| BackgroundHuman respiratory syncytial virus(RSV)is one of the most common viruses that cause lune and respiratory infections in children worldwide.Moreover,this virus can also infect adults,specially the elderly.Despite its high incidence,to date,ribavirin remains the only available FDA approved therapy to treat RSV infection in high risk patients.RSV infection is associated with high fever,rhinitis,pharyngitis,laryngitis,bronchiolitis,and pneumonia.Current immunization approaches,using denatured virus particles,fail to provide long-lasting immunity against subsequent RSV infections.Baicalin,flavonoids isolated from Scutellaria baicalensis,is well known for its antiviral activity and is one of the three active components along with baicalein and wogonin,of this medical plant.Baicalin has been documented for its pharmacologic antiinflammatory,anti-tumor,and anti-viral properties.Different studies,showed the potent inhibitory activity of baicalin or baicalein against RSV.Nevertheless,the role of baicalin in RSV is not yet completely understood,further studies are required to explore the precise mechanism of baicalin’s action against RSV.ObjectiveExploring the molecular mechanism of baicalin promoting host type Ⅰ IFN induction by inhibiting RSV NS protein function and speculating its binding site;exploring the biological process of baicalin affecting virus assembly and budding by inhibiting RSV M protein function.MethodsUsing a virus infection model,plaque reduction experiments,fluorescence detection technology and pathological detection of animal lung tissue were performed to verify the effect of baicalin on inhibiting RSV virus.Differential proteins before and after the action of baicalin were found by proteomic iTRAQ experiment.The effect of baicalin on RSV NS mRNA transcript level detected by RT-qPCR.Molecular simulation docking technology was used to find the binding site of baicalin and NS protein;surface plasmon resonance test was used to analyze the affinity.The effect of baicalin on the transcription level of RSV M mRNA was detected by nucleic acid method.The semiquantitative detection of M protein was carried out by Western Blot test,indicating whether baicalin inhibited the expression of M protein of RSV.Polysome profiling was performed to determine whether baicalin inhibits translation of the M protein RNA.ResultsThe TC50 of baicalin on HEp-2 cells was 0.449 mg/mL,and the IC50 of baicalin on RSV inhibition in vitro was 0.0894 mg/mL.In this experiment,the data detected by RTqPCR were normalized to Actb expression.In vitro experiment,the NS2 RNA level in HEp-2 cells infected with RSV treated with baicalin was detected by RT-qPCR method.The result of RSV control group was 1.06±0.06,and the result of NS2 RNA treated with 0.039 mg/mL,0.078 mg/mL,0.156 mg/mL and 0.312 mg/mL baicalin was 0.72±0.04,0.51±0.04,0.21±0.04 and 0.08±0.03,respectively.Compared with the RSV positive control group,NS2 RSV in each baicalin treatment group decreased significantly(P<0.0001),and baicalin inhibited the synthesis of NS2 RNA in a dosedependent manner.After baicalin treatment,the immunofluorescence signal of RSV N protein also decreased in a dose-dependent manner.In vivo experiment results showed that there was no significant difference between the weight of mice treated with baicalin and that of mice infected with the virus.The transcriptional level of RSV RNA in the lungs of mice infected with RSV was determined by Real-time PCR.The RSV RNA was 7895.41±2438.23 copies/mL;The RSV RNA results of lung tissues of mice treated with baicalin at 100 mg/kg and 200 mg/kg concentrations were 5311.34±1804.42 copies/mL and 4370.63±1854.41 copies/mL,respectively.Compared with RSV infected mice,high and low doses of baicalin decreased RSV RNA in lung tissue of mice,with a statistical difference(P<0.05),indicating that baicalin effectively inhibited virus replication in vivo.H&E staining results of mice lung tissues before and after baicalin treatment showed that baicalin improved RSV infection induced lung hyperplasia in a dose-dependent manner.These in vivo and in vitro results indicated that baicalin inhibits RSV replication.The results of proteomics analysis showed that baicalin had significant enrichment differences in the proteins related to the response to type I interferon.The detection results of type Ⅰ interferon(IFN-α and IFN-β),TNF-α,IL-10 and IL-6 in mice after baicalin treatment showed that the increase of IFN-α and IFN-β expression mediated by baicalin and the decrease of TNF-α,IL-10 and IL-6 expression were at the translation level.Furthermore,we studied the effect of baicalin on RSV genomic RNA by RT-qPCR.The results of mRNA detection of RSV NS1,NS2,N,P,M,SH,G,F,M2 genes in RSV infected mice were 1.14±0.16,1.11±0.11,0.96±0.03,1.06±0.15,1.03±0.18,1.11±0.11,1.24±0.10,1.10±0.12,1.14±0.12;The results of RSV NS1,NS2,N,P,M,SH,G,F,M2 gene mRNA of mice treated with baicalin at a concentration of 200 mg/kg were 0.47±0.06,0.58±0.07,1.07±0.15,1.04±0.10,1.24±0.10,1.31±0.08,1.18±0.22,1.22±0.85,1.29±0.05.The results showed that,compared with RSV infected mice,the RSV infected mice treated with baicalin only significantly reduced the mRNA levels of NS1 and NS2,with a statistical difference(P<0.001),indicating that baicalin inhibited the transcription of NS1 and NS2 RNA;The other virus genes N,P,M,SH,G,F,M2 of RSV infected mice treated with baicalin had no significant difference compared with RSV infected mice(P>0.05).Western blot results showed that NS1,NS2 and M proteins in lung homogenate of mice treated with baicalin decreased compared with untreated RSV infected mice.The viral protein M,the rate limiting factor for RSV replication,was also down regulated after baicalin treatment,but the level of M mRNA did not change.Therefore,we next analyzed the polyribosomes of viral RNA.The translation indexes of NS1,NS2 and M in lung homogenates of mice infected with RSV were 4.27±0.47,5.53±0.64,5.70±0.80;the translation indexes of mice lung homogenate NS1,NS2 and M treated with baicalin were 0.36±0.06,0.78±0.05,0.29±0.06.The experimental results showed that compared with RSV infected mice,the polyribosome ratio of NS1 and NS2 RNA in RSV infected mice treated with baicalin decreased significantly(P<0.001).Compared with RSV infected mice,the polyribosome ratio of viral RNA after baicalin treatment also decreased significantly(P<0.001).The experimental results showed that baicalin inhibited the translation of viral RNA.Affinity analysis based on molecular docking and surface plasma resonance showed that baicalin binds to the α3 helix of NS1 protein with an affinity of 1.119×10-5M via hydrogen bonding,π-π stacking and hydrophobic interactions,and binds to amino acids residues such as strong interaction Tyr125(tyrosine),Leu132(leucine)and Leu133 physically interact with the NS 1 protein.The analysis of viral protein M showed that RSV infection itself resulted in the release of L13a from ribosomes compared with uninfected mice.After baicalin treatment,L13 a was released more strongly;The results of immunohistochemistry showed that the expression of L13a and P-L13a protein in the lungs of RSV infected mice treated with baicalin increased,while the expression of NS1 and M protein of virus decreased.The results showed that baicalin inhibited the translation of viral RNA,which may be related to its promotion of the release of ribosomal protein L13a.ConclusionIt is an in-depth exploration which includes the effector mechanism of baicalin in changing the RSV life cycle,the molecular mechanism of promoting host type Ⅰ IFN induction and generation by inhibiting RSV NS protein function and the binding site,and the specific role of host type Ⅰ IFN induction and generation.It also identifies the biological process that baicalin maybe affect viral assembly and budding by inhibiting RSV M protein function.The results of this study further enrich the drug mechanism of baicalin and provide a scientific basis for its effective treatment of RSV infectious diseases. |
PDF Full Text Request