Inhibition of SARS Pseudovirus Cell Entry by Lactoferrin Binding to Heparan Sulfate Proteoglycans

It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease

Liquiritin exhibits anti-acute lung injury activities through suppressing the JNK/Nur77/c-Jun pathway

Abstract Background Licorice (Glycyrrhiza uralensis Fisch.), a well-known traditional medicine, is traditionally used for the treatment of respiratory disorders, such as cough, sore throat, asthma and bronchitis. We aim to investigate the effects of liquiritin (LQ), the main bioactive compound in licorice against acute lung injury (ALI) and explore the potential mechanism. Methods Lipopolysaccharide (LPS) was used to induce inflammation in RAW264.7 cells and zebrafish. Intratracheal instillation of 3 mg/kg of LPS was used for induction an ALI mice model. The concentrations of IL-6 and TNF-α were tested using the enzyme linked immunosorbent assay. Western blot analysis was used to detect the expression of JNK/Nur77/c-Jun related proteins. Protein levels in bronchoalveolar lavage fluid (BALF) was measured by BCA protein assay. The effect of JNK on Nur77 transcriptional activity was determined by luciferase reporter assay, while electrophoretic mobility shift assay was used to examine the c-Jun DNA binding activity. Results LQ has significant anti-inflammatory effects in zebrafish and RAW264.7 cells. LQ inhibited the expression levels of p-JNK (Thr183/Tyr185), p-Nur77 (Ser351) and p-c-Jun (Ser63), while elevated the Nur77 expression level. Inhibition of JNK by a specific inhibitor or small interfering RNA enhanced the regulatory effect of LQ on Nur77/c-Jun, while JNK agonist abrogated LQ-mediated effects. Moreover, Nur77-luciferase reporter activity was suppressed after JNK overexpression. The effects of LQ on the expression level of c-Jun and the binding activity of c-Jun with DNA were attenuated after Nur77 siRNA treatment. LQ significantly ameliorated LPS-induced ALI with the reduction of lung water content and BALF protein content, the downregulation of TNF-α and IL-6 levels in lung BALF and the suppression of JNK/Nur77/c-Jun signaling, which can be reversed by a specific JNK agonist. Conclusion Our results indicated that LQ exerts significant protective effects against LPS-induced inflammation both in vivo and in vitro via suppressing the activation of JNK, and consequently inhibiting the Nur77/c-Jun signaling pathway. Our study suggests that LQ may be a potential therapeutic candidate for ALI and inflammatory disorders

LF, heparin or enzymatic removal of cell surface HSPGs can prevent SARS pseudovirus entry into Vero E6 or Caco-2 cells.

<p>(<b>A</b>) Interference of the interaction between Vero E6 and SARS pseudovirus by LF, heparin and heparinase leads to reduction of viral infection. Vero E6 cells were treated by the same methods above with 10 µM LF, 10 µM heparin or 10 U of heparinase I, respectively. Then, the cells were incubated with SARS pseudovirus as described in Methods. GFP-expressing cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the control. Error bars represent the SD of three independent experiments. *** P<0.001 and **P<0.01. (<b>B</b>) Incubation with LF or heparin, or degradation of HSPGs by heparinase inhibits SARS pseudovirus infection of Caco-2 cells. The Caco-2 cells were treated by same methods as described above. *** P<0.001, **P<0.01 and *P<0.05.</p

Lactoferrin exerts its inhibitory effect on spike protein and SARS pseudovirus by binding to cell-surface HSPGs.

<p>(<b>A</b>) Heparin neutralizes the LF-mediated inhibition of S1190-Fc binding to HEK293E/ACE2-Myc cells. Before treatment with 10 µM LF for 1 h at 37°C, HEK293E/ACE2-Myc cells were incubated with heparin for 10 min at the concentration of 1 µM, 3 µM or 10 µM. S1190-Fc was added to each group to detect S1190-Fc binding to the cells as described above. Error bars represent the SD of three independent experiments. ***P<0.001, **P<0.01 and *P<0.05. (<b>B</b>) Inhibition of SARS pseudovirus infection of HEK293E/ACE2-Myc cells by LF can be partially neutralized by heparin. HEK293E/ACE2-Myc cells were treated with heparin for 10 min at the concentration of 1 µM, 3 µM or 10 µM. Then, 10 µM LF was added to each group and incubated at 37°C for 1 h. The GFP-expressing HEK293E/ACE2-Myc cells in the total population were analyzed as described above. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group with that of the BSA control. Error bars represent the SD of three independent experiments. *** P<0.001, ** P<0.01 and *P<0.05.</p

Lactoferrin localizes to the cell membrane by targeting HSPGs.

<p>(<b>A</b>) LF is present on the cell surface. Oregon Green-labeled LF localization was observed under confocal microscopy. LF untreated cells were used as control. Cell membrane and nuclei were stained with DiI and Hoechst33342, respectively. Scale bar, 8 µm. (<b>B</b>) Heparin inhibits LF binding to HEK293E/ACE2-Myc cells. HEK293E/ACE2-Myc cells were incubated with 0.5 µM Oregon Green-labeled LF at 4°C for 1 h after pretreatment with heparin at the concentration of 3 µM, 10 µM or 30 µM. The MFI was measured for each group by flow cytometry as described above. Error bars represent the SD of three independent experiments. **P<0.01.</p

Incubation with heparin and degradation of HS polysaccharides on the cell surface by heparinase inhibits SARS pseudovirus infection of HEK293E/ACE2-Myc cells.

<p>(<b>A</b>) Heparin inhibits SARS pseudovirus entry into HEK293E/ACE2-Myc cells. Before incubation with SARS pseudovirus at 37°C for 4 h, HEK293E/ACE2-Myc cells were treated with heparin for 10 min at the concentration of 0.625 µM, 1.25 µM, 2.5 µM, 5 µM or 10 µM. GFP-expressing HEK293E/ACE2-Myc cells in the total population were analyzed by flow cytometry. The relative viral infection ratio was measured by comparing the percentage of GFP expressing cells of each group to that of the BSA control. Error bars represent the SD of three independent experiments. (<b>B</b>) Lysis of cell-surface HS by heparinase blocks the infection of HEK293E/ACE2-Myc cells by SARS pseudovirus. After incubation with 10 U of heparinase I or chondroitinase ABC for 1 h at 37°C, the cells were treated with SARS pseudovirus as described above. The relative viral infection ratio was calculated by the same method. *P<0.05.</p