochondrial dysfunction and apoptosis resistance in NHLFs. CX3CL1 induced p65, IκBα, and IKKα phosphorylation in a time-dependent manner. Furthermore, CX3CL1-induced fibronectin expression and JC-1 monomer were decreased by p65 siRNA. TP213 reduced TGF-ß-induced p65 and α-SMA expression in NHLFs. CONCLUSIONS: These findings suggest that neutralizing CX3CL1 attenuates lung fibroblast activation and mitochondrial dysfunction. Understanding the impacts of CX3CL1 neutralization on fibroblast mitochondrial function could contribute to the development of therapeutic strategies for managing airway remodeling in severe asthma.

Endothelin-1 induces connective tissue growth factor expression in human lung fibroblasts by disrupting HDAC2/Sin3A/MeCP2 corepressor complex.

BACKGROUND: Reduction of histone deacetylase (HDAC) 2 expression and activity may contribute to amplified inflammation in patients with severe asthma. Connective tissue growth factor (CTGF) is a key mediator of airway fibrosis in severe asthma. However, the role of the HDAC2/Sin3A/methyl-CpG-binding protein (MeCP) 2 corepressor complex in the regulation of CTGF expression in lung fibroblasts remains unclear. METHODS: The role of the HDAC2/Sin3A/MeCP2 corepressor complex in endothelin (ET)-1-stimulated CTGF production in human lung fibroblasts (WI-38) was investigated. We also evaluated the expression of HDAC2, Sin3A and MeCP2 in the lung of ovalbumin-induced airway fibrosis model. RESULTS: HDAC2 suppressed ET-1-induced CTGF expression in WI-38 cells. ET-1 treatment reduced HDAC2 activity and increased H3 acetylation in a time-dependent manner. Furthermore, overexpression of HDAC2 inhibited ET-1-induced H3 acetylation. Inhibition of c-Jun N-terminal kinase, extracellular signal-regulated kinase, or p38 attenuated ET-1-induced H3 acetylation by suppressing HDAC2 phosphorylation and reducing HDAC2 activity. Overexpression of both Sin3A and MeCP2 attenuated ET-1-induced CTGF expression and H3 acetylation. ET-1 induced the disruption of the HDAC2/Sin3A/MeCP2 corepressor complex and then prompted the dissociation of HDAC2, Sin3A, and MeCP2 from the CTGF promoter region. Overexpression of HDAC2, Sin3A, or MeCP2 attenuated ET-1-stimulated AP-1-luciferase activity. Moreover, Sin3A- or MeCP2-suppressed ET-1-induced H3 acetylation and AP-1-luciferase activity were reversed by transfection of HDAC2 siRNA. In an ovalbumin-induced airway fibrosis model, the protein levels of HDAC2 and Sin3A were lower than in the control group; however, no significant difference in MeCP2 expression was observed. The ratio of phospho-HDAC2/HDAC2 and H3 acetylation in the lung tissue were higher in this model than in the control group. Overall, without stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex inhibits CTGF expression by regulating H3 deacetylation in the CTGF promoter region in human lung fibroblasts. With ET-1 stimulation, the HDAC2/Sin3A/MeCP2 corepressor complex is disrupted and dissociated from the CTGF promoter region; this is followed by AP-1 activation and the eventual initiation of CTGF production. CONCLUSIONS: The HDAC2/Sin3A/MeCP2 corepressor complex is an endogenous inhibitor of CTGF in lung fibroblasts. Additionally, HDAC2 and Sin3A may be of greater importance than MeCP2 in the pathogenesis of airway fibrosis.

Amphiregulin induces CCN2 and fibronectin expression by TGF-ß through EGFR-dependent pathway in lung epithelial cells.

BACKGROUND: Airway fibrosis is one of the pathological characteristics of severe asthma. Transforming growth factor (TGF)-ß has been known to promote epithelial-mesenchymal transition formation and to play a role in the progression of tissue fibrosis. Cellular communication network factor 2 (CCN2) and fibronectin (FN) are well-known markers of EMT and fibrosis. However, whether AREG is involved in TGF-ß-induced CCN2 and FN expression in human lung epithelial cells is unknown. METHODS: AREG and FN were analyzed by immunofluorescence staining on ovalbumin-challenged mice. CCN2 and FN expression were evaluated in human lung epithelial (A459) cells following TGF or AREG treatment for the indicated times. Secreted AREG from A549 cells was detected by ELISA. Cell migration was observed by a wound healing assay. Chromatin immunoprecipitation was used to detect the c-Jun binding to the CCN2 promoter. RESULTS: AREG and FN expression colocalized in lung tissues from mice with ovalbumin-induced asthma by immunofluorescence staining. Moreover, TGF-ß caused the release of AREG from A549 cells into the medium. Smad3 siRNA down-regulated AREG expression. AREG also stimulated CCN2 and FN expression, JNK and c-Jun phosphorylation, and cell migration in A549 cells. AREG small interfering (si) RNA inhibited TGF-ß-induced expression of CCN2, FN, and cell migration. Furthermore, AREG-induced CCN2 and FN expression were inhibited by EGFR siRNA, a JNK inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). EGFR siRNA attenuated AREG-induced JNK and c-Jun phosphorylation. Moreover, SP600125 downregulated AREG-induced c-Jun phosphorylation. CONCLUSION: These results suggested that AREG mediates the TGF-ß-induced EMT in human lung epithelial cells through EGFR/JNK/AP-1 activation. Understanding the role of AREG in the EMT could foster the development of therapeutic strategies for airway remodeling in severe asthma.

Thrombin induces IL-8/CXCL8 expression by DCLK1-dependent RhoA and YAP activation in human lung epithelial cells.

BACKGROUND: Doublecortin-like kinase 1 (DCLK1) has been recognized as a marker of cancer stem cell in several malignancies. Thrombin is crucial in asthma severity as it can promote IL-8/CXCL8 production in lung epithelial cells, which is a potent chemoattractant for neutrophils. However, the pathologic role of DCLK1 in asthma and its involvement in thrombin-stimulated IL-8/CXCL8 expression remain unknown. METHODS: IL-8/CXCL8, thrombin, and DCLK1 expression were observed in the lung tissues of severe asthma patients and ovalbumin (OVA)-induced asthmatic mice model. A549 and BEAS-2B cells were either pretreated with inhibitors or small interfering RNAs (siRNAs) before being treated with thrombin. IL-8/CXCL8 expression and the molecules involved in signaling pathway were performed using ELISA, luciferase activity assay, Western blot, or ChIP assay. RESULTS: IL-8/CXCL8, thrombin, and DCLK1 were overexpressed in the lung tissues of severe asthma patients and ovalbumin (OVA)-induced asthmatic mice model. Our in vitro study found that DCLK siRNA or LRKK2-IN-1 (DCLK1 inhibitor) attenuated IL-8/CXCL8 release after thrombin induction in A549 and BEAS-2B cells. Thrombin activated DCLK1, RhoA, and YAP in a time-dependent manner, in which DCLK1 siRNA inhibited RhoA and YAP activation. YAP was dephosphorylated on the Ser127 site after thrombin stimulation, resulting in YAP translocation to the nucleus from the cytosol. DCLK1, RhoA and YAP activation following thrombin stimulation were inhibited by U0126 (ERK inhibitor). Moreover, DCLK1 and YAP siRNA inhibited κB-luciferase activity. Thrombin stimulated the recruitment of YAP and p65 to the NF-κB site of the IL-8/CXCL8 promoter and was inhibited by DCLK1 siRNA. CONCLUSIONS: Thrombin activates the DCLK1/RhoA signaling pathway, which promotes YAP activation and translocation to the nucleus from the cytosol, resulting in YAP/p65 formation, and binding to the NF-κB site, which enhances IL-8/CXCL8 expression. DCLK1 might be essential in thrombin-stimulated IL-8/CXCL8 expression in asthmatic lungs and indicates a potential therapeutic strategy for severe asthma treatment.

MPT0E028, a novel pan-HDAC inhibitor, prevents pulmonary fibrosis through inhibition of TGF-ß-induced CTGF expression in human lung fibroblasts: Involvement of MKP-1 activation.

Histone deacetylase (HDAC) inhibitors are potential candidates for treating pulmonary fibrosis. MPT0E028, a novel pan-HDAC inhibitor, has been reported to exhibit antitumor activity in several cancer cell lines. In this study, we investigated the mechanism underlying the inhibitory effects of MPT0E028 on the expression of fibrogenic proteins in human lung fibroblasts (WI-38). Our results revealed that MPT0E028 inhibited transforming growth factor-ß (TGF-ß)-, thrombin-, and endothelin 1-induced connective tissue growth factor (CTGF) expression in a concentration-dependent manner. In addition, MPT0E028 suppressed TGF-ß-stimulated expression of fibronectin, collagen I, and α-smooth muscle actin (α-SMA). Furthermore, MPT0E028 inhibited the TGF-ß-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). MPT0E028 reduced the increase in SMAD3 and c-Jun phosphorylation, and SMAD3-and activator protein-1 (AP-1)-luciferase activities under TGF-ß stimulation. Transfection with mitogen-activated protein kinase phosphatase-1 (MKP-1) siRNA reversed the suppressive effects of MPT0E028 on TGF-ß-induced increases in CTGF expression; JNK, p38, and ERK phosphorylation; and SMAD3 and AP-1 activation. Moreover, MPT0E028 increased MKP-1 acetylation and activity in WI-38 cells. Pretreatment with MPT0E028 reduced the fibrosis score and fibronectin, collagen, and α-SMA expression in bleomycin-induced pulmonary fibrosis mice. In conclusion, MPT0E028 induced MKP-1 acetylation and activation, which in turn inhibited TGF-ß-stimulated JNK, p38, and ERK phosphorylation; SMAD3 and AP-1 activation; and subsequent CTGF expression in human lung fibroblasts. Thus, MPT0E028 may be a potential drug for treating pulmonary fibrosis.

High frequency of KRAS and EGFR mutation profiles in BRAF-negative thyroid carcinomas in Indonesia.

OBJECTIVE: Thyroid cancer incidence has steadily increased in Indonesia. However, data on Kirsten rat sarcoma virus (KRAS) and EGFR mutations in thyroid cancer in Indonesia remain unavailable, except for BRAF-V600E, the most common BRAF gene mutation. This study aimed to analyze KRAS and EGFR mutation profiles in BRAF-V600E negative thyroid cancer samples. RESULTS: BRAF-V600E mutations were found in papillary thyroid carcinomas in 40.3% patients with mean age of 53 years old. In BRAF-V600E-negative samples, 41.3% had KRAS mutations with mean age of 55.5 years old. KRAS mutation was found in 52.6% of follicular carcinomas and 47.4% of papillary thyroid carcinomas. Additionally, 45.7% had EGFR mutations in patients with mean age of 50.5 years old. EGFR mutation was found in 71.4% of papillary thyroid carcinoma and 28.6% of follicular carcinoma. Nearly half of the BRAF-V600E negative thyroid carcinoma samples harbored either KRAS or EGFR mutations. This finding suggests that in BRAF-V600E negative thyroid carcinoma samples, testing for RAS and EGFR mutation may be warranted for further therapeutic consideration.

Polyherbal formula (ASILACT®) induces Milk production in lactating rats through Upregulation of α-Lactalbumin and aquaporin expression.

BACKGROUND: Polyherbal formula (PHF) contains extract of Sauropus androgynous (L.) Merr., Trigonella foenum-graceum L., and Moringa oleifera Lam. considered to induce galactagogue activity. This research aimed to evaluate the galactagogue activity of PHF and its effects on α-lactalbumin (LALBA) as well as aquaporin (AQP) gene expression at messenger ribonucleic acid (mRNA) levels in mammary glands of lactating rats. METHODS: Thirty lactating Wistar rats were randomly divided into five groups (n = 6), each has 7 pups. Group I was treated orally with distilled water as negative control. Groups II, III, and IV were orally administered with PHF at 26.25, 52.5 and 105 mg/kg/day, respectively. Group V was treated with domperidone 2.7 mg/kg/day, orally as positive control. The treatment was performed at third day until fifteenth day of parturition. The observed parameters include the galactagogue activity indicating by milk yield of lactating rats, the pup weight changes and lactating rats body weight changes during lactating period, mRNA expression of LALBA and AQP using quantitative Real Time Polymerase Chain Reaction (qRT-PCR) and histopathological analysis of mammary glands at the end of treatment period. RESULT: The result showed that the PHF groups (52.5 and 105 mg/kg/day) and domperidone were significantly increased milk production of lactating rats (p < 0.05). The levels of mRNA expression of LALBA and AQPs were significantly upregulated by 105 mg/kg/day of PHF or 2.7 mg/kg of domperidone administration (p < 0.0001). Histopathological analysis of mammary glands shows that alveoli diameter was increase 14.59 and 19.33% at 105 mg/kg of PHF and 2.7 mg/kg of domperidone treatment, respectively. CONCLUSION: The study suggested that PHF has potentially to induce galactagogue activity on lactating period through upregulation of LALBA and AQP genes at the mRNA level.