| The difficulty of wound healing in diabetic patients is an urgent clinical problem to be solved.Diabetic patients have reduced insulin secretion and decreased ability to metabolize blood glucose,further complicating the wound healing process and forming chronic wounds,leading to stalled healing.Macrophages are major immune effector cells that play a crucial role in tissue homeostasis and injury.Studies have found that in the pathological state of diabetes,macrophages in the wound are in a continuous proinflammatory state,and then release pro-inflammatory factors,which seriously affects the transition of wound healing from inflammatory phase to proliferative phase,leading to prolonged inflammatory phase.Therefore,regulating the polarization of macrophages from a pro-inflammatory phenotype(M1)to an anti-inflammatory phenotype(M2)can improve the ability of diabetic wound healing.Tetramethylpyrazine is a component extracted from Ligusticum chuanxiong Hort.It has the effects of improving blood circulation and dispersing stasis,antiinflammatory and anti-oxidation,and has the ability to improve wound healing.Because the direct action of Chinese medicine monomer on the wound will cause irritation,which will hinder the wound healing.In recent years,because of its their uniform porous structure and suitable swelling rate,hydrogel can also be used as a carrier for drug release,which has attracted more and more attention in the field of regeneration.To sum up,we plan to deliver tetramethylpyrazine through hyaluronic acid hydrogel carrier to promote wound healing in diabetes.Firstly,the effect of TMP on cell viability was detected by MTT assay in this study,and it was confirmed that TMP would not cause toxicity to skin-related cells.Flow cytometry subsequently demonstrated TMP’s ability to regulate macrophage phenotype at a given concentration,that is,it decreased the proportion of M1-type macrophages(mainly regulated by STAT1 signaling pathway)while increasing the proportion of M2-type macrophages(mainly regulated by STAT6 signaling pathway).By Real Time fluorescence quantitative PCR(RT-qPCR),enzyme-linked immunosorbent assay,ELISA,Western Blotting and other experiments were conducted to study the mechanism of TMP’s regulation of macrophage polarization,indicating that TMP can up-regulate the release level of cytokines related to M1 macrophages,mRNA expression of key genes,and expression of signature proteins.At the same time,the release level of cytokines associated with M2-type macrophages,mRNA expression of key genes and signature proteins were down-regulated.Secondly,in order to improve the pharmaceutical performance of TMP in diabetes wound healing,hyaluronic acid hydrogel is proposed for follow-up research.It has been reported that hyaluronic acid hydrogels with different molecular weights have different physical and chemical properties.In this study,three kinds of hyaluronic acid with different molecular weights were oxidized with sodium periodate,and then chemical cross-linking agent Adipic dihydrazide(ADH)was added.The hydrogel with optimal physical and chemical properties was screened by the determination of swelling rate,sustained release ability,and resistance to enzymatic hydrolysis.Finally,the skin injury model of diabetic mice was established,and tetramethylpyrazine-loaded hyaluronic acid hydrogel(TMP-HA)was applied to the model.Through RT-qPCR,ELISA,WB,immunofluorescence staining,H&E staining and Masson staining of skin tissue,it was confirmed that TMP-HA could promote diabetic wound healing by regulating the phenotype of macrophages in the wound.This study demonstrated the ability of tetramethylpyrazin-loaded HA hydrogel to modulate macrophage phenotype,which is expected to become a novel therapeutic method for promoting diabetic wound healing. |
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