| Background:(1)The role of inflammation in Venous Thromboemlism(VTE)is a hot research topic in recent years.Previous published studies on the association between VTE and inflammatory genes are single gene,single point studies,and the results are inconsistent.It is of clinical significance to study the relationship between multiple inflammatory genes,multi-locus polymorphism and VTE.(2)IL-1 gene is not only involved in blood lipid,thrombosis and immune regulation,but also an inflammatory factor,which is an ideal candidate gene for VTE.The research on the correlation between IL-1 gene and VTE based on Fast Target region sequencing is helpful to discover the common and rare mutation sites in IL-1 gene region which are closely related to the occurrence and development of VTE.(3)NLRP3/IL-1/NF-?B signaling pathway and cytokines such as IL-1 regulated by macrophage polarization may be key indicators of prethrombotic state and the intrinsic mechanism of NLRP3/IL-1/NF-?B signaling pathway and macrophage polarization in connection with VTE is still unclear.Methods:(1)ELISA and i MLDR were used to detect the levels of IL-1,IL-6,TNF-?,SAA and CRP in 284 VTE patients and 268 control subjects whose general information matched,and their polymorphism at 14 loci.Univariate and multivariate Logistic regression analysis was carried out.(2)Target capture sequencing of IL-1? and IL-1? gene regions by Fast Target region sequencing technology.To analyze the common and rare variations in IL-1? and IL-1? gene regions.(3)ELISA,RT-PCR and Western-Blot method were used to detect the expression levels of NLRP3/IL-1/NF-?B signaling pathway key proteins IL-1??Caspase1?NLRP3 and NF-?B P65 in SD rats.Immunohistochemistry was used to detect the polarization of M1(CD86)and M2(CD206)macrophages.Results:(1)(1)The levels of IL-1,IL-6,TNF-?,SAA and CRP in VTE group were higher than those in the control group,and the difference was statistically significant(P<0.05).(2)Univariate Logistic regression analysis indicated that TT SNP with mutation at rs2234650 site of IL-1 gene had lower risk of VTE than those with wild type CC and heterozygous CT SNP(OR: 0.500;95%CI: 0.268–0.934).Compared with wild-type GG and heterozygous GA SNP,those with mutant AA SNP at rs11800587 site of IL-1 gene have higher risk of VTE(or: 4.444;95%CI: 1.466–13.470).(3)Multivariate Logistic regression analysis of VTE risk factors suggested that compared with CC wild-type SNP at rs2234650 of IL-1 gene,those with TT mutant SNP had higher VTE risk(OR: 2.086;95%CI: 1.091-3.985).Compared with AA mutant SNP at rs1800587 site of IL-1 gene,those with GG wild-type SNP have lower VTE risk(OR: 0.226;95%CI: 0.074-0.890).(4)Compared with VTE survivor group,the levels of IL-1,TNF-?,SAA and CRP in VTE endpoint group were significantly higher(P<0.05).(5)Compared with wild GG genotype at rs1800587 of IL-1 gene,patients with GA and AA mutation genotype had higher mortality(log Rank P<0.05).(6)COX regression analysis still showed that the mortality of the genotype with mutation at rs1800587 site of IL-1 gene increased significantly(HR=2.982;95%CI: 1.681-5.100).The mortality rate of the genotype with the mutation at rs1143634 site of IL-1 gene was lower(HR=0.294;95%CI: 0.132-0.652).(2)(1)A total of16 mutations were screened out,including 6 common mutations and 10 rare mutations.(2)The results of common mutation analysis: The risk of VTE was lower in those with AA SNP mutation at rs3783550 site of IL-1? gene(OR: 0.579;95%CI: 1.352-7.225).The VTE risk is lower in those who carry the heterozygous variant GA SNP at rs1143627 site of IL-1? gene(OR: 0.439;95%CI: 0.219-0.879).The AG SNP with heterozygous variant at rs16944 site of IL-1? gene has a lower risk of VTE(OR: 0.439;95%CI: 0.219-0.879).(3)Analysis results of rare mutations: 10 rare mutations were screened.Two of them are located in intron region,two in exon region,one in 3' untranslated region,one in clipping region,one in 5' untranslated region and two in upstream region of gene.Among the two rare mutations in exon region,one is non-synonymous mutation and the other is synonymous mutation.The rare mutation site may affect the expression and function of IL-1 gene,but the effect of this mutation on IL-1 gene and its role in VTE still need to be verified by expanding the sample size.(3)(1)The levels of IL-1 and TNF-? in VTE rats were significantly higher than those in inhibitor,sham and control group(P<0.05).(2)The m RNA expression of IL-1??Caspase1?NLRP3 and NF-?B P65 gene in VTE rats was significantly higher than that in inhibitor,sham and control group(P<0.05).(3)The expression levels of IL-1?,Caspase1?NLRP3 and NF-?B P65 protein in VTE rats were significantly higher than those in in inhibitor?sham and control group(P<0.05).(4)Six hours after the VTE model was established,a large number of cells in thrombus tissue were positively stained with CD86,while CD206,a polarization marker of M2 macrophages,showed less positive stained cells(P<0.05).Conclusions:(1)The levels of IL-1,IL-6,TNF-?,SAA and CRP in VTE patients were higher than those in the control group,suggesting that VTE patients may be in an inflammatory activation state.The mutation at rs2234650 of IL-1 gene is an independent risk factor for VTE,and the mutation at rs11800587 of IL-1 gene is an independent protective factor for VTE.During the follow-up months,21.83% of VTE patients had an end point event(all-cause death),and the median survival time was 49 months.The risk of all-cause death is increased in those who carry the mutant genotype of IL-1 gene at rs1800587.The risk of all-cause death is reduced in patients with IL-1 gene mutation at rs1143634.(2)A total of16 mutations were screened by Fast Target target region sequencing technology,including6 common mutations and 10 rare mutations.Among the common mutations,the mutations of rs3783550 of IL-1? gene,rs1143627 of IL-1? gene and rs16944 of IL-1? gene are closely related to the risk of VTE.In the analysis of rare mutations,10 rare mutations were screened.They are located in intron region,exon region,3' untranslated region,clipping region,5' untranslated region and gene upstream region.(3)After VTE modeling,the levels of IL-1 and TNF-? increased significantly,and the phenotype of macrophages was mainly pro-inflammatory M1 macrophages.The expression level of key proteins of NLRP3/IL-1/NF-?B signaling pathway IL-1?,Caspase1,NLRP3 and NF-?B P65 increased significantly after VTE modeling.Caspase-1 inhibitor(AC-YVAD-cmk)can reduce venous thrombosis area,IL-1 and TNF-? levels,and inhibit the expression of key proteins in NLRP3/IL-1/NF-?B signaling pathway.There were more positive cells of M1 type macrophages and less positive cells of M2 type macrophages.NLRP3/IL-1/NF-?B signaling pathway and macrophage polarization play an important role in the occurrence and development of VTE.Targeted regulation of NLRP3/IL-1/NF-?B signaling pathway and macrophage polarization may become a new target for prevention and treatment of VTE. |
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