The Rab27b Knockout Mouse Model Was Used To Study The Mechanism Of Jinhong Decoction Regulating Platelet Function In Sepsis Through The Rab27b Signaling Pathway

PART 1:Objective: To identify and validate the heredity of Rab27 b gene knockout mice model and observe its histopathological changes.Methods: Real time PCR was used to detect the expression of Rab27 b gene in CRISPR/Cas9-mediated Rab27b(-/-)transgenic mice.At the same time,HE staining was used to observe the pathological changes of heart,liver,spleen,lung,kidney and brain in wild type mice and knockout mice.Result: After identification and verification by PCR,F0 successful gene knockout mice could be steadily inherited to F1 generation,and the expanded Rab27 b gene knockout mice had no expression of Rab27 b protein.HE staining showed that Rab27 b gene knockout mice had no significant pathological changes in heart,liver,spleen,lung,kidney and brain tissues compared with normal mice,which could be completely used in experimental study.Conclusion: Rab27 b gene knockout mice model has been successfully constructed and has stable heredity.After expansion,the research can be carried out smoothly.PART 2:Objective: To observe the effect of Jinhong Decoction on sepsis model in Rab27 b knockout mice.Methods: 120 Rab27 b knockout mice were randomly divided into four groups: knockout gene Rab27b(-/-)group,Rab27b(-/-)sham group,Rab27b(-/-)+CLP group,Rab27b(-/-)+CLP + JHT group,30 mice in each group,30 mice in wild type WT group,and sepsis model of Rab27b(-/-)mice was established by cecal ligation(CLP).Rab27b(-/-)+CLP+JHT group was intragastrically administered with saline and other groups were observed.The changes of body weight,survival rate,PLT,PT,APTT,TT,FDP,ALT and CR were observed at 4 and 72 hours after operation.Results: Body weight,survival rate,PLT,PT,APTT,TT,FDP,ALT and Cr between the wild WT group,the knockout gene Rab27b(-/-)group and the Rab27b(-/-)sham group at 4 h and 72 h after surgery.There was no statistical difference(P>0.05).At 4h and 72 h after operation,the Rab27b(-/-)+CLP group and the Rab27b(-/-)+CLP+JHT group had a weak mental state,and the diet was small,inactive,and squatting.The body weights of Rab27b(-/-)+CLP group and Rab27b(-/-)+CLP+JHT were not significantly different among the groups(P>0.05).At 72 h postoperatively,the survival rate of Rab27b(-/-)+CLP group was 46.65%,and the survival rate of Rab27b(-/-)+CLP+JHTgroup was 63.35%,but there was no statistical difference between the two groups(P>0.05).At 72 h postoperatively,the PLT count of Rab27b(-/-)+CLP+JHT group was significantly higher than that of Rab27b(-/-)+CLP group(P<0.05);Rab27b(-/-)+CLP 4h after operation The PLT count of the group was higher than that of the 72-hour postoperative Rab27b(-/-)+CLP group,but there was no statistical difference(P>0.05).At 72 h after operation,the TT of Rab27b(-/-)+CLP group was significantly higher than other groups.(P<0.05);TT of Rab27b(-/-)+CLP+JHT group was significantly lower than that of Rab27b(-/-)+CLP group(P<0.05);72h postoperatively,Rab27b(-/-)+CLP The DD of the group was significantly higher than that of the other groups(P<0.05);the DD of the Rab27b(-/-)+CLP+JHT group was significantly lower than that of the Rab27b(-/-)+CLP group(P<0.05).Conclusion:The damage after knocking gene Rab27b(-/-)is mainly manifested by the significant decrease of platelet count,TT,DD,FDP and the damage of liver and kidney function was obvious.The weight of knockdown gene Rab27b(-/-)after modeling,The changes in PT and APTT are not very obvious.After treatment with Jinhong Decoction,it was found that the number of platelets,coagulation function and fibrinolysis function of 72 hours mice were significantly improved.PART 3:Objective:To observe the effect of Jinhong Decoction on platelet function in cecal ligation model of Rab27 b knockout mice.Methods: 120 Rab27 b knockout mice were randomly divided into four groups: Rab27b(-/-)knockout group,Rab27b(-/-)sham group,Rab27b(-/-)+ CLP group,Rab27b(-/-)+ CLP + JHT group,30 mice in each group,30 mice in wild type WT group.Rab27b(-/-)sepsis model was established by cecal ligation.Rab27b(-/-)+ CLP + JHT group was intragastrically administered with Jinhong Decoction after operation.The rest groups were given the same amount of saline after operation.The expression of P-selectin was detected by transmission electron microscopy,5-HT(secretory capacity of dense granules),flow cytometry,confocal laser microscopy,Real time PCR and Western blot were observed at 4h and72 h after operation.The change of Rab27 b was detected by blot technique.Results: At4 h and 72 h after operation,the results of electron microscopy showed that the wild-type WT,knockdown gene Rab27b(-/-)group,Rab27b(-/-)sham platelets had smooth surface,no pseudopod formation,and the alpha particles in the platelet cytoplasm were evenly distributed.There were no differences in 5-HT and P-selectin between wild-typeWT,knockout gene Rab27b(-/-)group and Rab27b(-/-)sham group(P>0.05);4 h and72 h after surgery,wild-type WT 5-HT concentration was significantly higher than all other groups(P<0.01);the 5-HT concentration of Rab27b(-/-)+CLP and Rab27b(-/-)+CLP+JHT group was significantly lower than Rab27b(-/-)and Rab27b(-/-)+sham(P<0.01);there was no significant difference in 5-HT concentrations between Rab27b(-/-)+CLP groups at 4h and 72 h postoperatively(P>0.05);At 72 h after,the 5-HT concentration in the Rab27b(-/-)+CLP+JHT group was significantly higher than that in the Rab27b(-/-)+CLP group(P<0.05).P-selectin presents a trend that is exactly the opposite of 5-HT.Using immunofluorescence technique,Real time PCR technique and Western blotting technique,the positive rate of platelet Rab27 b gene and protein in wild-type WT was significantly higher than that in other groups at 4h and 72 h after surgery;knocking gene Rab27b(-/-)group,Rab27b(-/-)Sham group,Rab27b(-/-)+ CLP group and Rab27b(-/-)+ CLP + JHT no rabbit27 b positive expression.Conclusion:The results of electron microscopy after knocking gene Rab27b(-/-)mainly showed that the number of platelet pseudopods increased,and the distribution of ?particles in platelet cytoplasm was uneven,moving to platelet membrane and releasing more ? particles;Rab27b(-/-)5-HT was significantly reduced after model establishment.After treatment with Jinhong Decoction,5-HT concentration increased,and the longer the treatment time,the better the effect.P-selectin presents a trend that is exactly the opposite of 5-HT.Knockout gene Rab27b(-/-)group,Rab27b(-/-)sham group,Rab27b(-/-)+ CLP group and Rab27b(-/-)+ CLP + JHT no rabbit27 b positive expression.There was no change after treatment with Jinhong Decoction,indicating that Rab27 b was not expressed on platelet after knockout,and there was no effect,indicating that the treatment of sepsis relied on the regulation of Rab27 b.