| 1.Objective:This study aims to observe the effect of Huangqin Qingre Chubi Capsule(HQC)on AS patients with damp-heat syndrome,and the possible relationship with the differential expressed CircRNA?0003307/miR-191-5p/CDK6 combination and immune inflammation changes.The whole RNA sequencing(RNA-seq)technology was used to analyse the transcriptome of peripheral blood mononuclear cells(PBMCs)of AS patients with damp-heat syndrome.Thus to clarify the intervention mechanism and impact on immune inflammatory response of HQC on AS patients with damp-heat syndrome from the perspective of competing endogenous RNAs(ce RNA).2.Methods:2.1 Research 1.Analyze the changes of CircRNA?0003307 in PBMCs from AS patients with damp-heat syndrome by RNA-seq,and research the clinical correlation.The key immune-inflammatory gene,CircRNA?0003307,from PBMCs of patients with damp-heat syndrome was screened by means of coupling total RNA-Seq,transcriptome reannotation and differential expression analysis.The target miRNA and m RNA were predicted by bioinformatics for constructing CircRNA?0003307 /miR-191-5p/CDK6 combination.To discuss the relationship between CircRNA?0003307 combination with the expression of NF-?B signaling pathway,cytokines,and define the measurable correlations between these indicators with disease activity,and self patient perception(SPP)in AS patients with dam-heat syndrome.2.2 Research 2.Data mining research for the effects of HQC on CircRNA?0003307/miR-191-5p/CDK6 combination and immune inflammation in AS patients with damp-heat syndrome.Patients information were gathered by electronic medical record system,including disease activity score,and the SPP index,a clinical quality of life instrument that includes symptoms and mental state assessment.Using data mining methods to measure the relationship between the changes of clinical indicators with NF-?B signaling pathway,cytokines after intervention.2.3 Research 3.Mechanism research based on the ce RNA network regulatory relationships of CircRNA?0003307/miR-191-5p/CDK6 combination,and the function in mediating macrophage immune inflammatory processes in AS patients with damp-heat syndrome.The PBMCs were isolated from peripheral blood of AS patients with damp-heat syndrome,than the macrophages were sorted by immunomagnetic and cultured in vitro.To observe the effects of CircRNA?0003307,miR-191-5p and CDK6 on macrophage immune inflammation and NF-?B signaling pathway.The interaction among CircRNA?0003307,miR-191-5p and CDK6 were verified by dual-luciferase reporter gene assay,RNA pull down assay,RNA immunoprecipitation assay(RIP)and cellular functional recovery test.2.4 Research 4.CircRNA overexpression used for investigating the inhibition function of CircRNA?0003307/miR-191-5p/CDK6 on macrophage immune inflammatory response,that can regulates NF-?B pathway,and may discussing the treatment approach of HQC.Prepared serum containing HQC drugs were used to culture the macrophages in vitro.To explore the effects of HQC containing serum on both CircRNA?0003307/miR-191-5p/CDK6 combination,NF-?B signaling pathway and immune inflammation indicators.Than in order to investigate circRNA function it is necessary to manipulate its expression.All expression were compared with before either.3.Results3.1 Research 1.Analyze the changes of CircRNA?0003307 in PBMCs from AS patients with damp-heat syndrome by RNA-seq,and research the clinical correlation.(1)131 differentially expressed circRNAs in AS group compared with the control group,89 circRNAs were up-regulated and 42 circRNAs were down-regulated.(2)Gene ontology(GO)enrichment analysis indicated that these differentially expressed genes(DEGs)were mainly related to the pathogenesis such as the positive regulation of epidermal growth factor-activated receptor activity,trophoblast giant cells differentiation and oxygen transport.Besides,Gen MAPP analysis indicated that those DEGs were concerned to TNF,PI3K-AKT and NF-?B pathways.(3)Compared with the control group,the levels of TNF-?,IL-1?,IL-23 and IL-17 were significantly increased,while IL-4,IL-10 were significantly decreased in AS group(P<0.01).The m RNA expressions of IKB? and NF-?B p65 in AS patients were significantly increased either(P<0.01).(4)Compared with the control group,the CircRNA?0003307 in AS patients were highly increased.Association rule analysis found that circRNA?0003307 was correlated with the rise of TCM syndrome score,CRP,IL-1?,TNF-?,ESR,BASDAI,NF-?B p65 and IL-17,and the decline of RP,RE,SF,VT,BP and GH score.The lift degree are greater than one,indicating a strong correlation between them.3.2 Research 2.Data mining research for the effects of HQC on CircRNA?0003307/miR-191-5p/CDK6 combination and immune inflammation in AS patients with damp-heat syndrome.(1)Compared with no treatment before,CircRNA?0003307 and CDK6 were significantly decreased,while miR-191-5p was significantly increased in both groups after treatment(P<0.01).Compared with the control group,CircRNA?0003307 was significantly decreased and miR-191-5p was significantly increased in the treatment group(P<0.05 or P<0.01).(2)Compared with no treatment before,IKB? and NF-?B p65 were significantly decreased in both groups after treatment(P<0.05 or P<0.01).Compared with the control group,NF-?B p65 was significantly decreased in the treatment group(P<0.05 or P<0.01).(3)Compared with no treatment before,TNF-?,IL-1?,IL-17,IL-23,ESR and CRP were significantly decreased,while IL-4 and IL-10 were significantly increased in both groups after treatment(P<0.01).Compared with the control group,IL-23 and CRP were significantly decreased,while IL-10 was significantly increased in the treatment group(P<0.05 or P<0.01).(4)Compared with no treatment before,disease activity score were significantly decreased in both groups after treatment(P<0.05 or P<0.01).Compared with the control group,BASDAI was significantly decreased in the treatment group(P<0.05).(5)Compared with no treatment before,indicators of SPP were significantly decreased in both groups after treatment(P<0.05 or P<0.01).Compared with the control group,RE,SF,BP and VT was significantly decreased in the treatment group(P<0.05 or P<0.01).3.3 Research 3.Mechanism research based on the ce RNA network regulatory relationships of CircRNA?0003307/miR-191-5p/CDK6 combination,and the function in mediating macrophage immune inflammatory processes in AS patients with damp-heat syndrome.(1)The levels of TNF-? and L-1? were significantly increased,IL-4 and IL-10 were significantly decreased in the macrophages of AS patients(P<0.01).The m RNA of CD80 and CD86 in macrophages of AS patients were significantly increased,the m RNA of CD163 and CD206 were significantly decreased(P<0.01).The expressions of CircRNA?0003307 and CDK6 were significantly increased,while the expression of miR-191-5p was significantly decreased(P<0.01).(2)After CircRNA?0003307 overexpression,the m RNA of IKB? and NF-?B p65 and the protein expression of p-IKB? and p-NF-?B p65 were significantly increased(P<0.01).After CircRNA?0003307 knockdown,m RNA expression of IKB?and NF-?B p65 and protein expression of p-IKB? and p-NF-?B p65 were significantly decreased(P<0.01).(3)After CircRNA?0003307 overexpression,the expressions of CD80 and CD86 in macrophages were significantly increased,while the expressions of CD206 and CD163 were significantly decreased(P<0.01).After CircRNA?0003307 knockdown,the expressions of CD80 and CD86 in macrophages were significantly decreased,while the expressions of CD206 and CD163 were significantly increased(P<0.01).(4)After CircRNA?0003307 overexpression,the expressions of TNF-? and IL-1? in macrophages were significantly increased,while the expressions of IL-4 and IL-10 were significantly decreased(P<0.01).After CircRNA?0003307 knockdown,the expressions of TNF-? and IL-1? in macrophages were significantly decreased,while the expressions of IL-4 and IL-10 were significantly increased(P<0.01).(5)Compared with the control probe,the CircRNA?0003307 positive probe significantly enriched CircRNA?0003307 and miR-191-5p,indicating that CircRNA?0003307 could bind to miR-191-5p.The WT luciferase activity of CircRNA?0003307 decreased after the addition of miR-191-5p mimics.There was no significant difference between the activity of Mut luciferase and that of the control group.The activity of WT luciferase in CDK6 decreased.There was no significant difference between the activity of Mut luciferase and the control group.The enrichment of miR-191-5p and CDK6 gene in AGO2 antibody group was significantly higher than that in Ig G antibody group.(6)CircRNA?0003307 overexpression can inhibit the expression of miR-191-5p and increase the expression of CDK6,CircRNA?0003307 knockdown can increase the expression of miR-191-5p and inhibit the expression of CDK6.After the expression of CircRNA?0003307 overexpression by pc DNA-CircRNA?0003307,the functions of CircRNA?0003307 and CDK6 were restored after the transfection of miR-191-5p mimics with miR-191-5p.(7)MiR-191-5p overexpression inhibited the expression of TNF-? m RNA and increased the expression of IL-10 m RNA.CDK6 knockingdown could inhibit the expression of TNF-? m RNA and increase the expression of IL-10 m RNA.After the expression of CircRNA?0003307 overexpression by pc DNA-circRNA?0003307,the functions of TNF-? and IL-10 m RNA were restored after the transfection of miR-191-5p mimics with miR-191-5p.(8)MiR-191-5p overexpression inhibited the expression of IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B protein.CDK6 knockingdown could inhibit the expression of IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B protein.After the expression of CircRNA?0003307 overexpression by pc DNA-CircRNA?0003307,the functions of IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B protein were restored after the transfection of miR-191-5p mimics with miR-191-5p.3.4 Research 4.CircRNA overexpression used for investigating the inhibition function of CircRNA?0003307/miR-191-5p/CDK6 on macrophage immune inflammatory response,that can regulates NF-?B pathway,and may discussing the treatment approach of HQC(1)CCK8 results showed that when the inhibition rate was close to 50%,20% of the drug serum group was selected,and 24 h was the optimal action time.(2)Compared with model group,CircRNA?0003307 and CDK6 were significantly decreased in HQC containing serum group,while miR-191-5p was significantly increased(P<0.01).After pc DNA-CircRNA?0003307 was transfected into AS macrophages,the expression of CircRNA?0003307 and CDK6 were significantly increased,and miR-191-5p was significantly decreased(P<0.01).When macrophages transfected with pc DNA-CircRNA?0003307 were co-cultured with HQC containing serum,CircRNA?0003307 and CDK6 were significantly decreased,and miR-191-5p was significantly increased(P<0.01).(3)Compared with model group,IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B p65 protein were significantly decreased in HQC containing serum group(P<0.01).After pc DNA-CircRNA?0003307 was transfected into AS macrophages,the expression of IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B p65 protein were significantly increased(P<0.01).When macrophages transfected with pc DNA-CircRNA?0003307were co-cultured with HQC containing serum,IKB?,NF-?B p65 m RNA and p-IKB?,p-NF-?B p65 protein were significantly decreased(P<0.01).(4)Compared with model group,CD80 and CD86 were significantly decreased in HQC containing serum group,while CD163 and CD206 were significantly increased(P<0.01).After pc DNA-CircRNA?0003307 was transfected into AS macrophages,the expression of CD80 and CD86 were significantly increased,and CD163 and CD206 were significantly decreased(P<0.01).When macrophages transfected with pc DNA-CircRNA?0003307 were co-cultured with HQC,CD80 and CD86 were significantly decreased,and CD163 and CD206 was significantly increased(P<0.01).(5)Compared with model group,TNF-? and IL-1? were significantly decreased in HQC containing serum group,while IL-4 and IL-10 were significantly increased(P<0.01).After pc DNA-CircRNA?0003307 was transfected into AS macrophages,the expression of TNF-? and IL-1? were significantly increased,and IL-4 and IL-10 were significantly decreased(P<0.01).Macrophages transfected with pc DNA-CircRNA?0003307 were co-cultured with HQC,TNF-? and IL-1? were significantly decreased,and IL-4 and IL-10 was significantly increased(P<0.01).4.Conclusion(1)CircRNAs was differentially expressed in PBMC of AS patients with damp-heat syndrome,and CircRNA?0003307 was correlated with NF-?B pathway activation,immune inflammatory response,and disease activity in AS patients with damp-heat syndrome.(2)HQC can decrease the expression of CircRNA?0003307 and CDK6,increase the expression of miR-191-5p,inhibit the activation of NF-?B pathway,and improve the immune inflammatory response in AS patients with damp-heat syndrome.(3)CircRNA?0003307 cloud competitively binded miR-191-5p to regulate CDK6/NF-?B and mediate macrophages immune inflammatory response in AS patients with damp-heat syndrome.(4)HQC can inhibit the overactivation of NF-?B signaling pathway and suppress the immune inflammatory response in AS patients with damp-heat syndrome by regulating CircRNA?0003307/miR-191-5p/CDK6 combination. |
PDF Full Text Request