Study On The Correlation Between Theaflavin Alleviated Atherosclerosis And Macrophage Pyroptosis

Objective:To investigate the effect of theaflavin on alleviating the development of Atherosclerosis through regulating the pyroptosis of macrophage.Methods:1.Construction of stable cell lines for Gsdmd knockout in macrophages.(1)Screening of effective sgRNA sequencesSgRNA(Single guide RNA)cloning into viral vector,identify sgRNA efficiency by using T7 nucleic acid endonuclease I(T7 endonuclease I,T7E1)enzymatic cleavage and sequencing.(2)Knockout of Gsdmd gene in macrophage Ana-1Stable cell lines were divided into three groups:Ttg-Scramble(control),Ttg-Gsdmd-Crl and Ttg-Gsdmd-Cr2.The mutation of the knockout cells were detected by T7 enzymatic digestion and sequencing.The knockout efficiency of target cells were detected by real time quantitative polymerase chain reaction(RT-qPCR)and Western blot(WB).2.Modulation of pyroptosis-related genes in macrophages by theaflavin under different stimuliEffects of different concentrations theaflavin on the genes and proteins of pyroptosis-related genes(1)Effects of pyroptosis-related genes and proteins in wild-type macrophage by theaflavin in different concentrationsAfter wild-type macrophage Ana-1 was induced by oxidized low density lipoprotein(ox-LDL),it was treated different concentrations of theaflavin(0,20,40,80,160μM).The mRNA expression of Caspase-1,Gsdmd,NLRP3,IL-1β and IL-lα pyroptosis-related genes and inflammatory genes were detected by RT-qPCR;WB was used to detect the expression of caspase-1 and NLRP3(2)Increaseing the effect of theaflavin on pyroptosis-related proteins by knockout of GsdmdControl group:Ana-1-Scramble-1,Ana-1-Scramble-2 and Ana-1-Scramble-3Gsdmd knockout group:Ana-1-Gsdmd-Cr 1,Ana-1-Gsdmd-Cr2 and Ana-1-Gsdmd-Cr 1-10-2(Ana-1-Gsdmd-/-).1)Theaflavin inhibited VBP-induced expression of pyroptosis associated proteins in Gsdmd knockout macrophageAfter induced by the pyroptosis inducer sulfonate(Val-boroPro,VBP),the control and two aforementioned groups of cell were treated with the vehicle and theaflavin,then the protein expression of Caspase-1 was detected by WB.2)Theaflavin inhibiting the expression of pyroptosis-related proteins in foam macrophageAll groups were stimulated with ox-LDL at the same time,the mRNA expression of Caspase-1,IL-1α,IL-1β,IL-18 and NLRP3 related to the pyroptosis were detected by RT-qPCR.Gsdmd knockout group cells were induced by ox-LDL and then treated with theaflavin and vehicle,and RT-qPCR was performed to detect pyroptosis-related genes and inflammatory genes:Caspase-1,IL-1β,IL-1α,IL-18 and NLRP3;WB was used to detected Caspase-1 expression.3.Inhibition of macrophage pyroptosis in vivo by thcaflavin slowed down the formation of Atherosclerosis in miceThe ApoE-/-Atherosclerosis(AS)mice were randomly divided into the PBS group and the theaflavin group.Oil red O(ORO)staining was performed to detect the formation of AS in the descending branches of the aorta of mousemice.The bone marrow macrophages were sorted by CD 14 magnetic bead and the proportion was detected by Flow cytometry.Bone marrow macrophages were collected from mice,the Caspase-1,Gsdmd,and IL-1αexpressions of Bone marrow macrophages were detected by RT-qPCR.Results:1.Screening to obtain valid sgRNA sequences(1)Two sgRNAs were successfully screened:cleavage by nucleic acid endonuclease T7 generated two bands of different sizes,and sequencing of the mutated region showed a large number of double peaks.(2)Constructed Gsdmd knockout cell lines in macrophages Ana-1The flow analysis results showed that the infection efficiency was above 85%,and the monoclonal cells had a common deletion of 330bp in both chromosomes;RT-qPCR results showed that the knockout efficiency of Ana-1-Gsdmd-Cr1,Ana-1-Gsdmd-Cr2 and Ana-1-Gsdmd-/-were 60.86%,61.86%and 99.76%respectively.WB results showed three cells knockout efficiencies of 50.69%,79.9%and close to 100%.2.The effect of theaflavin on the regulation of pyroptosis related genes in macrophages under different stimulus(1)Wildtype macrophages Ana-1 were treated with different concentrations of theaflavin after stimulated by ox-LDL,and RT-qPCR results showed that the expression of Caspase-1,Gsdmd,NLRP3,IL-1α and IL-1βwere significantly dccreased with increasing concentration of theaflavin(p<0.05);Meanwhile,WB results showed that the expression levels of Caspase-1 and NLRP3 protein were decreased.(2)Knockout of Gsdmd enhanced the regulatory effect of theaflavin on pyroptosis related proteins.After ox-LDL stimulation,RT-qPCR results showed that the expression of Caspase-1,IL-1α,IL-1β and NLRP3 in knockout cells were significantly decrease(P<0.05),and IL-18 showed an educed trend compared with control cells.Added the theaflavin in the knockout group cells after treated with ox-LDL,the RT-qPCR results showed that the levels of Caspase-1,IL-1α,IL-1β,IL-18 and NLRP3 showed a significantly decrease(p<0.05);WB results showed that the Caspase-1 expression was significantly decreased(P<0.05).3.Theaflavin inhibited macrophage pyroptosis in vivo to slow down the formation of Atherosclerosis in ApoE-/-miceCompared with control group,Oil red O staining results showed that Atherosclerosis formation were weaked in the theaflavin group in ApoE-/mice fed high-fat diet(HFD)caused Atherosclerosis;Mice bone marrow macrophages were sorted by CD 14 magnetic bead,and then determined the proportion of mice bone marrow macrophages were obtained 90%through flow cytometry;RT-qPCR results showed that Caspase-1,IL-1α and Gsdmd expression were significantly lower in bone marrow macrophages of the theaflavin group(P<0.05)than control group.Conclusion:1.GSDMD knockout macrophage cell lines can be successfully constructed by CRISPR/CAS9 technology.2.Theaflavin can alleviate ox-LDL induced macrophages,which was related to the pyroptosis pathway.3.Theaflavins slow down the development of Atherosclerosis may be related to the inhibition of the expression of factors such as GSDMD,NLRP3,and Caspase-1 in the macrophage scorch pathway.