Effect Of Ginsenoside Rb1on H9N2-SIV-Induced Pulmonary Oxidative Stress Injury And Toll-Like Receptor4Signaling Pathway In Mice

Influenza is an acute infectious respiratory disease caused by influenza virus,often causes regional and even global popularity. Influenza virus hemagglutinin andneuraminidase have variation characteristics, because the evolution and theemergence of new mutations causepeople concern that these viruses may cause humanflu pandemic. Pigs may serve as "mixing vessels" for the generation ofhuman-avianinfluenza A virus reassortants in influenza epidemiology, gets people attention due toclosely related to human influenza epidemics. Flu virus infracts mainly the respiratorysystem, causes acute lung injury.The pathophysiology of ALI is complex andinvolvesa complicated array of molecular, cellular, andphysiological mechanisms.However,studies have shown that oxidative stress and TLR4signaling pathways is akey pathway of acute lung injury. Based on this, studying the role of the resistance tooxidative stress and related signaling pathway on acute lung injury induced by virus，and screening safe and effective antiviral, anti-stress drugs become a starting point forthe prevention and treatment of influenza virus on the present study.Studies demonstrate that ginsenoside Rb1(G-Rb1)have a wide variety ofbiological activities including antioxidant, scavenge free radicals, improve immunityin many inflammatory injury diseases, but whether G-Rb1can protect against acutelung injury induced by virus, literature was reported rarely. Therefore, it has importantresearch significance that this paper discusses the effect of G-Rb1on free radical andrelated enzymes and the role of TLR4signaling pathways in acute lung injuryinducedby virus.Based on the above research background and analysis, in this study，establishedthe model of mice acute lung injury induced H9N2-SIV.To study effect of G-Rb1onALI induced oxidative stress,free radical and enzyme in lung tissue were tested; Toanalyse the role of TLR4signaling pathway in acute lung injury and G-Rb1on itsinfluence, TLR4in lung tissue was tested,for exploring G-Rb1role in acute lunginjury induced by virus to provide theoretical guidance.1. Model that acute lung injury induced virus replication: through chickenembryo culturing H9N2-SIV, and to determine the median lethal dose (LD50).SPF BALB/c mice were inoculated intranasally with H9N2-SIV diluted in sterile saline,vaccination dose was0.1mL (about1310-3LD50) per, taking lung tissue on the2nd,4th,6th,8th,14thday respectively, weighing and measuring lung coefficient, Grossanatomy was observed, the left lung tissue of the same position was made tissue sec-tions using HE staining, test wet-weight and dry-weightratio with right lung,evaluating index of lung injury comprehensively; At the same time point, taking miceventricular arterial blood for hemodynamics; Survival situationwererecorded andcumulative survival rateanalysiswas performed with the use of Kaplan-Meier analysis.The results showed,the beginning of the3rdday breathing was extreme difficulty,breath sounds were obvious, feed intakereduction and body weightloss obviously，anddeath.Pulmonary edema, hemorrhage, lung volume increased. Lung wet weight dryweight ratio and lung coefficient increased obviouslyon the2ndday, reaches thehighest on the6thday, declines on the8thday.Observating HE staining slice, on the2ndday,it is found alveolar, bronchioles and interstitial around small blood vesselsedema, inflammatory cells and exudate, on the4thday, it appeared interstitialpneumonia lesions, alveolar space smaller, hemorrhage,on the6thday, inflammatorycells exudation, it formed bronchial pneumonia. According to the results ofhemodynamics,blood oxygen partial pressure reduced and elevated CO2partialpressure, appeared severe hypoxemia, lack of oxygen exchange, Dysfunctionalventilatory. The model was builted successfully by the above index.2.To study the effect of G-Rb1on acute lung injury in mice induced oxidativestress byH9N2SIV, inhibition ability of hydroxyl radical (OH), malondialdehyde(MDA) and nitric oxide(NO) content of mouse lungs tissue were detected.Experimentset up three groups: The mice in control group were inoculated Intranasally with0.1mL dilution of noninfectiousallantoic; And that of acute lung injury group (ALI group)and ginsenoside Rb1(G-Rb1group) both were inoculated intranasally withH9N2-SIV diluted in sterile saline,and meanwhile, animals of G-Rb1group weretreated with G-Rb1(10mg·Kg-1bw) by oral for up to seven days.The mice were sacrificed on2nd,4th,6th,8th,14thday post inoculation respectively and their lungswere removed and processed as described below. wet/dry weight ratio of lung tissueand lung coefficient were determined，gross anatomy and histologic biopsy (HEdyeing tissue sections) were observed. Homogenates were used to test inhibitionability of OH,content ofMDAand NO.The results showed that the clinical symptoms of the ALI group mice, the grossanatomy and pathologic histology observation was similar to the result of replicationmodel, symptoms of the G-Rb1group was significantly lighter than that of the ALIgroup. Cumulative survival rate of the ALI group gradually declined from66.7%(the3rdday) to26.7%(the5thday); Cumulative survival rate of the G-Rb1group graduallydeclined from80%(the4thday) to53.3%(the6thday), survival time was prolongedsignificantly (P<0.05), and mortality was decreased;on the2ndday~6thday Afterlunginfection, lung wet/dry quality ratio of the ALI group and the G-Rb1group bothshowed a rising trend, lung wet/dry quality ratio of the ALI group was higher than thatof the control group significantly (P<0.01), lung wet/dry quality ratio of the G-Rb1group was extremely higher than that of the control group significantly (the4th~6thday, P<0.01), lung wet/dry quality ratio of the G-Rb1group was significantly smallerthan that of the ALI group (P<0.05); on the8thday after infection mice began torecover gradually, lung wet/dry quality ratio was lower, but there are still differencescompared with the control group significantly (P<0.01); Compared with the ALIgroup, difference was very significant. On the14thday after infection, the lung wet/dryquality ratio close to the normal. Lung coefficient and lung wet/dry quality ratio wasboth similarexcept that two groups compared with the control group had not obviousdifference on the2ndday.Compared with the control group,the content of ON were increased significantly(P<0.01) in the ALI group and the G-Rb1group mice from the2ndday to the6thday,and the content of NO was on the rise up to the8thday. The difference betweenthe ALI group and the G-Rb1group was significant, the content of NO in the ALI group washigher than that of the G-Rb1(P<0.01).Comparedwith control group, thechanging tendency of the MDA content and inhibition ability of OH were similar tothe changing tendency ofNO content, the content of the MDA and NO, inhibitionability of OH in the ALI group were significantly higher than that of G-Rb1groupfrom the4thday to the6thday (P<0.01). On the14thday, the content of NO, MDA andinhibition ability of OH in two groups tend to be normal.3. To Study effect of ginsenoside Rb1on antioxidant enzyme in acute lung injuryof mice induced by A/swine/HeBei/012/2008influenza virus,at the same time, the ac-tivity of T-SOD, MPO, CAT and GSH-PX both were detected in mouse lungs,in orderto evaluating protective effects of G-Rb1on the mice lung injury caused by oxidativestress.The results showed,from the2ndday to6thday post inoculation, the activity ofT-SOD in both the ALI group and the G-Rb1group mice were decreased, the activityof T-SODbegan to increase on the8thday. on the2ndday,the activity of T-SOD wassignificantly decreased in the G-Rb1group compared with that of the control groupmice depression (P<0.05), On the4th,6thand8thday after infection,the activity ofT-SOD,CAT both were significantly decreased (P<0.01) in the ALI group and theG-Rb1group mice compared with that of the control group mice,on the14thday,thedifference have not significantly. But from the4days to6days, the activity of T-SODof the G-Rb1groupwas higher than that of the ALI group (P<0.01); From the2nddayto the6thday, the activity of CAT of both the G-Rb1group and the ALI group weresignificantly decreased (P<0.01),and the activity of CAT began to increase on the8thday. Compared with the control group,the activity of CAT of both the G-Rb1groupand the ALI group were significantly decreased on the4th,6th,8thdays (P<0.01),Butthe active of CAT in the G-Rb1group was higher than that of the ALI group,Thedifferences had significant statistically significant (P<0.01).From the2ndday to14thday, the activity of GSH-PX in the ALI group were significantly decreasedcompared with that of the control group (P<0.01),on the contrary, the activity of GSH-PX in the G-Rb1group were significantly increased (P<0.01),and the activity ofGSH-PX in the G-Rb1group was higher than that of the ALI group (P<0.01),thedifference had significant statistically significant (P<0.01). Compared with the controlgroup,the activity of MPO was significantly increased in the ALI group(P<0.01),despite the activity of MPO was increased from the8thday to the14thday,but still higher than that of the control group.The activity changes of MPO in theG-Rb1group was similar to the ALI group,the activity of MPO was significantlyincreased in the G-Rb1compared with that of the control group on the4thday,the6thday and the8thday (P<0.01),but the degree improved was lower than the ALI group(P<0.05).4. To study effect of G-Rb1on ALI mediated by oxidative stress and TLR4signaling pathway, TLR4protein levels and TLR4mRNA expression in the lungtissue were detected by Western blot and real-time quantitative PCR technologyrespectively.The results showed,the TLR4mRNA expressiondisplayed the evident increasingtendency in the ALI group and the G-Rb1group from the4thday to the6thday,and theTLR4mRNA expression in the ALI group and the G-Rb1group are higher than thatof the control group, with significant statistical significance (P<0.01).the G-Rb1group expression was belower than the ALI group, with significant statisticalsignificance (P<0.05); On the8thdays,the TLR4mRNA expressiondisplayed theevident increasing tendency in the ALI group and the G-Rb1group,but the TLR4mRNA expressionin the ALI group and the G-Rb1group still are both higher than thatof the control group,with significant statistical significance(P<0.01);The TLR4mRNA expression in the G-Rb1group was lower than that of the ALI group,whichhas significant statistical significance (P<0.01); On the14thday, the TLR4mRNAexpression of two groups has tended to be normal.TLR4protein expression and TLR4mRNA expression have almost the sametrend.the TLR4protein expression in the lung tissue of the ALI group and the G-Rb1 group were obviously increased than that of the control group from4thday to8thdayspost inoculation, which had significant difference (P<0.01), and showed obviousrising trend; After the8thdays the TLR4protein expression both in two group began tofalling, on the14thday it returns to normal. The TLR4protein expression in theG-Rb1group was obviously lower than that of the ALI group from4thday to8thdays,which hadsignificant statistical significance (P<0.01).In summary, Infection of A/masty swyn/Hebei/012/2008(H9N2) virus couldinduceacute lung injury in BALB/c mice,the BALB/c mice may constitute a suitablemodel for the study of swine influenza virus infections; Oxidative stress is a majorcause of acute lung injury.Active oxygen free radicals, such as NO, OH,overproduction of ROS causes oxidative stress, and leading to lung tissue damage.G-Rb1can scavenge reactive oxygen free radicals such as OH and inhibit thegeneration of reactive oxygen free radicals such as NO; G-Rb1can regulate enzymereaction system,balance between production and scavenging of free radicalscausesoxidative stress，G-Rb1can increase the activity of SOD, GSH-PX and CAT anddecrease the activity of MPO; TLR4signaling pathway is also involved in the acutelung injury induced by oxidative stress, G-Rb1can decreases the levels of TLR4protein and mRNA in mice lung tissue. To a certain extent, G-Rb1can attenuate acutelung injury caused by oxidative stress via swine influenza virus.