| 1 ObjectiveExploring Jianpi Qingre Tongluo prescription(JQP)via long noncoding RNANONHSAT227927.1/janus kinase 2/signal transducers and activators of transcription 3(NONHSAT227927.1/JAK2/STAT3)combination on the mechanism of immune inflammatory response and patient perception(SPP)in patients with ankylosing spondylitis(AS)damp-heat obstruction syndrome.2 Methods2.1 Study 1.Based on retrospective data mining to explore the changes of SPP,immune and inflammatory indexes and JQP intervention research in patients with AS damp heat obstruction syndrome.In this paper,using the method of self-control research,113 patients with ankylosing spondylitis with damp heat obstruction syndrome treated by JQP were screened from the database of Anhui Provincial Hospital of Traditional Chinese Medicine,and the immune and inflammatory indexes and SPP indexes of the sample patients were collected.By selecting the serum of 50 AS patients before and after treatment and 30 healthy volunteers from the Physical Examination Center of Anhui Provincial Hospital of Traditional Chinese Medicine during the same period,peripheral blood mononuclear cells(PBMCs)were extracted,and then the blood samples were analyzed by RT-PCR and ELISA.The correlation analysis and association rule analysis methods were used to explore the correlation between SPP and lncRNA NONHSAT227927.1 and inflammatory indicators;the binary logistic regression method was used to explore the risk factors of SPP indicators;the association rule analysis was used to explore the relationship between JQP and SPP,lncRNA NONHSAT227927.1,The correlation of immune and inflammatory indicators;the diagnostic utility of NONHSAT227927.1 for AS was evaluated based on the receiver operating characteristic curve(ROC).2.2 Study 2.Based on the interference/overexpression of NONHSAT227927.1,to investigate the inhibition of synovial fibroblast inflammatory response by JQP drug-containing serum through the combination of NONHSAT227927.1/JAK2/STAT3.Extract AS-PBMCs from patients with AS damp heat obstruction syndrome,purchase AS fibroblast-like synoviocytes(FLS),and construct a co-culture model of AS-FLS and AS-PBMCs in a transwell chamber;construct NONHSAT227927.1 Expression and small interfering plasmids were transfected into AS-FLS;cell counting kit-8(CCK-8)was used to detect the viability of AS-FLS cells;RT-qPCR was used to detect the expression of NONHSAT227927.1,The expressions of IL-17,IL-6,IL-10 and TNF-α were detected by ELISA;the expressions of JAK2,phosphorylation(p)-JAK2,STAT3 and p-STAT3 proteins were detected by western blot(WB).The optimal concentration and time of JQP drug-containing serum were screened,and the effects of JQP on NONHSAT227927.1,JAK2/STAT3 pathway proteins,cell viability and inflammatory cytokines were observed.Through functional rescue experiments,JQP-containing serum was added to the transfection of pcDNA3.1-lncRNA NONHSAT227927.1 to further explore the mechanism of JQP interfering with the inflammatory response of AS-FLS.3 Results3.1 Study 1:Based on retrospective data mining to explore the changes of SPP,immune and inflammatory indexes and JQP intervention research in patients with AS damp heat obstruction syndrome.(1)The general conditions of 50 AS patients with damp heat obstruction syndrome and 30 normal controls were compared,and there was no significant difference in age,gender,height and weight between the two groups(P>0.05).(2)NONHSAT227927.1 is highly expressed in AS patients with damp heat obstruction syndrome,and the area under the ROC curve(AUC)is 0.8463[95%confidence interval(CI):0.7514-0.9413,P<0.001],suggesting that NONHSAT227927.1 has a role in AS Significant diagnostic value.(3)SPP indexes(RP,GH,BP,PF,SF,MH,VT,RE were significantly decreased;SAS,VAS,SDS,damp heat obstruction syndrome scores were significantly increased),clinical indicators(ESR,hs-CRP,C3,C4,IgA,IgG,IgM elevated)abnormal(P<0.05).Compared with the normal control group,the expression of NONHSAT227927.1,IL-17,and IL-23 in PBMCs of AS patients with damp heat obstruction syndrome increased(P<0.05),and the expression of IL-10 decreased(P<0.05).(4)Effects of NONHSAT227927.1 on AS-FLS cell viability,inflammatory cytokines and JAK2/STAT3 pathway:Overexpression,small interfering RNA model was constructed,and the results of CCK8 experiments showed that overexpression of NONHSAT227927.1 can promote AS-FLS cell viability,Knockdown of NONHSAT227927.1 could inhibit the cell viability of AS-FLS.ELISA results showed that overexpression of NONHSAT227927.1 could inhibit the level of IL-10 and promote the levels of IL-17,IL-6 and TNF-α(P<0.001),Knockdown of NONHSAT227927.1 can inhibit the levels of IL-17,IL-6 and TNF-α,and promote the level of IL-10.The Western Blot technique demonstrates that overexpressing NONHSAT227927.1 can increase the expression of the proteins p-JAK2 and p-STAT3.,while The opposite was true for knockdown of NONHSAT227927.1(P<0.001).(5)Association rule analysis of AS patients with damp heat obstruction syndrome and NONHSAT227927.1 and inflammatory indicators:C4 is strongly correlated with VAS,SDS,SF,SAS,ESR is strongly correlated with SDS,SAS,hs-CRP is strongly correlated with RP,SF,SDS There was a strong correlation,and IL-23 was strongly correlated with SF.(Confidence>45%,Rule Support>30%,Lift>1).(6)Logistic regression analysis of SPP,NONHSAT227927.1,and immune inflammation indexes in AS patients with damp heat obstruction syndrome:Under the conditions of B>0,P<0.05,|OR|>1,IL-17(P=0.017),IgG(P=0.047)and NONHSAT227927.1(P=0.0023)were risk factors for AS-SPP,and IL-10(P=0.024)was a protective factor for AS-SPP.(7)JQP has an improving effect on SPP,NONHSAT227927.1 and immune inflammation indexes:Compared with AS patients with damp heat obstruction syndrome before treatment,the scores of damp heat obstruction syndrome,SAS,SDS,VAS,and scores of AS patients after treatment decreased(P<0.05,P<0.001);BP,VT,MH,PF,RP and GH scores increased(P<0.05,P<0.001);ESR,hs-CRP,C3,C4,IgA,IgG and IgM indicators decreased(P<0.05,P<0.001);the expression of NONHSAT227927.1 decreased(P<0.001);the inflammatory cytokines IL-17 and IL-23 decreased(P<0.001),and the anti-inflammatory factor IL-10 increased(P<0.001);RE,SF,IgG,C3 did not significantly decrease before and after treatment(P>0.05).(8)Association rule analysis of JQP on the feelings of AS patients with damp heat obstruction syndrome NONHSAT227927.1 and inflammatory indicators:there is a strong correlation between JQP and SPP indicators:the improvement of BP,PF and VAS;the relationship between JQP and the decrease of NONHSAT227927.1 expression There is a strong correlation between JQP and the improvement of immune inflammation indicators:IL-17,IL-10,ESR,hs-CRP(confidence>45%,rule support>40%,Lift>1).3.2 Study 2:Based on the interference/overexpression of NONHSAT227927.1,to investigate the inhibition of synovial fibroblast inflammatory response by JQP drug-containing serum through the combination of NONHSAT227927.1/JAK2/STAT3.(1)Construct the best co-culture model of AS-FLS and AS-PBMCs,using CCK8 In the experiment,the concentration and time were screened,and it was finally determined to use the cell model with a concentration of 3:1 and a treatment time of 48 h for subsequent experiments.(2)Effect of AS-PBMCs and AS-FLS co-culture model on NONHSAT227927.1 and inflammatory cytokines:RT-qPCR experiment results showed that the expression of NONHSAT227927.1 in the co-culture model increased(P<0.001),ELISA experiment results showed that,The anti-inflammatory factor IL-10 decreased,and the pro-inflammatory factors IL-6,IL-17,and TNF-α increased(P<0.001).(3)Effect of NONHSAT227927.1 on AS-FLS cell viability,cytokines and JAK2/STAT3 signaling pathway:overexpression,small interfering RNA model,CCK8 experiment results show that overexpression of NONHSAT227927.1 can promote AS-FLS cell viability,knock-out Down-regulation of NONHSAT227927.1 could inhibit the cell viability of AS-FLS.ELISA results showed that over-expression of NONHSAT227927.1 could inhibit the level of IL-10 and increase the level of IL-17,IL-6,and TNF-α(P<0.001),knocking out Down-regulation of NONHSAT227927.1 can inhibit the levels of IL-17,IL-6 and TNF-α,and promote the level of IL-10.Western Blot method shows that over-expression of NONHSAT227927.1 can promote the expression of p-JAK2 and p-STAT3 proteins,while knockdown The opposite was true for NONHSAT227927.1(P<0.001).(4)The optimal concentration of serum containing JQP was screened out.The results of CCK8 experiment showed that the serum group containing 20%JQP had the strongest inhibitory effect on cell viability at 48h(P<0.001).Therefore,we chose the serum containing 20%JQP Subsequent verification experiments were carried out at 48 h.(5)Effect of JQP on NONHSAT227927.1,JAK2/STAT3 and inflammatory cytokines:RT-qPCR experiment results showed that JQP could inhibit the expression of NONHSAT227927.1 in AS-FLS(P<0.001).The results of ELISA experiments showed that JQP could promote the expression of IL-10 in AS-FLS and inhibit the expression of IL-17,IL-6 and TNF-α in AS-FLS(P<0.001).After treatment,the expressions of p-JAK2 and p-STAT3 proteins in AS-FLS decreased(P<0.001).After transfection with pcDNA3.1-NONHSAT227927.1,the expression of anti-inflammatory cytokine IL-10 decreased,and the protein expression of IL-17,IL-6,TNF-α,p-JAK2 and p-STAT3 increased(P<0.05),In functional rescue experiments,JQP-containing serum could reverse the increase of IL-17,IL-6,TNF-α,p-JAK2 and p-STAT3,and IL-10 levels in AS-FLS by overexpression of NONHSAT227927.1 reduced impact(P<0.05).4 Conclusions(1)There is a decrease in SPP and an imbalance of immune and inflammatory responses in patients with AS damp heat obstruction syndrome.,and there is a significant correlation and correlation between the decrease in SPP and the high expression of NONHSAT227927.1 and the increase of immune and inflammatory indicators.JQP improves SPP,down-regulates the expression of NONHSAT227927.1,and inhibits the immune inflammatory response in AS patients with damp-heat-bi-blocking syndrome,and the use of JQP is strongly correlated with the improvement of SPP.(2)JQP medicated serum can inhibit the inflammatory response and cell viability of AS-FLS through the combination of NONHSAT227927.1/JAK2/STAT3,thereby improving the SPP of AS patients with damp heat obstruction syndrome. |
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