| ObjectiveDelayed wound healing in diabetes leads to severe diabetic complications, such as chronic wounds, diabetic foot, which acting as a major cause of death and disability in diabetic patients. The causes of diabetic refractory wound are very complex. The typical features include decreased angiogenesis, delayed inflammation resolusion, and reduced extracellular matrix(ECM) deposition. Tuo-Li-Xiao-Du-San is a traditional Chinese medicine formula consisting of four herbs: Danggui(Radix Angelica sinensis), Huangqi(Radix Astragali), Baizhi(Angelica dahurica), and Zaojiaoci(thorns of Gleditsia sinensis). It is derived from “orthodox manual of surgery”(“Wai Ke Zheng Zong” in Chinese) formulated by a famous TCM physicians Shigong Chen and has been used for the treatment of various refractory wounds. In Chinese medicine theory, TLXDS includes the therapeutic method of “TUO” represented by Radix Angelica sinensis and Radix Astragali and the therapeutic method of “TOU” represented by Angelica dahurica and thorns of Gleditsia sinensis. Therapeutic method of “TUO” means raising “Qi”(vital energy)and nourishing “Blood”(body circulation), while therapeutic method of “TOU” refers to cleansing wound environment and eliminating toxins. There is possibility that TLXDS might be suitable for the treatment of diabetic wounds. In this study, a diabetic animal model was adopted to observe the therapeutic effects of TLXDS complete formula and its decomposed recipes—TUO method and TOU method on healing diabetic wounds through assessing healing rate, angiogenesis, inflammation resolution, and ECM deposition of the wound. Furthermore, we used cell models to explore the underlying mechanisms of TLXDS.Methods1. SD rats were induced diabetic through a single high dose of intravenous injection of STZ. The rats subsequently underwent procedures of dorsal skin puncturing to establish a diabetic animal model of cutaneous wound. Rats were randomly assigned to normal control group(NC), diabetic group(DM), TUO group(DM + TUO group),TOU group(DM + TOU group), and TLXDS group(DM + TT). Rats were sacrificed5 days, 8 days, 11 days, and 14 days after wounding respectively to acquire the wound tissues for analysis. We used hematoxylin and eosin stain and Masson’s trichrome staining to observe the morphological changes of wounds and employed immunohistochemistry, q PCR, and Western Blotting to assess the influence of drugs intervention on the following aspects both on protein and m RNA levels:(1) wound vascularization and expression of angiogenic factors;(2) resolution of inflammation and expression of inflammatory cytokines;(3) collagen deposition and expression of related regulatory factors.2. To evaluate the in vitro angiogenic potential of the ethanoic extracts of four herbs in TLXDS, we used CCK-8 assay, wound healing assay, and matrigel assay to investigate their effects of on the proliferation, migration, and tube formation of human umbilical vein endothelial cells(HUVEC), and adopted aortic ring assay to assess their abilities on stimulating endothelial sprouting. To further clarify the underlying mechanisms, we detected the activation of key angiogenesis pathways including ERK1/2, PI3K/Akt, and e NOS/NO signaling pathways upon herb extracts treatment, and observe the effect of particular signaling pathway inhibitors on their angiogenic activities. q PCR, Western Blotting, and cell immunofluorescence techniques were used to determine the effects of four herb extracts on HIF-1αexpression and transcriptional activity in hypoxia HUVEC.3. We applied LPS-stimulated mouse macrophage(RAW264.7) as a cell model of inflammation. Analyze the effects of ethanoic extracts of Angelica dahurica and thorns of Gleditsia sinensis on the expression and activity of NLRP3/ASC/caspase-1/IL-1β inflammasome in LPS-activated macrophage cultured in high glucose medium. Detect the ROS content and TXNIP m RNA and protein expression in macrophage to to clarify its possible mechanisms of regulating NLRP3 inflammasome.(1). Animal modeling situations: After STZ injection, rats developed typical diabetic symptoms with higher blood glucose levels and rapid weight loss. The healing of DM group was significant delayed compared with NC group(P <0.05). Typical pathological features of diabetic skin ulcers such as decreased angiogenesis, delayed inflammation resolusion, and reduced ECM deposition were observed in diabetic rats.(2). Healing rate and general indicators: The healing rates of DM group were significantly decreased compared with NC group at each time point. The healing rate of TUO group was significantly higher than DM group since 6 days after wounding,while TOU group exceeded DM group since 8 days after wounding and catched up with TUO group since days 10. The healing rates of TLXDS group surpassed DM group and stayed ahead of TUO and TOU group since 4 days after wounding. There was no difference in blood glucose level between DM group and intervening groups during the entire treatment period. By the end of experiment, the body weight of intervening groups was significantly higher than DM groups.(3). Wound vascularization and growth factor expression: CD31 and desmin staining,expression of VEGF, VEGFR-2, PDGF, PDGFR-β of DM group were significantly decreased compared with NC group. CD31 and desmin staining, expression of VEGF,VEGFR-2, PDGF, PDGFR-β of TUO group, TOU group, and TLXDS group were significantly higher than DM group, with the best effect by TLXDS treatment,followed by TUO and TOU treatment. The expression of HIF-1α in DM group was significantly reduced compared with NC group on days 5, which was rescued by TUO and TLXDS treatment.(4). Inflammation resolution, collagen deposition and related factors expression: On days 11, the CD68 and MPO staining, proinflammatory mediators IL-1β, TNF-α and protease MMP-9 expression of DM group were significantly increased compared with NC group, while the TGF-β1 expression was significantly reduced. TOU and TLXDS treatment significantly decreased the CD68 and MPO staining, proinflammatory mediators IL-1β, TNF-α and protease MMP-9 expression. TUO, TOU, and TLXDS treatment all increased the expression of TGF-β1. On days 14, the COL Ⅰstaining of Results1. Results of animal experimentsDM group was significantly reduced, which was corrected by TUO, TOU, and TLXDS treatment, with the best effect by TLXDS treatment, followed by TOU and TUO treatment.2. Results of cell experiments(1). The effect of the four herb ethanoic extracts on endothelial cells in vitro angiogenesis: The extracts of Angelica sinensis, Radix Astragali, Angelica dahurica,and thorns of Gleditsia sinensis all can increased the proliferation of HUVEC. The extracts of Radix Angelica sinensis, Radix Astragali, and Angelica dahurica can significantly improve the migration and tube formation ability of HUVEC. The extracts of Radix Astragali and Angelica dahurica can stimulate the sprouting of aortic ring. The extracts of Radix Astragali and Angelica dahurica can activated ERK1 / 2, PI3 K / Akt, e NOS / NO signaling pathway in HUVEC. Their angiogenic abilities can be significantly inhibited by special pathway inhibitors.(2). The effects of the four herb ethanoic extracts on HIF-1α expression and function in HUVEC cultured under hypoxia condition: There is no significant difference in HIF-1α m RNA levels betweent low glucose group and high glucose group, but the protein level and the nuclear translocation of HIF-1α in high glucose group was significantly decreased compared with low glucose group. The extracts of Radix Angelica sinensis and Radix Astragali can significantly increased the m RNA and protein level of HIF-1α and promote its translocation toward nucleus and transcriptional activity.(3). The effects of ethanoic extracts of Angelica dahurica and thorns of Gleditsia sinensis on NLRP3 inflammasome in macrophage: High glucose increases the expression of pro-caspase-1 and pro-IL-1β, and the cleavage of pro-caspase-1 and pro-IL-1β in both non-activated macrophages and LPS-activated macrophages.Glucose concentration has no effects on the expression of NLRP3 and ASC.Pretreatment with extracts of Angelica dahurica and thorns of Gleditsia sinensis can significantly decreased the m RNA and protein level of pro-IL-1β. And extracts of Angelica dahurica can reduce the expression of pro-caspase-1 and the cleavage ofpro-caspase-1 and pro-IL-1β. High glucose treatment significantly increases ROS content in macrophage and the expression of TXNIP, which can be ameliorated by pretreatment with extracts of Angelica dahurica.Conclusions1. TLXDS complete formula and its decomposed recipes—TUO method and TOU method can promote the healing of diabetic wounds. In the early phase of healing,TUO method has significant effects, while TOU method catches up in middle-late stages. TLXDS complete formula has the best effects throughout the observation period.2. TUO method increases angiogenesis and maturation of new blood vessels, the mechanism of which may be associated with upregulation of pro-angiogenic factors through improving the expression and transcriptional function of HIF-1α.3. Among the four herbs in TLXDS, Radix Astragali and Angelica dahurica have remarkable angiogenic activities. Those effects are associated with their activation of ERK1 / 2, PI3 K / Akt and e NOS / NO signal pathway.4. TOU method accelerates the resolution of inflammation, improves the healing microenvironment. The mechanism might be associated with decreased release of IL-1β through inhibition of the overactive NLRP3 inflammasome in diabetes. |
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