Ziegler + Müller In vivo Inhibition of S180 Tumors by the SynergisticEffect of the Chinese Medicinal Herbs Coptis chinensisand Evodia rutaecarpa Xiao-na Wang1, Li-na Xu1, Jin-yong Peng1,2, Ke-xin Liu1, Li-hua Zhang2, Yu-kui Zhang2 1 College of Pharmacy, Dalian Medical University, Dalian, P. R. China2 National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, The Chinese Academy of
Microsoft word - 3-dr pazoki-okReceived: 27.10.2009 Accepted:
Original Article The Effects of Finasteride and Azelaic Acid on Skin Flap Viability in Rats
Marjan Ajami1, Mohamad Ali Nilforoushzadeh2, Shahab Babakoohi3, Rouhollah Habibey4, Fatemeh Banimostafa Arab3, Niloofar Pazoki-Toroudi3, Mehdi Rashighi-Firoozabadi3, Alireza Firooz3, Yahya Dowlati3, Hamidreza Pazoki-Toroudi5 Abstract
Background: Skin flaps still play a crucial role in reconstructive surgery but they are frequently at the risk of ischemia lead-
ing to distal necrosis. The aim of this study was to assess the effects of pre-treatment with azelaic acid and finasteride on
skin flap viability and impact of L-NAME on the function of these two drugs.
Methods: Five treatment groups and one control group of 200-250 gram male rats were used. In 3 treatment groups, finas-
teride (1 mg/flap, subcutaneous), azelaic acid (100 mg/flap, topical) and L-NAME (20 mg/kg, intraperitoneal) were admin-
istered, respectively, before random skin flap elevation. In two other treatment groups, L-NAME was administered half an
hour before application of finasteride and azelaic acid, respectively. Flap samples were used for measurement of necrotic
Results: Skin flap necrotic area decreased from 48.17% ± 2.22% in control group to 35.0% ± 1.59% and 35.0% ± 02.55% in
finasteride and azelaic acid treated groups, respectively (P < 0.05). L-NAME abolished the effects of finasteride and azelaic
acid on flap survival (P < 0.001 and P < 0.01, respectively).
Conclusion: In conclusion, finasteride and azelaic acid increased the survival of skin flaps via nitric oxide dependent path-
way that was hampered by L-NAME.
Key words: Azelaic acid, Finasteride, Nitric oxide, L-NAME, Skin flap.
Skin & Leishmaniasis 2010; 1(1): 12-17 ntil the dream of skin reproduction by tions and morbidity.3 Previous studies have tissue engineering of skin grafts as a demonstrated that application of specific phar- U routine clinical practice comes true1, macologic agents or physical conditions can skin flap transposition is considered the main- protect tissue against ischemic injury.4,5 Mean- stay in many reconstructive surgeries for deep while, using pharmacological treatments to skin and soft tissue injuries.2 In spite of all mod- survive skin flaps has largely been an issue of ern surgical techniques, using skin flaps to interest in literature.4 A myriad of substances obtain appropriate tissue coverage has a pivotal have been under study for their effects on role, because these flaps are frequently at the ischemia reperfusion injury including inducible risk of ischemia which leads to major complica- nitric oxide synthase (iNOS) and L-Arginine6, 1 Department of Nutrition, Iran University of Medical Sciences, Tehran, Iran. 2 Skin Diseases & Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan and Center for Research and Training in Skin Diseases and Leprosy, Medical Sciences/University of Tehran, Tehran and Skin Diseases & Stem Cell Research Center, Tehran, Iran. 3 Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran. 4 Department of Physiology, Iran University of Medical Sciences, Tehran, Iran. 5 Center for Research and Training in Skin Diseases and Leprosy and Nano Vichar Pharmaceutical Ltd, Tehran University of Medical
Sciences, Tehran, Iran.
Correspondence to: Hamidreza Pazoki-Toroudi, Tehran University of Medical Sciences, Tehran, Iran.
Email: [email protected]
Journal of Skin & Leishmaniasis, Spring / Summer 2010; Vol 1, No 1. Finasteride and Azelaic acid effects on skin flap Ajami
pentoxyfylline,7 hemoglobin vesicles,8 erythro- Dawley male rats (200-250 grams) were chosen poietin9 and enalapril.10 Among all these, the for random pattern cranial based skin flap nitric oxide (NO) has been the target of most elevation. All along the study period, the rats interest and controversy. While some studies were situated in single cages supplied with am- advocate harming effects of nitric oxide on flap ple water and food. survival, many others suggest protective role for it.11-15 NO concentration was increased in Experimental design
rabbit Epigastric Island flaps whereas NO syn- The rats were divided into six equal number thase inhibitors like L-NAME ameliorated the groups (7 rats in each) including one control ischemia induced injury which suggested det- and five treatment groups whose flap survivals rimental effect of NO for flap vitality.11 Con- were evaluated under receiving different single versely, inhibition of NO synthase by L-NAME or combination treatments. Intraperitoneal and aminoguanidine has attenuated oedema administration of a mixture of ketamine and and hyperaemia during one day after flap ele- xylazine (50 and 10 mg/kg, respectively, Pake- vation in rats.12-15 However, plenty of studies do Davis Pharmaceutical Co., Cambridge, UK) was not support the aforementioned ideas about performed for induction of general anesthesia. NO. Nitric oxide plays a role in maintenance of The experimental groups included; 1) control basal blood flow in dorsal flaps of the rats and group that received normal saline by subcuta- its inhibition by L-NAME decreased flap blood neous injection (SC), 2) topical application of flow and survival.13 NO precursor, L-arginine, azelaic acid (100 mg/flap, Merck), 3) SC injec- has shown that promoted angiogenesis and tion of finasteride (1 mg/flap, Reddy, India) at consequently, improved flap microcirculation spots 5.5, 6.5 and 7.5 cm distant from caudal and survival14 by decreased oedema, neutro- margin of flap, 4) intraperitoneal (IP) injection phils infiltration and necrosis.15 of L-NAME (20 mg/kg, Sigma), 5) L-NAME + We focused our study on two substances not azelaic acid [20 mg/kg (IP) and 100 mg/flap tested up to now for their potential effects on skin (topical), respectively] and 6) L-NAME + finas- flap survival, finasteride and azelaic acid. Finasteride teride [20 mg/kg (IP) and 1 mg/flap (SC), is a 5α-reductase inhibitor which decreases dihydro- respectively). In the last two groups, L-NAME testosterone (DHT) and is used in treatment of an- was injected prior to azelaic acid or finasteride drogenic alopecia.16 In a recent study, DHT re- duction by finasteride has stimulated expres- sion of iNOS and as a result, NO production.17 Skin flap surgery and calculation of necrotic
Azelaic acid, an aliphatic dicarboxylic acid is area
used in treatment of acne18, also has inhibitory Half an hour after drug administration, flap effect on 5α-reductase which is compatible with its elevation was performed by making two paral- effect against acne vulgarise, an androgen de- lel caudal to cephalad incisions extending from pendent pathology.19 This similarity in proper- inferior angle of scapulae to superior border of ties of finasteride and azelaic acid encouraged pelvic bones with distance of 3 cm from each us to evaluate their effects on skin flap survival other on the mice backs; so, 8×3 cm flaps were and determine the possible role of NO in this created after final incision connecting distal ends of two parallel cuts and leaving a 3 cm base connected to body for blood supply. After raising the flap and detaching it from underly- This experiment received approval of ethical ing fascia, an impermeable plastic barrier simi- committee of the Center for Research and Train- lar in size to the flap (8×3 cm) was interposi- ing in Skin Disease and Leprosy, Tehran Uni- tioned between the flap and its corresponding versity of Medical Sciences. Forty two Sprauge- bed.20 On the seventh post-operative day, the Journal of Skin & Leishmaniasis, Spring / Summer 2010; Vol 1, No 1. Finasteride and Azelaic acid effects on skin flap Ajami
percentage of skin flap necrosis area was calcu- for data analysis. One way analysis of variance lated using paper template method. The boun- (ANOVA) test was used for comparison of flap dary between viable and necrotic tissue was survival that followed by Tukey test for inter- demarcated considering rosy color, skin soft- group comparisons. P value less than 0.05 was ness, warmth and having hair as markers for considered statistically significant. viability and skin darkening, stiffness, coolness and being devoid of hair as signs of necrosis.21 To calculate the percentage of necrotic area, a As shown in Figure 1, administration of azelaic mould of total flap was drawn and an equal size acid and finasteride kept the random skin flaps transparent paper (representing both viable and from jeopardy. In control group, flap necrotic necrotic skin) was cut and weighed. Then, the area was 48.17% ± 2.22% while for azelaic acid extent of necrosis was outlined on the transpa- and finasteride, the average necrotic areas were rent paper template and was cut and weighed. 32.5% ± 02.55% and 35.0% ± 1.59%, respectively By dividing the weight of paper template of flap (P < 0.05 for both, vs. control group). L-NAME necrosis by that of whole area of flap, the per- treated flaps showed 50% ± 2.75% necrotized centage of necrosis area of flap was obtained. area which was not statistically significant from Immediately after finishing the study, the ani- the control group (P > 0.05). Pre-treatment with mals were killed by intracardiac ketamine injec- L-NAME in azelaic acid treated flaps increased tion (150 mg/kg) and flap tissue samples pre- average necrotic area to 48.0% ± 3.52% (P < 0.01 pared for measurement of necrotic area. vs. azelaic acid) that was not significant com- pared with the control group. Similarly, in the Statistical analysis
case of finasteride, pre-treatment with L-NAME SPSS statistical package (version 14) was used increased necrotic area (P < 0.001 vs. finasteride). Figure 1. Effects of different types of treatments on skin flap survival. Data are shown as mean ±
standard error of mean (SEM) of percent of necrotic area for each group. Azela: azelaic acid, Finas: finasteride. *P < 0.05 vs. control group; #P < 0.01 vs. azelaic acid; and †P < 0.001 vs. finasteride. Journal of Skin & Leishmaniasis, Spring / Summer 2010; Vol 1, No 1. Finasteride and Azelaic acid effects on skin flap Ajami
filling molecule between finasteride inhibitory This study for the first time evaluated the effects effects on 5-alpha-reductase28-29 and protection of finasteride or azelaic acid 20% on skin flap that was induced by finasteride. Finasteride viability after ischemic injury and showed that primarily was marketed for the treatment of both treatments significantly reduced the ne- benign prostatic hypertrophy30, and this was crotic area of skin flap. Second part of the pre- derived from its inhibitory effects on 5-alpha- sent study suggested involvement of NO as a reductase activity.31 Enzyme 5-alpha-reductase possible second messenger in this protective is involved in catalyzing the testosterone to pathway, because pre-treatment with L-NAME, DHT conversion.32 Inhibition of DHT led to a non-selective iNOS inhibitor, abolished finas- increased expression of iNOS in testis and teride and azelaic acid 20% induced protection. epididymis of rats.17 In the present study, There are different molecules that are involved administration of L-NAME prior to the finas- in protection of different tissues against teride blocked its effects on reducing the flap ischemic injury and one of well-known path- necrotic area that suggested involvement of NO ways is induction of inducible NO synthase in this pathway. The role of nitric oxide in pro- (iNOS) and production of NO.22-23 Most of these tective effects of azelaic acid against ischemia studies carried on the cardiac tissue and dem- reperfusion injury in skin flap model also was onstrated that increased expression of inducible assessed and revealed that production of NO is iNOS and subsequent production of NO pro- essential for this effect, because this effect was tected against ischemia reperfusion injury.23 abolished by administration of L-NAME just However, in presence of L-NAME, this type of before topical application of azelaic acid. There tissue protection was lost.24 One of the best are some studies that ultimately support this studied pathways include elevated expression finding. An in vitro study on human skin using of nitric oxide synthase (NOS) and subsequent assay with 1,2[3H]-testosterone as substrate, has production of NO that consequently protects demonstrated that application of azelaic acid tissue by opening the ATP sensitive potassium induced potent inhibitory effect on 5-alpha- channels (KATP channels) and inducing of anti- reductase activity and this effect was detectable apoptotic proteins.25 The role of NO in protec- at concentrations as low as 0.2 mmol/l and was tion of skin flap against ischemic injury has complete at 3 mmol/l.19 A study conducted by been demonstrated in a study by Claytor RB et Kuntscher MV et al., to evaluate the effects of al., which demonstrated that increased tissue NO donor on skin flap protection, demon- survival is correlated with elevated levels of p38 strated that more flap survival can be achieved MAP kinase phosphorylation and interestingly, by intravenous administration of sper- this effect was inhibited by L-NAME that mine/nitric oxide complex before ischemia, prevents NO production.26 Involvement of NO while no protection produced when sper- in skin flap protection was demonstrated by mine/nitric oxide complex administered at the Kuntscher MV et al. in another study that end of the ischemic period or in the early reper- showed administration of NO-donor prior to fusion period.33 In the present study, blocking ischemia protected skin tissue and decreased the iNOS expression and NO production by L- necrotic area whereas L-NAME blocked NO NAME in azelaic acid or finasteride treated synthesis and abolished its' protective effects.27 flaps were correlated with increased flap ne- Similarly, application of L-NAME before azelaic crotic area in these groups. Therefore, it seems acid or finasteride, inhibited their protective that finasteride or azelaic acid by inhibition of properties and increased necrotic area of flaps 5-alpha-reductase activity and decreasing the (Figure 1). Next to the results of skin flaps pre- testosterone to DHT conversion has eliminated pared from finasteride treated and untreated the inhibitory effects of DHT on iNOS expres- rats that showed significant protection by finas- sion, and as a result, this NO dependent path- teride, we hypothesized nitric oxide as a gap way of protection has been triggered. Journal of Skin & Leishmaniasis, Spring / Summer 2010; Vol 1, No 1. Finasteride and Azelaic acid effects on skin flap Ajami
In summary, increased skin flap survival by tials for application in clinic on one hand and finasteride or azelaic acid pre-treatment which highlighted NO as an important signalling requires NO production, suggested their poten- molecule for flap protection on the other hand. References
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J. ENTOMOL. SOC. BRIT. COLUMBIA 100, DECEMBER 2003 Testing an attracticide hollow fibre formulation for control of Codling Moth, Cydia pomonella ALAN L. KNIGHT YAKIMA AGRICULTURAL RESEARCH LABORATORY, AGRICULTURAL RESEARCH SERVICE, USDA. 5230 KONNOWAC PASS RD., WAPATO, WA 98951 Laboratory and field tests were conducted to evaluate the use of an experimentalsprayable formulation of chopped hollow fibres loaded with codlemone and mixed with1.0% esfenvalerate and an adhesive to control codling moth, Cydia pomonella (L.)(Lepidoptera: Tortricidae). Moths were not repelled by the addition of the insecticide tothe adhesive and were rapidly killed following brief contact. A significantly greaterproportion of male moths flew upwind and contacted individual fibres for a longerperiod of time when fibres had been aged > 7 d versus fibres 0 – 7 days-old in flighttunnel tests. Field tests using sentinel fibres placed in 10.0 mg drops of adhesive onplastic disks stapled to the tree found that fibres were not touched until they had aged >8 d. Conversely, moth mortality following a 3-s exposure to field-collected fibresdeposited on the top of leaves was low in bioassays with fibres aged > 8 d. Thedeposition and adhesion of fibres within the apple canopy appear to be two majorfactors influencing the success of this approach. Fibres were found adhering to foliage,fruit, and bark within the orchard; however, visual recovery of fibres following each ofthe three applications was < 5.0%. Both the substrate and the positioning of the fibre onthe substrate influenced fibre retention. The highest proportion of fibres was foundinitially on the upper surface of leaves and this position also had the highest level offibre retention. Fibres on the underside of leaves or partially hanging off of a substratewere dislodged within two weeks.