Effects Of Berberine On The NLRP3 Inflammasome In Influenza Virus-infected Macrophages

Viral pneumonia is an acute respiratory virus infection disease,one of the most serious complications after influenza virus infection.As a clinical common disease and frequently occurring disease,viral pneumonia has the characteristics of varity and change quickly.In addition to the symptoms of cyanosis,continuous high fever and breathing difficulties,it may lead to respiratory distress syndrome,shock and even death.Berberine is a kind of isoquinoline alkaloid,which extracted and separated from Chinese herbal medicine,has extensive pharmacological functions,especially its anti-inflammatory function attract widely attention.Our preliminary results indicated that,Berberine has a therapeutic effect on mice with influenza viral pneumonia and significantly inhibited effect on a variety of inflammatory substances produced by the lung after mice infected with influenza virus.Recently NLRP3 inflammasome become research focus in the role of inflammatory.Objectives1、This study base on previous studies,RAW264.7 macrophages infected by influenza virus PR8 was employed as a model,NLRP3 inflammasome as a pivotal point,to research the molecular mechanism of berberine against influenza viral infections and provide theoretical and experimental basis for the interpretation of the molecular mechanism of berberine inhibit excessive inflammasome and treat viral pneumonia.2、in order to further study the precise mechanism of berberine effects on NLRP3 inflammasome,NLRP3 shRNA recombinant plasmids were constructed and NLRP3 gene was cloned,respectively prepare for the establishment of NLRP3 silencing cell model and NLRP3 overexpressing cell model.MethodsThis study was divided into two parts:Part one The effects of berberine on NLRP3 inflammasome in macrophages infected by influenza virus1、MTT was used to detect the maximum non-toxic concentration of berberine on macrophages,which was 6.25ug/mL.TCID50 was detected by cytopathic effect after Influenza virus PR8 infected MDCK cells,which was 10-4.6/mL.Macrophage cells(RAW264.7)was employed as a model,and divided into normal group,virus infection group and berberine intervention group.Viral infection group was treated with 100TCID50 influenza virus for 24 hours,meanwhile berberine intervention group was treated with 100TCID50 influenza virus and the maximum non-toxic concentration of berberine at the same time,the cells were same time,the cells were harvested for detection after 24 hours.2、Cellular RNA were extracted by Trizol method,Real-time PCR was used to detect the effect of berberine on the mRNA expression of IL-1 β、IL-18 and NF-κB in macrophages.3、Total RNA was recovered from RAW264.7 cells using Trizol reagent,Real-time PCR analysis of the effect of berberine on NLRP3 inflammasome key molecules in macrophages infected by influenza virus.4、Extracted protein from RAW264.7 cells,Caspase-1 activity test kit was used to detect the effect of berberine on Caspase-1 activity in macrophages which infected by influenza virus.5、Extracted cells protein,Western blot was used to detect the effect of berberine on the expression of NLRP3 inflammasome and IL-1β protein in macrophages infected with influenza virus.Part two NLRP3 gene cloning and construction of recombinant plasmid of nlrp3 shRNAExtracted RNA from cells,Reverse transcription reaction was performed to obtain cDNA.Use this cDNA as a template,after 1st and 2nd PCR amplification,the PCR product was recovered and connected with the T carrier.Then the product was sequenced,and the sequence of NLRP3 gene cloning was successful.Use OligoEngine’s shRNA design software,siRNA target sequences of the mouse gene NLRP3 were scanned,and four simple target secondary structure,high specificity and the reasonable GC value of siRNA sequences were selected from the candidate siRNA sequences,to design a corresponding shRNA which convenient expression vector construction insertion,and the synthesis of single stranded oligonucleotides can be complementary,but at the ends of the HandⅢ and XhoⅠ enzyme cleavage sites.Two complementary oligonucleotide chains annealed in PCR instrument,a double stranded DNA with a sticky end was formed after annealing,and then connected pGHl expression vector to downstream enzyme cutting sites of H1 promoter,constructed expression vector which can be sustained and stable express the corresponding shRNA,four shRNA expression vectors were sequenced and identified to ensure that the sequences of shRNA expression vector were constructed correctly.Results1、Compared with normal group,the mRNA expression of inflammatory cytokine IL-1β、IL-18 and transcription cytokine NF-κB are significantly increased,Respectively had a relative expression of 4.76 times,6.3 times and 4.5 times compared with that in the normal group.Berberine intervention group compared with viral infection group,cytokine IL-1 beta,IL-18 and nf-kappa BmRNA expression were decreased,respectively had a relative expression of 1.2 times,2.2 times and 1 times compared with that in the normal group.The difference was statistically significant.2、levels of NLRP3 inflammasome(mRNA)were higher in the virus infection group than in the normal group,NLRP3、ASC and Caspase-1 mRNA respectively had a relative expression of 2.9、2.86 and 2,55 times in the virus infection group compared with that in the normal group.The mRNA expression of NLRP3、ASC and Caspase-1 in berberine intervention group was lower,0.55 times,1.5times and 1.32 times as much as that in the normal group.There were significant differences between groups.These results showed that berberine could reduce the mRNA expression of NLRP3、ASC and Caspase-1.3、compared with the normal group,Caspase-1 activity was higher in the viral infection group,2.22 times as much as that in the normal group.Berberine intervention group was 1.07 times as much as that in the normal group,sifnificantlt lower than that of the virus infection group,and the difference was statistically significant.4、compared with normal group,virus infection group of Caspase 1 protein expression increased;Compared with viral infection group,berberine intervention group protein expression is reduced,and the difference was statistically significant.NLRP3、ASC and IL1β protein expression,compared with normal group,Viral infection group were increased,and the difference is statistically significant;Compared with viral infection group berberine intervention groups were decreased,but the difference was not statistically significant.5、The sequence of NLRP3 gene cloning was successful.6、The sequences of shRNA expression vector were constructed correctly.Conclusion and SignificanceThe above results suggests that,NLRP3 inflammasome formatted in macrophages after infected with influenza virus,resulting in Caspase-1 activation and produce a large numbers of IL-1βand IL-18.Berberine may through interventing NLRP3 inflammasome formation and inhibiting Caspase-1 activation,thus reduce IL-1β and IL-18 secretion in macrophages which infected by influenza virus.This study provide a certain theoretical and experimental basis for elucidating the mechanism of berberine against influenza virus infection.This study also successfully cloned NLRP3 gene and constructed shRNA recombinant plasmids targeted on NLRP3.Which would provide basis for establishing NLRP3 overexpression cell model and NLRP3 gene silencing cell model,and further studying the effect of influenza virus and berberine on NLRP3 inflammasome.