Study On The Effect And Mechanism Of Bushen Yijing Therapy Mediating MicroRNA Treatment Of SSc

Objective1、Screening significantly different expression miRNAs in serum exosomes between systemic sclerosis(SSc)patients and health people provides scientific basis for the the screen of diagnostic markers for diseases and follow-up study.2、To observe the effect of Chinese herbal compound of Bushen Yi jing Therapy on skin fibrosis in mice with SSc and to explore its mechanism of action.Methods1、Collect serum exosomes from patients with SSc and normal subjects,extract exosome by precipitation method.Then separate and recover small RNA,filter to get miRNA.Finally,establish a database and screen differential miRNAs by DEGseq and DESeq2 software to observe and compare differentially expressed miRNAs.2、SSc mice model were established and intervened with Astragaloside IV,Bushen Yijing Recipe containing serum,Bushen Yijing Recipe containing serum+miR-26a antagomir respectively.The skin tissues of mice were collected for histopathological examination and observe the thickness of dermis and collagen fibers in scleroderma mice.The expression of miR-26a,FLI1,CollagenI and Fibronectin mRNA and protein were detected by QPCR and WB.Results1、Compared with the normal controls,there were 1337 miRNAs differentially expressed in serum exosomes of SSc patients,of which 366 were up-regulated and 971 were down-regulated.Among them,miR-126-3p and 5p have obvious up-regulation.2、Compared with the normal group,HE staining and Masson staining showed that the thickness of dermis and the number of collagen fibers were ignificantly increased in SSc mice model group.The results of QPCR showed that compared with the normal group,the expression of miR-26a,Fibronectin,Collagen Ⅰ mRNA was up-regulated and the expression of FLI1 mRNA was down-regulated in the model group(P<0.05).Compared with the model group,the expression of miR-26a mRNA could be down-regulated after adding astragaloside Ⅳ or Bushen Yi jing recipe(p>0.05),but the expression of FLI1 mRNA was up-regulated significantly(p<0.05),Fibronectin,).The expression of Collagen I mRNA decreased significantly(p<0.05),the expression of miR-26a,Fibronectin,Collagen Ⅰ mRNA decreased significantly(p<0.05)and the expression of FLI1 mRNA increased significantly(p<0.05)in the Bushen Yijing+miR-26a antagomir group.Compared with Bushen Yijing recipe group,the expression of miR-26a,Fibronectin mRNA in Bushen Yijing+miR-26a antagomir group was down-regulated,but there was no significant difference(p>0.05).The results of WB showed that compared with the normal group,the expression of FLI1 protein decreased significantly(P<0.05)and the expression of Fibronectin,Collagen Ⅰ protein increased significantly(P<0.05)in the model group.Compared with the model group,the expression of FLI1 protein was significantly up-regulated(p<0.05)after astragaloside Ⅳ was added,but there was no significant difference in FLI1 protein expression after adding Bushen Yijing recipe or Bushen Yijing recipe(miR-26a antagomir)(p>0.05).Compared with Bushen Yijing recipe group,there was no significant difference in FLI1 mRNA expression between miR-26a antagomir group and Bushen Yijing recipe group(p>0.05).Compared with the model group,the expression of,Fibronectin,Collagen Ⅰ protein was significantly down-regulated after adding astragaloside Ⅳ,Bushen Yijing recipe or Bushen Yijing recipe(miR-26a antagomir).Compared with Bushen Yijing recipe group,the expression of Fibronectin,Collagen Ⅰ protein in miR-26a antagomir group was signif icantly lower than that in Bushen Yijing recipe group(p<0.05).Conclusion1、There is differential expression of miRNAs in serum exosomes of SSc patients,and the expression of miR-126 is increased,which may be associated with SSc vascular disease.2、Aastragaloside Ⅳ,Bushen Yijing traditional Chinese medicine compound can alleviate skin fibrosis in SSc mice,and its mechanism may be related to down-regulation of miR-26a and promotion of FLI1 expression.