Pterostilbene Alleviates Oxidative Stress And Airway Inflammation In Asthmatic Mice Through AMPK/Sirt1/Nrf2/HO-1 Signaling Pathway

Objective: To study the pharmacological effect and mechanism of pterostilbene(Pts)on oxidative stress and airway inflammation in asthmatic mice.Methods:In vivo experiment: 1.Observe the effect of different concentrations of Pts(25,50 μ M)on the lung histopathology of asthmatic mice;2.The number of inflammatory cells was determined by Diff-Quik staining;3.The levels of cytokines in BALF were detected by ELISA;4.The changes of airway hyperresponsiveness(AHR)were measured by airway high reactivity assessment;5.Colorimetry was used to detect SOD and CAT activity in serum and MDA content;6.Western blot: the relative expression of AMPK,Sirt1,Nrf2,HO-1 were detected;7.Immunohistochemistry: the expression levels of p-AMPK,Sirt1,Nrf2,HO-1 were detected;8.Immunofluorescence: the expression level of p-AMPK protein was detected.In vitro experiments: 1.LPS was used to stimulate 16 HBE cells to produce inflammatory response.MTT method was used to set up different concentrations of Pts(15,30 μ M),and the effects of these on the activity of 16 HBE cells were analyzed;2.The cytokines PGE2,TNF-α and IL-1 β were detected by ELISA;3.NO content was detected by Griess method;4.Flow cytometry was used to detect ROS expression;5.Western blot was used to detect the relative expression of i NOS,COX-2,AMPK,p-AMPK,Nrf2,HO-1 and other proteins;6.The expression levels of ROS and p-AMPK protein were detected by immunofluorescence.Results:The results of in vivo experiment were as follows:1.Pts can inhibit inflammatory cell infiltration and goblet cell proliferation in the lung of asthmatic mice(P < 0.05);2.The number of inflammatory cells in BALF decreased in the Pts group,and the number of inflammatory cells in the BALF of asthmatic mice decreased in dose-dependent manner,and the number of inflammatory cells in OVA induced inflammatory response was decreased by Pts(P < 0.05);3.Pts significantly reduced the levels of IL-4,IL-5,IL-13,total Ig E and OVA specific Ig E in BALF,but the expression level of IFN-γ was opposite(P < 0.05);4.Pts decreased AHR(P < 0.05);5.Pts can improve SOD and CAT activity,but it has negative effect on the increase of MDA expression(P < 0.05),and Pts reduces oxidative stress level of asthma mice caused by OVA;6.Pts promoted the expression of p-AMPK,Sirt1,Nrf2 and HO-1 proteins(P <0.05).The results of in vitro experiments were as follows:1.The concentration of Pts had no effect on the activity of 16 HBE cells;2.Pts can inhibit the expression of IL-6,TNF-α and IL-1 β in 16 HBE cells(P <0.05),which is beneficial to the reduction of inflammatory response in 16 HBE cells;3.Pts is not conducive to the expression of NO,PGE2,i NOS and COX-2(P <0.05),which can reduce the activation of 16 HBE cells treated by LPS;5.Pts can reduce the ROS expression level of 16 HBE cells,which is not conducive to the occurrence of oxidative stress,thus enhancing the ability of antioxidation;6.Pts is beneficial to the expression of p-AMPK,Sirt1,Nrf2 and HO-1(P <0.05).Conclusion:1.Pts may relieve airway inflammation in asthma by regulating Th1/Th2 immune imbalance;2.Pts may activate AMPK/Sirt1/Nrf2/HO-1 signaling pathway to reduce oxidative stress and airway inflammation in asthma model mice.