Analysis method for relieving diabetic foot wound scars in tibia transverse movement

Abstract

The invention provides an analysis method for relieving diabetic foot wound scars through tibia transverse movement, and belongs to the technical field of postoperative analys.The method comprises the steps that a New Zealand rabbit serves as an experimental subject, a diabetic rabbit model is built through alloxan, and a DFU wound model is built through femoral artery ligation; bone transport and VEGF (vascular endothelial growth factor) are used for intervention, and the following conclusions are obtained by observing changes of general conditions, pathological changes of skin tissues, inflammatory factors of skin tissues of New Zealand rabbits and expression levels of related genes and proteins in TGF-beta1 / Smad3 signaling pathways: 1, bone transport can inhibit expression levels of inflammatory factors IL-6, IL-18 and TNF-alpha; therefore, the infiltration degree of inflammatory cells in skin tissues can be reduced, and local inflammation and scarring can be effectively improved. 2, the bone transport can promote the expression of related protein on a TGF-beta1 / Smad3 signal channel, so that the effects of promoting muscle fiber contraction, revascularization and reducing scar generation are achieved;

Description

Technical Field

[0001] The invention relates to the technical field of postoperative analysis, in particular to an analysis method for alleviating scars on diabetic foot wounds during tibial transverse transport. Background Art

[0002] Diabetic foot (DFU) is a foot infection, ulceration, and / or deep tissue damage caused by vascular and neuropathy in the lower extremities of diabetic patients. The high amputation and mortality rates impose a significant burden on patients and society. Currently, the medical community lacks effective treatments for DFU, and limb salvage remains a global challenge. The primary cause of lower limb amputation in patients with DFU is tissue damage caused by peripheral neuropathy and peripheral vascular disease, as well as accelerated vascular damage and impaired collagen synthesis, which compromise wound healing.

[0003] As early as the 1950s, renowned scholar Ilizarov proposed the "tension-stress law," which states that slow, steady, and continuous tension in biological tissues stimulates rapid cell division, tissue regeneration, and active growth. Based on this law, bone transfer technology offers a new option for treating diabetic foot. The tibial transverse bone transfer technique utilizes the "time variable," moving the osteotomized bone fragment at a rate of 1 mm per day. This chronic tension and compressive stress stimulation can maximize the regeneration and repair functions of human tissues.

[0004] To further investigate the mechanism of transverse tibial bone transport in the treatment of diabetic foot and verify its effectiveness, researchers developed a small transverse tibial transport experimental device for animal use, providing the necessary tools for simulation experiments in rabbits and other animals. However, existing transverse tibial transport experimental devices are still simple medical devices that have been scaled down, making them difficult to adapt to animal experiments. Animals are active and prone to licking their wounds. This makes the wounds of experimental animals and the transverse tibial transport experimental device susceptible to external forces, resulting in detachment or drift in transverse distance, resulting in loss of experimental specimens and reduced reliability of experimental results.

[0005] The effectiveness of the existing Ilizarov tibial transverse transfer technique has not been analyzed in detail. For example, whether the Ilizarov tibial transverse transfer can activate the PI3K-Akt-mTOR signaling pathway by influencing Exos, accelerating endothelial cell proliferation, collagen deposition, and neovascularization, thereby promoting wound healing. Whether the Ilizarov tibial transverse transfer can regulate the transformation of macrophage M1 and M2 phenotypes by influencing Exos, thereby alleviating the inflammatory state of DFUs, and whether the Ilizarov tibial transverse transfer can dynamically regulate the expression levels of proteins related to the above pathways, affecting the proliferation and crawling of epidermal cells, and regulating the re-epithelialization process to reduce scar formation in diabetic foot ulcer wounds are still unknown. Existing technologies have not yet been analyzed and verified in detail. Therefore, it is necessary to design an analytical method for the reduction of scarring in diabetic foot wounds by tibial transverse transfer. Summary of the Invention

[0006] The purpose of the present invention is to provide an analysis method for reducing scars on diabetic foot wounds by tibial transverse transfer, and to solve the technical problem of related analysis of reducing scars on diabetic foot wounds by tibial transverse transfer.

[0007] Through experimental analysis, we further clarify the therapeutic effect of bone transfer on DFU and the specific reasons why it plays a role in reducing wound scarring, thereby providing a theoretical basis for bone transfer treatment of DFU.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] An analysis method for reducing scarring on diabetic foot wounds by tibial transverse transfer, the method comprising the following steps:

[0010] Step 1: Granulation tissue from the wound edge of the Xilan white rabbit tibia was collected for protein extraction at seven time points: before, before, and 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after tibial transverse transfer. Granulation tissue was collected from the same site of the wound surface of each group at these seven time points, deep into the fascia layer.

[0011] Step 2: Wound healing analysis;

[0012] Step 3: Histomorphological observation: HE and Sirius staining of skin tissue were performed to observe the morphological changes of wound tissue structure;

[0013] Step 4: Detection of inflammatory factors in skin tissue;

[0014] Step 5: Immunohistochemical detection of TNF-α, IL-1β, IL-6, and IL-18 proteins;

[0015] Step 6: Western Blot was used to detect the expression levels of PI3K, Akt, mTOR, type I collagen fibers, type III collagen fibers, and HIF1a protein;

[0016] Step 7: Observe the distribution characteristics of angiogenesis using electron microscopy and perform morphological analysis of Exos using electron microscopy;

[0017] Step 8: ELISA was used to detect inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-18 to analyze the anti-inflammatory effect of Ilizarov tibial transfer.

[0018] Step 9: Immunofluorescence imaging experiments were used to systematically analyze the temporal and spatial distribution characteristics of HIF1a, Exos, type I collagen fibers, and type III collagen fibers.

[0019] Furthermore, the specific process of step 2 is:

[0020] Wound photographs were taken on days 3, 7, 10, and 14 after tibial transverse transfer. The wound healing rate at each time phase was calculated using Image J. The wound area was measured using NIH Image J image analysis software, and the wound healing percentage was calculated using the following formula: wound healing rate = (initial wound area - wound area on the observation day) / initial wound area × 100%. The relationship between the healing rate and the number of Exos was evaluated.

[0021] Furthermore, in step 4, IL-18, IL-6, and TNF-α inflammatory factors were analyzed by ELISA, and PI3K, Akt, and mTOR mRNA expression levels were analyzed by qRT-PCR.

[0022] Ilizarov tibial transverse transfer technique alleviated scar healing. Scar healing in a New Zealand rabbit diabetic foot ulcer model was observed. ImageJ software was used to demarcate the scar area and measure the area. HE and Masson staining were used to observe the changes in wound tissue morphology, granulation formation, and collagen fibers in each group.

[0023] Ilizarov tibial transverse transfer promoted the release of Exos. Electron microscopy was used to observe the distribution characteristics of fibroblasts and angiogenesis. The number and morphology of Exos were analyzed using electron microscopy.

[0024] Ilizarov tibial transverse transport reduces the inflammatory response of the wound. ELISA was used to detect inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-18 to analyze the anti-inflammatory effect of Ilizarov tibial transverse transport. Immunohistochemistry was used to detect the distribution of inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-18.

[0025] The effect of Ilizarov tibial transverse transfer on the expression of genes related to wound healing of diabetic foot ulcers was investigated. The mRNA expressions of PI3K, Akt, mTOR, HIF1a, type I collagen fibers, type III collagen fibers, and α-SMA were detected using RT-PCR technology.

[0026] The effect of Ilizarov tibial transverse transfer on the expression of proteins related to wound healing in diabetic foot ulcers was studied. The protein expressions of PI3K, Akt, mTOR, type I collagen fibers, type III collagen fibers, HIF1a, and α-SMA were detected by Western blot. Immunofluorescence imaging was used to analyze the temporal and spatial distribution characteristics of HIF1a, Exos, type I collagen fibers, type III collagen fibers, and α-SMA proteins.

[0027] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0028] The present invention uses New Zealand rabbits as experimental subjects, employs alloxan to construct a diabetic rabbit model, and constructs a DFU wound model by ligating the femoral artery. Bone transfer and VEGF intervention are then used to observe changes in general condition, pathological changes in skin tissue, inflammatory factors in New Zealand rabbit skin tissue, and the expression levels of related genes and proteins in the TGF-β1 / Smad3 signaling pathway. The following conclusions are drawn: 1. Bone transfer can inhibit the expression levels of inflammatory factors IL-6, IL-18, and TNF-α, thereby reducing the infiltration of inflammatory cells in skin tissue and effectively improving local inflammation and scarring. 2. Bone transfer can promote the expression of related proteins in the TGF-β1 / Smad3 signaling pathway, thereby promoting muscle fiber contraction, angiogenesis, and reducing scar formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing changes in wound surface at various time phases of the present invention;

[0030] Figure 2 This is a graph showing the IL-6 content in skin tissue of each group at different time phases of the present invention;

[0031] Figure 3 This is the HE staining picture of the skin tissue 3 days after surgery;

[0032] Figure 4 This is the HE staining picture of the skin tissue of the present invention 7 days after surgery;

[0033] Figure 5 This is the HE staining picture of the skin tissue of the present invention 10 days after surgery;

[0034] Figure 6This is the HE staining picture of the skin tissue of the present invention 14 days after surgery;

[0035] Figure 7 This is the Sirius staining picture of the skin tissue 3 days after surgery;

[0036] Figure 8 This is the Sirius staining image of the skin tissue 7 days after surgery;

[0037] Figure 9 This is a Sirius staining image of the skin tissue of the present invention 10 days after surgery;

[0038] Figure 10 This is a Sirius staining image of the skin tissue of the present invention 14 days after surgery;

[0039] Figure 11 This is a graph showing the IL-6 content in skin tissue of each group at different time phases of the present invention;

[0040] Figure 12 This is a graph showing the IL-18 content in skin tissue of each group at different time phases of the present invention;

[0041] Figure 13 This is a graph showing the TNF-α content in skin tissue of each group at different time phases of the present invention;

[0042] Figure 14 This is a graph showing changes in HIF-1α and TGFβ1 mRNA expression levels in skin tissue on day 7 of the present invention;

[0043] Figure 15 This is a graph showing changes in HIF-1α and TGFβ1 mRNA expression levels in skin tissue on day 10 of the present invention;

[0044] Figure 16 This is a graph showing changes in the relative expression levels of TNF-α and VEGF proteins in skin tissue on day 3 of the present invention;

[0045] Figure 17 This is a graph showing changes in the relative expression of TNF-α and VEGF proteins in skin tissue on day 7 of the present invention;

[0046] Figure 18 This is a graph showing changes in the relative expression levels of TNF-α and VEGF proteins in skin tissue on day 10 of the present invention;

[0047] Figure 19 This is a graph showing changes in the relative expression of TNF-α and VEGF proteins in skin tissue on day 14 of the present invention;

[0048] Figure 20 This is a graph showing the changes in the relative expression levels of HIF-1α and TGFβ1 proteins in skin tissue on day 3 of the present invention;

[0049] Figure 21This is a graph showing changes in the relative expression levels of HIF-1α and TGFβ1 proteins in skin tissue on day 7 of the present invention;

[0050] Figure 22 This is a graph showing changes in the relative expression levels of HIF-1α and TGFβ1 proteins in skin tissue on day 10 of the present invention;

[0051] Figure 23 This is a graph showing changes in the relative expression levels of HIF-1α and TGFβ1 proteins in skin tissue on day 14 of the present invention; DETAILED DESCRIPTION

[0052] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and by way of preferred embodiments. However, it should be noted that many of the details listed in this specification are merely provided to help the reader gain a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be practiced even without these specific details.

[0053] Thirty healthy New Zealand white rabbits aged 6 months, weighing 2.5 kg to 3.0 kg, were randomly divided into a blank group, a control group, a model group, an Ilizarov group, and a Beifuxin group, with 6 rabbits in each group. The blank group only had skin prepared without any injury. The New Zealand rabbits in the control group were used to establish an acute wound model: anesthesia was induced by injecting 2% sodium pentobarbital into the ear vein at a dose of 1.5 ml / kg, and anesthesia was maintained by injecting a mixture of linalool and ketamine (volume ratio 1:1) at a dose of 0.2 ml / kg intramuscularly. After anesthesia, a 1.5 cm diameter stamp was used to mark the wound. Under sterile conditions, a full-thickness dorsal foot skin defect with a diameter of 1.5 cm was made along the mark, reaching the fascia. After the full-thickness skin defect model was completed, no intervention was performed. The diabetic foot ulcer wound models of the remaining three groups of rabbits were established according to the following methods: (1) The diabetic foot modeling method of Tan and Meng Jianbo was used. Preparation of a rabbit diabetes model: 1g of alloxan was dissolved in 10ml of double-distilled water to prepare a 100mg / ml aqueous solution, which was then filtered and sterilized. Twenty-four New Zealand white rabbits were fasted for 12 hours and then injected with 150mg / kg of alloxan aqueous solution into the marginal ear vein. Fasting marginal ear venous blood was collected 1 day before modeling, and 48 hours, 1 week, and 4 weeks after modeling. Blood glucose was measured using a rapid glucometer. A fasting blood glucose level >15mmol / L after 1 week was considered a successful diabetes model. Following anesthesia as described above, the right medial thigh of the hind limb was shaved with an electric razor. The surgical field was disinfected, and a longitudinal incision was made to expose the vascular sheath. The femoral artery and its branches were carefully isolated, ligated, and the subcutaneous tissue and skin were sutured. The animals were housed at 28-30°C. After spontaneous recovery, they were allowed to eat and drink freely. Penicillin was administered for 5 days postoperatively to prevent infection, and the wound dressing was disinfected and changed daily. Two weeks later, local anesthesia was performed with 2% lidocaine. The modeling area was marked with a 1.5 cm diameter stamp. Under sterile conditions, a full-thickness skin defect with a diameter of 1.5 cm was surgically created on the dorsum of the foot, reaching the fascia. This was then covered with a layer of sterile vaseline gauze and two layers of sterile dry gauze. This established a diabetic foot ulcer wound model. The interventions in the model, Ilizarov, and Beifuxin groups were as follows:

[0054] (1) Model group: The wound was covered with two layers of saline gauze, one layer of sterilized vaseline gauze, and then covered with a layer of sterilized dry gauze and fixed with adhesive tape.

[0055] (2) Ilizarov group: One week after modeling, the Ilizarov group was anesthetized again, and a 10mm×8mm bone block was made on the medial side of the tibial tubercle of the upper tibia on the affected side. A tibial transverse transport bracket was installed and the wound was sutured. The wound surface was cleaned with 1 / 5000 furazolidone solution before the first dressing change, and then with normal saline before each dressing change, twice a day. Three days after the bracket was installed, the bone block was moved laterally by 3.5mm within one week through the traction of the bracket, 0.5mm per day, completed in two times, at 8 am and 8 pm respectively. Thereafter, it was moved in the opposite direction within one week. The foot wound was covered with a layer of sterile vaseline gauze and two layers of sterile dry gauze, and fixed with adhesive tape.

[0056] (3) Beifuxin group: After the wound was disinfected, a layer of human recombinant bovine basic growth factor gel (Beifuxin) was applied, and a layer of sterilized vaseline gauze and two layers of sterilized dry gauze were applied to the wound, and fixed with adhesive tape.

[0057] Protein was extracted from the granulation tissue at the wound edge before surgery, before horizontal transfer, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after horizontal transfer. Granulation tissue was collected from the same part of the wound surface of each group at these 7 time points, deep into the fascia layer.

[0058] Wound healing rate: Wound photos were taken on the 3rd, 7th, 10th and 14th days after surgery, and the wound healing rate at each time phase was calculated using Image J. Figure 1-2 A is the 3rd day; B is the 7th day; C is the 10th day; D is the 14th day. * indicates the difference between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001. n indicates no statistical significance between the model group and the normal group.

[0059] Day 3: Compared with the model group, there was no significant difference in the wound healing rate among the groups (P>0.05).

[0060] Day 7: Compared with the model group, the wound surface of the acute group and the horizontal movement group was smaller, but the difference was not statistically significant; the wound healing of the VEGF group was significantly accelerated (P < 0.001).

[0061] On the 10th day, compared with the model group, the wound surface of the acute group and the horizontal movement group was smaller, but the difference was not statistically significant; the wound healing of the VEGF group was significantly accelerated (P < 0.01).

[0062] Day 14: Compared with the model group, the wound healing in the acute group, the horizontal movement group and the VEGF group was significantly accelerated (P < 0.001).

[0063] With the increase of treatment days, the wound healing rate of VEGF group was at a higher level in each phase. The wound of horizontal movement group healed relatively quickly after 14 days and was basically healed.

[0064] HE and Sirius staining of skin tissue: HE staining of skin tissue, such as Figure 3-6 shown.

[0065] Day 3: Compared with the normal group, the main features of the lesions in each group were inflammatory exudation, and the structure of the model group was relatively disordered.

[0066] Day 7: Compared with the normal group, the model group had disordered structure, obvious red exudate, and the arrangement of the ducts was obviously more disordered than before; a small amount of compensatory vascular proliferation was seen in the acute group and the Beifuxin group; and the horizontal movement group still had obvious exudate.

[0067] Day 10: In the model group, a red exudate was evident at the incision site; the subcutaneous tissue showed numerous inflammatory cell infiltrations; basophilic granules increased significantly; sebaceous glands, sweat glands, and ducts became disorganized and morphologically altered; and some sweat glands and ducts showed red blood cell infiltration. In the acute group, a small amount of exudate was observed at the incision site, with significantly fewer inflammatory cells than in the control group. Numerous new sebaceous glands, sweat glands, and ducts were observed, but their arrangement was disordered and their morphology was irregular. In the VEGF group, a small amount of red exudate was still observed at the incision site, with fewer new sweat glands and ducts. The basal layer, stratum spinosum, granular layer, stratum lucidum, and a small amount of stratum corneum were visible. In the horizontal movement group, no red exudate was evident at the incision site. Granulation tissue formed in the dermis, with numerous new sebaceous glands, sweat glands, and ducts, but their morphology was irregular.

[0068] Day 14: In the model group, a red exudate was evident at the incision site, with a large number of inflammatory cells infiltrating the subcutaneous tissue. The sebaceous glands, ducts, and sweat glands were severely damaged, and no significant regeneration of the full thickness of the skin was observed. In the acute group, a small amount of red exudate was still evident at the incision site, but the skin tissue structure was clear, with a small amount of neovascularization. The morphology of the sweat glands and ducts was normal. In the VEGF group, a slight exudate was observed at the incision site, with a clear skin tissue structure, normal arrangement of ducts and sweat glands, and a small amount of neovascularization within the ducts. In the horizontal movement group, no exudate was observed at the incision site, and no inflammatory cell infiltration was observed in the subcutaneous tissue. The basal layer, stratum spinosum, granular layer, stratum lucidum, and a small amount of stratum corneum were visible. A distinct papillary layer was present between the dermis and epidermis, the reticular layer was dense, and abundant blood flow signals were observed within the ducts.

[0069] The results of Sirius staining of skin tissue are shown in Figures 7-10.

[0070] On the 3rd day, the model and VEGF groups were primarily composed of collagen fibers, with no myofiber hyperplasia observed. However, the acute and horizontal movement groups showed abundant myofiber deposition in yellow areas. Myofiber, the initiator of wound healing and scar hyperplasia, was abundant in the acute and horizontal movement groups, indicating the onset of wound healing and scar formation. Figure 7 In the figure, red represents collagen fibers and yellow represents muscle fibers.

[0071] On the 7th day, the model group and VEGF group mainly began to show a large number of yellow area muscle fiber proliferation, while the acute group and the horizontal movement group still had a small amount of yellow area muscle fiber deposition. The muscle fiber expression in the model group and VEGF group indicated that the wound contraction and healing began to be induced in these groups around the 7th day. On the 7th day, the acute group and the horizontal movement group were mainly composed of collagen fibers, which was the normal structure of skin tissue. Figure 8 In the figure, red represents collagen fibers and yellow represents muscle fibers.

[0072] On the 10th day, muscle fiber deposition was observed in all groups. In the acute group, model group, and VEGF group, a large number of muscle fibers in the yellow area proliferated, indicating that scar proliferation was the main feature at this time. In the VEGF group and horizontal movement group, a small amount of muscle fiber deposition in the yellow area was still observed, and collagen fibers were mainly expressed in the red area. Figure 9 In the figure, red represents collagen fibers and yellow represents muscle fibers.

[0073] On the 14th day, the acute group, model group, and VEGF group showed a large amount of muscle fiber proliferation in the yellow area, far exceeding the collagen fiber, indicating that the group was mainly characterized by scar proliferation at this time; the horizontal movement group had no obvious muscle fiber deposition, and the collagen expression level was close to normal at this time, indicating that the scar on the wound surface was significantly reduced compared with the previous time. Figure 10 As shown, red is collagen fiber and yellow is muscle fiber.

[0074] As time went on, myofiber hyperplasia occurred in the acute group and the horizontal movement group on the third day, inducing wound contraction and healing, while the model group and the VEGF group began to induce wound healing around the seventh day; there was no obvious myofiber deposition in the horizontal movement group on the 14th day, indicating that the wound was close to in situ healing without obvious scarring, while the other three groups were still dominated by scar hyperplasia on the 14th day.

[0075] Detection of inflammatory factors in skin tissue

[0076] The inflammatory factors IL-18, IL-6 and TNF-α were detected by ELISA, and the results are shown below.

[0077] 2.5.1 Expression of IL-6 inflammatory factor

[0078] (1) Analysis of inflammatory factor expression levels on the 3rd day

[0079] The results of IL-6 inflammatory factor expression are shown in Figure 11 A, Compared with the normal group, the expression of IL-6 inflammatory factor in skin tissue of the model group was significantly increased (P < 0.001); compared with the model group, the expression of IL-6 inflammatory factor in the acute group was significantly increased (P < 0.001), and the expression of IL-6 inflammatory factor in the horizontal moving group was significantly decreased (P < 0.01).

[0080] (2) Analysis of inflammatory factor expression levels on day 7

[0081] The results of IL-6 inflammatory factor expression are shown in Figure 11 B, Compared with the normal group, the expression of IL-6 inflammatory factor in skin tissue of the model group was significantly increased (P < 0.001); compared with the model group, the expression of IL-6 inflammatory factor in the VEGF group was significantly increased (P < 0.001), and the expression of IL-6 inflammatory factor in the acute group and the horizontal movement group was significantly decreased (P < 0.001).

[0082] (3) Analysis of inflammatory factor expression levels on day 10

[0083] The results of IL-6 inflammatory factor expression are shown in Figure 11 C, Compared with the normal group, the expression of IL-6 inflammatory factor in the skin tissue of the model group was not significant (P>0.05); compared with the model group, the expression of IL-6 inflammatory factor in the acute group was significantly increased (P<0.001).

[0084] (4) Analysis of inflammatory factor expression levels on day 14

[0085] The results of IL-6 inflammatory factor expression are shown in Figure 11 D, Compared with the normal group, the expression of IL-6 inflammatory factor in the model group's skin tissue was significantly increased (P < 0.001); compared with the model group, the expression of IL-6 inflammatory factor in the VEGF group, acute group and horizontal movement group was significantly decreased (P < 0.01 or P < 0.001).

[0086] As the number of treatment days increased, the expression of IL-6 inflammatory factor in the serum of experimental animals after horizontal movement treatment gradually decreased, and the expression was lowest on the 7th and 14th days, indicating that the anti-inflammatory effect was better at these two time points.

[0087] like Figure 11 Figure 2 shows the IL-6 content in skin tissue of each group at different time points. A is the 3rd day; B is the 7th day; C is the 10th day; D is the 14th day.

[0088] * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistical significance between the model group and the normal group.

[0089] IL-18 inflammatory factor expression

[0090] (1) Analysis of inflammatory factor expression levels on the 3rd day

[0091] The results of IL-18 inflammatory factor expression are shown in Figure 12A, Compared with the normal group, the expression of IL-18 inflammatory factor in skin tissue of the model group had no significant change (P>0.05). Compared with the model group, the expression of IL-18 inflammatory factor in the VEGF group was significantly decreased (P<0.01), and the expression of IL-18 inflammatory factor in the horizontal moving group was significantly increased (P<0.01).

[0092] (2) Analysis of inflammatory factor expression levels on day 7

[0093] The results of IL-18 inflammatory factor expression are shown in Figure 12 B, Compared with the normal group, the expression of IL-18 inflammatory factor in skin tissue of the model group was significantly decreased (P < 0.001); compared with the model group, the expression of IL-18 inflammatory factor in skin tissue of the VEGF group and the horizontal movement group was significantly increased (P < 0.001 or P < 0.05).

[0094] (3) Analysis of inflammatory factor expression levels on day 10

[0095] The results of IL-18 inflammatory factor expression are shown in Figure 12 C, Compared with the normal group, the expression of IL-18 inflammatory factor in the skin tissue of the model group was significantly increased (P < 0.001); compared with the model group, the expression of IL-18 inflammatory factor in the acute group, VEGF group and horizontal movement group was significantly decreased (P < 0.001).

[0096] (4) Analysis of inflammatory factor expression levels on day 14

[0097] The results of IL-18 inflammatory factor expression are shown in Figure 12 D, Compared with the normal group, the expression of IL-18 inflammatory factor in skin tissue of the model group had no significant change (P>0.05); compared with the model group, the expression of IL-18 inflammatory factor in the acute group and VEGF group was significantly decreased (P<0.001 or P<0.01).

[0098] Summary: With the increase of treatment days, the expression of IL-18 inflammatory factor in the serum of experimental animals after horizontal movement treatment gradually decreased, and the expression was the lowest on the 10th day, indicating that the anti-inflammatory effect was better at this time point. Figure 12 As shown, A is the 3rd day; B is the 7th day; C is the 10th day; D is the 14th day, * is the comparison between the model group and the normal group, # is the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n is no statistically significant difference between the model group and the normal group.

[0099] TNF-α inflammatory factor expression

[0100] (1) Analysis of inflammatory factor expression levels on the 3rd day

[0101] The results of TNF-α inflammatory factor expression are shown in Figure 13 A, Compared with the normal group, the expression of TNF-α inflammatory factor in skin tissue of the model group had no significant change (P>0.05); compared with the model group, the expression of TNF-α inflammatory factor in the VEGF group and the horizontal movement group was significantly increased (P<0.001 or P<0.05).

[0102] (2) Analysis of inflammatory factor expression levels on day 7

[0103] The results of TNF-α inflammatory factor expression are shown in Figure 13 B, Compared with the normal group, the expression of TNF-α inflammatory factor in skin tissue of the model group was significantly increased (P < 0.01); compared with the model group, the expression of TNF-α inflammatory factor in skin tissue of the acute group, VEGF group and horizontal movement group had no significant change (P > 0.05).

[0104] (3) Analysis of inflammatory factor expression levels on day 10

[0105] The results of TNF-α inflammatory factor expression are shown in Figure 13 C, Compared with the normal group, the expression of TNF-α inflammatory factor in skin tissue of the model group was significantly increased (P<0.01); compared with the model group, the expression of TNF-α inflammatory factor in the VEGF group and the horizontal movement group was significantly increased (P<0.05).

[0106] (4) Analysis of inflammatory factor expression levels on day 14

[0107] The results of TNF-α inflammatory factor expression are shown in Figure 13 D, Compared with the normal group, the expression of TNF-α inflammatory factor in skin tissue of the model group was significantly increased (P<0.001); compared with the model group, the expression of TNF-α inflammatory factor in the horizontal moving group was significantly decreased (P<0.01).

[0108] As the number of treatment days increased, the expression of TNF-α inflammatory factor in the serum of experimental animals after horizontal movement treatment decreased, and was at a lower level than that of other groups on the 14th day, indicating that the anti-inflammatory effect was better at this time point. Figure 13 As shown, A is the normal group; B is the acute group; C is the model group; D is the VEGF group; E is the horizontal movement group, *P<0.05; **P<0.01; ***P<0.001.

[0109] HIF-1α and TGFβ1 mRNA levels in skin tissue

[0110] (1) Analysis of HIF-1α and TGFβ1 mRNA expression levels on day 7

[0111] The results of HIF-1α mRNA expression levels are shown in Figure 14 A, Compared with the normal group, the HIF-1α mRNA expression level in the model group was significantly decreased (P < 0.001); compared with the model group, the mRNA expression levels in the VEGF group, acute group and horizontal movement group had no significant changes (P > 0.05).

[0112] The results of TGFβ1 mRNA expression levels are shown in Figure 14 B, Compared with the normal group, the expression level of TGFβ1 mRNA in the model group was significantly decreased (P<0.001); compared with the model group, the expression level of TGFβ1 mRNA in the horizontal moving group was significantly increased (P<0.01), while there was no significant change in the expression level of TGFβ1 mRNA in the VEGF group and the acute group (P>0.05).

[0113] like Figure 14 As shown, * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates there is no statistical significance between the model group and the normal group.

[0114] (2) Analysis of HIF-1α and TGFβ1 mRNA expression levels on day 10

[0115] The results of HIF-1α mRNA expression levels are shown in Figure 15 A, Compared with the normal group, the HIF-1α mRNA expression level in the model group had no significant change (P>0.05); compared with the model group, the HIF-1α mRNA expression level in the acute group was significantly increased (P<0.01), while the mRNA expression levels in the VEGF group and the horizontal movement group had no significant change (P>0.05).

[0116] The results of TGFβ1 mRNA expression levels are shown in Figure 15 B, Compared with the normal group, the expression level of TGFβ1 mRNA in the model group was significantly decreased (P<0.0,1); compared with the model group, the expression level of TGFβ1 mRNA in the VEGF group, acute group and horizontal movement group had no significant change (P>0.05).

[0117] like Figure 15 As shown in the figure, the changes in HIF-1α and TGFβ1 mRNA expression levels in skin tissue on the 10th day are compared between the model group and the normal group, # represents the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n represents no statistical significance between the model group and the normal group.

[0118] The expression levels of HIF-1α and TGFβ1 mRNA were low at the 7th and 10th day time points, and the expression level of TGFβ1 mRNA was high at the 7th day; HIF-1α was oxygen-dependent, and it is not ruled out that it had already decreased when RNA was extracted, which will be further verified in the future.

[0119] VEGF and TNF-α protein expression levels in skin tissue

[0120] (1) Analysis of protein expression levels on the 3rd day

[0121] The results of VEGF protein expression are shown in Figure 16 A, Compared with the normal group, the relative expression of VEGF protein in the model group was significantly decreased (P < 0.001); compared with the model group, the expression of VEGF protein in the VEGF group, acute group and horizontal movement group was significantly increased (P < 0.05 or P < 0.001).

[0122] The results of TNF-α protein expression are shown in Figure 16 B, Compared with the normal group, the relative expression of TNF-α protein in the model group was significantly increased (P < 0.001). Compared with the model group, the expression of TNF-α protein in the VEGF group and the acute group was significantly increased (P < 0.01 or P < 0.001), and the expression of TNF-α protein in the horizontal moving group was significantly decreased (P < 0.05).

[0123] like Figure 16 As shown in the figure, the changes in the relative expression of TNF-α and VEGF proteins in skin tissue on the 3rd day, * represents the comparison between the model group and the normal group, # represents the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n represents no statistical significance between the model group and the normal group.

[0124] Analysis of protein expression levels on day 7

[0125] The results of VEGF protein expression are shown in Figure 17 A, Compared with the normal group, the relative expression of VEGF protein in the model group was significantly increased (P < 0.001); compared with the model group, the expression of VEGF protein in the VEGF group and the acute group was significantly decreased (P < 0.001), and the expression of VEGF protein in the horizontal moving group was significantly increased, and the differences were statistically significant (P < 0.001).

[0126] The results of TNF-α protein expression are shown in Figure 17B, Compared with the normal group, the relative expression of TNF-α protein in the model group's skin tissue was increased (P < 0.001). Compared with the model group, the expression of TNF-α protein in the VEGF group and the acute group was increased (P < 0.001), while the expression of TNF-α protein in the horizontal movement group was significantly decreased (P < 0.001).

[0127] like Figure 17 The graph shows the changes in the relative expression levels of TNF-α and VEGF proteins in skin tissue on the 7th day. * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistical significance between the model group and the normal group.

[0128] Analysis of protein expression levels on day 10

[0129] The results of VEGF protein expression are shown in Figure 18 A, Compared with the normal group, the relative expression of VEGF protein in the model group was significantly decreased (P < 0.001); compared with the model group, the expression of VEGF protein in the horizontal moving group, VEGF group and acute group was increased (P < 0.001).

[0130] The results of TNF-α protein expression are shown in Figure 18 -B, compared with the normal group, the relative expression of TNF-α protein in the model group's skin tissue did not show significant changes (P>0.05); compared with the model group, the expression of TNF-α protein in the VEGF group did not show significant differences (P>0.05), the expression of TNF-α protein in the acute group was significantly increased (P<0.001), and the expression of TNF-α protein in the horizontal moving group was significantly decreased (P<0.001). Figure 18 As shown, * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates there is no statistical significance between the model group and the normal group.

[0131] Analysis of protein expression levels on day 14

[0132] The results of VEGF protein expression are shown in Figure 19 A, Compared with the normal group, the relative expression of VEGF protein in the model group was significantly decreased (P < 0.05); compared with the model group, the expression of VEGF protein in the VEGF group had no significant change (P > 0.05), while the expression of VEGF protein in the horizontal moving group and the acute group was significantly increased (P < 0.01 or P < 0.05).

[0133] The results of TNF-α protein expression are shown in Figure 19A, Compared with the normal group, the relative expression of TNF-α protein in the model group was significantly increased (P < 0.05); compared with the model group, the expression of TNF-α protein in the VEGF group and the acute group was significantly increased (P < 0.01 or P < 0.001), and the expression of TNF-α protein in the horizontal movement group was significantly decreased (P < 0.001).

[0134] like Figure 19 As shown, * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates there is no statistical significance between the model group and the normal group.

[0135] As the number of treatment days increased, the expression of VEGF protein in the skin tissue after horizontal moving treatment was higher than that in the model group. On the 14th day, the VEGF expression in the horizontal moving group was higher than that in the other groups. The expression of TNF-α protein in each phase after horizontal moving treatment was lower than that in the model group, which was particularly obvious on the 10th and 14th days.

[0136] HIF-1α and TGFβ1 protein expression levels in skin tissue

[0137] To determine the effect of transverse tibial bone transport on DFU, we performed immunofluorescence analysis; the results are shown below:

[0138] (1) Immunofluorescence analysis of HIF-1α and TGFβ1 protein expression levels on day 3

[0139] Representative images of HIF-1α immunofluorescence are shown in Figure 20 A; Compared with the normal group, the relative expression of HIF-1α protein in the model group had no significant change (P>0.05); compared with the model group, the expression of HIF-1α protein in the VEGF group had no significant change (P>0.05), while the expression of HIF-1α protein in the acute group and the horizontal movement group was significantly increased (P<0.05 or P<0.001).

[0140] Representative images of TGFβ1 immunofluorescence are shown in Figure 20 B; Compared with the normal group, the relative expression of TGFβ1 protein in the model group was significantly decreased (P<0.01); compared with the model group, the expression of TGFβ1 protein in the VEGF group, acute group, and horizontal movement group had no significant changes (P>0.05).

[0141] like Figure 20Figure 3: Changes in the relative expression of HIF-1α and TGFβ1 proteins in skin tissue on day 3. A: Representative images of HIF-1α and TGFβ1 proteins in each group, ×400; B, C: Fluorescence intensity statistics of HIF-1α and TGFβ1 proteins. * indicates the comparison between the model group and the normal group, # indicates the comparison between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistically significant difference between the model group and the normal group.

[0142] Immunofluorescence analysis of HIF-1α and TGFβ1 protein expression levels on day 7

[0143] Representative images of HIF-1α immunofluorescence are shown in Figure 21 A; Compared with the normal group, the relative expression of HIF-1α protein in the model group's skin tissue had no significant change (P>0.05); compared with the model group, the expression of HIF-1α protein in the VEGF group had no significant change, and the difference was not statistically significant (P>0.05). The expression of HIF-1α protein in the acute group and the horizontal movement group was significantly increased (P<0.05 or P<0.001).

[0144] Representative images of TGFβ1 immunofluorescence are shown in Figure 21 B; Compared with the normal group, the relative expression of TGFβ1 protein in the model group was significantly decreased (P<0.001); compared with the model group, the expression of TGFβ1 protein in the VEGF group and the acute group had no significant change (P>0.05), while the expression of TGFβ1 protein in the horizontal moving group was significantly increased, and the difference was statistically significant (P<0.01). Figure 21 A: Representative images of HIF-1α and TGFβ1 proteins in each group, ×400; B, C: Fluorescence intensity statistics of HIF-1α and TGFβ1 proteins. * indicates the difference between the model group and the normal group, # indicates the difference between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistically significant difference between the model group and the normal group.

[0145] Immunofluorescence analysis of HIF-1α and TGFβ1 protein expression levels on day 10

[0146] Representative images of HIF-1α immunofluorescence are shown in Figure 22 A; Compared with the normal group, the relative expression of HIF-1α protein in the model group's skin tissue had no significant change (P>0.05). Compared with the model group, the expression of HIF-1α protein in the VEGF group was significantly increased (P<0.05), while that in the horizontal movement group was significantly decreased (P<0.05).

[0147] Representative images of TGFβ1 immunofluorescence are shown in Figure 22B: Compared with the normal group, the relative expression of TGFβ1 protein in the model group skin tissue had no significant change (P>0.05); compared with the model group, the expression of TGFβ1 protein in the VEGF group, acute group, and horizontal movement group had no significant change (P>0.05). Figure 22 Figure 2: Representative images of HIF-1α and TGFβ1 proteins in each group, ×400. B and C: Fluorescence intensity statistics of HIF-1α and TGFβ1 proteins. * indicates the difference between the model group and the normal group, # indicates the difference between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistically significant difference between the model group and the normal group.

[0148] Immunofluorescence analysis of HIF-1α and TGFβ1 protein expression levels on day 14

[0149] Representative images of HIF-1α immunofluorescence are shown in Figure 23 A; Compared with the normal group, the relative expression of HIF-1α protein in the model group's skin tissue had no significant change (P>0.05); compared with the model group, the expression of HIF-1α protein in the VEGF group and the acute group had no significant change (P>0.05), while the expression of HIF-1α protein in the horizontal moving group was significantly increased (P<0.05).

[0150] Representative images of TGFβ1 immunofluorescence are shown in Figure 23 B; Compared with the normal group, the relative expression of TGFβ1 protein in the model group skin tissue did not change significantly (P>0.05); compared with the model group, the expression of TGFβ1 protein in the VEGF group and the horizontal movement group did not change significantly (P>0.05), and the expression of HIF-1α protein in the acute group was significantly increased (P<0.05). Figure 23 Figure 2: Representative images of HIF-1α and TGFβ1 proteins in each group, ×400. B and C: Fluorescence intensity statistics of HIF-1α and TGFβ1 proteins. * indicates the difference between the model group and the normal group, # indicates the difference between each group and the model group; * / #: P < 0.05, ** / ##: P < 0.01, *** / ###: P < 0.001, n indicates no statistically significant difference between the model group and the normal group.

[0151] As the number of treatment days increased, the expression of HIF protein in the skin tissue after horizontal moving treatment was higher than that in the model group. On the 14th day, the expression of VEGF in the horizontal moving group was higher than that in the other groups. The expression of TNF-α protein in each phase after horizontal moving treatment was lower than that in the model group, which was particularly obvious on the 10th and 14th days.

[0152] Alloxan was used to establish a diabetic rabbit model, and a DFU wound model was established by ligating the femoral artery. Bone transposition and VEGF intervention were then used. By observing changes in general condition, skin histopathological changes, inflammatory factors in New Zealand rabbit skin tissue, and the expression levels of related genes and proteins in the TGF-β1 / Smad3 signaling pathway, the following conclusions were drawn:

[0153] 1. Bone transfer can inhibit the expression levels of inflammatory factors IL-6, IL-18 and TNF-α, thereby reducing the infiltration of inflammatory cells in skin tissue and effectively improving local inflammation and scarring.

[0154] 2. Bone transfer can promote the expression of related proteins in the TGF-β1 / Smad3 signaling pathway, thereby promoting muscle fiber contraction, angiogenesis and reducing scar formation.

[0155] Matters not covered by the present invention are known technologies.

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An analytical method for reducing scarring on diabetic foot wounds using tibial transverse transfer, characterized by: The method comprises the following steps: Step 1: Granulation tissue from the wound edge of the Xilan white rabbit tibia was collected for protein extraction at seven time points: before, before, and 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks after tibial transverse transfer. Granulation tissue was collected from the same site of the wound surface of each group at these seven time points, deep into the fascia layer. Step 2: Wound healing analysis; Step 3: Histomorphological observation: HE and Sirius staining of skin tissue were performed to observe the morphological changes of wound tissue structure; Step 4: Detection of inflammatory factors in skin tissue; Step 5: Immunohistochemical detection of TNF-α, IL-1β, IL-6, and IL-18 proteins; Step 6: Western Blot was used to detect the expression levels of PI3K, Akt, mTOR, type I collagen fibers, type III collagen fibers, and HIF1a protein; Step 7: Observe the distribution characteristics of angiogenesis using electron microscopy and perform morphological analysis of Exos using electron microscopy; Step 8: ELISA was used to detect inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-18 to analyze the anti-inflammatory effect of Ilizarov tibial transfer. Step 9: Immunofluorescence imaging experiments were used to systematically analyze the temporal and spatial distribution characteristics of HIF1a, Exos, type I collagen fibers, and type III collagen fibers.

2. The method for analyzing scar reduction of diabetic foot wounds by tibial transverse transfer according to claim 1, characterized in that: The specific process of step 2 is: Wound photographs were taken on days 3, 7, 10, and 14 after tibial transverse transfer. The wound healing rate at each time phase was calculated using Image J. The wound area was measured using NIH Image J image analysis software, and the wound healing percentage was calculated using the following formula: wound healing rate = (initial wound area - wound area on the observation day) / initial wound area × 100%. The relationship between the healing rate and the number of Exos was evaluated.

3. The method for analyzing scar reduction of diabetic foot wounds by tibial transverse transfer according to claim 1, characterized in that: In step 4, IL-18, IL-6, and TNF-α inflammatory factors were analyzed by ELISA, and the mRNA expression levels of PI3K, Akt, and mTOR were analyzed by qRT-PCR.

Images