| Respiratory syncytial virus (respiratory syncytial virus, RSV) infection can causesevere bronchiolitis, pneumonia and even death in infants and young children, but alsoinduced an important risk factor for asthma in children. However, there is no specificantiviral drug for the treatment of RSV infection, so exploring a safe and effectiveantiviral drug can be used to prevent and (or) be in the treatment of RSV infection is stillan urgent need.Recombinant human interferon-α1b (recombinant human interferon-α1b, rhIFN-α1b)is a class of successful genetic engineering of production approved by the China, and hascurrently used in the treatment of chronic hepatitis B, chronic hepatitis C and somemalignant tumors, with significant anti-viral, anti-tumor and immune modulating function.However, about the report of rhIFN-α1b curing children with RSV infection is limited in our country, lacking formal uniform standardized treatment guidelines, and nationalexperts’ consensus that “α-interferon as an antiviral commonly used drug, has clinicalexperience, but still doesn’t have inhalation recommended dose for children, and itseffectiveness should be further confirmed.”Our RSV series of experiments preliminary studied the effect of rhIFN-α1binhalation on mice with RSV infection, as well as tolerance and safety of rhIFN-α1binjection in children with viral pneumonia. The former study was founded that therhIFN-α1b12μg by inhalation was effective to treat the RSV-infected mice, and the latterstudy was founded that the application of rhIFN-α1b injection at single doses of0.3~2.0μg/kg is safe and tolerable for children in recovery from viral pneumonia. Therefore, thisexperiment studied the effect of rhIFN-α1b of multi-dose, high-dose by inhalation onRSV-infected mice, trying to further investigate the antiviral and immune modulatoryeffects of rhIFN-α1b on RSV-infected mice.Objective:This study was designed to use two methods of rhIFN-α1b different doses byinhalation and by intraperitoneal injection to intervene the mice with RSV infection. Bylight microscopy and transmission electron microscopy observing lung tissue lesions,fluorescence quantitative RT-PCR analyzing RSV viral load of lung tissue, flow cytometryanalyzing the T lymphocyte subsets of peripheral blood and ELISA detecting cytokines ofblood serum, to explore the antiviral and immune modulatory effects of rhIFN-α1b onmice with RSV infection, and to provide experimental evidences of rhIFN-α1b for thetreatment of children with RSV infection on respiratory tract.Methods:1) Cultivated Hep-2cells, proliferated RSV, and collected the viral suspension.2) Extracted the RNA of liquid virus, and was identified by the fluorescentquantitative RT-PCR.3) After identification of RSV, assayed TCID50virus drops.4) BALB/c mice were randomly divided into10groups,10in each group. Blankcontrol group (1group), negative control group (1group), RSV infection model group (1 group), rhIFN-α1b (3.125μg,12.5μg,25μg, and50μg) aerosol inhalation group (4groups),ribavirin aerosol inhalation group (1group), rhIFN-α1b intraperitoneal injection group (1group) and ribavirin intraperitoneal injection group (1group).5) RSV virus droplets in the nose, successfully established RSV infection model onmice, according to the grouping, giving each group mice (atomization inhalation orintraperitoneal injection) intervention treatment for continuous5days (except the blankcontrol group).6) Collected the whole blood of mice, detected CD3+CD4+and CD3+CD8+levels byflow cytometry, and calculated the ratio.7) Separated the serum of mice, detected IFN-γ, IL-4and IL-17levels by ELISA.8) Dissected the lung tissue of mice, made HE staining slice, observed thehistopathology change, and calculated the histopathological scores under the lightmicroscopy.9) Prepared the ultrathin sectioning, and observed the ultrastructure changes of lungtissue under transmission electron microscopy.10) Detected RSV viral load in lung of mice by the fluorescence quantitativeRT-PCR.11) Statistical analysis.Results:1) The progress of Hep-2cell’s culture and RSV’s proliferation was smooth.2) Fluorescence quantitative RT-PCR establishing a standard curve: slope-2.62,Y-axis intercept of39.5, the correlation coefficient0.990, the baseline range6to15, thethreshold value of0.16, the standard curve equation Y=-2.62X+39.5. The virus solutionnucleic acid testing positive, identified as RSV, can be used for subsequent experiments.3) According to the Reed-Muench formula, calculated RSV virus solutionTCID50=10-4.6.4) RSV-infected mice showed varying degrees of mental malaise, bradykinesia,eating less, shortness of breath, hair erected dull, some performance coughing andsneezing symptoms, suggesting that RSV infection in the mouse model was successfully established.5) Light microscopy showed large number of RSV infection model mice lung tissuebronchi and bronchioles week lymphocytic infiltration within the lumen of a large numberof secretions oozing, significantly widened alveolar septa, alveolar space narrowing ordisappearance, alveolar space visible red blood cells and short red transparent edema fluid.RSV infection model group mice lung histopathology score (24.600±1.342points) andnegative control group (1.600±2.191) and blank control group (0.800±1.789points)significantly increased, the difference was statistically significant (P＜0.05). rhIFN-α1bdifferent doses of aerosol inhalation group, with the multiplier of the inhalation dose, thedegree of inflammatory injury of the lung tissue of mice decreased lung histopathologicalscores showed a decreasing trend, four dose groups the difference was statisticallysignificant (P＜0.05), with RSV infection model group, the difference was statisticallysignificant (P＜0.05), except the rhIFN-α1b3.125μg inhalation group. Ribavirininhalation mice lung tissue inflammatory injury than of rhIFN-α1b25μg inhalation group,ribavirin intraperitoneal injection group lung tissue inflammatory injury severity wasslightly heavier than the rhIFN-α1b intraperitoneal injection group.6) By transmission electron microscopy visible RSV infection model mice, alveolarseptal thickening, congestion, hemorrhage, edema, pulmonary consolidation performance,reduction or disappearance of the alveolar space, the cavity visible red blood cells andnecrosis, cell debris shedding; type I alveolar epithelial cells as early apoptotic changes inthe nucleus shaped set of edges in the nuclear chromatin condensation, cytoplasmicvesicle number significantly reduced, the secondary lysosomes increase in the number ofdecrease in the number of mitochondria, the cell surface micro-fluff degeneration andnecrosis, chaotically distributed, the formation of collagen, the cell connections loosesection has been completely off; rare in type II alveolar epithelial cells early apoptoticperformance, little change in the nucleus to the cytoplasm of an increase in the number ofmitochondria, osmiophilic layer The corpuscle increase in the number of secretion,exocytosis active phenomena. rhIFN-α1b different doses of inhalation were observed ininflammatory injury of the lung tissue of mice was reduced significantly. Ribavirin inhalation group showed varying degrees of high electron density dense depositsphenomenon, connected into a linear or the ribbon evenly distributed in the alveolar septabasement membrane zone, while the rhIFN-α1b atomization inhalation group and twointraperitoneal injection group were not observed this type of phenomenon.7) Fluorescence quantitative RT-PCR test results showed that the negative controlgroup and blank control group were not detected RSV in lung tissue of mice. RSVinfection model mice lung tissue RSV viral load (153.706±18.982) copies/mg, with therest groups were statistically significant (P＜0.05), except rhIFN-α1b3.125μg inhalationgroup and ribavirin intraperitoneal injection group. The rhIFN-α1b different doses ofinhalation group results showed that RSV viral load of lung tissue showed a decreasingtrend with the inhalation dose doubled, the rhIFN-α1b25μg atomization inhalation groupand rhIFN-α1b50μg atomization inhalation group antiviral activity were statisticallysignificant (P＜0.05). The rhIFN-α1b intraperitoneal group and ribavirin intraperitonealinjection group lung tissue of mice RSV viral load was not statistically significant (P>0.05).8) Flow cytometry results showed that RSV infection model mice peripheral bloodCD3+CD4+levels (78.820%±0.973%), CD3+CD8+levels (18.020%±1.628%) the ratio(4.404±0.415) was significantly higher compared with the negative control group andblank control group, the difference was statistically significant (P＜0.05). The rhIFN-α1bdifferent doses of inhalation group results showed that with the inhalation dose doubledmouse peripheral blood CD3+CD4+levels gradually decline, CD3+CD8+levels graduallyrise, the ratio of the two reduced, rhIFN-α1b50μg inhalation group and the rest of thedrug intervention group difference were statistically significant (P＜0.05), the rhIFN-α1b25μg inhalation group except. The difference of rhIFN-α1b intraperitoneal injection groupand ribavirin intraperitoneal injection group was not statistically significant (P>0.05).9) ELISA test results showed that RSV infection model mice group’s levels of serumof IFN-γ (39.386pg/mL±11.942pg/mL), IL-4(131.241pg/mL±15.388pg/mL) and IL-17(132.080pg/mL±17.659pg/mL), compared with negative control group and blank controlgroup, the difference was statistically significant (P＜0.05). The rhIFN-α1b different doses of inhalation group results showed that with the doubling of the inhalation dose,mice gradual recovery in the serum of IFN-γ, IL-4and IL-17levels gradually decreased,and the difference between the rhIFN-α1b50μg inhalation group and other groups wasstatistically significant (P＜0.05). The difference between rhIFN-α1b intraperitonealinjection group and ribavirin intraperitoneal injection group was not statisticallysignificant (P>0.05).Conclusion:1) The rhIFN-α1b by aerosol inhalation has good inhibitory effect on viral load ofthe lung tissue on mice with RSV infection.2) The rhIFN-α1b by aerosol inhalation has good regulatory effect on peripheralblood of T lymphocyte subsets imbalance on mice with RSV infection.3) The rhIFN-α1b by aerosol inhalation has good regulatory effect on serum ofIFN-γ, IL-4and IL-17levels on mice with RSV infection.4) There is a dose-effect relationship on the antiviral and immune modulatory effectsof different doses of rhIFN-α1b by aerosol inhalation on mice with RSV infection.5) The antiviral and immune modulatory effects of rhIFN-α1b50μg by aerosolinhalation on mice with RSV infection are best, and better than ribavirin by inhalation andrhIFN-α1b by intraperitoneal injection.6) There is no significant difference between the rhIFN-α1b by intraperitonealinjection and ribavirin by intraperitoneal injection on the antiviral and immune modulatoryeffects on mice with RSV infection.7) For the first time to study the antiviral and immune modulatory effects ofrhIFN-α1b of multi-doses and high-dose by aerosol inhalation on mice with RSV infection,and the efficacy compared with rhIFN-α1b by intraperitoneal injection. And laid theexperimental basis for further study of rhIFN-α1b’s clinical application to therapy theRSV infection. |
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