Berberine Induces Mitochondrial Autophagy To Interfere With The Mechanism Of ROS-NLRP3 Inflammasome Pathway Activated By Influenza Virus

Background and ObjectViral pneumonia is a common clinical disease caused by a variety of respiratory viruses,influenza virus is one of the most common respiratory viruses.Viral pneumonia is a leading complication related to influenza virus infection,which are commonly associated with high mortality in infected patients.At present,the main pathogenesis of influenza virus pneumonia is the excessive inflammatory response and immune pathological damage caused by excessive protective response from the host.This is also the common pathogenesis of clinical severe viral pneumonia such as new coronary pneumonia and SARS.At present,for the treatment of influenza virus pneumonia,there is still no exclusive special treatment drug.Traditional Chinese medicine has the characteristics of multiple targets,multiple mechanisms and multiple links.It has a significant effect on anti-inflammatory and immune regulation,and has obvious advantages in the prevention and treatment of viral pneumoniaBerberine(BBR)is an isoquinoline alkaloid extracted from several Chinese herbs such as Coptis chinensis Franch.,Phellodendron chinense Schneid,and it is currently recognized as a drug with wide anti-inflammatory and immunomodulatory effects.Our previous studies have demonstrated the inhibitory effect of BBR on lung inflammatory injury and the decrease in the mortality of mice with viral pneumonia after BBR treatment,and also significantly reduce the reactive oxygen species(ROS)levels in macrophages infected by influenza virus.However,the precise mechanism underlying these effects is yet unclear.The recent argument of autophagy against inflammatory responses has aroused wide concerns.And related studies have shown that autophagy is involved in the inflammatory response of lung,and the excessive activation of the NLRP3 inflammasome also plays an important role in the inflammatory response of influenza A viral pneumonia.The major source of cellular ROS is mitochondria.Several studies have described the fundamental role of reactive oxygen species(ROS)in the activation of NLRP3 inflammation.mitophagy is the main way to remove damaged mitochondria and prevent the accumulation of ROS.Therefore,this study focuses on the ROS-NLRP3 pathway to investigate whether BBR inhibits NLRP3 inflammasome activation by inducing mitophagy to clear ROS accumulated in tissues or cells infected by influenza virus,thereby suppressing influenza Inflammation caused by the virus to further explore the mechanism of berberine inhibiting excessive inflammation induced by influenza virus.Methods1.Experiments in vitro:Construction of J774A.1 macrophage model infected with influenza virus PR8(1)Set the normal group,model group(PR8,MOI=1),MSU(150μg/mL)group,BBR(16.8 μM)group,PR8+BBR group,PR8+MSU group and PR8+MSU+BBR group.Western blot was used to detect the expression of NLRP3 protein;flow cytometry was used to detect the activity of Caspase1;ELISA was used to detect the level of IL-1β secretion.(2)The intervention group of BBR and the intervention group of Mito-TEMPO(mitochondrial-specific antioxidants,500μM)were set up.Mitochondrial membrane potential(MMP),mitochondrial ROS(mtROS)release levels and caspase1 activity were detected by flow cytometry;Simultaneous detection ofNLRP3 protein expression and IL-1β secretion level.(3)Set the normal group,model group(PR8,MOI=1),PR8+BBR(16.8μM)group,PR8+MSU group and PR8+MSU+BBR group.At the same time set up BBR control group and 3-MA control group.Western blot was used to detect the expression of LC3II and P62 protein;Immunofluorescence was used to detect the co-localization of autophagosome-mitochondrial fluorescence;The structure of autophagy was observed by transmission electron microscopy.(4)The experiment group is the same as(3).Flow cytometry was used to detect the release level of MMP and mtROS and the activity of Caspase1;meanwhile,the expression of NLRP3 protein and the secretion level of IL-1β were detected.(5)The normal group,model group,PR8+BBR group,PR8+CsA(5 mM)group and PR8+CsA+BBR group were set,and the BBR control group and CsA control group were also set.Detect the expression of LC3II and BNIP3 protein in cells of each group by Western blot,and detect the co-localization of autophagosome-mitochondrial-BNIP3 fluorescence by immunofluorescence.Meanwhile,J774A.1 macrophages transfect BNIP3 siRNA to knockdown of BNIP3 gene,Western blot detection of LC3II,BNIP3 protein expression.2.Experiment in vivoThe mouse influenza virus pneumonia model was constructed and divided into normal group,model group,BBR(10mg/kg/d)treatment group and BBR+3-MA(15mg/kg/d)treatment group.Observe the gross lung lesions and lung index of mice in each group.Western blot detected the expression of LC3II,P62 and BNIP3 protein.Flow cytometry was used to detect the ROS release levels in the lung tissues of mice in each group;Western blot was used to detect the expression of Caspase1p20 and NLRP3 protein;Simultaneous detection and detection of NLRP3 protein expression and IL-1β secretion level.Results1.As a result,we found that PR8 infection induced NLRP3 inflammasome activation,as evident from the increase in the expression of NLRP3 protein,induction of caspase-1 activation,and secretion of mature IL-1β.However,BBR treatment decreased NLRP3 protein expression,suppressed caspase-1 activation and IL-1β secretion.Our results indicate that BBR could inhibit the activation of NLRP3 inflammasome.2.Influenza virus infection cause mitochondrial damage,mtROS accumulation and NLRP3 inflammasome activation.However,BBR and Mito-TEMPO can alleviate mitochondrial damage caused by influenza virus infection,reduce mtROS release,inhibit NLRP3 inflammasome activation.Thus,NLRP3 inflammasome activation was associated with mtROS levels.on the other hand,it further showed that the mechanism of BBR inhibiting the activation of NLRP3 inflammasome in macrophages infected with influenza virus may be related to the reduction of damaged mitochondria and the accumulation of intracellular mtROS related.3.BBR treatment significantly upregulated the expression level of LC3Ⅱ and downregulated the protein level of p62.At the same time,we also observed the colocalization of mitochondria and LC3 with confocal microscopy,and transmission electron microscopy can observe mitochondrial autophagosome structure.Taken together,BBR clearly and specifically induced mitophagy in J774A.1 macrophages infected with influenza virus.4.Autophagy/mitochondrial autophagy inhibitor 3-MA can aggravate mitochondrial damage of influenza virus-infected macrophages,increase mtROS accumulation,and further promote the activation of NLRP3 inflammasome;Mitochondrial damage reduces the release of mtROS and reverses the excessive activation of the NLRP3 inflammasome.It indicates that the mechanism of BBR reducing mtROS release and thus inhibiting the excessive activation of NLRP3 inflammasome may be related to its induction of mitophagy.5.BBR can increase the expression of BNIP3 and LC3II proteins and reduce the expression of p62 protein in macrophages intervened by influenza virus,CsA and BNIP3 siRNA.At the same time,BBR treatment enhanced the colocalization of mitochondria,LC3,and BNIP3.the knockdown of BNIP3 expression attenuated the effects of BBR on mitophagy induction to some extent,suggesting that the BBR-induced mitophagy may be,at least in part,mediated in a BNIP3-dependent manner,and may be related to its increased expression of BNIP3 protein.6.Berberine significantly reduce the inflammatory damage of lung tissue in mice with influenza virus pneumonia,increase the expression of LC3Ⅱ and BNIP3 protein in the lung tissue of viral pneumonia mice,and reduce the expression of p62 protein;meanwhile,berberine can reduce the release of ROS in the lung tissue of mice,Reduce Caspase1p20,NLRP3 protein expression and IL-1β secretion levels,and autophagy inhibitors can reverse the effect of berberine.The in vivo experiments verified the results of the in vitro experiments,indicatingthat the mechanism of berberine alleviating the inflammatory damage to the lungs of mice with influenza virus pneumonia is related to its induction of mitochondrial autophagy,reducing the release of ROS,and thus inhibiting the excessive activation of NLRP3 inflammasome.ConclusionsOur results demonstrated that BBR suppressed the activation of NLRP3 inflammasome,which is correlated with the elimination of damaged mitochondria,inhibition of mtROS production and induction of mitophagy,and thus inhibit the excessive inflammation caused by influenza virus response to reduce inflammatory damage caused by influenza virus infection.Berberine-induced mitophagy is partially dependent on the BNIP3 pathway.This study provides a new way to suppress the occurrence of excessive inflammation of influenza virus pneumonia,and provides a new theoretical and experimental basis for the study of the mechanism of berberine treatment of influenza virus inflammation.