Isoliquiritin Pre-Treatment Promotes Multi-Territory Perforator Flap Survival in Rats: An Experimental Study

Background The present study was design to investigate the effect of isoliquiritin (ISL) pretreatment on multi-territory perforator ap survival and blood vessels of Choke II zone in rats. Methods A total of 80 adult Sprague-Dawley (SD) rats were randomly divided into ISL group and normal saline group, and subsequently and subjected to multi-territory perforator ap operations on the left ank. Afterwards, rats in ISL group were intraperitoneally injected with ISL, and rats from normal saline group were intraperitoneally injected with equal amount of normal saline. After seven days, the surviving ap area was calculated, the density of microvessels and (vascular endothelial growth factor,VEGF)were measured in Choke II zone. In addition, blood vessels of the ap were subjected to lead oxide-gelatin radiography. Results The ap survival area was signicantly enhanced in rats from the ISL group compared with that from the saline group (P < 0.01). HE staining indicated signicantly higher microvascular density in Choke II zone in the ISL group (P < 0.01). Immunohistochemistry and Western blot assays showed that the expression of VEGF in the ISL group was signicantly higher than that in the control group (P < 0.01). Moreover, ISL comparison those the normal saline group.


Background
Multi-territory perforator aps are commonly used for soft tissue defect repair in the extremities [1], however, distal necrosis of the ap remains challenging. The angiosomes are the basic anatomical unit of the perforator aps [2], which can be divided into three regions, including the anatomical region from the source artery, the dynamic region adjacent to the anatomical region and the potential region adjacent to the dynamic region. The ap is relatively stable in the anatomical and dynamic regions, while necrosis generally occurs in the potential region. A choke vessel is de ned as the vascular connection between adjacent angiosomes with gradually decreased caliber, which is of great signi cance to the multi-territory perforator ap survival. The occluded vascular anastomosis area between the anatomical region and the dynamic region is called as the Choke I zone, and the occluded vascular anastomotic area between the dynamic area and the potential area is called as the Choke II zone. The vascular dilation and insu cient angiogenesis of the Choke II zone is one of the important potential reasons for ap necrosis [3]. Thus, improving blood supply in the Choke II zone plays a critical role in ap survival [4][5][6].
In recent years, to improve the survival rate of multi-territory perforator aps, delayed surgery [7,8] drug intervention [4][5][6] and etc., have been proposed to dilate vessels and/or promote angiogenesis in the Choke II zone, to improve the blood supply in the Choke II zone, thereby increasing the ap survival area.
However, delayed surgery has the disadvantage of increasing the number of operations and increasing Page 3/17 pain [8]. In addition, drug intervention has become a research hotspot due to high safety, small trauma and little pain [4][5][6].
Liquorice is a widely-used herb in southern Europe and certain parts of Asia [9]. Isoliquiritin (ISL) is a water-soluble iso avone component in liquorice root. ISL has been reported to exert an additional proangiogenic effect on damaged angiogenesis in zebra sh embryos, which might be potentially used to treat disorders caused by insu cient angiogenesis [9]. Liu YY, et al. [10] have investigated the effect and mechanism of ISL on wound healing activity in zebra sh, revealing that ISL can promote in ammatory response and angiogenesis, which plays a key role in promoting wound healing in zebra sh. Therefore, ISL is a promising candidate to promote wound healing. However, it remains largely unde ned whether ISL could promote angiogenesis in Choke II zone of the aps to subsequently improve the survival of distal multi-territory perforator aps in rats. Compared with vertebrates, mammalian skin wound healing is a complicated, multi-step process that involves various stages, including formation of blood clot, in ammation, re-epithelialization, formation of granulation tissue, neovascularization and remodeling, generally with scar left [11][12]. To this end, the present study was design to assess the effects of ISL pretreatment on the survival of ap transplantation and angiogenesis in Choke II zone of the ap in rats by establishing the multi-territory perforator ap model.

Animals and group assignments
Adult Sprague-Dawley (SD) female rats (weight 210-240 g) were purchased from the Experimental Animal Center of Suzhou University. Rats were housed in the single cage, fed with standard rat chow at an appropriate temperature (23-25 °C).
In total, 80 rats were randomly divided into ISL group (n = 40) and normal saline group (n = 40). All surgical procedures and protocols in this study were approved by the Animal Ethics Committee of Suzhou University. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals described by the National Research Council Drug Administration.

Operative technique
All rats were anesthetized with 2% pentobarbital sodium (40 mg/kg, intraperitoneal injection). After anesthesia, rats were xed in the prone position on the sterile operating table, hair was removed using a pet electric shaver, and rats were deiodinated with alcohol after iodophor disinfection. The dorsal ap was designed according to Miyamoto [11]. To be speci c, a trivascular perforator ap based on a perforating artery of deep iliac circum ex artery was design on the left dorsal of the rat, including two choke zones. In addition, the ap borders were as follows: medial border, the midline of the dorsum; the cranial border, subscapular angle; caudal border, the spina iliaca posterior superior, and the dorsal ap measuring 10 × 2.5 cm was designed. Firstly, the skin was cut along the medial border of the ap, and the subcutaneous tissue was separated just from the meat layer. The ap was gradually lifted to separate trivascular pedicles. The arteriathoracodorsalis perforator and the intercostal artery perforator were ligated. Finally, the arteriae circum exa ilium profunda perforator was preserved and the aps were completely separated, followed by identi cation of Choke I and II zone (Fig. 2) and corresponding marking on the epidermis using a marker pen (Fig. 2). After complete hemostasis, the ap sutured back into its original position with 4 − 0 silk. Wounds were post-operatively coated with chlortetracycline ointment to prevent infection. The ap was resected by the same surgeon to ensure the uniform thickness of each ap and to reduce the factors that might affect ap survival due to the different thickness of the ap.

Dosage and administration of drug
Rats in the experimental group were intraperitoneally injected with ISL solution at a dose of 100,200 µg/kg two days before operation, two hours before operation and two days after operation according to the pre-experiment results. Rats in the control group were intraperitoneally injected with the same volume of saline solution accordingly.

Gross observation of ap and calculation of ap survival rate
All aps were grossly observed and recorded 1, 3, 5, and 7 days after operation, including the ap color, tissue elasticity, texture, skin and hair growth, infection, necrosis, and etc. After post-operative observation for seven days,ten rats were anesthetized and high-quality photographs of ap were obtained using a digital camera and imported into Image-Pro Plus v6.0 software to calculate the percentage of ap survival area. The ap survival rate in each group was calculated according to the following formula: ap survival rate = ap survival area / total ap area x100%. The standard of ap necrosis was as follows: the ap color was black, the tissue was hard, shrunk, dry necrosis, inelastic, and no blood out ow when cutting.

Perforator ap angiography
On the 7th day after operation, 10 rats were randomly selected from the control and ISL groups (n = 10 each, 20 in total), and subjected to gelatin-lead oxide angiography of the ap vessels. (1) Preparation of the perfusion solution: The preparation ratio of perfusion solution was: industrial gelatin 1 g, warm water 20 ml 40 ℃, lead oxide 20 g. First, the gelatin was dissolved in warm water for 3-4 h. After the gelatin was completely dissolved, lead oxide was added and stirred. Because the gelatin-lead oxide mixture was prone to solidify under low temperature, the entire procedure must be carried out in water bath at 40 ℃. The amount of perfusion depended on the size of rats, generally ranging from 40-50 ml / kg, not exceeding 50 ml / kg. (2) Perfusion process: rats were xed in a supine position after anesthesia, and unilateral carotid artery was bluntly separated. Afterwards, No.24 indwelling catheter was inserted into the carotid artery, the catheter was ligated with silk thread r xation. The blood from was completely drained from the rat, rinsed with heparin saline. When the e uent was clear, the prepared gelatin-lead oxide mixture was slowly and uniformly injected into the carotid artery until the sclera and the distal limbs of the rat showed dot-like or patchy orange-red. Afterwards, rats were placed in a supine position in the refrigerator at 4 ℃ for about 24 h for gelatin solidi cation. (3) X-ray radiography: 24 hours after perfusion, the entire back skin of the rat was peeled off and photographed under X-ray camera (40kv, 50 mA and 100 ms exposure time). (4) Image analysis: The PACS system of our hospital was used to visualize and assess the blood vessels in Choke II zone of the aps in rats from both groups. 2.7 Histological examination of microvessel density (MVD) in the Choke II zone of the ap Seven days after operation, six rats from each group were anesthetized according to the previously described procedure. The ap tissue (2.0 cm × 0.5 cm in size) of Choke II zone was obtained, xed in 40 g/L paraformaldehyde, routinely dehydrated, sliced into 5 µm-thick sections and subjected to HE staining. Afterwards, the staining results of the tissue sections were observed under the microscope. As a result, the cytoplasm was stained as pink by eosin, and the nuclei were stained as blue by hematoxylin. Firstly, the dense area of microvessels was found under 4 × 10 low magni cation. Afterwards, ve elds of view were randomly selected under 10 × 10 magni cation microscope, and the number of blood vessels was counted manually for average to calculate the number of microvessels per unit area (number/mm 2 ), which was used an indicator for MVD.

Immunohistochemistry (IHC) for VEGF expression in Choke II zone of the ap
The sections prepared in Sect. 2.7 (one for each rat) were routinely dewaxed, dehydrated, microwaved for antigen retrieval, incubated with 3% (volume fraction) peroxide at 37 ° C in water bath for 10 min to inactivate endogenous peroxidase, blocked with 5% (volume fraction) goat serum at 37 ° C for 30 min. The sections were subsequently incubated with anti-rat VEGF primary antibody (dilution 1:200) at 4 ° C overnight, reacted with HRP-conjugated goat anti-rat IgG secondary antibody at 37 ° C for 1 h, and incubated with HRP-conjugated streptomycin avidin at 37 ° C for 45 min. Afterwards, the sections were visualized by DAB, counterstained with hematoxylin, dehydrated with ethanol, transparented with xylene, sealed with neutral resin. The distribution of VEGF (brown yellow particles) in Choke II zone of the ap was observed under light microscope at 400 magni cation and photographed under ve randomly selected elds of view. Images were imported into Image Pro Plus 6.0 software to detect VEGF expression and the results were shown as integral absorbance values.
2.9 Western blot for VEGF expression in Choke II zone of the ap Seven days after operation, total tissue protein was extracted from the Choke II zone of the ap from ten rats in each group. After adjusting protein concentration, equal amount protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes and blocked with 50 g / L skim milk solution at room temperature for 2 h. The membranes were incubated with mouse anti-rat VEGF primary antibody (dilution 1:300), rabbit anti-mouse GAPDH primary antibody (dilution 1:500) overnight at 4 °C, followed by incubation with HRP-conjugated goat anti-mouse IgG secondary antibody (dilution 1:4000) or HRP-conjugated goat anti-rabbit IgG secondary antibody (dilution 1:5000) at room temperature for 2 h. Afterwards, the membranes were visualized using chemiluminescence, and grayscale scanning analysis was performed using gel image analysis system. The band intensity of VEGF was quanti ed and presented relative to GADPH (internal control). This assay was performed in triplicate.

Treatment Of Rats After Experiment
All rats were killed by cervical dislocation after the experiment.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 statistical software package (SPSS Inc., Chicago, IL, USA). All normally distributed data were expressed as mean standard deviation (SD). Independent student-t test was used for comparison between two groups when the data were normally distributed. Otherwise, Wilcoxon rank sum test was used. A P value 0.05 was considered as statistically signi cant.

Gross observation and ap survival rate
Seven days after operation, rats in both groups were alive, without ap infection. There was no abnormal behavior of rats throughout the experiment, and the body weight of rats were not signi cantly changed before and after the experiment. Seven days after operation, the necrotic area of the ap of rats in the ISL group was approximately located far away from the thoracodorsal artery perforator, the scar was dark yellow, with soft texture and easy to peel off. The necrotic area of the ap of rats in the control group was approximately located far away from the Choke II zone, and the scar was brownish black, with hard texture and di cult to peel off (Fig. 3). Seven days after operation, the ap survival rate was signi cantly higher in the ISL group (89 ± 2%) than that in the saline solution group (81 ± 3%) (P < 0.01).

Vascular condition in Choke II zone of ap and potential region
Seven days after operation, the vascular structure of Choke II zone of ap of rats in the ISL group was relatively clear, with more neovascularization and relatively complete vascular structure in the potential region. However, in the saline solution group, the vascular structure of Choke II zone of ap of rats was ambiguous, with less neovascularization and disordered or disappeared vascular structure in the potential region (shown in Fig. 4).

MVD of Choke II zone of ap
Seven days after operation, there was more neovascularization in the Choke II zone of ap in rats of the ISL group, with MVD of 30.2 ± 2.2/mm2. In rats of saline solution group, there was fewer neovascularization in the Choke II zone of ap, with MVD of 21.5 ± 3.2/mm2. The difference was statistically signi cant between groups (P < 0.01) (shown in Fig. 5).

VEGF expression in Choke II zone of ap
Seven days after operation, IF showed that the expression of VEGF in the Choke II zone of ap in rats from ISL group was 7050 ± 143, which was signi cantly higher than that of the saline solution group (3512 ± 146) (P < 0.01) (shown in Fig. 6).
Seven days after operation, Western blot showed that the expression of VEGF in the Choke II zone of rats from the ISL group was 0.52 ± 0.02, which was signi cantly higher than that of the saline solution group (0.24 ± 0.03) (P < 0.01) (shown in Fig. 7).

Discussion
In this study, we validated for the rst time that ISL pretreatment can promote multi-territory perforator ap survival in rats and promote angiogenesis in the Choke II zone of the ap. Vascular endothelial growth factor (VEGF) is one of the most important regulatory factors in angiogenesis, which can speci cally stimulate the proliferation and regeneration of vascular endothelial cells. VEFG is generally considered as an improving factor of the survival ability of ischemic aps [12]. In addition, VEGF has been validated to induce angiogenesis and improve the survival rate of muscle aps [13]. Fichter, et al. [14] have demonstrated that single dose injection of VEGF into the ap wound area can signi cantly decrease the necrotic area. Kane, et al. [15] have demonstrated that NO from iNOS can promote ischemic ap survival through angiogenesis, possibly by increasing the expression of VEGF in mast cells in the angiogenesis zone. In this study, Western blot and IHC staining were used to detect the expression of VEGF in Choke II zone of the aps in rats, both of which showed that the expression of VEGF in the Choke II zone of the rat ap in the ISL group was signi cantly higher than that in the normal saline group. Therefore, we consider that ISL can induce angiogenesis in the Choke II zone by promoting VEGF expression, thereby promoting ap survival.
Skin tissue, blood vessels and other accessory structures are generally formed seven days after ap transplantation [16][17][18], therefore, this time point was selected for observation. Zhao G, et al. [16] has con rmed that irisin can protect the perforator ap from ischemia-reperfusion injury by promoting the proliferation of vascular endothelial cells. In their study, the ap survival area after ischemia-reperfusion treatment in rats was signi cantly increased, compared with the saline group. Tao XY, et al. [17] have investigated the effects of iNOS on the multi-territory perforator ap survival and the blood vessels in the Choke II zone in rats, revealing that ap survival area was signi cantly enhanced in the iNOS intervention group than the control group. Cao B, et al. [18] have observed the effect of lidocaine on the ap survival area in rats, who found that the ap survival area in the lidocaine group was signi cantly larger than that in the control group. Herein, in the present study, we aimed to evaluate whether intraperitoneal injection of ISL could promote ap survival in rats. The calculating method of ap survival rate was consistent with the above studies. As a result, the ap survival area of the ISL group was signi cantly larger than that of the control group.
The blood vessels in the Choke II zone of the aps were macroscopically observed by lead oxide radiography, and microvascular density was determined by histological detection. As a result, the microvascular density of the aps was signi cantly enhanced in rats from the ISL group than that of the normal saline group; and lead oxide radiography showed that the vascular structure of the aps was clearer, and the vascular structure in the potential area was more complete in rats from the ISL group compared to those from the normal saline group. Xie LZ, et al. [19] improved the survival of rat aps by local injection of bone marrow mesenchymal stem cells (BMSCs)-derived exosomes. In their study, HE staining of Choke II zone showed higher microvascular density and more angiogenesis in the exosome group. By using the macroscopic observation via lead oxide angiography for vascular structure in the Choke II zone in previous literature [20,5], we demonstrate that ISL can promote angiogenesis in the Choke II zone of rat aps.
In this study, we have validated that ISL promotes the multi-territory perforator ap survival by promoting angiogenesis in the Choke II zone in rats. There was no rat death or ap infection in this study, no abnormal behavior in rats from the ISL group. The body weight of rats was basically the same at the beginning and the end of the experiment, indicating the safety and tolerability of ISL. However, there are still certain limitations in this study. We failed to investigate the speci c mechanism of ISL in promoting angiogenesis in Choke II zone after ap transplantation. Previous studies have shown that the upregulation of VEGF/VEGFR2 and Ang/Tie signaling pathways by ISL may be associated with the angiogenic activity observed on zebra sh embryos [10]. Therefore, in the future, we will further study the speci c mechanism of ISL in promoting ap survival. Although accumulative evidence has supported the bene cial effects of ISL on the cardiovascular system, the potential negative effects still need to be explored. Our present ndings only suggest that ISL has a certain role in promoting the perforator ap survival in rodents, which should by cautiously extrapolated to humans. In addition, the dose, route of administration, and timing of ISL for ap transplantation in clinical practice should also be further investigated. Nevertheless, our study provides a potential bene cial drug to promote ap survival.

Conclusion
In this study, we show for the rst time that ISL can promote the survival of multi-territory perforator aps in rats and promote angiogenesis in the Choke II zone of the aps, which has enriched the medicinal value of liquorice again. Hopefully, our ndings could provide a potential drug candidate to promote ap survival.  Figure 1 Chemical structure of isoliquiritin (ISL).

Figure 2
Schematic diagram of dorsal perforator ap in rats. The ap side was exposed to show the deep circum ex artery perforator (↑), intercostal artery perforator (↑), thoracodorsal artery perforator (↑), three vascular pedicles, Choke I zone (between anatomical region and dynamic region), Choke II zone (between dynamic region and potential region). The midlines of the three vascular pedicles and two choke zones were marked in the ap surface using a marker pen, and the flap sutured back into its original position.

Figure 3
Gross observation of aps in two groups of rats seven days after operation. A. The necrotic area of the ap of rats in the ISL group was approximately located far away from the thoracodorsal artery perforator, and the scar was dark yellow. B. The necrotic area of the ap of rats in the saline solution group was approximately located far away from the Choke II zone, and the scar was brownish black. C. The ap survival rate of rats was signi cantly higher in the ISL group than that in the saline group; *Statistical signi cance (P<0.01) Figure 4 Radiography of aps in rats from both groups seven days after operation. A. In the ISL group, the vascular structure of Choke II zone of ap of rats was relatively clear, with more neovascularization and relatively complete vascular structure in the potential region B. In the saline solution group, the vascular structure of Choke II zone of ap of rats was ambiguous, with less neovascularization and disordered or disappeared vascular structure in the potential region.

Figure 5
Histological observation of the neovascularization of Choke II zone of ap in rats of both groups seven days after operation. HE ×100. A. There was more neovascularization in the Choke II zone of ap in rats from the ISL group; B. There ws fewer neovascularization in the Choke II zone of ap in rats from the saline solution group; C. The MVD in the irisin group was signi cantly higher than that in the saline solution group seven days after operation. * Statistical signi cance (P <0.01) Figure 6 Seven days after operation, IF showed the expression of VEGF in the Choke II zone of rats from two groups (brown), diaminobenzidine-hematoxylin x100. A: The expression of VEGF was obvious in ISL group; B: the expression of VEGF in saline solution group was weaker than that in ISL group.