Research On The Inhibitory Effect Of Human β Defensin-3 Against Influenza A Virus Based On The Oxidative Stress

Objective:To investigate the inhibitory effect of humanβdefensin 3（HBD3）on influenza A virus（IAV）A/PR8/34/H1N1 infection human bronchial cells（BEAS-2B）based on oxidative stress.Methods:HBD3 was intervened into 100×TCID₅₀IAV infected BEAS-2B cells for 12 h,24h,and 48 h,respectively.The experiment was divided into control group,IAV group,and IAV+HBD3 group.（1）CCK8 method was used to detect the effect of HBD3intervention on the activity of BEAS-2B cells infected by IAV.（2）JC-1 probe was utilized to detect variations in mitochondrial membrane potential（MMP）.（3）DCFH-DA probe was used to determine intracellular ROS content.（4）The content of malondialdehyde（MDA）,glutathione（GSH）and the activity of catalase（CAT）were detected by TBA,DTNB,and peroxidase methods,respectively.（5）Detect the changes of the expression level of Nrf2 pathway related molecules in BEAS-2B cells infected by IAV after HBD3 intervention:（1）The m RNA and protein levels of Nrf2,Keap1,and HO-1 were measured using RT-q PCR and western blotting,respectively.（2）Using Western blotting,the expression of the Nrf2 protein in the nucleus and cytoplasm was determined.2.BEAS-2B cells were pretreated for 2 h with 20 mol/L of the Nrf2inhibitor ML385,and then IAV-infected BEAS-2B cells were cultured for 48 hours with 5 mol/L of HBD3.The experiment was divided into Control group,IAV group,IAV+HBD3 group,ML385 group,ML385+IAV group,ML385+IAV+HBD3 group.（1）The intracellular ROS production was detected using the DCFH-DA probe;（2）The content of MDA and CAT in cells was detected using the MDA and CAT detection assays;（3）Western blotting was utilized to detect the protein expression of Nrf2,Keap1,and HO-1.Results:（1）CCK8 results showed that cell viability was significantly decreased in the IAV group compared to the control group（P<0.01）.Cell viability was significantly increased in the IAV+HBD3 group compared to the IAV group（P<0.05 or P<0.01）.（2）The results of mitochondrial membrane potential detection by JC-1 probe showed that compared with the control group,red/green fluorescence ratio was significantly reduced（P<0.01）in the IAV-infected 24 h and 48 h groups.Compared with the IAV group,HBD3intervention significantly increased red/green fluorescence ratio（P<0.05 or P<0.01）.（3）Detection of intracellular ROS,the results revealed that the ROS content was substantially increased compared with the control group（P<0.01）,while HBD3intervention resulted in a statistically significant reduction in ROS generation（P<0.01）.（4）Intracellular MDA content was found to have significantly increased（P<0.05 or P<0.01）after 12 h,24 h,and 48 hof IAV infection,while CAT activity and GSH content were found to have significantly diminished（P<0.05 or P<0.01）.After HBD3intervention,MDA content significantly decreased（P<0.05 or P<0.01）,CAT activity and GSH content significantly increased（P<0.05 or P<0.01）.（5）The results of the RT-q PCR experiment demonstrated that the expression of Nrf2 m RNA was significantly reduced（P<0.05 or P<0.01）and the expression of the Keap1 gene was significantly increased（P<0.05 or P<0.01）after 12 h,24 h,and 48 h of IAV infection compared with the control group.After 12 h of infection with IAV,there was no discernible change in the transcript of the HO-1 gene（P>0.05）.Following an infection with IAV for 24 h or48 h,the expression of the HO-1 gene was found to be substantially reduced（P<0.05or P<0.01）.In the IAV+HBD3 group,the expression of the Nrf2 gene significantly increased（P<0.05 or P<0.01）while the expression of the HO-1 gene significantly increased（P<0.05 or P<0.01）,Keap1 gene expression significantly decreased（P<0.05or P<0.01）compared with the IAV group.（6）The protein expressions of Nrf2 and HO-1 were significantly decreased（P<0.05 or P<0.01）after 12 h,24 h,and 48 h of IAV infection compared to the control group,while the protein expressions of Nrf2 and HO-1 were substantially increased after HBD3 treatment（P<0.05 or P<0.01）.The expression of Keap1 did not change significantly after 12 h of IAV infection（P>0.05）,but it dramatically increased after 24 h and 48 h（P<0.01）,,and it decreased significantly after HBD3 treatment（P<0.01）.（7）After 48 h of IAV infection,compared with the control group,the expression of Nrf2 protein in the cytoplasm had no significant change（P>0.05）,but the expression of Nrf2 protein in the nucleus was significantly reduced（P<0.05）,HBD3 intervention can significantly increase the Nrf2 protein expression in the nucleus（P<0.01）.2.（1）Comparatively to the control group,Nrf2 and HO-1 protein levels in the IAV,ML385 and ML385+IAV groups were significantly decreased（P<0.01）.The Nrf2 and HO-1 protein levels were substantially decreased（P<0.05 or P<0.01）and Keap1 protein expression was significantly increased（P<0.01）in the IAV+HBD3+ML385 group compared to the IAV+HBD3 group.（2）The intensity of ROS showed that ROS production was significantly increased in the IAV group,ML385 group,and ML385+IAV group compared with the control group（P<0.01）.Compared with the IAV+HBD3 group,the ROS production substantially significantly in IAV+HBD3+ML385 group（P<0.01）;（3）In the IAV group,ML385 group,and ML385+IAV group,the MDA content was substantially increased（P<0.01）and the CAT activity was significantly reduced（P<0.01）compared with the control group.Comparing to the IAV+HBD3 group,the MDA concentration increased（P<0.01）and the CAT activity（P<0.05）decreased significantly in the IAV+HBD3+ML385 group.Conclusions:1.HBD3 has anti IAV（A/PR/8/34）activity,which is related to the reduction of mitochondrial membrane potential and oxidative stress induced by IAV infection in BEAS-2B cells.2.HBD3 can reduce the expression of Keap1 in BEAS-2B cells after IAV infection,dissociate Nrf2 protein from the Nrf-Keap1 complex,promote Nrf2protein entry into the nucleus,and alleviate the oxidative stress response induced by IAV（A/PR/8/34）infection.3.HBD3 alleviates the oxidative stress induced by IAV（A/PR/8/34）infection in BEAS-2B cells,which depends on the Nrf2 signaling pathway.