CN111888349B

CN111888349B - Action of nimbin in preparing medicine for promoting survival of ischemic superlong random skin flap

Info

Publication number: CN111888349B
Application number: CN202010747603.3A
Authority: CN
Inventors: 林丁盛; 何纪兵; 方妙杰; 马心怡; 李文杰; 刘莹莹
Original assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Current assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2021-10-22
Anticipated expiration: 2040-07-29
Also published as: CN111888349A

Abstract

The invention relates to an effect of azadirachtin in preparing a medicine for promoting survival of an ischemic random skin flap, wherein an oral administration mode is adopted in the process of promoting survival of the ischemic random skin flap, the using method is that the oral administration dosage is 100mg/kg or 200mg/kg every day, 5-7 days are a treatment course, and a conclusion is drawn through a large number of animal experiments.

Description

Action of nimbin in preparing medicine for promoting survival of ischemic superlong random skin flap

Technical Field

The invention relates to a new application of azadirachtin, in particular to an effect of azadirachtin in promoting survival of ischemic ultralong random skin flaps.

Background

In recent years, industrial, traffic, energy and the like have been rapidly developed, and various wounds such as large-area wounds and tissue defects have been increasingly serious. Such wounds often leave varying degrees of deformity and dysfunction after repair, particularly traumatic deformities of the hands and face. The requirements of patients on perfect appearance and functional recovery are increasingly raised, and the repair technology and treatment means of wound surface problems become an important thinking of medicine. The random flap has great flexibility, so that the random flap is most widely applied to the repair of tissue defects and deformities in plastic surgery. However, distal flap necrosis is a common complication in plastic surgery due to the lack of the arteriovenous system of the voluntary flap, relying primarily on perfusion of the subcutaneous capillary network, and the formation of new vascular ganglia starting distally from the flap pedicle bed. In order to enable the flap to survive smoothly in the transfer process, the flap is designed with certain limitation on the length and width ratio, the length-width ratio exceeds the specified limit of 1.5-2: 1, the necrosis caused by the nutrient metabolic disorder of the flap tissue at the distal part can occur after the flap transfer, and the flap transfer operation in the prior art has many limitations because the survival rate of the flap is low, so the flap transplantation operation cannot be applied to more scenes.

Factors that affect flap survival include blood circulation within the flap, metabolic factors, tolerance of the tissue to conditions such as ischemic hypoxia. Promoting angiogenesis, improving local blood circulation, inhibiting the generation of inflammatory mediators, relieving ischemia-reperfusion injury, inhibiting apoptosis and the like become key factors for improving the success rate of random flap surgery. How to cooperatively regulate and control the factors can effectively reduce the possible skin flap necrosis, become one of the core problems in the treatment and repair of the wound surface, and have strong social value, economic value, theoretical value and practical research value.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide the function of promoting the survival of ischemic overlong random skin flaps.

Further, the specification of the ischemia overlong random skin flap is 3cm X9 cm.

Furthermore, the azadirachtin comprises the medicinal application of preparing the medicine for improving the expression quantity of VEGF in the ischemic overlong random skin flap.

Furthermore, the azadirachtin is used for preparing a medicine for promoting the regeneration of blood vessels in the ischemic overlong random skin flap.

Furthermore, the azadirachtin has the function of preparing a medicament for promoting the proliferation of fiber cells in the ischemic ultralong random flap.

Further, the azadirachtin comprises the application of preparing a medicament for inhibiting the expression of inflammatory factors in the ischemic overlong random flap, wherein the inflammatory factors comprise IL-6 and/or IL-1 beta and/or TNF-alpha and/or NF-kB and/or TLR 4.

Further, the azadirachtin is used for preparing a medicament for promoting the anti-oxidation capacity of the ischemic ultralong random flap, wherein the anti-oxidation capacity comprises the capacity of resisting lipid peroxidation.

Further, the azadirachtin is used for preparing a medicine for reducing tissue edema in the ischemic overlong random skin flap.

Furthermore, the application method is that the daily oral dosage is 100-200mg/kg of azadirachtin, and 5-7 days are a course of treatment.

Further, the azadirachtin is administered in the form of a solvent having a content of 10mg/ml, and the solvent of the azadirachtin solvent is physiological saline.

The invention has the following advantages: the application of azadirachtin in the treatment process of the ischemic ultralong random skin flap is concluded through a large number of animal gastric lavage experiments, the survival area ratio of the ultralong skin flap can be obviously improved by using the azadirachtin, the survival rate of the skin flap is greatly improved by the following improvements, on one hand, the expression quantity of VEGF expression quantity can be well promoted, the proliferation of fiber cells of the skin flap is improved, the blood vessel density and the blood flow of the skin flap are improved, on the other hand, the inflammatory response of the skin flap can be reduced and the antioxidant capacity can be enhanced by using the azadirachtin, the damage of the cell of the skin flap is reduced, the edema degree of the ischemic ultralong random skin flap is reduced, and the healing speed of the skin flap is further promoted.

Drawings

FIG. 1 shows the chemical structure of azadirachtin;

FIG. 2A. ischemia arbitrary skin flap model establishment; 2B, establishing a self-made headgear by an ischemia random flap model;

FIG. 3A is an appearance comparison graph of the survival condition of skin flaps on the back of rats 7d after the operation, and 3B is a comparison of the average survival rate of the skin flaps;

FIG. 4A. TNF- α expression levels detected by ELISA diabody method, IL-6 expression levels detected by ELISA diabody method;

FIG. 5A. postoperative 7d laser Doppler blood flow assessment; 5B, comparing the average blood perfusion level of the skin flap;

FIG. 6 comparison of skin flap lead oxide-gelatin angiography of control and experimental groups seven days after surgery;

FIG. 7A is a microscopic (100 times) observation of HE staining of skin flaps in the middle area of the experimental group and the control group; 7B, comparing the density of the neutrophils at the middle end of the flap; 7C, comparing the average density of the capillaries at the middle end of the flap;

FIG. 8A integrated absorbances of skin flaps VEGF, TLR4, NF-kB, IL-1 β, IL-6 and TNF- α expression (immunohistochemistry X400) in azadirachtin experimental group and saline control group, middle-end VEGF, TLR4, NF-kB, IL-1 β, IL-6 and TNF- α in 8B skin flaps;

fig. 9A and 9B are respectively: comparing the SOD value and MDA content of skin flap in the azadirachtin experimental group and the normal saline control group.

Detailed Description

The present invention will be further described in detail with reference to examples and effect examples, but the scope of the present invention is not limited thereto.

Establishment of ischemia random skin flap model

Healthy male Sprague Dawley rats were selected 54, provided by the university of Wenzhou medical laboratory animal center, at a clean grade, weighing 200-. Rats were divided into high-dose azadirachtin group, low-dose azadirachtin group and normal saline control group according to the random number table method, and each group had 18 animals.

The formula proportion of the azadirachtin solvent is as follows: 1g of azadirachtin powder is dissolved in 100ml of normal saline to prepare a 10mg/ml solution, and the chemical structural formula of azadirachtin is shown in figure 1.

The rats were injected with 1% sodium pentobarbital normal saline solution in the abdominal cavity and anesthetized at a dose of 40 mg/kg. After the anesthesia takes effect, the hair on the back trunk of the rat is primarily shaved by a shaver, the hair which is not completely removed is moistened by distilled water, and the depilatory cream is uniformly smeared. After waiting 3 minutes, the cream was gently removed with a plastic sheet and the process was repeated until the hair was completely removed. Designing a modified McFarlane skin flap with the longitudinal axis of the skin flap taking the connecting line of the two iliac crests as a base and the parallel dimension of the long axis of the back of the rat as 3cm X9 cm in the middle of the back of the rat: marking the sides with Melan solution, cutting skin layer by layer along the marking lines during operation, separating subcutaneous tissue to deep fascia shallow layer, exposing the wound surface active bleeding part while electrocoagulation or ligation, completely separating skin flap and deep fascia, cutting off two symmetrical sacral arteries at the pedicle of the flap with an operation knife, and keeping capillary network under dermis to stop bleeding thoroughly. Finally, the skin flap was intermittently sutured in situ using 4-0 medical mousse nylon suture. After operation, 4 ten thousand units of gentamicin are injected intraperitoneally to each group of rats, and iodophor is used for disinfection and erythromycin ointment is applied to prevent infection. To facilitate postoperative observation, the flap was trisected into a proximal flap region (region I), a middle flap region (region II) and a distal flap region (region III) according to the blood supply characteristics of the flap (fig. 2A). Rats were housed in a single cage. Water and food for rats are supplied from the laboratory, and the temperature (25 ℃ and humidity 40-60%) and lighting conditions in the laboratory are controlled to be appropriate and the same. Normal saline (50ml/kg) was injected intraperitoneally to resist shock.

High and low dose azadirachtin groups: perfusing with 200mg/kg and 100mg/kg azadirachtin respectively; saline control group: gavage with the same dose, 1 time daily for 7 consecutive days. As the rats have self-mutilation, in order to avoid the skin flap survival from being influenced by the back bite of the rats after the operation, the rats are provided with a neck sleeve (figure 2B) for preventing self-mutilation and fed with a single cage. To reduce the error caused by the operation, all the operations are completed by 1 person.

Example 1: flap survival area ratio and flap survival status detection

After the operation for 1-7 days, the color, texture and tissue elasticity of the skin flap of the rat and the existence or nonexistence of the hair growth or necrosis phenomenon are observed by naked eyes every day and recorded. On day 7, the rats of each group accurately measured the area of skin flap survival and necrosis with cellophane under anesthesia, cut into two parts of survival and death, and weighed with an electronic scale respectively. The percentage of flap survival area was calculated [ mass of flap survival area transparency paper ÷ mass of flap total surface area transparency paper × 100% ].

Standard of skin flap necrosis: the skin flap is black in color, the tissue is retracted, the elasticity is poor, the texture is hard, and the cut tissue does not bleed. The pathological manifestations are tissue disintegration, nucleus disappearance, inflammatory cell extensive infiltration and focal bleeding.

The results are shown in fig. 3, 7 days after operation, the middle and proximal regions of the skin flap in the experimental group were light red, no crusty skin formed on the surface, better elasticity, and black color at the distal region, indicating crusty skin, poor elasticity, active bleeding of the skin flap, high bleeding amount, no hematocele under the flesh membrane, effusion and abundant blood vessels when the skin flap is lifted in situ. The middle and distal end regions of the flaps of the normal saline control group all turn black, the surfaces of the flaps are formed with crusts, the elasticity is extremely poor, the flaps are lifted in situ and have less bleeding, more inflammatory secretion under the periosteum is generated, and the blood vessels are relatively sparse (fig. 3A). The skin flap survival rates were 50.16 + -2.32%, 71.19 + -2.50% and 90.83 + -2.26% for the control, low azadirachtin and high azadirachtin dose groups, respectively, with statistical differences (p < 0.01; FIG. 3B).

Example 2: flap neovascularization condition detection

The laser Doppler blood flow instrument is used in detecting the neovascular state of skin flap and monitoring the perfusion of microcirculation blood flow in animal or human body tissue based on the laser Doppler principle. The subject laboratory has the instrument and is matched with operating software to collect and analyze blood flow signals. On day 7 post-surgery, microcirculation blood flow was measured using a laser doppler flow meter. Each group of 6 rats was anesthetized and scanned using Laserflo BPM (Vasamedic, Saint Paul, MN, United States) with an area of 15cm × 15cm and 256 × 256 pixels. LDBF generally provides deeper penetration, enhances the visualization of small vessels beneath the tissue surface, and is well suited for angiogenesis assessment. Blood supply was visualized using LDBF strong signals (green, yellow and red), and its area quantified using ImageJ software (NIH, Bethesda, MD, United States). Rats were analyzed for dorsal flap survival by comparing blood flow in the high azadirachtin dose group, the low azadirachtin dose group and the control group. The percentage calculation of the flap area with blood supply is: strong signal area x 100%/, total area.

As shown in fig. 5, the laser doppler blood-flow meter showed significantly more blood flow in the skin flap of the experimental group than the control group after 7 days, and was dose-dependent (fig. 5A). The blood perfusion amount of the skin flap of the azadirachtin high-dose group is (300.32 +/-45.25 PU), the blood perfusion amount of the skin flap of the azadirachtin low-dose group is (263.15 +/-55.29 PU), the blood perfusion amount of the skin flap of the control group is (50.73 +/-12.70 PU), and compared with the blood perfusion amount of the skin flaps of the three groups, the difference has statistical significance (P is less than 0.01; fig. 5B).

On day 7 post-surgery, 6 rats per group were anesthetized and pretreated, with bilateral cannulation of the proximal and distal femoral artery on one side, while the femoral vein on the same side was dissected. The isoosmotic potassium chloride solution with the temperature of 37 ℃ is perfused from the femoral artery to continuously wash the blood vessels of the whole body, control the temperature and prevent the vasospasm of the rat. After the fluid flowing out of the femoral vein is clear, the contrast agent (5 g of 300Bloom gelatin, 100ml of warm water at 37 ℃ and 100g of lead oxide) is continuously and manually injected from the far side and the near side by an injector, and the injection amount is 20-30 ml/kg. When the color of the spot or patch-like perfusate appears at the extremities, face, sclera, etc., the perfusion is stopped. The specimens were then refrigerated to facilitate gelatin coagulation. Angiography with X-rays was performed on day 2.

As shown in fig. 6, the angiography showed higher quality of revascularization in the experimental group and better revascularization at the receptor site and at the flap margin than in the control group, especially in the high dose group.

Example 3: histological examination

7 days post-surgery, rats were sacrificed and tissue specimens were dissected from the middle of the skin flap, fixed in 4% (v/v) paraformaldehyde for 24 hours, and embedded in paraffin for cross-sectioning. Sections (4 μm thick) were mounted on poly-L-lysine coated slides for H & E staining. The thickness of granulation tissue, edema and angiogenesis were observed under an optical microscope (magnification 20 times). In addition, the neutrophil density and the number of microangioses per unit area (/ mm2) were calculated for each group.

The results are shown in fig. 7, where the high and low dose groups of azadirachtin showed significant subcutaneous fibroblast proliferation, thin granulation tissue, mild tissue edema, and significant diffuse subcutaneous bleeding. The control flap was thicker, with less fibroblast proliferation and neovascularization, and more severe edema and inflammatory cell infiltration (fig. 7A). The neutrophil density at the middle of the flap in the experimental group (high dose 22.03. + -. 4.33/mm 2; low dose 37.46. + -. 3.09/mm2) was lower than that in the control group (66.14. + -. 4.44/mm2) (FIG. 7B). The density of the microvasculature at the middle end of the skin flap in the experimental group (high dose 35.98 + -3.99/mm 2; low dose 23.16 + -2.98/mm 2) is higher than that in the control group (12.46 + -2.88/mm 2). The differences were all statistically significant (p < 0.01; FIG. 7C).

Example 4 immunohistochemical observations

Paraffin sections were stained in the Elivison two-step method. Dropping normal goat serum confining liquid into each section, standing for 20min at room temperature, blocking non-specific sites, and then incubating with primary antibodies of Vascular Endothelial Growth Factor (VEGF), Toll-like receptor 4(TLR4), nuclear factor-kB (NF-kB), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) overnight at 4 ℃. On day 2, the slides were rewarmed at room temperature (37 ℃) for 45 minutes, incubated with specific secondary antibody at room temperature for 2 hours, washed 3 times with PBS, and developed with diaminobenzidine for 5-10 min. The dense protein expression area is searched under a low power lens and then observed by a high power lens. 5 fields of view are selected from each section, and the section is shot and stored. The expression level of each protein was calculated.

As shown in FIG. 8, immunohistochemical analysis showed VEGF levels in the control, azadirachtin low dose and azadirachtin high dose groups to be (1522.00 + -312.56 IA), (2956.67 + -351.41 IA) and (4450.50 + -288.81 IA), respectively. The level of TLR4 for each group was (5142.17 + -458.47 IA), (3118.00 + -215.51 IA) and (1117.00 + -157.09 IA), respectively. NF-kB levels of each group were (5222.17. + -. 438.09IA), (2570.33. + -. 246.92IA) and (850.50. + -. 98.08IA), respectively. The IL-1 β levels for each group were (2667.33. + -. 349.93IA), (1397.67. + -. 244.81IA) and (622.83. + -. 97.02IA), respectively. The IL-6 levels in each group were (3637.83 + -244.24 IA), (2052.50 + -132.85 IA) and (736.50 + -71.94 IA), respectively. TNF- α levels in each group were (3118.33 + -249.91 IA), (1426.00 + -212.30 IA) and (813.00 + -125.53 IA), respectively. These results indicate that azadirachtin can dose-dependently up-regulate the expression of Vascular Endothelial Growth Factor (VEGF) and down-regulate the expression of pro-inflammatory cytokines (TLR4, NF-kB, IL-1 β, IL-6 and TNF- α). The differences between the groups were statistically significant (p < 0.01).

Example 5 detection of expression of superoxide dismutase (SOD), Malondialdehyde (MDA)

After 2 days of operation, 6 rats are randomly selected from each group, each part of the skin flap of each rat is homogenized, and the contents of the rats and the skin flap are detected according to the specification of an SOD detection kit and an MDA detection kit.

As shown in FIG. 9, the content of superoxide dismutase (SOD) in the high azadirachtin dose group was (62.84. + -. 2.99units/mg protein), the content of SOD in the low azadirachtin dose group was (52.41. + -. 2.75units/mg protein), and the content of SOD in the control group was (23.89. + -. 1.96units/mg protein). The content of Malondialdehyde (MDA) in the high-dose group of azadirachtin is (21.36 +/-3.00 nmol/mg protein), the content of Malondialdehyde (MDA) in the low-dose group of azadirachtin is (44.25 +/-3.60 nmol/mg protein), and the content of MDA in the control group is (70.16 +/-5.21 nmol/mg protein). The differences of SOD and MDA contents in three groups have statistical significance (P is less than 0.01).

The levels of TNF-. alpha.and IL-6 in the serum of rats were measured by enzyme-linked immunosorbent assay (ELISA), and blood was obtained from each group of rats and centrifuged at 5000 Xg for 15 minutes. Then, the supernatant was collected, and the levels of tumor necrosis factor alpha (TNF-. alpha.) and interleukin 6(IL-6) in serum were measured using an ELISA kit. The contents of all standards and samples (wavelength 450 nm) were measured with a microplate reader.

As shown in FIG. 4, the ELISA kit assay showed that the mean level of tumor necrosis factor alpha (TNF-alpha) in the high-dose group of azadirachtin was (79.06. + -. 8.75pg/ml), and the mean level of interleukin 6(IL-6) was (28.57. + -. 2.41 pg/ml); the average level of TNF-alpha in the low-dose group of azadirachtin is (116.31 +/-15.41 pg/ml), and the average level of IL-6 is (46.82 +/-3.58 pg/ml); controls were (198.24. + -. 13.55pg/ml) and (87.84. + -. 8.36pg/ml), respectively. Three groups of differences were statistically significant.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. The application of the nimbin in preparing the medicine for promoting the survival of the ischemic ultralong random skin flap is characterized in that the specification of the ischemic ultralong random skin flap is 3cm X9 cm.

2. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong random flap, according to claim 1, wherein said azadirachtin is used to increase the amount of VEGF expression in an ischemic ultralong random flap.

3. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong voluntary flap, according to claim 1, wherein the azadirachtin is used for promoting revascularization in an ischemic ultralong voluntary flap.

4. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong voluntary flap, according to claim 1, wherein said azadirachtin is used as a medicament for promoting proliferation of fibroblasts in an ischemic ultralong voluntary flap.

5. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong random flap, according to claim 1, wherein said azadirachtin is used to inhibit the expression of inflammatory factors in an ischemic ultralong random flap, said inflammatory factors comprising IL-6 and/or IL-1 β and/or TNF- α and/or NF-kB and/or TLR 4.

6. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong voluntary flap, according to claim 1, wherein the azadirachtin is used for promoting antioxidant capacity, including anti-lipid peroxidation capacity, in an ischemic ultralong voluntary flap.

7. The use of azadirachtin in the preparation of a medicament for promoting survival of an ischemic ultralong random flap, according to claim 1, wherein said azadirachtin is used to reduce tissue edema in an ischemic ultralong random flap.

8. The use of azadirachtin according to claim 1 for the preparation of a medicament for promoting survival of ischemic ultralong voluntary flaps, characterized in that: the application method comprises orally taking 100-200mg/kg azadirachtin per day, and 5-7 days as a course of treatment.

9. The use of azadirachtin according to claim 8 in the preparation of a medicament for promoting survival of an ischemic ultralong voluntary flap, wherein: the azadirachtin is administered in the form of a solvent having a content of 10mg/ml, and the solvent of azadirachtin is physiological saline.