| Objective: Under the guidance of vessel collateral theory, the present study established a rat model of diabetic foot using high-fat diet in combination with streptozotocin(STZ) as well as ligating and cutting off femoral artery and vein, in order to observe changes in rat’s blood glucose, blood fat and imaging of lower-limb blood flow after treatment with Tongxinluo(TXL) combined with peripheral blood derived mesenchymal stem cells(PB-MSCs). By-observing indicators of inflammation and oxidative stress, changes in advanced glycation end products(AGEs) and expressions of angiogenesis-related factors, this study also investigated potential mechanism of action for treating diabetic foot and promoting angiogenesis by TXL combined with PB-MSCs transplantation. The above-mentioned research, aimed to provide new treatment for diabetic foot.Methods:1 Theoretical exploration of diabetic foot under the guidance of vessel collateral theoryVessel collateral theory, a systematic theory proposed by my supervisor, is used for guiding the prevention and treatment of vasculopathy. It mainly researches thoracic obstruction, cardiodynia, stroke, palpitation, heart accumulation, cardiac edema, heart impediment, diabetes mellitus and gangrene, and involves many modern major diseases, such as cardio-cerebrovascular disease, diabetes and its vascular complications, and peripheral vascular disease. Guided by vessel collateral theory, this paper put forward a new concept of “vessel collateral-vascular system disease” based on close relationship between meridian of traditional Chinese medicine(TCM) and blood vessel of Western medicine, between vessel collateral and small-medium blood vessel, as well as between minute collateral of vessel collateral end and microvessel/ microcirculation. Diabetic foot is a complication caused by long-term effect of diabetes mellitus(DM) on large, medium, small and micro vessels of limbs. It is within the scope of “symptom-complex of excessive eating and gangrene” in TCM. Apparently, diabetic foot is also included in the research on vessel collateral-vascular system disease. On the basis of vessel collateral theory, this study provided a theoretical guidance for treatment of diabetic foot with combined application of TXL and stem cells by exploring improvement in microenvironment of the ischemic area, acceleration of angiogenesis and establishment of collateral circulation.2 Research on role of TXL combined with PB-MSCs transplantation in the treatment of rats with diabetic footDiabetic rat model was established using high-fat diet combined with-intraperitoneally injected STZ of 35mg/Kg. And diabetic foot rat model was then made by ligating and cutting femoral artery and vein in rat’s right hind limb. Hind limb ischemia model of normal rat from the same batch was used as the control. The two models were categorized into Normal group and Control(Surgery) group. Rats with diabetic foot were randomly divided into six groups: diabetic foot model(Model), Tongxinluo(TXL), Cilostazol, PB-MSCs(stem cells), TXL combined with PB-MSCs(T-MSCs), and Cilostazol combined with PB-MSCs(C-MSCs). Stem cell transplantation was performed in PB-MSCs, T-MSCs and C-MSCs. On the third day after surgery, 0.05 m L of cell suspension(PB-MSCs 1.0×106/m L) was injected into eight 5mm-spaced sites respectively in subcutaneous muscular layer of ischemic hind limb(avoiding blood vessels); rats in the remaining groups were injected, an equal volume of sterile RPM1640 medium. On the third day after surgery, intragastric administration of 0.4g/kg TXL was initiated in TXL group and T-MSCs group, 18mg/kg cilostazol in Cilostazol group and C-MSCs group, and 0.9%CMC-Na of the same volume in the remaining groups. All these agents were administered once a day for consecutive 28 days. Abdominal aortic blood was sampled on the 7th day and the 28 th day respectively to separate serum, and rats’ blood glucose and blood fat were determined. Radioimmunoassay(RIA) was used to detect fasting insulin(FINS) for calculating insulin resistance index(HOMA-IR) and insulin sensitivity index(Ln(ISI)). Laser Doppler Perfusion Imager(LDPI) was used for detecting hind-limb blood flow. Arteriography was also performed to test changes in hind-limb blood vessels.3 Effects of TXL combined with PB-MSCs transplantation on inflammatory response, oxidative stress and AGEs in rats with diabetic footModeling method was identical to section 2, so did grouping, drug administration and sampling of diabetic-foot rats. Hind limb ischemia model of normal rat from the same batch served as control group(surgery group). RIA method was employed to determine serum interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) levels. A chemical method was used for detection of serum superoxide dismutase(SOD), malonaldehyde(MDA) and AGEs levels. Changes in muscular tissue structures of rats of each group were observed through HE staining and transmission electron microscope. Western Blot was implemented to detect protein expression of intercel-lular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1).4 Study on mechanism of proangiogenic effect of TXL combined with PB-MSCs transplantation on diabetic foot ratsExperimental modeling, grouping, drug administration and sampling methods were the same as those in section 3. Enzyme linked immunosorbent assay(ELISA) was conducted for determining serum vascular endothelial growth factor-A(VEGF-A) and hypoxia-inducible factor-1α(HIF-1α) levels. State of vascular endothelial cell in muscular tissue was observed under transmission electron microscope. Real-time PCR was used to detect gene expression in VEGF-A and cell cycle proteins(cyclin D1). Western Blot was carried out for detection of gene expression on CD34, CD105, VEGF-A, vascular endothelial growth factor receptor 2(VEGF-R2), p-Akt, Phosphorylati-on of glycogen synthase kinase-3β(p-GSK-3β) and cyclin D1.Results: 1 Research on role of TXL combined with PB-MSCs transplantation in the treatment of diabetic foot rat 1.1 Changes in blood glucose, blood fat, FINS, HOMA-IR and Ln(ISI) of rats from each groupOn the 7th day and the 28 th day during treatment, compared with control group, model group showed significantly enhanced blood glucose, significantly decreased serum total cholesterol(TC), triglyceride(TG) and low density lipoprotein cholesterol(LDL-C) levels, significantly declined high density lipoprotein cholesterol(HDL-C) level, reduced serum FINS, increased HOMA-IR and reduced Ln(ISI). All these differences had statistical significance(P<0.01). Rats of each treatment group had blood glucose level lower than model group but such difference was not statistically significant(P>0.05). Changes in serum FINS had no statistical significance(P>0.05); TXL group and T-MSCs group presented decreased serum TC, TG and LDL-C levels, enhanced HDL-C level, reduced HOMA-IR and increased Ln(ISI)(P<0.05, P<0.01). Compared with PB-MSCs group, TXL group and T-MSCs group had significantly higher serum HDL-C level on the 7th day(P<0.01) as well as lower serum TG and LDL-C levels and higher serum HDL-C level on the 28 th day(P<0.05, P<0.01). On the 28 th day of treatment, T-MSCs group exhibited decline in serum TG level and increase in HDL-C level compared with C-MSCs group(P<0.05, P<0.01). 1.2 Changes in blood flow different ratio of each group at different time pointsBefore surgery, blood flow different ratio between each group had no statistical significance(P>0.05). Immediately after the surgery, control group, model group and each treatment group had significantly lower ischemic hind-limb blood perfusion and significantly higher blood flow different ratio than normal group(P<0.01). On the 7th day, blood flow different ratio of control group was significantly higher than normal group(P<0.01); compared with model group, each treatment group had significantly increased ischemic hind-limb blood perfusion and significantly decreased blood flow different ratio(P<0.01); T-MSCs was superior to TXL and Cilostazol(P<0.05). On the 28 th day, blood flow different ratio of control group was obviously higher than that in normal group(P<0.01); compared with model group, each treatment group showed significant increase in ischemic hind-limb blood perfusion and significant decline in blood flow different ratio(P<0.01); among all treatment groups, T-MSCs group had ischemic hind-limb blood perfusion better than that of TXL, Cilostazol and PB-MSCs(P<0.05, P<0.01). 1.3 Changes in hind-limb arteriography of rats from each groupArteriography of normal group indicated unobstructed blood flow, uniform arterial wall thickness, clearly shown blood vessels and clear terminal arteriole imaging. Arteriography of model group revealed slow blood flow, partially uneven arterial wall thickness, formation of a few collateral circulations, which were interconnected as a capillary network, and weakened imaging of terminal arteriole. Compared with model group, all treatment groups were demonstrated in arteriography to have varying-degree improvement in vascular wall morphology and blood flow. Arteriography of T-MSCs showed unobstructed blood flow, collateral circulation(interconnected as a capillary network) formation and clear terminal arteriole imaging. 2 Effects of TXL combined with PB-MSCs transplantation on inflammatory response, oxidative stress and AGEs in diabetic foot rats 2.1 Pathological changes in ischemic hind-limb muscle tissue of rats from each groupCommon optical microscopy of HE staining revealed:Control group: slightly disordered myofibrillar arrangement, occasional muscular interstitial fibrosis, and mild inflammatory cell infiltration; model group: obviously disordered myofibrillar arrangement, partial muscular interstitial fibrosis, and remarkable leukocyte infiltration; all treatment groups: alleviated myofibrillar arrangement disorder, muscular interstitial fibrosis and leukocyte infiltration compared with model group, with the greatest decrease in TXL and T-MSCs group. 2.2 Transmission electron microscopic observation of ultrastructure of muscle tissues of ischemic hind-limb in each groupControl group: Regular arrangement and clear structure of myomere and myofilament; mitochondria and glycogen particles were visible; some mitochondria swelled mildly; a small number of double membrane structures, crest structures and intercristal spaces were blurry; sarcoplasmic reticulum expanded slightly, with a few cavitation bubbles and complete myolemma. Model group: Partial lysis and dilation of myofilament; lipid droplet and glycogen accumulation; sparse myofibril; widened space with breakage; mitochondria edema; crest structure breakage and lysis of some mitochondria; vacuolar degeneration; corpus-medullae-like change; local lysis and disappear-ance of adventitia; dilation of endoplasmic reticulum; widening of muscular cell nucleus gap. Each treatment group: Varying-degree reduction in ultrastruc-ture of above-mentioned muscle tissues, with the most significant reduction in T-MSCs group. 2.3 Comparison of serum IL-1, IL-6 and TNF-α levels between rats of each groupSerum IL-1, IL-6 and TNF-α levels of model group were significantly higher than those in control group(P<0.01). Compared with model group, PB-MSCs group showed a trend of decreased serum IL-1, IL-6 and TNF-α levels but these differences were not statistically significant(P>0.05). The remaining treatment groups all had obviously decline in serum IL-1, IL-6 and TNF-α levels(P<0.05, P<0.01). 2.4 Comparison of serum SOD and MDA levels between rats of each groupCompared with control group, model group had significantly lower serum SOD level and significantly higher MDA level(P<0.01). Compared with model group, all treatment groups showed an obvious increase in serum SOD level and a remarkable decrease in MDA level(P<0.05, P<0.01). 2.5 Comparison of serum AGEs level between rats of each groupSerum AGEs level of model group was significantly higher than that of control group(P<0.01). Serum AGEs level of PB-MSCs group was lower than model group but such difference had no statistical significance(P>0.05); the remaining treatment groups presented an obviously reduced serum AGEs level(P<0.05). 2.6 Comparison of ICAM-1 and VCAM-1 protein in ischemic hind-limb muscle tissue between each groupCompared with control group, model group had significantly enhanced ICAM-1 and VCAM-1 protein levels in muscle tissue(P<0.01). Compared with model group, ICAM-1 and VCAM-1 protein levels were decreased in PB-MSCs group, which was no statistically significant(P>0.05). ICAM-1 and VCAM-1 protein expression of muscle tissue was reduced in all remaining treatment groups(P<0.05, P<0.01), with the lowest level in T-MSCs group. 3 Study of mechanism for proangiogenic effect of TXL combined with PB-MSCs transplantation on diabetic-foot rats 3.1 Transmission electron microscopic observation of microvascular endothelial cell ultrastructure of ischemic hind-limb muscle of diabetic foot ratsControl group: Complete endothelial cell with normal shape and clear structure; mitochondria and pinocytosis vesicles were observed; intercellular junction was close; some extracellular matrix membranes were vague, with slightly narrow lumen. Model group: Mild atrophy and damage of endothelial cell; partial crest and membrane integration of mitochondria in the cell; visible cavitation-like change; mild dilation of intercellular space; complete intercellular junction; loose and discontinuous extracellular matrixes; lysis of partial region. All treatment groups: Regenerative vascular endothelial cells were seen in PB-MSCs, T-MSCs and C-MSCs groups; in PB-MSCs group, overall cellular structure was not normal yet and close vascular connection was incomplete. All remaining treatment groups had varying-degree decrease in ultrastructure. 3.2 Comparison of CD34 and CD105 protein expression in ischemic hind-limb muscle tissue between rats of each group Compared with control group, model group had significantly reduced CD34 and CD105 protein expression in muscle tissue(P <0.01). Compared with model group, all treatment groups presented significantly increased CD34 and CD105 protein expression in muscle tissue(P<0.01). T-MSCs group had the highest CD34 and CD105 protein expression in muscle tissue. 3.3 Comparison of serum VEGF-A and HIF-1α levels between rats of each groupCompared with control group, model group had significantly lower serum VEGF-A and HIF-1α levels(P<0.01). Each treatment group showed obvious enhancement in serum VEGF-A level(P<0.05, P<0.01), compared with model group. Serum HIF-1α levels of T-MSCs and C-MSCs groups were significantly increased(P<0.01). HIF-1α levels of T-MSCs and C-MSCs groups were remarkably higher than those in PB-MSCs group(P<0.05). 3.4 Comparison of VEGF-A and VEGF-R2 protein expression in ischemic hind-limb muscle tissue between rats of each groupCompared with control group, model group had significantly decreased VEGF-A and VEGF-R2 protein expression in muscle tissue(P<0.01). Compared with model group, all treatment groups exhibited significant increase in VEGF-A and VEGF-R2 protein expression in muscle tissue(P<0.01). Comparison between treatment groups indicated that VEGF-A and VEGF-R2 protein expression levels of PB-MSCs group were higher than Cilostazol group(P<0.05); muscle-tissue VEGF-A and VEGF-R2 protein expression levels of muscle tissues in T-MSCs and C-MSCs groups were higher than those in TXL, Cilostazol and PB-MSCs groups(P<0.01). 3.5 Comparison of VEGF-A and cyclin D1 m RNA protein expression in muscle tissues of ischemic hind-limb of rats in each groupCompared with the control group, significantly reduced VEGF-A and cyclin D1 m RNA protein expression of muscle tissue were observed in the model group(P<0.01). Compared with the model group, there was significantly increased VEGF-A and cyclin D1 m RNA expression of muscle tissue in each treatment group(P<0.01). Comparison between treatment groups indicated higher VEGF-A and cyclin D1 m RNA expression of muscle tissue in PB-MSCs group than those in TXL and Cilostazol group(P<0.05). Compared with TXL and Cilostazol and PB-MSCs group, VEGF-A and cyclin D1 m RNA expression of muscle tissue were higher in T-MSCs and C-MSCs group(P<0.01). 3.6 Comparison of cyclin D1, p-Akt and p-GSK-3β protein expressions in muscle tissue of ischemic hind-limb in each groupCompared with control group, model group had significantly-reduced cyclin D1 and p-Akt protein expressions as well as significantly-enhanced p-GSK-3β protein expression in muscle tissue(P<0.01). Compared with model group, each treatment group had significantly-increased cyclin D1 and p-Akt protein expressions and significantly-decreased p-GSK-3β protein expression(P<0.01). According to comparisons between treatment groups, cyclin D1 and p-Akt protein expressions of muscular tissue in PB-MSCs group were higher than those in TXL and Cilostazol groups(P<0.01, P<0.05); T-MSCs and C-MSCs groups showed higher muscular-tissue cyclin D1 and p-Akt protein expressions as well as lower p-GSK-3β protein expression than TXL, Cilostazol and PB-MSCs groups(P<0.05, P<0.01).Conclusions:1 Under the guidance of vessel collateral theory and from the perspective of “collateral-vessel” protection, the present study proposes that application of dredging collaterals in prevention and treatment of diabetic foot can be effective in the treatment by promoting improvement in local microenvironment of ischemic area and facilitating therapeutic angiogenesis and collateral circulation establishment.2 TXL or TXL combined with PB-MSCs transplantation can increase body weight and ISI, reduce HOMA-IR and lipid metabolism disorder, promote therapeutic angiogenesis and collateral circulation establishment, and improve blood supply of ischemic hind limb in rats with diabetic foot.3 TXL can lower level of inflammation, enhance anti-oxidative capacity, reduce accumulation of AGEs, decrease pathological and ultrastructural changes in muscular tissue, protect and improve microenvironment of stem cell transplantation, and thus facilitate angiogenesis after local PB-MSCs transplantation.4 PB-MSCs can promote endothelial cell proliferation and differentiation to generate new vessels by improving VEGF-A and VEGF-R2 gene and protein expression, activating p-Akt protein expression, inhibiting p-GSK-3β protein expression and up-regulating cyclin D1 protein level. Combined application of TXL and PB-MSCs transplantation has above-mentioned effects, which is superior to stem cell transplantation alone, indicating TXL can enhance endothelial cell proliferation and differentiation after stem cells transplantation. |
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