摘要
ObjectiveTo investigate the promotional effect of astragaloside on the repair and healing of chronic non-healing wounds and its mechanism. MethodA total of 60 male SD rats were constructed with full-layer skin defect wounds on the back， and except for the control （Con） group， the rest were constructed with non-healing wounds， which were then randomly divided into the sham-operation （sham） group， the low-dose astragaloside group， the high-dose astragaloside group， the astragaloside + LY294002 ［phosphatidylinositol 3-kinase （PI3K） inhibitor］ group， and the astragaloside + EX527 ［silencing regulatory protein 1 （SIRT1） inhibitor］ group. The percentage of wound area in each group was observed on the 2nd， 4th， 6th， and 8th days after wound molding. Collagen type Ⅰ alpha 1 （COL1A1） and alpha smooth muscle actin （α-SMA） expressions in the wound tissue were detected by immunofluorescence. Hematoxylin and eosin （HE） staining was performed to determine the pathological structure of the wound. The mRNA expression of inflammatory factors in the wound was measured by real-time polymerase chain reaction （Real-time PCR）， and the expression of proteins related to the SIRT1/ nuclear factor （NF）-κB and PI3K/protein kinase B （Akt） signaling pathways in the wound was tested by Western blot. ResultCompared with the sham group， the percentage of postoperative wound area of rats in both low-dose and high-dose astragaloside groups gradually decreased with time， and the efficacy of the high-dose astragaloside group was better. Compared with the Con group， the fluorescence intensity of COL1A1 in wound tissue of the sham group decreased， while the expression of α-SMA increased. The epithelial tissue was severely damaged， with an increase in the thickness， and a large number of inflammatory cells were seen in the infiltration. The mRNA expression of tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and inducible nitric oxide synthase （iNOS） was elevated. The protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was elevated， while SIRT1 expression was decreased （P<0.05）. Compared with the sham group， the fluorescence intensity of COL1A1 and α-SMA increased after astragaloside treatment. The number of epithelial cells increased， and the thickness decreased. The inflammatory cells decreased， and the amount of collagen increased. The mRNA expression of TNF-α， IL-1β， IL-6， and iNOS was decreased， and the protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was decreased. SIRT1 was elevated， and the effect was better in the high-dose astragaloside group （P<0.05）. Compared with the high-dose astragaloside group， inhibition of the PI3K/Akt and SIRT1 pathways by LY294002 and EX527 prevented the therapeutic efficacy of astragaloside on chronic non-healing wounds. ConclusionThe topical application of astragaloside significantly promotes the healing of chronic non-healing wounds in rats， and the mechanism may be related to the activation of the PI3K/Akt pathway and the SIRT1/NF-κB pathway.
摘要
BACKGROUND:Cartilage degeneration and subchondral bone damage are the main pathological features of osteoarthritis,and treatment based on this pathological feature will be a promising improvement for osteoarthritis. OBJECTIVE:To design and study an annotated strontium ranelate-loaded drug delivery system and to observe its therapeutic effect on promoting cartilage repair and improving subchondral bone structure in osteoarthritis. METHODS:(1)In vitro experiment:Strontium ranelate was loaded into sodium alginate/collagen hydrogel matrix to construct in situ drug delivery system,and the in vitro slow release performance of the system was characterized.Strontium ranelate-loaded sodium alginate/collagen hydrogel(experimental group)and alginate sodium/collagen hydrogel(control group)were co-cultured with bone marrow mesenchymal stem cells,respectively,and cultured cells were used as a blank control group to detect cell proliferative activity.After chondroblast-induced differentiation,saffron O staining,Alcian blue staining and RT-qPCR were performed respectively.The two hydrogels were co-cultured with osteoblasts,and the cultured cells were used as a blank control group for immunofluorescence staining and RT-qPCR.(2)In vivo experiment:A total of 18 adult SD rats were selected and the model of right posterior knee osteoarthritis was established by the method of medial meniscectomy.After 1 week,the rats were divided into three groups by the random number table method:The blank group did not receive any treatment.The control group was injected with sodium alginate/collagen hydrogel in the knee,and the experimental group was injected with strontium ranelate-loaded sodium alginate/collagen hydrogel,with 6 rats in each group.After 6 weeks,the samples were subjected to Micro-CT scanning,hematoxylin-eosin staining,saffron O-solid green staining and immunofluorescence staining. RESULTS AND CONCLUSION:(1)In vitro experiment:Strontium ranelate-loaded sodium alginate/collagen hydrogel had porous microstructure and sustainable release of strontium ranelate.At 21 days,the cumulative release reached(60.89±0.58)%.Bone marrow mesenchymal stem cell staining showed that both hydrogels had good cytocompatibility.The results of the CCK-8 assay demonstrated that strontium ranelate-loaded sodium alginate/collagen hydrogel could promote the proliferation of bone marrow mesenchymal stem cells.The results of Safranin O staining,Alcian blue staining,immunofluorescence staining and RT-qPCR exhibited that strontium ranelate-loaded sodium alginate/collagen hydrogel could promote chondrogenic differentiation of bone marrow mesenchymal stem cells.Immunofluorescence staining and RT-qPCR revealed that strontium ranelate-loaded sodium alginate/collagen hydrogel could decrease bone resorptivity by increasing the ratio of osteophosphorin/nuclear factor κB receptor activator ligand.(2)In vivo experiment:Micro-CT scan verified that compared with the blank group and control group,the subchondral bone volume fraction and bone mineral density of the knee of rats were increased in the experimental group(P<0.05,P<0.01).Histological staining displayed that compared with the blank group and control group,the knee cartilage injury was significantly reduced;the expression of type II collagen was promoted,and the expression of matrix metalloproteinase 2 protein was inhibited in the experimental group(P<0.05,P<0.01).(3)These results confirm that the strontium ranelate-loaded sodium alginate/collagen hydrogel can promote the repair of cartilage defects in osteoarthritis and reconstruct the complex interface between cartilage and subchondral bone.