Chin J Integr Med 2009 Aug;15(4):279-283

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ORIGINAL ARTICLE
Clinical Study of External Application of Qiyu Oil () Gauze for Promoting Post-operational Healing in Patients with Anal Fistula
LI Shao-tang ()1, CAO Bo ( )2, DENG Wen-Ling ()2, and LI Zhi ( )2

ABSTRACT Objective: To observe the effects of the external application of Qiyu oil () gauze (QYOG) for promoting post-operational healing in patients with anal fistula and to explore its mechanism of action so as to provide a beneficial scientific basis for its wide use. Methods: Sixty patients with anal fistula scheduled to receive simple low anal fistulectomy were equally assigned, according to the sequence of hospitalization, to the tested group and the control group, and their wounds were classified according to longitudinal diameter into three grades (Grade with a diameter below 2 cm; Grade , 2-5 cm; and Grade , over 5 cm). After the operation was completed and the operational wound was sterilized with benzalkonium bromide, the wound substratum was packed with QYOG in the test group and with vaseline gauze in the control group. The packing gauze was changed every day till the wound was healed. The healing time of the patients was observed, and the number of capillaries and positive cell percentages of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in wound granulation tissues were counted on the 5th day after the operation. Results: The wound healing time was 17.805.46 days in the test group, which was significantly shorter than that in the control group (21.906.32 days, P <0.01). The number of capillaries and positive cell percentages of VEGF and bFGF in wound granulation tissues on the 5th day in the tested group were higher than those in the control group (P <0.01), though the difference in EGF between the two groups was insignificant (P >0.05). Conclusion: QYOG could shorten the wound healing time after anal fistulectomy, which suggests that it participates in the stimulation of wound granulation tissues to produce VEGF and bFGF, and thus promotes capillary genesis and improves blood circulation in wounds so as to promote wound healing. KEY WORDS Qiyu oil gauze, anal fistula, wound healing, vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, number of blood capillary

Anal fistula is a frequently encountered disease in the anorectal department, and the basic treatments for the illness at present are surgical excision, incision, or incision combined with the thread-drawing method. Since the operational wound after anorectal surgery is easily infected due to the long course of the disease and the large operational wound surface, the healing of the wound is often protracted or even results in disunion. Research on wound union is an important topic in medical science, and Chinese medicine has shown potential advantages in this field. Although much has been won in animal experiments, a clinical practical Chinese herbal preparation usable specially for promoting anorectal post-operational healing is still rare. Qiyu oil ( ) gauze (QYOG) used in this study was formulated according to the clinical experience of Dr. PENG Xian-guang, a specialist

in anorectal diseases well known across the whole country and working in the Guiyang College of Traditional Chinese Medicine, which has won distinct efficacy in clinical use. In order to explore the mechanism of action of QYOG, this study was carried out to observe the effect of QYOG on wound healing time and related indexes, the number of capillaries, and the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and epidermal growth
Supported by the Funds of Guiyang College of Traditional Chinese Medicine (No. 200607) 1. Fujian Medical University, Fuzhou (350004), China; 2. Department of Anorectal Diseases, the First Affiliated Hospital of Guiyang College of Traditional Chinese Medicine, Guiyang (550001), China Correspondence to: Dr. LI Shao-tang, Tel: 86-591-26560185, E-mail: lishaotang163@163.com DOI: 10.1007/s11655-009-0279-5

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Table 1.
Group Test Control Case 30 30

factor (EGF) in wound granulation tissues.

General Materials of the Two Groups

Sex (Case, M/F) 23/7 23/7 Age (Year, s ) 38.011.2 37.011.0 Grade of wound (Case) 4 3 17 20 9 7

METHODS
Standards for Diagnosis, Inclusion, Exclusion, and Elimination
The clinical diagnosis was made according to the standard for middle and low position simple anal fistula in the "Standard for Diagnosis and Efficacy Evaluation of Traditional Chinese Medicine Diseases and Syndromes"(1) promulgated by the State Administration of Traditional Chinese Medicine, P.R. China, 1994. The subjects enrolled in this study were those who had a history of anal carbuncle, certifiable focal outer opening, channel, and inner opening of a single anal fistula positioned below the anorectal ring, and they signed the written informed consent. Patients meeting the following conditions would be excluded: (1) accompanied by tuberculous anal fistula, severe malnutrition, diabetes mellitus, severe cardiocerebral vascular disease, hepatopathy, or nephropathy; (2) unable to endure surgical operation; (3) women in pregnancy stage; and (4) complicated with other severe diseases like pulmonary infection, central respiratory failure, electrolytes disorder, metabolic acidosis, high fever, etc. The elimination criteria were: (1) those who were not treated according to the regulation; and (2) those asked to quit halfway through the trial.

QYOG Preparation
The prescription of the QYOG was: raw milkvetch root 120 g; unprepared burnet root 60 g, Chinese angelica root tip 60 g, red sage root 30 g, gromwell root 30 g, bletilla tuber 30 g, boswellin 30 g, myrrh 30 g, and dragon's blood 30 g. All the drugs were immersed in 1 000 g of benne oil for 24 h, then fried in low fire to dry, with the waste slags discarded and filtrated with double-layer gauze to obtain 1 000 mL of filtration. Then, after the fire-toxin was eliminated by soaking in water for 48 h, they were plunged in an aluminum lunch box, in which gauze dressing blocks of various sizes were placed in advance to fullly saturate them with the oil but not to the point of dripping. Finally, QYOG was sterilized by 30-min autoclaving and put on standby for use.

Treatment
Skin preparation and cleaning enema were conducted before the operation. Anal fistulectomy was performed under caudal or peridorsal anesthesia with wound margin pruning to form an upper wide, lower narrow, "V"-shaped incision for smooth drainage, which was kept unsutured at the time. The longitudinal diameter of the wound was measured. Then the wound was sterilized with 3 benzalkonium bromide, and the wound substratum was packed with QYOG in the test group and with vaseline gauze in the control group. The gauze was changed every day until the wound was healed.

Subjects
All the 60 patients enrolled were inpatients hospitalized in the anorectal department, First Affiliated Hospital of Guiyang College of Traditional Chinese Medicine, from January 2007 to December 2007, whose clinical symptoms, signs, results of anoscopy or probe examination, and post-operational retrospective diagnosis all matched the diagnosis of simple low anal fistula. The longtitudinal diameter of the wound was graded by the following criteria: grade denoted the wound diameter was below 2 cm, grade denoted it was within 2-5 cm, and grade denoted it was over 5 cm. The patients were assigned to the test group and the control group by sequence of hospitalization, 30 in each group. The differences between the two groups in terms of sex, age, and grade of wound were deemed statistically insignificant (P >0.05) through a 2 test and t -test for groups. The details are listed in Table 1.

Items and Methods of Observation Wound Healing Time

The wound healing time was estimated by visual inspection, and a completely epithelized wound surface was regarded as wound healing.

Counting of Capillary in Wound Surface Granulation Tissues

The counting was carried out on the 5th day after the operation by cutting 1.5 mm 1.5 mm in size fresh granulation tissue from the wound

Chin J Integr Med 2009 Aug;15(4):279-283 surface, which was then fixed with 10% formalin and embedded in paraffin. A quarter of the paraffin block was taken for counting through a sliced sheet with hematoxyllin-eosin stain. The counting was carried out by a pathologist, who had no knowledge of the experimental grouping, under a microscope (100-fold, type BX60F5, Olympus Co., Japan). The number of capillaries in each slide in 3 visual fields was counted, and their mean values were used for analysis.

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from 5 randomly selected visual fields of a slice, and the positive cell percentage was calculated. Staining and counting were performed throughout by the same responsible technician.

Statistical Analysis
The software SPSS 10.0 was used to manage the measurement data with t -test.

RESULTS
Comparison of Wound Healing Time
The wound healing time in the test group was 17.805.46 days, which was shorter than that in the control group, 21.906.32 days, showing statistical significance (P <0.01, Table 2).

Immunohistochemcal Analysis
The positive cell percentages of VEGF, bFGF, and EGF on the 5th day's wound surface granulation tissues were detected by immunohistochemcal analysis of the thick slices (4 m) made from the above-mentioned paraffin block with corresponding multi-colony antibody and SP testing kits purchased from Beijing Zhongshan Co. of Bioengineering, according to the instruction and through the following procedures. The paraffin slice was dewaxed and hydrated according to routine, washed with PBS 3 times, and incubated at room temperature for 10 min with 1 drop of dioxygenase blocking agent. Then, the slice was washed with PBS 3 times, and the serum was discarded. The first antibody was added and incubated under 4 overnight, and after PBS washing for three times, the second antibody was added and incubated under 37 for 10 min. Afterwards, the slice was washed 3 times with PBS, and a streptomycin-avidindioxygenase solution was added before incubation under 37 for 10 min. Finally, DAB coloration, hematoxyllin counterstain, gradient alcohol dehydration, and xylene transparency were done successively, and mounting was done with neutral gum. The prepared slide was observed under a 400-fold light microscope. Cells with brown-stained cytoplasm and/or nuclei were regarded as positive. The number of positive cells in 100 cells was read

Comparison of Number of Capillary and Related Indexes in Wound Granulation Tissue between Groups
The measurements on the 5th day after the operation showed that, in the wound granulation tissue, the number of capillary and positive cell percentages of VEGF, bFGF, and EGF in the tested group were all higher than those in the control group, showing statistical significance (P <0.01). However, the comparison of the EGF expression between groups showed no significance (P >0.05, Table 2, Figure 1).

DISCUSSION
Anal fistula is a frequently encountered disease in the anorectal medical department; it is called hemorrhoid-complicated anal fistula, leaky sore, or anal leakage in Chinese medicine. Chinese medicine holds that it is caused either by the invasion of external evil dampness-heat, the transition of affected dampness to heat, or by excessive ingestion of piquant, fatty, and spicy food, which damage the ability of the Pi ()-Wei () to cause dampness to accumulate and change into heat. The evil dampnessheat flow downward to the anal rectal region and block the Meridian-Collaterals, and its long-time

Table 2.
Group Test Control

Comparison of Wound Healing Time and Related Indexes in Wound Granulation Tissue ( s )
Wound healing time (Day) 17.805.46 21.906.32 5.03 No. of capillary (No./field) 32.134.48 14.673.59 14.94 VEGF (%) 47.133.14 15.173.45 37.24 bFGF (%) 41.004.30 14.073.02 24.45 EGF (%) 13.572.82 13.402.95 0.20

Case 30 30

t value

Note: P <0.01, compared with the control group

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A
Figure 1.

Number of Capillary in Wound Granulation Tissue on the 5th Day after Operation in the Two Groups (HE Staining, 100)

Notes: A: the test group; B: the control group

accumulation will transform it to toxin, form pus, and lead to disunion fistula. In sum, the pathogenesis of anal fistula is mainly the accumulation of dampnesstoxin and rooted in the deficiency of qi-blood, so it is really a syndrome of both excessive evil pathogens and essential deficiency. Although anal fistula can be excised by surgical operation, the injuries from long-term illness and bleeding caused by the operation would inevitably induce qi-blood deficiency in patients at the postoperational stage; therefore, the post-operational treatment of the disease should chiefly reinforce the qi, activate blood circulation, dispell putrefaction, and generate new tissues. In the prescription of QYOG, a large dose of milkveteh is used as the monarch drug, which highly supplements the qi of the Pi-Wei and enhances the circulation of qi-blood to remove stasis and dredge the Collaterals. Burnet root can cool and activate blood circulation, remove putrefaction, and restrain the wound; Chinese angelica root-tip is excellent in activating blood circulation and dissolving stasis without damaging the blood. These two drugs are used as adjuvant drugs. Red sage root, frankincense, and myrrh can assist the action of Chinese angelica root-tip on the blood and stasis, while gromwell, bletilla, and dragon's blood have effects in getting rid of putrefaction and restraining the wound; all of them are assistant drugs. Therefore, as a whole, QYOG could supplement qi, activate blood circulation, dispel putrefaction, and generate new tissues. It is able to advance wound healing after fistulectomy and improve the symptoms in post-operational patients.

Modern medical study has proven that milkvetch root possesses good angiogenesis-promoting action(2) and could improve blood circulation on the wound surface to advance the repair of the wound (3). The external application of QYOG could form a closed moist circumstance on the wound surface, which has been proven to be able to promote the release of various growth factors from macrophage, platelet, fibroblast, and neutrophil. Also, it could regulate or stimulate the proliferation of macrophage (4-7). A closed moist circumstance is not only beneficial for various cells to maintain their activity but also to cell groups, which is most important for the restoration of defensive functions and wound healing, such as the accumulation of macrophages. These cells will release a large amount of growth factors to create a circumstance that could expedite angiogenesis and fibrinolysis so as to accelerate wound healing. Moreover, the external application of oil gauze could form an oil film on the wound surface that will prevent the surface from drying quickly, thus keeping the drugs acting longer and reducing the friction of the surface to protect it well. It has been found in this study that the external application of QYOG could stimulate the synthesis of VEGF and bFGF in wound surface granulation tissue. VEGF is a special mitogen of vascular endothelial cells, which could specially promote the cleavage and proliferation of vascular endothelial cells, and strongly speed up angiogenesis through binding with the special receptor to induce their dimerization and the phosphorylation of tyrosine kinase, as it is the most important growth factor for accelerating angiogenesis and maintaining the normal condition of vessels(8,9). Owing to the higher expression of VEGF, it could also

Chin J Integr Med 2009 Aug;15(4):279-283 promote the genesis of capillaries, improve blood circulation on the wound surface, and thus advance wound healing. bFGF could act to advance the process of wound healing either by way of increasing the expression of VEGF or through enhancing the activity of cell proliferation(10,11). While EGF could advance wound healing and membranous cell repair by means of an endocrinal mechanism, including auto-secretion and parasecretion(12,13), its expression has been found. In this study, however, it is not significantly different between the two groups. In sum, the external application of QYOG could shorten the wound healing time after fistulectomy, and its mechanism of action might be through promoting capillary genesis and improving blood circulation on the wound surface by way of participating in the excitation of VEGF and bFGF synthesis in wound granulation tissues.

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burn. Med J Chin People Liber Army (Chin) 2003;28:470. 6. Zhang J, Niu XT, Li D. Comparative research of the donor site wound healing in occlusive and dry environments. Chin J Repar Reconstr Surg (Chin) 2004;18:152-155. 7. Hu Y. An analysis of 112 cases treated with closed moist therapy. Acta Univ Med Nanjing (Chin) 2003;23:183-184. 8. Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Sterm Cells 2002;20:530-541. 9. Toma JG, Akhavan M, Fernandes KJ, Barnab-Heider F, Sadikot A, Kaplan DR, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001;(9):778-784. 10. Seghezzi G, Pate S, Ren CJ, Gualandris A, Pintucci G, Robbins ES, et al. Fibroblast growth factor-2 (FGF-2) induced vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries an autocrine mechanism contributing to angiogenesis. J Cell Biol 1998;141:1659-1673. 11. Numata Y, Terui T, Okuyama R, Hirasawa N, Sugiura Y, Miyoshi I, et al. The accelerating effect of histamine on the cutaneous wound-healing process through the action of basic fibroblast growth factor. J Invest Dermatol 2006; 126:1403-1409. 12. Yanaka A, Suzuki H, Shibahara A, Matsui H, Nakahara A, Tananka N. EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells. Am J Physiol Gestrointest Liver Physiol 2002;282:G866-876. 13. Liu Y, Zhong XL, Tian WY. Mechanism of action to accelerate second deep degree scald wound healing by endogenous EGF. Med J Natl Defend Forces Northwest China (Chin) 2001;22:214-217. (Received November 6, 2008) Edited by WANG Wei-xia

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