Study On The Regulatory Effects Of Huiyang Shengji Decoction And Jixueteng On The Chemotaxis And Migration Of MSCs During The Healing Process Of Chronic Skin Ulcers

Background:Chronic skin ulcer is a common and frequently-occurring disease in surgery,often complicated by diabetes,radiotherapy and chemotherapy,and peripheral vascular disease.In particular,the wound without healing for a long time,shows cold wound,thin pus,purple and dark warm symptoms around the sore,which is called the "yin syndrome ulcers"in traditional Chinese medicine(TCM),and it is the most difficult to treat.In recent years,Mesenchymal Stem Cells(MSCs)based cell therapy has made great progress in chronic wound repair.However,the treatment of ulcer repair by implanting MSCs has disadvantages such as unstable cell source,complex operation,adverse reactions and high cost,which makes it difficult to use and promote in clinical practice.Chronic skin ulcers always belong to the category of TCM chuang yang.Chronic skin ulcers with Yin syndrome have the characteristics of "deficiency of kidney essence".Professor Zhao Bingnan,the founder of the Department of Dermal Surgery of Beijing Hospital of Traditional Chinese Medicine,and his successors,Professor Wang Yuzhang and Professor Lv Peiwen,treated Yin syndrome of chronic skin ulcer with Huiyang Shengji Decoction(HYSJD),which is based on tonifying kidney,filling essence,promoting blood circulation and dredging collaterals,is safe and effective.As the main component of HYSJD for promoting blood circulation and dredging collaterals,Spatholobi Caulis has been widely reported in promoting hematopoietic function of bone marrow,but there are few studies on Spatholobi Caulis used in wound repair.Stem cells have the characteristics of "the essence of the kidney" in TCM.Therefore,it is hypothesized that HYSJD can promote the proliferation of endogenous bone marrow MSCs,enhance their chemotaxis and migration ability,and achieve the transformation of Qi and blood,the growth of muscle and flesh,and promote the wound healing of chronic skin ulcers through tonifying kidney and filling essence,activating blood circulation and dredging collaterals.Therefore,it is hypothesized that HYSJD promotes the proliferation of endogenous bone marrow mesenchymal stem cells(BMSCs),and promote the wound healing of chronic skin ulcers by supplementing kidney essence and promoting blood circulation,thereby enhancing the chemotaxis and migration ability of MSCs.This study will be verified through in vivo and in vitro experiments to explore the target of HYSJD and its main component,Spatholobi Caulis,in treating Yin syndrome of chronic skin ulcer.Objective:To study the effects and mechanisms of HYSJD and Spatholobi Caulis on the proliferation,chemotaxis and migration of BMSCs in the process of promoting the healing of chronic skin ulcers,and to provide experimental and theoretical basis for the clinical prevention and treatment of chronic skin ulcers in vascular surgery of traditional Chinese medicine.Methods:1.The analysis of blood components of Huiyang Shengji Decoction and water extract of Spatholobi Caulis:BALB/C mice were given medicine intragastric administration to prepare drug-containing serum.Ultra-performance liquid chromatography combined with tandem mass spectrometry(UPLC-MS/MS)was used to detect the blood components of HYSJD and Spatholobi Caulis water extract.2.In vivo experiment:(1)db/db diabetic mouse wound model:Diabetic wound model was prepared by the method of full thickness skin perforation in the middle part of back of db/db diabetic mice,and HYSJD was used for intervention.The wound size was recorded by digital camera and the wound healing rate was analyzed.Hematoxylin-eosin staining(HE)was used to observe the histological morphology of the wound.The whole bone marrow cells were extracted and the number of bone marrow cells was observed under microscope.The proportion of Stem cell markers CD29,CD44,Stem cell surface antigen 1(Sca-1)and CD117 positive cells in bone marrow were detected by flow cytometry.Immunofluorescence staining was used to detect the number of MSCs marker CD29 and CD271 positive cells in the wounds.The expression of Interleukin 3(IL-3),IL-6,IL-13 and other inflammatory cytokines in the wound were detected by inflammatory factor microarray.(2)Wound model of myelosuppressive rats:Doxorubicin was injected intravenously(8 mg/kg)in combination with full-thickness skin perforation in the middle part of the back to make a myelosuppressive rat model.HYSJD and water extract of Spatholobi Caulis were used for intervention.The number of white blood cells in peripheral blood was detected.The weight of thymus and spleen were weighed and the thymus index and spleen index were calculated.The wound healing rate was observed.The concentrations of stromal cell-derived factor-1(SDF-1)and granulocyte-colony Stimulating factor(G-CSF)in rat serum were detected by enzyme-linked immunosorbentassay(ELISA).The histological morphology and collagen content of the wound were observed by HE and Masson staining.The number of CD34 and CD 133 positive cells in skin lesions was detected by immunofluorescence staining.The proportion of CD29,CD90,CD34 and CD 133 positive cells in bone marrow and peripheral blood was detected by flow cytometry.The BMSCs were purified by whole bone marrow cell adherent culture.The proliferation ability of BMSCs was detected by EdU staining.The migration and chemotactic ability of BMSCs was detected by scratch test and transwell chemotactic assay.The relative expression levels of SDF-1 and CXC chemokine receptor 4(CXCR4)proteins in BMSCs were detected by Western Blot.3.In vitro experiment:BMSCs were purified by whole bone marrow cell adherent culture,and identified by flow cytometry,adipogenic and osteogenic differentiation.BMSCs model of oxidative stress was established by stimulating BMSCs with H2O2.CCK-8 was used to detect the proliferation of BMSCs.The migration and chemotactic ability of BMSCs was detected by scratch test and transwell chemotactic assay.The protein expressions of SDF-1 and CXCR4 in BMSCs were detected by Western Blot.Results:1.19 compounds including flavonoids,iridoids,terpenes,triterpenes,steroids,saponins,and carbohydrate compounds were obtained by analyzing the components of HYSJD.The higher contents were sucrose,formononetin,baicalein,strychnine,(6?R,11?R)-10-hydroxy-3,9-dimethoxy rosandalane,methyl 16-oxyacetyl porycoic acid,mononoside,riboflavin,?-sitosterol,oldolatine-7-O-?-D-glucoside,formononetin,and calynisoflavone glycoside,respectively.Mainly from Achyranthis bidentatae radix(Niuxi),Spatholobi Caulis(Jixueteng),Atractylodis Rhizoma(Cangzhu),Corni Fructus(Shanzhuyu),Astragali Radix(Huangqi),and Poria(Fuling).According to the analysis of the blood components of the aqueous extract of Spatholobi Caulis,8 compounds were obtained,with the contents ranging from high to low as formononetin,Afrormosin,isoglycyrrhizin,daidzein,3,7-dihydroxy-6-methoxydihydroflavanol,vermicidin,prixanthin,and kayanin,all of which were belong to flavonoids.2.In vivo experiment:(1)db/db diabetic mouse wound model:Overall efficacy observation showed that compared with model group,the wound healing rate of mice in Huiyang Shengji Decoction group was increased.In the wound,compared with the model group,the growth of granulation tissue on the wound increased,the number of stem cell markers CD29 and CD271 positive cells increased,and the expression of IL-3,IL-6,IL-15 and Fas ligand(Fas L)increased.The expression of IL-13,Tumor necrosis factor-?(TNF-?),intercellular adhesion molecule-1(ICAM-1),Eotaxin and LIX/CXCL5 decreased.In the bone marrow,compared with the model group,the number of bone marrow cells in the Huiyang Shengji Decoction group increased,and the proportion of CD29,CD44 and Sca-1 positive cells in the bone marrow increased.(2)Wound model of myelosuppressive rats:The observation of overall efficacy showed that compared with model group,HYSJD and water extract of Spatholobi Caulis significantly improved the wound healing rate of rats,and significantly increased the thymus index and spleen index.In the wound,compared with the model group,the number of inflammatory cells on the wound in the HYSJD and water extract of Spatholobi Caulis group was significantly less,the synthesis of collagen was significantly increased,and the number of CD34 and CD 133 positive stem cell markers was significantly increased.The proportion of CD29,CD90,CD34 and CD133 positive cells increased significantly in bone marrow.In the peripheral blood,compared with the model group,the Serum concentrations of SDF-1 and G-CSF,and the number of white blood cells and the proportion of CD29,CD90,CD34 and CD133 positive cells were increased in the groups of HYSJD and water extract of Spatholobi Caulis.After drug intervention,primary rat BMSCs were isolated and extracted.Compared with the model group.the number of proliferating cells,migration and chemotaxis ability of BMSCs were significantly increased in the HYSJD and water extract of Spatholobi Caulis group,and the protein expressions of SDF-1 and CXCR4 were significantly increased.3.In vitro experiment:BMSCs were spindle-shaped,swirling and sheetlike,and had good refractive properties.The expression of CD29 and CD44 in the third generation BMSCs was positive,but CD45 was almost not expressed.The proliferation activity,chemotaxis and migration of BMSCs were decreased under oxidative stress.Compared with the model group,the proliferation activity,migration and chemotaxis of BMSCs were increased,and the protein expressions of SDF-1 and CXCR4 were increased in the HYSJD group and Spatholobi Caulis group.Conclusion:1.Flavonoids are the main ingredients in the blood of HYSJD and Spatholobi Caulis,suggesting that flavonoids play an important role in the efficacy of HYSJD and Spatholobi Caulis.2.HYSJD can promote the proliferation of bone marrow stem cells in mice,increase the number of stem cells in the wound,reduce the inflammatory response of the wound,promote the regeneration of granulation tissue,and thus accelerate the wound healing.3.HYSJD and Spatholobi Caulis can can promote the secretion of SDF-1 and G-CSF in rat serum,and activate the SDF-1/CXCR4 signaling axis,promote the proliferation,migration and chemotaxis of BMSCs,mobilize stem cells into peripheral blood,increase the number of wound stem cells,promote collagen synthesis and angiogenesis,and thus accelerate wound repair.4.HYSJD and Spatholobi Caulis can activate the SDF-1/CXCR4 signal axis in vitro,protect the activity of BMSCs with oxidative injury,and enhance the migration and chemotaxis of BMSCs under oxidative injury.