Protection Functions And Mechanisms Of IFN-PDS On Virus Pneumonia In Mice Infected With A Type Influenza Virus

Severe Acute Respiratory Syndrome(SARS) is caused by a kind of newCoronavirus(CoV) infection. It is a kind of malignant and infective diseasealso called lethal pneumonia with the main characteristics of high fever andsevere lung pathological changes. In 2003, SARS broke out in our country andmany other nations and areas, caused startle and attention of the whole world.During the period of SARS epidemic, the pathogen of SARS was confirmed tobe a variation of Coronavirus by WHO according to the isolation andidentification results of a great part of medical organizations, also called SARSrelated Coronavirus (SARS-CoV) which genome sequence determination hasbeen completed. Faced to sudden and unexpected epidemic of SARS, withouteffective treatment measure, a great deal of glucocorticoid treatment helps thepatient survive from the critical stage. But the serious side effects ofglucocorticoid made the researchers worry about. Therefore, to explore aneffective treatment drug becomes the hotspot of the study.Interferon(IFN) is a kind of cell regulate protein that can stimulate cellproduce many kinds of anti-virus proteins to kill the virus after combined withthe receptors on cell surface. Therefore IFN is generally acknowledged to be adrug with anti-virus effect. Previously study confirmed that IFN-a can inhibitCoV proliferation, reduce the nasal CoV infection rate administered in nasalcavity and has prevention function. During the period of untypical pneumoniaepidemic, recombined human IFN a-2b has been approved to be used for highrisk persons prevention by National Food and Drug Administration.Changchun biological product research institute is an important base ofresearch and production.SARS develops very emergently and seriously. It can cause death ofpatient in the early stage. The reason is a great deal of CoV variation invasionthrough the airway activate monocyte-macrophage and neutrophil system, notonly develop rapidly into difficult to controlled pneumonia, but also lead toserious anoxia and respiratory failure. Because of the excessive anduncontrolled release of endogenesis anti-inflammatory medium, inflammatorycells activated extensively and a great deal of inflammatory medium releasedinto blood cause inflammatory of far-between position furthermore developinto systemic inflammatory response syndrome(SIRS) which accelerate patientdeath in the early stage. In the study of PDS anti-shock effects, author and ourdepartment find paraxadiol saponins(PDS) has anti-shock effect similar toglucocorticoid but has not side effect of glucocorticoid hormone. PDS hasprotective effect to endotoxic shock induced by LPS. The mechanism ofprotective effect of PDS is reducing expression of NF-κB mRNA and protein,improving the self-control of inflammatory medium release, lighteningpathogenic changes of lung and systemic inflammatory response syndrome.Therefore, PDS combined with virus killer-IFN may become the new methodto treat SARS.Purpose:1. Influenza virus A type (A/fm/1/47(H1N1)) was used toestablish a mice influenza virus pneumonia model in order to simulate the lunginjury model caused by SARS-CoV. 2. Study the protection effect andmechanism of PDS and IFN in influenza virus infected mice, evaluate thepredominance and significance of PDS-IFN combined use.Methods:1.model establishment, grouping.Clean grade KM mice were infected with influenza virusA(A/fm/1/47(H1N1)) administered through nasal cavity,10 times of LD50every mouse to establish a influenza virus pneumonia model.Medicines and administration method : ① Control group: do not infectedwith virus, treated with physical water .② virus infected group(VIG): sameamount of virus was used to establish lung injury model, mice were treatedwith same dosage menstruum. ③different dosages group of IFN: recombinedhuman IFN a-2b (4000IU/ml,40000IU/ml,400000IU/ml). ④different dosagesof PDS(5mg/kg,10mg/kg,20mg/kg). ⑤I FN-PDS mixture: (IFN4000IU/PDS5mg/kg, IFN4000IU/PDS10mg/kg, IFN4000IU/PDS20mg/ kg). Medicine wasadministered once a day 3 days before infection and 10 days after infection,observed for 15 days after infection.2.Protective effect and mechanism of IFN-PDS mixture on mice infectedwith influenza virus.2.1 common index: ① death rate: according to the number of deathbefore put the mice to death ②l ung coefficient, the mg/ g weight × 100.2.2 pathology observations of the lung tissueTake off the cervical vertebra to kill the small rat. The right inferior lungwas taken out and fixed in 10% neutral formaldehyde, embedded in theparaffin, sliced in the normal regulations slice and dyed with HE. Observe themorphological changes of lung tissue under optical microscope.2.3functions of macrophage: measurement of IL-1 level in supernatant ofceliac macrophage culture: IMDM culture fluid was injected into celiac cavity.Celiac cells were collected and cultured. The cells stick the wall ismacrophage. LPS(20 ug/ ml) was added into the culture fluid as a stimulator.The supernatant was collected respectively 8hrs and 48hrs after culture. Twoantibodies clip-ELISA method was used to measure IL-1 level.2.4researches of the immunity organs and its functions(1)thymus gland and spleen coefficient: thymus gland and spleen weightwas measured by precise electronics weighing scales, the results express withspleen coefficient and the thymic coefficients. (2) self-proliferation andmitogenesis induced by ConA of thymic cell and spleen cell was measuredthrough MTT method. (3) measurement of IL-2 level in supernatant fluid ofspleen cell culture: use two antibodies clip-ELISA method to examine IL-2level. (4) immunity organization chemistry of IL-2 in thymus gland andlungs tissue : SABC immunity organization chemistry dye method. (5)Western blot analysis of IL-2 expression in lung tissue.2.5 NF-κBp65,NF-κBp50 expression in lung and thymus gland:Western blot and immunity organization chemistry.2.6 Caspase-3 expression in lung tissue: Western blot and immunityorganization chemistry.2.7 measurement of AST, ALT and LDH level in blood serum :Bloodwas collected through eyes before sentence to death. Centrifuge to separate theblood serum. EOS-880 auto bio-chemical analysis instrument was used tomeasure AST, ALT and LDH level in blood serum.2.8 Assay of SOD and MDA level in blood serum: colorimetry methodwas used to measure serum SOD and MDA level.Results and discussion:1. Death rate compare with each group 15d after infection: ① death rateof VIG group is up to 75% . ②In IFN4000IU group and PDS20mg/kg group,the death rate is 35%. ③ The death rate in IFN-PDS group is low to 10%. Theresults show that both IFN and PDS can increase the survival rate of influenzavirus infected mice and lower the death rate. The effect is more obviousespecially in IFN-PDS group.2. Pathologic histological observation : ① VIG group: large area oflung change solid, the lung alveolar interval width increases obviously, greatquantities lymphcyte soakage, alveolar endothelium cell proliferation, thealveolus become smaller, air contained in alveolus decrease, local effusion andbleeding, presenting a serious interstitial pneumonia change. Moreover, giantcell proliferation can be seen in some part of lung tissue also presenting achange of giant cell pneumonia. ② In IFN treatment group, the lung injuryis lighter than in VIG group. But lung alveolar interval width increase andgreat quantities lymphcyte soakage also can be seen, the alveolus containingmore air can be seen. ③ In PDS treatment group, lung pathological changesare more lighter than in IFN group. The lung alveolar interval width slightlyincreased. A small quantity of lymphcyte soakage. Air contained in a greatnumber of alveolus is enough. Individual lung alveolus contains edemafluid inside. The protection effect of PDS to lung tissue showed dependenceon dosage. ④ The lung pathogenic changes in PDS-IFN group is lighterthan in IFN group and in PDS group. It shows a light change of interstitialpneumonia.3. Functions of macrophage in different group: Measurement of IL-1level in supernatant fluid of celiac macrophage culture: 15ds after A/fm/1/47( H1N1) virus infection, IL-1 activity in VIG before LPS stimulation ishig hest, but the increase range of IL-1 activity after LPS stimulation is farinferior to that in PDS group and in PDS-IFN group. The results suggest thatmacrophage is highly activated and release large amount of IL-1, its reserveability descend. However, PDS can repress the excessive activation ofmacrophage to protect its reserve ability. Therefore, IL-1 activity in PDS andPDS-IFN group after LPS stimulate is obviously higher than before LPSstimulate.4. immunity organs and its functions.(1)thymus gland coefficient and spleen coefficient:15ds after A/fm/1/47( H1N1) virus infection, the thymus gland weight descend(2.28+1.58)obviously in VIG, but has no difference in PDSG(5.12+2.24) and PDS-IFNG(3.84+0.95) compared with Control group. The thymus gland weight inIFN group also descend after infection compared with control group. Thedifference is significant. The variety trend of spleen coefficient is similar tothe thymus glands. It is obvious that PDS can raise thymus gland weightobviously and has obvious protection functions to immunity organs.(2) Mitogenesis of spleen and thymic cell induced by ConA: 15ds afterthe A/ fm/1/47( H1N1) virus infection, the self-proliferation of spleen cell andthymic cell in VIG is higher than other groups, but mitogenesis induced byConA lowers obviously. This results prompt that PDS and PDS-IFN treatmentcan reduce IL-1 level.(3) IL-2 level in supernatant fluid of spleen cell culture in differentgroups: 15ds after A/ fm/1/47( H1N1) virus infection, IL-2 level beforestimulation by ConA in VIG is higher than in control group significantly, butafter stimulation, IL-2 level in VIG has no elevation. The results shows thatthe spleen cells are activated continuously to release great quantities of IL-2and other cytokines which have already had no storage ability. The PDS andPDS-IFN have no obviously promotion to IL-2 level.(4)Immunity organization chemistry of IL-2 in thymus gland andlungs tissue: 15ds after A/ fm/1/47( H1N1) virus infection, macrophage ofthymus gland and lung in VIG shows strong positive staining and activatedstate. But IL-2 expression level in PDS and PDS-IFN group show a lowerstaining than in VIG. The IL-2 staining density in IFN group is a little bitlower than in VIG, but stronger than in PDS and PDS-IFN group.5. Effects of IFN, PDS and PDS-IFN on NF-κBp65,NF-κBp50 expresslevel in lung and thymus gland.The results of Western blot analysis and immunity organization chemistryshowed that 15ds after A/ fm/1/47( H1N1) virus infection, macrophage andlyphocyte of thymus gland and lung in VIG shows strong positive staining andactivated state. But NF-κBp65 expression level in PDS and PDS-IFN groupshow a lower staining than in VIG. The NF-κBp65 staining density in IFNgroup is a little bit stronger than in VIG, but very stronger than in PDS andPDS-IFN group. The results of NF-κBp50 immunity organization chemistryshowed that NF-κBp50 expressed mainly in bronchial epithelium. The resultsof NF-κBp50 Western blot analysis showed that PDS and IFN-PDS mixturecould increase NF-κBp50 expression.6.Caspase-3 expression in lung showed strong possive staining afterinfluenza virus infection and also in IFN treatment group. But in PDS andIFN-PDS treatment group, the expression of caspase-3 decreased. The resultsshowed PDS could inhibit the apoptosis induced by influenza virus infection.7. Effects of IFN, PDS and PDS-IFN on blood serum AST, ALT andLDH level:Serum AST, ALT and LDH level increased obviously in VIG, promptthat virus infection cause the injury of cell membrane structure . IFN also hasthe effect of increase blood serum enzymes activity in same level to VIGgroup. The increasing range of blood serum enzymes has dependence on thedosage which suggest that IFN has no effect to stabilize cell membranestructure. However, three kinds of blood serum enzymes are lowersignificantly in different dosages of PDS groups than in VIG. The resultssuggest that PDS has strong ability to protect the function to the cellmembrane structure. The application of PDS-IFN unites show complementeffect to each other.8. Effects of IFN, PDS and PDS-IFN on blood serum SOD and MDAlevel(1)15ds after the A/ fm/1/47( H1N1) virus infection, SOD activity inVIG is obviously lower than in control group( P<0.05). The SOD level in lowdosage IFN group is close to control group and higher than VIGgroup( P<0.05). The SOD level in middle and high dosage IFN group isobviously lower than in the control group, has no significant difference to thatin VIG( P>0.05). In low,middle and high three different PDS groups, SODlevel is lower than in Control group, but all higher than in VIG. The SOD levelin high, medium and low IFN-PDS group are all obviously higher than inVIG( P<0.05 or<0.01). The results suggest that IFN-PDS raise the function ofSOD activity remarkably better than two kinds of singles. The results showstwo medicines have cooperative effects raise the activity of SOD and improveoxygen free radicals clearance.(2)The blood serum MDA content in VIG is higher than in Controlset( P<0.05). IFN-PDS is better than PDS in the effect of lowering MDA level.The middle and high dosage IFN has the similar effect of increment oxygenfree radicals to VIG group so that promotes lipid superoxidation and cause thecell membrane structure injury. It is obvious that PDS has the effects toeliminate oxygen free radicals and stabilize cell membrane, showing strongcell protection function, thus easing the cell injuty caused by virus infection.Therefore, PDS-IFN consociation application in the prevention andtreatment of A/ fm/1/47( H1N1) virus pneumonia provide experiment andtheoretical support to the prevention and cure of flue and SARS.Conclusions:1. We use 10 times LD50 A/ fm/1/47( H1N1) virus successfullyduplicate a progressive interstitial pneumonia model, not only expressingtypical pathologic and histological changes, but also very obvious excessiveinflammatory response.2. A series experiment confirmed that IFN-PDS mixture showed anobvious cooperative effect with each other, not only for the protection functionof the lung histology, but also repressing the excesssive excitement to miceimmune system after virus infection and easing the systemic inflammatoryresponse. It is better than single IFN or PDS treatment .3. IFN has obvious and exact anti-virus effect. But high dosage IFN haseffects of inducing excessive inflammatory reaction including IL-2, IL-1activity and NF-κBp65 protein expression increasing, similar to influenzavirus infection. In the process of killing virus, IFN also caused increment ofthe oxygen free radicals and the lipid of cell membrane structuresuperoxidization so much as organs dysfunction. It suggested that the bestdosage of IFN is 4000IU/Kg.4. It has been confirmed that PDS has no direct anti-virus effects inprevious vitro experiment. But PDS has functions to repress NF-κBp65protein excessive expression, decrease expression and activity of IL-2, reduceIL-1 level during inflammatory reaction, thus inhibit excessive stressfulreaction. At the same time, PDS reduced the oxygen free radicals creation,increased SOD vitality and eased lipid superoxidization. PDS reducedcaspase-3 expression in lung tissue and inhibit apoptosis after influenza virusinfection to protect the function of cells and organs. Thus, PDS eased the sideeffect of IFN.Therefore, the consociation application of the IFN-PDS showed that twokinds of medicine is in conjunction with the functions, will become idealmedicine to treat and prevent virus pneumonia and SARS.