| Objective To construct gout air pouch model and acute peritonitis model on the basis of PRKG2 monocyte conditional knock out(cKO)and conditional knock in(cKI)mice,and to study the effect of gout pathogenic gene PRKG2 on the expression of inflammatory cytokines in monocyte and macrophage.Methods(1)PRKG2 monocyte cKI mice were constructed by inducing and regulating PRKG2 gene based on Cre loxP system.The gene type and expression of PRKG2 in monocyte of offspring mice were identified by PCR and Western Blotting.The expression level of PRKG2 in kidney,small intestine and other main expression organs of mice was observed by immunohistochemistry,and the success of construction of cKI mice with PRKG2 gene of monocyte was verified.The serum uric acid(sUA),liver function,kidney function,fasting blood glucose(Glu),cholesterol(TC),triglyceride(TG)and other biochemical indexes were detected in cKO mice(which had been successfully constructed by the research group),cKI and their respective control mice.Hematoxylin eosin(HE)staining was used to observe the morphology of the kidney and small intestine of mice,and to clarify the effect of PRKG2 gene knockout or knockin on the important indexes related to uric acid and organs.(2)Male CKO,CKI and their respective control mice aged 8-12 weeks were used to construct gout pouch and acute peritonitis models(each group > 3 mice).The amount of inflammatory cell infiltration was measured by cell counter.The levels of IL-1?,IL-8,IL-6 and TNF-? were measured by ELISA.The relative expression of Pro-IL-1? and NALP3 were measured by qRT-PCR.Results(1)The results of gene identification showed that cKI and control mice expressed related target bands,and PRKG2 protein level of monocyte increased significantly after gene insertion;there was no significant difference in the expression of PRKG2 in kidney and small intestine,suggesting that the construction of monocyte PRKG2 gene cKI mice was successful;at the same time,the results of cKO mice gene and protein suggested that PRKG2 gene of monocyte was knocked out.(2)The results of biochemical indexes and pathological examination of small intestine and kidney showed that there was no significant difference in sUA,liver function,kidney function,Glu,TC,TG between cKO mice and cKI mice(P > 0.05);the morphology of small intestine and kidney in PRKG2 gene modified mice did not change significantly.(3)The results showed that the levels of IL-1?,IL-8 and TNF-? in the peritoneal lavage fluid of cKO mice were significantly decreased(IL-1?: 66.78±6.38 vs.82.83±3.82 pg/mL,P<0.01;IL-8: 113.2±8.96 vs.144.9±10.91 pg/mL,P<0.05;IL-6: 79.17±8.77 vs.79.90±6.55 pg/mL,P>0.05;TNF-?: 288.2±11.03 vs.323.4±9.86 pg/m L,P<0.01),the expression of IL-1?,IL-8 and IL-6 of cKI mice was significantly increased(IL-1?: 78.89±8.07 vs.64.26±10.67 pg/mL,P<0.05;IL-8: 139.1±3.24 vs.128.3±10.31 pg/m L,P<0.05;IL-6: 110.3±7.08 vs.103.8±5.72 pg/mL,P<0.05;TNF-?: 288.2±11.03 vs.282.8±18.48 pg/mL,P>0.05),and the expression of these inflammatory cytokines was similar in the air pouch model of mice.Further studies showed that Pro-IL-1? and NALP3,the key molecules that affect the expression of IL-1?,the initiating inflammatory factor of gout,were significantly reduced in the monocytes of peritonitis in cKO mice,but significantly increased in cKI mice.Conclusion The results of gene and protein indicated that PRKG2 gene cKI mice were successfully constructed,and PRKG2 gene(knockout or knock in)in monocytes had no significant effect on the biochemical indexes such as sUA,Glu,TC,TG and the expression of organs such as small intestine and kidney,suggesting that cKO and cKI mice of PRKG2 gene were ideal models for the study of gouty induced inflammation and its mechanism.The results of gout air pouch model and peritonitis model showed that PRKG2 was involved in regulating the expression of IL-1?,IL-8,IL-6 and TNF-?,which might regulate the expression of Pro-IL-1? and NALP3 in monocytes at transcription level,promote the release of IL-1? and start gout inflammatory reaction. |
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