Clinical characteristics and related risk factors of disease severity in 101 COVID-19 patients hospitalized in Wuhan, China.

Coronavirus disease 2019 (COVID-19) broke out in December 2019. Due its high morbility and mortality, it is necessary to summarize the clinical characteristics of COVID-19 patients to provide more theoretical basis for future treatment. In the current study, we conducted a retrospective analysis of the clinical characteristics of COVID-19 patients and explored the risk factors for the severity of illness. A total of 101 COVID-19 patients hospitalized in Leishenshan Hospital (Wuhan, China) was classified into three sub-types: moderate (n = 47), severe (n = 36), and critical (n = 18); their clinical data were collected from the Electronic Medical Record. We showed that among the 101 COVID-19 patients, the median age was 62 years (IQR 51-74); 50 (49.5%) patients were accompanied by hypertension, while 25 (24.8%) and 22 (21.8%) patients suffered from diabetes and heart diseases, respectively, with complications. All patients were from Wuhan who had a definite history of exposure to the epidemic area. Multivariate logistic regression analysis revealed that older age, diabetes, chronic liver disease, percentage of neutrophils (N%) > 75%, CRP > 4 mg/L, D-dimer > 0.55 mg/L, IL-2R > 710 U/mL, IL-8 > 62 pg/mL, and IL-10 > 9.1 pg/mL were independent variables associated with severe COVID-19. In conclusion, we have identified the independent risk factors for the severity of COVID-19 pneumonia, including older age, diabetes, chronic liver disease, higher levels of N%, CRP, D-dimer, IL-2R, IL-8, and IL-10, providing evidence for more accurate risk prediction.

Chronic microaspiration of bile acids induces lung fibrosis through multiple mechanisms in rats.

Gastroesophageal reflux (GER) and microaspiration of duodenogastric refluxate have been recognized as a risk factor for pulmonary fibrosis. Recent evidence suggests that bile acid microaspiration may contribute to the development of lung fibrosis. However, the molecular evidence is scarce and the underlying mechanisms remain to be elucidated. We have recently demonstrated that bile acids induce activation of alveolar epithelial cells (AECs) and lung fibroblasts in vitro In the present study, a rat model of bile acid microaspiration was established by weekly intratracheal instillation of three major bile acids including chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA). Repeated microaspiration of CDCA, DCA, and LCA caused fibrotic changes, including alveolar wall thickening and extensive collagen deposition, in rat lungs. Bile acid microaspiration also induced alveolar epithelial-mesenchymal transition (EMT), as indicated by up-regulation of mesenchymal markers α-smooth muscle actin (α-SMA) and vimentin, as well as down-regulaton of epithelial markers E-cadherin and cytokeratin in alveolar epithelium of rat lungs. The expression of fibrogenic mediators, including transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and periostin, was significantly elevated in rat lungs exposed to microaspiration of bile acids. Furthermore, microaspiration of bile acids also induced p-Smad3 and farnesoid X receptor (FXR) expression in rat lungs. Our findings suggest that microaspiration of bile acids could promote the development of pulmonary fibrosis in vivo, possibly via stimulating fibrogenic mediator expression and activating TGF-ß1/Smad3 signaling and FXR.

Bile acids induce activation of alveolar epithelial cells and lung fibroblasts through farnesoid X receptor-dependent and independent pathways.

BACKGROUND AND OBJECTIVE: The roles of bile acid microaspiration and bile acid-activated farnesoid X receptor (FXR) in the pathogenesis of idiopathic pulmonary fibrosis (IPF) remain unclear. We hypothesized that bile acids activate alveolar epithelial cells (AECs) and lung fibroblasts, which may be regulated by FXR activation. METHODS: Human AECs and normal or IPF-derived lung fibroblast cells were incubated with the three major bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The AECs injury indices, epithelial-mesenchymal transition (EMT) and lung fibroblast activation were evaluated. FXR expression in IPF lungs and the roles of FXR and FXR-independent pathways in bile acid-induced profibrotic effects were also investigated. RESULTS: LCA, DCA and CDCA reduced cell viability and increased intracellular reactive oxygen species (ROS) production in A549 cells. They all induced EMT, as shown by enhanced α-SMA and vimentin and decreased E-cadherin levels. LCA directly induced differentiation of lung fibroblasts to myofibroblasts. All three bile acids promoted cellular migration but not proliferation of lung fibroblasts. FXR expression was upregulated in IPF lungs, and inhibition of FXR restrained the bile acid-induced EMT and lung fibroblast activation. Differentiation and proliferation were enhanced in lung fibroblasts exposed to conditioned medium from bile acid-stimulated A549 cells, which contained increased levels of profibrotic factors. TGF-ß/Smad3 signaling was also involved in the bile acid-induced EMT and lung fibroblast differentiation. CONCLUSION: Bile acid microaspiration may promote the development of pulmonary fibrosis by inducing activation of AECs and lung fibroblasts via FXR-dependent and independent pathways.

Overexpression of farnesoid X receptor in small airways contributes to epithelial to mesenchymal transition and COX-2 expression in chronic obstructive pulmonary disease.

BACKGROUND: Epithelial-mesenchymal transition (EMT) and cyclooxygenase-2 (COX-2) contribute to airway remodelling and inflammation in chronic obstructive pulmonary disease (COPD). Recent data suggest that the farnesoid X receptor (FXR), a nuclear receptor traditionally considered as bile acid-activated receptor, is also expressed in non-classical bile acids target tissues with novel functions beyond regulating bile acid homeostasis. This study aimed to investigate the potential role of FXR in the development of COPD, as well as factors that affect FXR expression. METHODS: Expression of FXR, EMT biomarkers and COX-2 was examined by immunohistochemistry in lung tissues from non-smokers, smokers, and smokers with COPD. The role of FXR in TGF-ß1-induced EMT and COX-2 expression in human bronchial epithelial (HBE) cells was evaluated in vitro. Factors regulating FXR expression were assessed in cultured HBE cells and a cigarette smoke-induced rat model of COPD. RESULTS: Expression of FXR, EMT markers and COX-2 was significantly elevated in small airway epithelium of COPD patients compared with controls. The staining scores of FXR in small airway epithelium were negatively related with FEV1% of predicted of smokers without and with COPD. FXR agonist GW4064 remarkably enhanced and FXR antagonist Z-Guggulsterone significantly inhibited EMT changes in TGF-ß1-treated HBE cells. Both chenodeoxycholic acid (CDCA) and GW4064 increased COX-2 expression in HBE cells, whereas Z-Guggulsterone dramatically restrained CDCA-induced COX-2 expression. Finally, FXR expression is induced by IL-4 and IL-13 in HBE cells, as well as by cigarette smoke exposure in a rat model of COPD. CONCLUSIONS: Overexpression of FXR in small airway may contribute to airway remodelling and inflammation in COPD by regulating EMT and COX-2 expression.

Selective Cox-2 inhibitor celecoxib induces epithelial-mesenchymal transition in human lung cancer cells via activating MEK-ERK signaling.

Increasing evidence has suggested that high expression level of cyclooxygenase-2 (Cox-2) is associated with the malignancies of non-small cell lung cancer (NSCLC), leading to a rationale of applying Cox-2 inhibitors as adjuvant therapy in the treatment of NSCLC. However, the addition of celecoxib, a selective Cox-2 inhibitor, to chemotherapy in clinical trials failed to benefit the survival of NSCLC patients, which urges the investigation to re-evaluate this strategy for NSCLC treatment. In this study, we observed that celecoxib treatment at clinically relevant concentrations induced epithelial-mesenchymal transition (EMT) in NSCLC cells regardless of Cox-2 status, which, however, was not recapitulated using another Cox-2 inhibitor, etodolac. Celecoxib-stimulated EMT in turn promoted cell invasion and rendered cells resistant to chemotherapy. Further mechanistic investigation by disrupting the integrity of signaling pathways using specific inhibitors or RNA interference revealed that celecoxib-induced EMT in NSCLC cells is indispensable of transforming growth factor-ß1/Smad signaling. Instead, the activated MEK/ERK/SNAIL1 signaling largely accounted for celecoxib-induced EMT. Taken together, our study reveals the diverse impacts of Cox-2 inhibitors on EMT in NSCLC cells independent of Cox-2 inhibition, where celecoxib treatment leads to metastasis and chemoresistance via EMT induction. These findings reveal the increased risks of cancer metastasis and chemoresistance by applying Cox-2 inhibitors, celecoxib in particular, in clinical trials of NSCLC treatment and urge intensive preclinical assessment before proceeding to clinical application.

Sulfated polymannuroguluronate inhibits Tat-induced SLK cell adhesion via a novel binding site, a KKR spatial triad.

AIM: Sulfated polymannuroguluronate (SPMG), a candidate anti-AIDS drug, inhibited HIV replication and interfered with HIV entry into host T lymphocytes. SPMG has high binding affinity for the transactivating factor of the HIV-1 virus (Tat) via its basic domain. However, deletion or substitution of the basic domain affected, but did not completely eliminated Tat-SPMG interactions. Here, we sought to identify other SPMG binding sites in addition to the basic domain. METHODS: The potential SPMG binding sites were determined using molecular simulation and a surface plasmon resonance (SPR) based competitive inhibition assay. The effect of SPMG on Tat induced adhesion was evaluated using a cell adhesion assay. RESULTS: The KKR domain, a novel high-affinity heparin binding site, was identified, which consisted of a triad of Lys12, Lys41, and Arg78. The KKR domain, spatially enclosed SPMG binding site on Tat, functions as another binding domain for SPMG. Further functional evaluation demonstrated that SPMG inhibits Tat-mediated SLK cell adhesion by directly binding to the KKR region. CONCLUSION: The KKR domain is a novel high-affinity binding domain for SPMG. Our findings provide important new insights into the molecular mechanisms of SPMG and a potential therapeutic intervention for Tat-induced cell adhesion.

[The effect of biofilms on the production of beta-lactamases in Pseudomonas aeruginosa].

OBJECTIVE: To investigate the effect of formation of biofilms on the expression of beta-lactamases in Pseudomonas aeruginosa. METHOD: Four strains of Pseudomonas aeruginosa producing TEM and CARB extended spectrum beta-lactamases were induced by 3 antimicrobials (Ciprofloxacin, Ceftazidime and Imipenem) to generate beta-lactamases before and after the formation of biofilms. Different expression of beta-lactamases-mRNA was determined by relatively quantitative polymerase chain reaction. RESULTS: Before biofilm formation, the relative level of mRNA expression of TEM beta-lactamase was 1.000 +/- 0.495 before and 2.038 +/- 0.036, 3.308 +/- 0.010, and 8.654 +/- 0.026 after induction with the 3 antimicrobials. After biofilm formation, the relative level of mRNA expression of TEM beta-lactamase was 1.766 +/- 0.026 before and 13.849 +/- 0.004, 16.372 +/- 0.044 and 34.581 +/- 0.143 respectively after antimicrobial induction with the above 3 antimicrobials (t = 6.94 - 582.14, P = 0.000). Before biofilm formation, the relative level of mRNA expression of CARB beta-lactamase was 1.000 +/- 0.069 before and 3.506 +/- 0.036, 5.878 +/- 0.190, and 8.771 +/- 0.066 after induction with the 3 antimicrobials; while for biofilm strains, the relative level of mRNA expression of CARB beta-lactamase was 2.937 +/- 0.164 before and 9.528 +/- 0.050, 10.844 +/- 0.062, and 21.900 +/- 0.087 respectively after induction with the 3 antimicrobials (t = 5.77 - 20.03, P = 0.000). Before biofilm formation, the increase of relative level of TEM mRNA expression after induction by antimicrobials was lower than that of CARB mRNA (5.49 +/- 4.32 vs 6.15 +/- 2.26; t = 3.31, P = 0.05), while after biofilm formation, the increase of the relative level of TEM mRNA after antimicrobial induction was higher than that of CARB mRNA (12.40 +/- 6.95 vs 4.92 +/- 2.33; t = 5.94, P = 0.01). CONCLUSIONS: The formation of biofilms could influence the ability of Pseudomonas aeruginosa in expressing different beta-lactamases; and the amplitude of TEM beta-lactamase was higher than that of CARB beta-lactamase after biofilm formation, suggesting that the formation of biofilms may have different effects on Pseudomonas aeruginosa strains with different genotypes.

MS80, a novel sulfated oligosaccharide, inhibits pulmonary fibrosis by targeting TGF-beta1 both in vitro and in vivo.

AIM: The pro-fibrogenic cytokine transforming growth factor-beta 1 (TGF-beta1) has attracted much attention for its potential role in the etiology of idiopathic pulmonary fibrosis (IPF). Here, we demonstrate that MS80, a novel sulfated oligosaccharide extracted from seaweed, can bind TGF-beta1. The aim of the present study was to determine whether MS80 is capable of combating TGF-beta1-mediated pulmonary fibrotic events both in vitro and in vivo, and to investigate the possible underlying mechanisms. METHODS: Surface plasmon resonance was used to uncover the binding profiles between the compound and TGF-beta. MTT assay, flow cytometry, Western blot analysis, BCA protein assay and SDS-PAGE gelatin zymography were used to probe the antifibrotic mechanisms of MS80. The in vivo fibrotic efficacy was evaluated in a bleomycin instillation-induced rat model. RESULTS: We report that MS80, a new kind of sulfated oligosaccharide extracted from seaweed, inhibits TGF-beta1-induced pulmonary fibrosis in vitro and bleomycin-induced pulmonary fibrosis in vivo. Our results indicated that MS80 competitively inhibited heparin/HS-TGF-beta1 interaction through its high binding affinity for TGF-beta1. Moreover, MS80 arrested TGF-beta1-induced human embryo pulmonary fibroblast (HEPF) cell proliferation, collagen deposition and matrix metalloproteinase (MMP) activity. Intriguingly, MS80 deactivated both the ERK and p38 signaling pathways. MS80 was also a potent suppressor of bleomycin-induced rat pulmonary fibrosis in vivo, as evidenced by improved pathological settings and decreased lung collagen contents. CONCLUSION: MS80 in particular, and perhaps oligosaccharide in general, offer better pharmacological profiles with appreciably few side effects and represent a promising class of drug candidates for IPF therapy.Acta Pharmacologica Sinica (2009) 30: 973-979; doi: 10.1038/aps.2009.86; published online 22 June 2009.

[Therapeutic effect and mechanism of sulfate polysaccharide of algae on lung cancer].

OBJECTIVE: To study the effect of sulfate polysaccharide of algae (SPA) on lung carcinoma and it's mechanism. METHODS: (1) C57BL/6 mice implanted with Lewis lung carcinoma were used as experimental animal model. The mice were randomly divided into a control group, SPA groups (3 groups) and a FT-207 group. After inoculation with Lewis lung carcinoma, the control group was treated with NS, the 3 SPA groups were treated with SPA 20 mg, 40 mg, and 80 mg/kg respectively, and the FT-207 group with FT-207 150 mg/kg for 10 days. The inhibiting activity of SPA on Lewis lung carcinoma was assayed, and proliferation of A549 tumor cells treated with SPA was detected by MTT assay. (2) New Zealand rabbits were randomly divided into a control group, a SPA 500 mg/kg group and a CTX 100 mg/kg group. Serum pharmacological method, and both in vivo and in vitro anti-tumor experiments were used in the study. After incubating A549 with SPA containing serum at different concentrations, the growth and apoptosis of cells, cell cycle, apoptosis rate and expression of apoptosis associated genes such as p53 and bcl-2 were detected by flow-cytometric assay. RESULTS: (1) SPA significantly inhibited the growth of implanted Lewis lung carcinoma in vivo and the effect had a dose dependent manner. The inhibitory rates of SPA on C57BL mice implanted Lewis lung carcinoma were 35.27% (P < 0.01), 48.29% (P < 0.01), and 65.41% (P < 0.01) respectively, which showed dose-dependent effects. SPA directly added to the culture medium neither induced A549 lung cancer cell apoptosis nor inhibited its proliferation in vitro. (2) SPA containing serum significantly induced A549 lung cancer cell apoptosis and inhibited its proliferation, with an increased expression of p53 and decreased expression of bcl-2 positive proteins, showing time and dose-dependent relationship. CONCLUSIONS: SPA possessed remarkable inhibitory activity against lung neoplasia in animal models and lung cancer cell strain. The anti tumor activity of SPA was considered to be derived from apoptosis induction, which might be associated with the increased expression of p53 and decreased expression of bcl-2 positive proteins.