Effects Of Chinese Medicine For Tonifying Deficiency And Removing Blood Stasis On MiRNA Expression Profile And Its Mechanism Of Action In Rats With Diabetic Ulce

Objective:The purpose of this study is to verify the healing effect of Buxuhuayu traditional Chinese medicine(TCM)on diabetic ulcer(DU)rats,screen the differentially expressed microRNAs of DU rats,and observe the effect of Buxuhuayu TCM in different ways of use on miRNA and its regulatory effect on miRNA-21-5p/TIMP-3/TGF-β axis and HIF-1α/miR-210-3p/E2F3 axis.Method:Streptozotocin(STZ)intraperitoneal injection and surgical removal of the full thickness of the skin were used to establish a DU model;After the model is successfully established,the drug is administered once a day until the wound surface is reepithelialized and Wound healing time(WHT)is recorded;Observing the tissue morphology,collagen deposition and microvessel density(MVD)on the 3rd,7th,and 14th day after administration;Gene chip was used to detect wound tissue on the 7th day,and differentially expressed miRNAs were screened out,then target genes for KEGG and GO enrichment analysis were predicted;Constructing a co-transfection plasmid of miRNA-21-5p and TIMP-3,and measuring the activity of dual luciferase reporter gene;Fluorescence in situ hybridization and qPCR were used to detect the localization and expression of miRNA-21-5p and miR-210-3p;qPCR and Western bloting were used to detect the expression of TIMP-3,TGF-β,HIF-1α,VEGF,and E2F3 in the local wounds after treatment.Results:1.Healing effects of Buxuhuayu TCM in DUs rats:: WHT and wound healing rate(WHR): Compared with the control group(NU),the WHR of the model group(DU)was reduced(P<0.01),WHT(P < 0.05);Compared with the DU,WHR of the Internal treatment group(DU+BXHYD),external treatment group(DU+FHSJYCO),combination treatment group(DU+Comb)and Positive control group(DU+rh-b FGF)were increased(P < 0.01),WHT shortened(P < 0.01).Comparison between the intervention groups: On the 7th day of medication,the WHR of the DU+Comb was higher than that of the DU+BXHYD and DU+FHSJYCO(P<0.05);on the 14th day of the medication,the WHR of the DU+Comb was higher than that of the DU+BXHYD(P<0.05).Histomorphology: On the 7th day of medication,the inflammation in DU was heavier than that in the other groups;In each intervention group,the newly formed fibroblasts were abundant and the cell bodies were large,approaching the epidermis,and some degree of epithelial creeping could also be observed.On the 14th day of medication,there was still some inflammation on the wound in the DU and the wound edge was not completely epithelialized and closed;The inflammatory cells at the wound edge of each intervention group gradually disappeared and all re-epithelialized and closed.Among them,new organelles appeared in the NU,DU+Comb,and DU+rh-b FGF.Masson staining: On the 3rd,7th,and 14th days of treatment,the collagen fibers in the DU were significantly less than those in the other groups,and there was no collagen deposition with the healing time;On the 7th day,the collagen fibers in each intervention group were arranged effectively,and scattered cells were rarely seen;On the 14th day,the collagen fibers of each intervention group were arranged neatly and tightly,which was similar to the wound surface of the NU;Compared with the DU,each intervention group increased significantly at 3,7,and 14 days(P<0.01).2.Effects of Buxuhuayu TCM on MicroRNAs in DUs rats: The gene chip screened a total of 185 DEMs,of which 128 DEMs were regulated in the internal treatment group,accounting for 69.19%;The external treatment group adjusted a total of 175 DEMs,accounting for 94.59%;The internal and external treatment groups coordinated to mediate 126 DEMs,accounting for 68.11%;Rt-PCR verified the expression of 8 miRNAs,and the trend was consistent with the gene chip results(P <0.01);The R package predicted a total of 858 target genes,of which GO enrichment analysis yielded a total of 234 biological functions(P<0.01),and KEGG enrichment analysis yielded a total of 56 signal pathways(P<0.05).3.Effects of Buxuhuayu TCM on miRNA-21-5p/TIMP-3/TGF-β axis:Luciferase results showed that compared with the blank group,the luciferase activity of TIMP3 wild-type 3’UTR was significantly inhibited by miR-21-5p(P < 0.01),indicating that the two have a targeting relationship;miRNA21-5p is mainly widely expressed in the dermis of DUs rats.As the wound heals,its expression gradually increases from the 3rd to the 7th day,and gradually decreases from the 7th to the 14th day;Compared with the NU,the miRNA21-5p level of DU was inhibited(P<0.01),the TIMP-3 m RNA level and protein expression increased(P<0.01),and the TGF-βm RNA level and protein expression decreased(P<0.01);Compared with the DU,the level of miRNA21-5p in each intervention group increased(P<0.01),the m RNA level and protein expression of TIMP-3 decreased(P<0.05),and the m RNA level and protein expression of TGF-β increased(P < 0.05);Comparison between the intervention groups: On the 7th day of medication,DU+Comb promoted the expression of miRNA21-5p and TGF-β,and inhibited the expression of TIMP-3 stronger than DU+BXHYD(P<0.05).4.Effects of Buxuhuayu TCM on HIF-1 / miRNA-210-3p/VEGF axis: miR-210-3p was mainly widely expressed in the dermis of DUs rats;Compared with the NU,the miR-210-3p level of DU increased(P<0.01),and the E2F3,HIF-1α,VEGF m RNA level and protein expression decreased(P<0.01);Compared with DU,the level of miR-210-3p in each intervention group decreased(P<0.01),the m RNA level and protein expression of E2F3,HIF-1α,and VEGF decreased(P<0.05);Comparison between the intervention groups: on the 7th day of medication,t DU+Comb had a stronger effect on promoting E2F3 m RNA level and protein expression than DU+BXHYD and DU+FHSJYCO(P<0.05).Conclusion:1.Buxuhuayu TCM can effectively promote wound healing of diabetic rats.In terms of WHR,the combination of internal and external use is better than single medicine.2.Buxuhuayu TCM can coordinately regulate 126 differential miRNAs in the wounds of DUs rats.The target genes of these differential miRNAs may be involved in a variety of biological processes and signal pathways related to wound repair.3.Buxuhuayu TCM can promote wound healing by affecting miRNA-21-5p/TIMP-3/TGF-β1 axis and HIF-1α/miR-210-3p/E2F3 axis;Among them,on the 7th day of administration,the combined use has better regulation of miRNA-21-5p/TIMP-3/TGF-β1 axis than internal treatment alone.