Research On The Effect Mechanism Of Qinghuang Powder Regulating TRIF Signaling Pathway In The Treatment Of Myelodysplastic Syndrome

Myelodysplastic syndrome(MDS)is a group of malignant clonal hematopoietic stem cell diseases with obvious heterogeneity.It is characterized by dysplasia of myeloid hematopoietic cells,decrease of peripheral blood cells of single lineage or multilineage,and increased risk of transformation to acute myeloid leukemia.The expansion of malignant clones in the bone marrow of MDS patients may be closely related to the pathological mechanism of immune compromise.Qinghuang Powder has shown definite and obvious therapeutic effect in clinical practice for decades,which can improve the peripheral blood cell counts and reduce the load of malignant clones in the bone marrow.Previous studies have shown that Qinghuang Powder can promote the mRNA and protein expression of TRIF signaling pathway related genes and increase the expression of type I cytokine interferon(IFN)in human MDS cell line MUTZ-1,suggesting that Qinghuang Powder may take clinical effects by regulating TRIF signaling pathway and enhancing the immune surveillance against bone marrow malignant clones.This study firstly analyzed the effects of Qinghuang Powder on the mRNA expression of TRIF signaling pathway related genes and epigenetic regulatory genes in bone marrow cells of MDS patients.The activation of TRIF signaling pathway may be one of the important mechanisms of clinical efficacy of Qinghuang Powder.Thus it is necessary to further explore which factors may influence the regulatory effect of Qinghuang Powder on TRIF signaling pathway in cellular experiment.Therefore cellular experiments were carried out in this study,in which human MDS cell line MUTZ-1 was treated with realgar and indirubin,the effective components of Qinghuang Powder.Clinical studies have shown that the clinical efficacy of Qinghuang Powder was related to the treatment time,suggesting that the exposure period might affect the regulatory effect of Qinghuang Powder on TRIF signaling pathway,thus the cellular experiments were conducted to explore the activating effect of realgar and indirubin of the same ratio on TRIF signaling pathway at different treatment time.Clinical studies have shown that different proportions of Qinghuang Powder had different clinical efficacy,suggesting that the proportion relationship may affect the regulatory effect of Qinghuang Powder on TRIF signaling pathway,thus the cellular experiments were conducted to explore the activating effect of realgar and indirubin of different ratios on TRIF signaling pathway at the same treatment time.Part 1 Clinical study of Qinghuang Powder regulating TRIF signaling pathway in the treatment of MDSObjective:To explore the effect of Qinghuang Powder on mRNA expression of TRIF signaling pathway related genes and epigenetic regulatory genes in bone marrow cells of MDS patient.Methods:From September 2018 to December 2019,a total of 48 patients were enrolled in this study,who were treated in the department of hematology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,including 39 MDS patients and 9 non-malignant hematological patients.All patients underwent iliac bone marrow puncture to obtain bone marrow aspiration samples.The mRNA expression levels of TRIF signaling pathway related genes and epigenetic regulatory genes were measured by qPCR.They were divided into the following three groups:untreated group,16 MDS patients were sampled and measured when they did not take Qinghuang Powder,and then began to take Qinghuang Powder after sampling;treated group,23 MDS patients were sampled and measured when they took Qinghuang Powder for 3 months;control group,9 patients with non-malignant hematological diseases did not need to take Qinghuang Powder.Mann Whitney U test was used to compare the mRNA expression differences of target genes among the three groups.Results:(1)Differences in mRNA expression of TRIF signaling pathway related genes among three groups:?Compared with the control group:in the untreated group the mRNA expression of IRF3 gene was significantly decreased(P<0.01),the mRNA expression of TRIF,TRAF3 and IFN genes were decreased but there was no significant difference(P>0.05);in the treated group the mRNA expression of TRIF,TRAF3,IRF3 and IFN had no significant differences(P>0.05);?Compared with the untreated group:in the treated group the mRNA expressions of IRF3 and IFN were significantly increased(P<0.01),the mRNA expression of TRIF was increased but there was no significant difference(P>0.05),and the mRNA expression of TRAF3 was decreased but there was no significant difference(P>0.05);(2)Differences in mRNA expression of epigenetic regulatory genes among three groups:?Compared with the control group:in the untreated group the mRNA expression of IDH1,IDH2,SF3B1,ZRSR2,U2AF1,RUNX1,EZH2,BCOR genes were significantly decreased(P<0.01,P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,P<0.05),there was no significant differences in the mRNA expression of other epigenetic regulatory genes(P>0.05);in the treated group the mRNA expression of SRSF2 was significantly increased(P<0.01),the mRNA expression of U2AF1 was significantly decreased(P<0.05),there was no significant differences in the mRNA expression of other epigenetic regulatory genes(P>0.05).?Compared with the untreated group:in the treated group the mRNA expressions of DNMT3A,IDH1,IDH2,SF3B1,ZRSR2,TP53,RUNX1,EZH2,BCOR genes were significantly increased(P<0.05,P<0.01,P<0.01,P<0.05,P<0.05,P<0.05,P<0.001,P<0.01,P<0.01),there was no significant differences in the mRNA expression of other epigenetic regulatory genes(P>0.05).Conclusions:(1)The mRNA expression levels of TRIF signaling pathway related genes were down-regulated in bone marrow cells of MDS patients,and Qinghuang Powder might promote the mRNA expression of TRIF signaling pathway related genes;(2)Qinghuang Powder might promote the mRNA expression of DNA methylation modification(DNMT3A,IDH1,IDH2),histone modification(EZH2),DNA transcription(RUNX1,TP53,BCOR),mRNA precursor splicing(SF3B1,ZRSR2)and other epigenetic regulatory genes.Part 2 Effects of realgar and indirubin on TRIF signaling pathway in human MDS cell line at different treatment timeObjective:To explore the regulatory effects of realgar and indirubin on the expression of TRIF signaling pathway genes in human MDS cell line MUTZ-1 at different treatment time.Methods:In this study,human MDS cell line MUTZ-1 was treated with the same ratio of realgar and indirubin(35?g/L realgar+70?g/L indirubin)for 48h,72h and 96h respectively.Drug treatment group and blank control group were set up at each treatment time point.The mRNA and protein expression of TRIF signaling pathway genes and NQO1 gene were detected by qPCR and Western Blot respectively,and the expression of cytokine type I IFN was detected by ELISA.Results:(1)Determination of oxidative stress related products:at 48h,72h and 96h,the content of GSH,MDA and the activity of SOD in the drug treatment group were significantly higher than those in the control group(P<0,01),and were significantly higher at 72h than those in 48h and 96h(P<0.05);(2)qPCR:in the drug treatment group,the mRNA expressions of TRAF3,IRF3 and IFN-? reached the maximum at 48h(P<0.05),the mRNA expressions of TBK1,IKK? and NQO1 reached the maximum at 72h(P<0.05),and the mRNA expressions of TRIF and IFN-? reached the maximum at 96h(P<0.05);(3)Western Blot:in the drug treatment group,the relative values of protein hybridization intensity of TRIF,TRAF3,TBK1,IKK? and IRF3 reached the maximum at 48h(P<0.05),and that of NQO1 reached the maximum at 96h(P<0.001);(4)ELISA:The relative contents of IFN-? and IFN-? at 96h were higher than those at 48h and 72h(P<0.05),and at each time point,the relative content of IFN-? was significantly higher than that of IFN-?(P<0.01).Conclusions:Qinghuang Powder might induce intracellular oxidative stress and activate TRIF signaling pathway,and promote the mRNA and protein expressions of TRIF,TRAF3,TBK1,IKK? and IRF3 genes.The expression of cytokine type I IFN might be time-dependent and increase with time,and the expression of IFN was mainly IFN-? in the early stage of activation.Part 3 Effects of realgar and indirubin of different ratios on TRIF signaling pathway in human MDS cell lineObjective:To explore the regulatory effects of realgar and indirubin of different ratios on the expression of TRIF signaling pathway genes in human MDS cell line MUTZ-1.Methods:In this study,human MDS cell line MUTZ-1 was treated with realgar and indirubin of different ratios and 10 treatment groups were set up:group A(blank control group),group B(35?g/Lrealgar),group C(70?g/L indirubin),group D(35?g/L realgar+35?g/L indirubin),group E(35?g/L realgar+52.5?g/L indirubin),group F(35?g/L realgar+70?g/L indirubin),group G(35?g/L realgar+315?g/L indirubin),group H(175?g/L realgar+70?g/L indirubin),group I(350?g/L realgar+70?g/L indirubin),and group J(antioxidant group,35?g/L realgar+70?g/L indirubin+antioxidant NAC).After 72 hours of treatment,the mRNA and protein expressions of TRIF signaling pathway genes and NQO1 gene of the above groups were measured by qPCR and Western Blot,and the expression of cytokine type I IFN was measured by ELISA.Results:(1)Determination of oxidative stress related products:the content of GSH reached the maximum in group F(P<0.001),and was significantly lower in group J with the antioxidant than in group B-I(P<0.001);the content of MDA was the highest in group H(P<0.001),and the lowest in group J(P<0.001);the activity of SOD reached the maximum in group B(P<0.05)and the minimum in group J(P<0.05);(2)qPCR:the mRNA expression of TRIF reached the maximum in group E(P<0.01);the mRNA expressions of IKK?,IRF3 and IFN-? genes reached the maximum in group B(P<0.05);the mRNA expressions of TRAF3 and TBK1 genes reached the maximum in group F(P<0.05);the mRNA expression of IFN-a reached the maximum in group C(P<0.05);the mRNA expression of NQO1 reached the maximum in group D(P<0.05).The mRNA expressions of TBK1,IKK?,IRF3,IFN?,IFN? and NQO1 genes reached the minimum in group J with the antioxidant(P<0.05);the mRNA expressions of TRIF and TRAF3 in group J were significantly lower than those in group A-G(P<0.01)but there was no significant differences compared with those in group H and group I(P>0.05).(3)Western Blot:the protein expressions of TRIF,TRAF3 and TBK1 in group C were higher than those in other groups(P<0.05),the protein expressions of IKK? and NQO1 in group D were higher than those in other groups(P<0.05),the protein expression of IRF3 in group F was higher than those in other groups(P<0.001).The protein expression of TRIF in group E was lower than that in other groups(P<0.01),the protein expression of TRAF3 in group A was lower than that in other groups(P<0.01),the protein expression of TBK1 in group G was lower than that in other groups(P<0.01),the protein expression of IKKs in group J was lower than that in other groups(P<0.01),the protein expression of IRF3 in group H was lower than that in other groups(P<0.01),and the protein expression of NQO1 in group A was lower than that in other groups(P<0.01).(4)ELISA:IFN-? and IFN-? reached the maximum in group H(P<0.05,P<0.01),and in group H the relative content of IFN-? was significantly higher than that of IFN-?(P<0.01).(5)TOPSIS comprehensive evaluation and analysis:the mRNA expression of TRIF signaling pathway genes altogether reached the maximum in group F and reached the minimum in group J;the protein expression of TRIF signaling pathway genes altogether reached the maximum in group D and reached the minimum in group I,and group J was close to group I.Conclusions:(1)When the ratio of realgar and indirubin was 1:1,the protein expression of genes in TRIF signaling pathway could be promoted to the greatest extent;(2)Oxidative stress induced by realgar and indirubin might activate the TRIF signaling cascade and promote the expression of cytokine type I IFN;(3)The cellular oxidative stress induced by realgar and indirubin might influence the expression of cytokine IFN-? and IFN-? at the end of TRIF signaling pathway.