The Effect Of Resveratrol On Airway Inflammation And AHR Induced By RSV And Its Mechanism

PARTⅠTHE ANIMAL MODEL ESTABLISHMENT OF CYP INDUCED IMMUNOCOMPRISED MICE WITH RSV INFECTIONObjective: To establish suitable animal model with RSV infection, cyclophosphamide (CYP) induced immunocompromised BALB/c mice used to explore whether they were susceptible to RSV infection or not.Methods: BALB/c mice were divided Control (PBS) group, PBS+RSV group, and CYP+RSV group, and were treated with CYP intraperitoneally 100mg/kg. After five days, mice were infected intranasally with 3.4×10~5 PFU of RSV in a volume of 100μl. One set of animals was monitored for weight loss from CYP treatment to 15 days after RSV infection. Others were sacrificed five days after infection. Their bronchoalvo larlavage fluid (BALF) was for total cells and styles, and lungs were for determination of RSV titers with plaque assay, histopathology, as well as lymphocyte styles with flow cytometry. Results: Weight loss of CYP-treated mice with RSV infection was lower than untreated mice with RSV infection. A decreased percentage of macrophages and an increased number of total cells and percentage of lymphocytes in the BALF of CYP-treated mice compared to untreated mice. The CYP-treated mice after RSV infection exhibited augmented pulmonary inflammation compared to PBS-treated mice after RSV infection. CYP-treated mice with RSV infection showed higher RSV titers in their lungs of than PBS-treated with RSV infection. The results of flow cytometry showed that percentage of CD19 was significantly decreased after CYP-treated, and percentage of CD49b had an increase, but there were no differences in CD3 percentage. Interestingly, after RSV infection, there was a significant decreased number of CD19 in lungs of CYP+RSV group compared to PBS+RSV group.Conclusion: Immunocompromised BALB/c mice treated by CYP with RSV infection showed that more serious pulmonary inflammation, higher virus titer, which was related with B cell decreased after CYP-treated, resulted in BALB/c mice immunocompromised and reduced antibody production. PARTⅡTHE EFFECT OF RESVERATROL ON AIRWAY INFLAMMATION AND AHR INDUCED BY RSVObjective: To observe the impact of resveratrol to airway inflammation and AHR induced by RSV.Methods: BALB/c mice were divided into Control group, RSV+PBS group and RSV+RES group, treated with CYP. After 5 days, RSV was inoculated intranasally, one or two hours later, mice were received intraperitoneal injections of resveratrol 30mg/kg/day or placebo PBS, and treatments were repeated daily for 5 days. After treatment 5 days, AHR was measured by means of whole-body plethysmography. Their BALF was removed for total cells and leukocytes classified. Lungs were used to measure RSV titer with plaque assay and histopathology with H&E.Results: After resveratrol treatment to mice with RSV infection, RSV titer was lower than placebo group. A decreased percentage of lymphocyte and a decreased number of total cells, but an increased percentage of macrophage in BALF of RSV+RES group compared to RSV+PBS group. RSV+RES group exhibited fewer leukocytes in pulmonary inflammation with histopathology compared to RSV+PBS group. The Penh value after RSV infection was significantly higher than that of Control group at concentration between 12.5-50.0mg/ml of methacholine. In RSV+RES group, the Penh value was significantly reduced compared with that of RSV+PBS group at concentration between 12.5-50.0mg/ml, but was a little higher than Control group.Conclusions: Resveratrol can reduce RSV titer in lung tissue of mice with RSV infection, inhibit RSV replication in mice, and attenuate airway inflammation and AHR induced by RSV. PARTⅢMECHANISM OF INHIBITION OF RESVERATROL ON AIRWAY INFLAMMATION AND AHR INDUCED BY RSVObjective: To explore the mechanism of inhibition of resveratrol on airway inflammation and AHR after RSV infection.Methods: BALB/c mice were divided into Control group, RSV+PBS group and RSV+RES group, treated with CYP. After 5 days, RSV was inoculated intranasally, one or two hours later, mice were received intraperitoneal injections of resveratrol 30mg/kg/day or placebo PBS, and treatments were repeated daily for 5 days. After 5 days, mice were sacrificed. Cytokines expression was detected by ELISA. TLR3 and M2 receptor gene expression were measured with Q-PCR. Western blot was used to test TRIF protein expression. To further assess the effect of IFN-γon airway inflammation and AHR during RSV infection, treated RSV-infected mice with a neutralizing antibody against IFN-γ. Detected total cells and leukocytes styles in BALF, and measured AHR with whole-body plethysmography.Results: After RSV infection, IFN-γlevel was higher than Control group, but there were no differences in IL-17, IL-4, IL-10, IL-12 and IL-6. Resveratrol treated mice after RSV infection, IFN-γlevel in RSV+RES group was decreased compared to RSV+PBS group, no difference between RSV+ RES and Control group. Against IFN-γafter RSV infection, total cells and percentage of lymphocytes were reduced, but percentage of macrophage was increased. The Penh value was significantly low at concentration 12.5-50.0mg/ml of methacholine, but a little higher than Control group. Q-PCR showed that resveratrol can down high TLR3 expression and up regulate low M2 receptor expression after RSV infection, but M2 receptor was not changed after treating with anti-IFN-γcompared to RSV+PBS group. Western blot found that TRIF expression was up regulated after RSV infection, and resveratrol treated mice, TRIF expression was inhibited in RSV+RES group compared to RSV+PBS group.Conclusion: IFN-γplayed an important role in airway inflammation and AHR induced by RSV. Resveratrol reduced IFN-γproduction after RSV infection, which may inhibit TRIF expression of TLR3/4 signaling pathway, to prevent airway inflammation and AHR induced by RSV.